Note: Descriptions are shown in the official language in which they were submitted.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
COMPOUNDS FOR INHIBITING BETA-AMYLOID PEPTIDE RELEASE AND/OR ITS SYNTHESIS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No:
60/-, , which was converted pursuant to 37 C.F.R. ~ 1.53(b) from U.S.
Patent Application No. 09/102,728, filed Iune 22, 1998; the disclosure of
which
is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to compounds which inhibit (3-amyloid peptide
release and/or its synthesis, and, accordingly, have utility in treating
Alzheimer's
disease.
References
The following publications, patents and patent applications are cited in
this application as superscript numbers:
' Glenner, et al. , Biochem. Biophys. Res. Commun. ( 1984)
120:885-890.
U.S. Patent No. 4,666,829, issued May 19; 1987, to G. G.
Glenner et al., entitled "Polypeptide Marker for Alzheimer's
Disease and Its Use for Diagnosis."
Selkoe, Neuron. ( 1991 ) 6:487-498.
Goate, et al., Nature (1990) 349:704-706.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 2 __
Chartier Harlan, et al., Nature (1989) 353:844-846.
Murrell, et al., Science (1991) 254:97-99.
' Mullan, et al., Nature Genet. (1992) 1:345-347.
Schenk, et al., International Patent Application Publication No.
WO 94/10569, "Methods and Compositions for the Detection of
Soluble ~Amyloid Peptide ", published 11 May 1994.
Selkoe, Scientific American, "Amyoid Protein and Alzheimer's
Disease", pp. 2-8, November, 1991.
'° Yates et al., U.S. Patent No. 3,598,859.
" Tetrahedron Letters 1993, 34(48), 7685.
'z R. F. C. Brown et al., Tetrahedron Letters 1971, 8, 667-670.
" A. O. King et al., J. Org. Chem. 1993, 58, 3384-3386.
'a U.S. Provisional Application Serial No. 60/019,790, filed June
14, 1996.
'S R. D. Clark et al., Tetrahedron 1993, 49(7), 1351-1356.
'6 Citron, et al., Nature (1992) 360:672-674.
' " P. Seubert, Nature (1992) 359:325-327.
'g Hansen, et al., J. Immun. Meth. (1989) 119:203-210.
'9 Games et al. , Nature ( 1995) 373:523-527.
'-° Johnson-Wood et al., PNAS USA (1997) 94:1550-1555.
All of the above publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as if each
individual
publication, patent or patent application was specifically and individually
indicated to be incorporated by reference in its entirety.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 3 __
State of the Art
Alzheimer's Disease (AD) is a degenerative brain disorder characterized
clinically by progressive loss of memory, cognition, reasoning, judgment and
emotional stability that gradually leads to profound mental deterioration and
ultimately death. AD is a very common cause of progressive mental failure
(dementia) in aged humans and is believed to represent the fourth most common
medical cause of death in the United States. AD has been observed in races and
ethnic groups worldwide and presents a major present and future public health
problem. The disease is currently estimated to affect about two to three
million
individuals in the United States alone. AD is at present incurable. No
treatment
that effectively prevents AD or reverses its symptoms and course is currently
known.
The brains of individuals with AD exhibit characteristic lesions termed
senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood
vessels) and neurofibrillary tangles. Large numbers of these lesions,
particularly
amyloid plaques and neurofibrillary tangles, are generally found in several
areas
of the human brain important for memory and cognitive function in patients
with
AD. Smaller numbers of these lesions in a more restrictive anatomical
distribution are also found in the brains of most aged humans who do not have
clinical AD. Amyloid plaques and amyloid angiopathy also characterize the
brains of individuals with Trisomy 21 (Down's Syndrome) and Hereditary
Cerebral Hemorrhage with Amyloidosis of the Dutch Type (HCHWA-D). At
present, a definitive diagnosis of AD usually requires observing the
aforementioned lesions in the brain tissue of patients who have died with the
disease or, rarely, in small biopsied samples of brain tissue taken during an
invasive neurosurgical procedure.
The principal chemical constituent of the amyloid plaques and vascular
amyloid deposits (amyloid angiopathy) characteristic of AD and the other
disorders mentioned above is an approximately 4.2 kilodalton (kD) protein of
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/14007
__ 4 __
about 39-43 amino acids designated the ~i-amyloid peptide (~3AP) or sometimes
A~i, A~iP or p/A4. ~i-Amyloid peptide was first purified and a partial amino
acid
sequence was provided by Glenner, et al.' The isolation procedure and the
sequence data for the first 28 amino acids are described in U.S. Patent No.
4,666,8292.
Molecular biological and protein chemical analyzes have shown that the
~i-amyloid peptide is a small fragment of a much larger precursor protein
termed
the amyloid precursor protein (APP), that is normally produced by cells in
many
tissues of various animals, including humans. Knowledge of the structure of
the
gene encoding APP has demonstrated that ~i-amyloid peptide arises as a peptide
fragment that is cleaved from APP by protease enzyme(s). The precise
biochemical mechanism by which the ~i-amyloid peptide fragment is cleaved from
APP and subsequently deposited as amyloid plaques in the cerebral tissue and
in
the walls of the cerebral and meningeal blood vessels is currently unknown.
Several lines of evidence indicate that progressive cerebral deposition of
~3-amyloid peptide plays a seminal role in the pathogenesis of AD and can
precede cognitive symptoms by years or decades. See, for example, Selkoe'.
~ The most important line of evidence is the discovery that missense DNA
mutations at amino acid 717 of the 770-amino acid isoform of APP can be found
in affected members but not unaffected members of several families with a
genetically determined (familial) form of AD (Goate, et al 4; Chartier Harlan,
et
al.s; and Murrell, et al.b) and is referred to as the Swedish variant. A
double
mutation changing lysine595-methionines~ to asparagine59s-leucine596 (with
reference to the 695 isoform) found in a Swedish family was reported in 1992
(Mullan, et al.'). Genetic linkage analyses have demonstrated that these
mutations, as well as certain other mutations in the APP gene, are the
specific
molecular cause of AD in the affected members of such families. In addition, a
mutation at amino acid 693 of the 770-amino acid isoform of APP has been
identified as the cause of the p-amyloid peptide deposition disease, HCHWA-D,
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 5 __
and a change from alanine to glycine at amino acid 692 appears to cause a
phenotype that resembles AD is some patients but HCHWA-D in others. The
discovery of these and other mutations in APP in genetically based cases of AD
prove that alteration of APP and subsequent deposition of its ~3-amyloid
peptide
fragment can cause AD.
Despite the progress which has been made in understanding the
underlying mechanisms of AD and other ~3-amyloid peptide related diseases;
there remains a need to develop methods and compositions for treatment of the
disease(s). Ideally, the treatment methods would advantageously be based on
drugs which are capable of inhibiting ~i-amyloid peptide release and/or its
synthesis in vivo.
Compounds which inhibit ~i-amyloid peptide release and/or its synthesis
in vivo are disclosed in U.S. Patent Application No. 08/996,422, filed
December
22, 1997 (Attorney Docket No. 002010-062) and entitled "Cycloalkyl, Lactam,
Lactone and Related Compounds, Pharmaceutical Compositions Comprising
Same, and Methods for Inhibiting (3-Amyloid Peptide Release, andlor its
Synthesis by Use of Such Compounds," the disclosure of which is incorporated
herein by reference in its entirety. The present invention is directed to
deoxy
derivatives of such compounds.
30
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 6 __
SUMMARY OF THE INVENTION
This invention is directed to the discovery of a class of compounds which
inhibit (3-amyloid peptide release andlor its synthesis and, therefore, are
useful in
the prevention of AD in patients susceptible to AD and/or in the treatment of
patients with AD in order to inhibit further deterioration in their condition.
Accordingly, in one of its composition aspects, the present invention
provides compounds of formula I:
J _ NH ~ Y ~W I
m n
wherein
W is a cyclic group selected from the group consisting of:
A B C N A
N~ ; N~ ;
R3 O R3
(R4)f
A
~R4)f ~Ra)f ~R4)f
l
N
~f~3 ; Z3 ;
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
~R4)f l~?41_
A
(R4)f
N ' '
S ~R3
R3
R3
N A
O O
a N
w R3
~R4)f
05 R5
~R4)r
~R4)f Q A
N ' '
~ Rs
O
and
wherein
ring A, together with the atoms to which it is attached, forms a
carbocyclic or heterocyclic ring selected from the group consisting of aryl,
cycloalkyl, substituted cycloalkyl, cycioalkenyl, substituted cycloalkenyl,
heteroaryl and heterocyclic;
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ g __
ring B, together with the atoms to which it is attached, forms a
carbocyclic or heterocyclic ring selected from the group consisting of aryl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl and heterocyclic;
ring C, together with the atoms to which it is attached, forms a heteroaryl
or heterocyclic ring;
Y is represented by the formula:
R2 I 2,
CH-C-NH or (CH)a NH
O
provided that at least one Y is -(CHRZ')~-NH-;
R' is selected from the group consisting of alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkenyl, substituted
alkynyl, substituted cycloalkyl, substituted cycioalkenyl, aryl, heteroaryl
and
heterocyclic;
R'- is selected from the group consisting of alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, aryl,
heteroaryl and heterocyclic;
each RZ' is independently selected from the group consisting of hydrogen,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted
alkynyl,
cycloalkyl, aryl, heteroaryl and heterocyclic;
R' is selected from the group consisting of hydrogen, alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, aryl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl and heterocyclic;
each R° is independently selected from the group consisting of alkyl,
substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,
aryl,
CA 02325388 2000-09-21
WO 99/67220 PCTNS99/14007
__ g __
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl and heterocyclic;
RS is selected from the group consisting of alkyl, substituted alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted amino,
aryl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, heterocyclic, thioalkoxy and substituted thioalkoxy;
Q is oxygen, sulfur, -S(O)- or -S(O~- ;
Z is represented by the formula -T-CX'X"C(O)-, wherein T is selected
from the group consisting of a bond covalently linking R' to -CX'X"-, oxygen,
sulfur and -NR6, wherein R6 is hydrogen, acyl, alkyl, aryl or heteroaryl
group;
X' is hydrogen, hydroxy or fluoro,
X" is hydrogen, hydroxy or fluoro, or X' and X" together form an oxo
group;
a is an integer from 2 to about 6;
f is an integer from 0 to 2;
m is an integer equal to 0 or 1;
n is an integer equal to 1 or 2; and pharmaceutically acceptable salts
thereof.
This invention also provides for novel pharmaceutical compositions
comprising a pharmaceutically acceptable carrier and a compound of the formula
I above.
Additionally, in one of its method aspects, this invention is directed to a
method for inhibiting ~i-amyloid peptide release and/or its synthesis in a
cell
which method comprises administering to such a cell an amount of a compound
or a mixture of compounds of formula I above effective in inhibiting the
cellular
release and/or synthesis of ~3-amyloid peptide.
Because the in vivo generation of (3-amyloid peptide is associated with the
pathogenesis of AD8~9, the compounds of formula I can also be employed in
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 10 --
conjunction with a pharmaceutical composition to prophylactically and/or
therapeutically prevent and/or treat AD. Accordingly, in another of its method
aspects, this invention is directed to a prophylactic method for preventing
the
onset of AD in a patient at risk for developing AD which method comprises
administering to said patient a pharmaceutical composition comprising a
pharmaceutically inert carrier and an effective amount of a compound or a
mixture of compounds of formula I above.
In yet another of its method aspects, this invention is directed to a
therapeutic method for treating a patient with AD in order to inhibit further
deterioration in the condition of that patient which method comprises
administering to said patient a pharmaceutical composition comprising a
pharmaceutically inert carrier and an effective amount of a compound or a
mixture of compounds of formula I above.
In formula I above, rings A and B may be the same or different and are
preferably independently selected from the group consisting of aryl,
cycloalkyl,
cycloalkenyl, heteroaryl and heterocyclic. More preferably, rings A and B are
independently selected from the group consisting of aryl and cycioalkyl. Still
more preferably, rings A and B are independently aryl.
Particularly preferred A and B rings include, by way of example, phenyl,
substituted phenyl, including fluoro-substituted phenyl, cyclohexyl and the
like.
Preferred C rings include, by way of example, pyrrolidinyl, piperidinyl,
morpholino and the like.
When m is zero (i.e., there is a covalent bond from R' to NH}, R' is
preferably aryl (including substituted aryl) or heteroaryl (including
substituted
heteroaryl). In this embodiment, further preferred R' groups include
(a) phenyl,
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-11--
(b) a substituted phenyl group of the formula:
Rb,
R
Rb
wherein R~ is selected from the group consisting of acyl, alkyl, alkoxy,
alkylalkoxy, azido, cyano, halo, hydrogen, vitro, trihalomethyl, thioalkoxy,
and wherein Rb and R' are fused to form a heteroaryl or heterocyclic ring with
the phenyl ring wherein the heteroaryl or heterocyclic ring contains from 3 to
8
atoms of which from 1 to 3 are heteroatoms independently selected from the
group consisting of oxygen, nitrogen and sulfur;
Rb and Rb' are independently selected from the group consisting of
hydrogen, halo, vitro, cyano, trihalomethyl, alkoxy, and thioalkoxy with the
proviso that when R' is hydrogen, then Rb and R"~ are either both hydrogen or
both substituents other than hydrogen,
(c) 2-naphthyl,
(d) 2-naphthyl substituted at the 4, 5, 6, 7 and/or 8 positions with 1 to 5
substituents selected from the group consisting alkyl, alkoxy, halo, cyano,
vitro,
trihalomethyl, thioalkoxy, aryl, and heteroaryl,
(e) heteroaryl,
(f) substituted heteroaryl containing 1 to 3 substituents selected from the
group consisting of alkyl, alkoxy, aryl, aryloxy, cyano, halo, vitro,
heteroaryl,
thioalkoxy, thioaryloxy provided that said substituents are not ortho to the
heteroaryl attachment to the -NH group, and
(g) alkyl.
When m is zero, particularly preferred substituted phenyl R' groups
include mono-, di- and tri-substituted phenyl groups including 3,5-
disubstituted
phenyls such as 3,5-dichlorophenyl, 3,5-difluorophenyl, 3,5-
di(trifluoromethyl)-
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 12 --
phenyl, etc.; 3,4-disubstituted phenyls such as 3,4-dichlorophenyl, 3,4-
difluorophenyl, 3-(trifluoromethyl)-4-chlorophenyl, 3-chloro-4-cyanophenyl, 3-
chloro-4-iodophenyl, 3,4-methylenedioxyphenyl, etc.; 4-substituted phenyls
such
as 4-azidophenyl, 4-bromophenyl, 4-chlorophenyl, 4-cyanophenyl, 4-
ethylphenyl, 4-fluorophenyl, 4-iodophenyl, 4-(phenylcarbonyl)phenyl, 4-(1-
ethoxy)ethylphenyl, etc., 3,4,5-trisubsituted phenyls such as 3,4,5-
trifluorophenyl, 3,4,5-trichlorophenyl, etc.
Specific R' groups for when m is zero include 3,4-dichlorophenyl, 4-
phenylfurazan-3-yl, and the like.
When m is zero, other preferred R' substituents include, by way of
example, 2-naphthyl, quinolin-3-yl, 2-methylquinolin-6-yl, benzothiazol-6-yl,
5-
indolyl, phenyl, and the like.
When m is one, preferred R' groups include unsubstituted aryl groups
such as phenyl, 1-naphthyl, 2-naphthyl, etc.; substituted aryl groups such as
monosubstituted phenyls (preferably substituents at 3 or 5 positions);
disubstituted phenyls (preferably substituents at 3 and 5 positions); and
trisubstituted phenyls (preferably substituents at the 3,4,5 positions).
Preferably,
the substituted phenyl groups do not include more than 3 substituents.
Examples
of substituted phenyls include, for instance, 2-chlorophenyl, 2-fluorophenyl,
2-
bromophenyl, 2-hydroxyphenyl, 2-nitrophenyl, 2-methylphenyl, 2-
methoxyphenyl, 2-phenoxyphenyl, 2-trifluoromethylphenyl, 4-fluorophenyl, 4-
chlorophenyl, 4-bromophenyl, 4-nitrophenyl, 4-methylphenyl, 4-hydroxyphenyl,
4-methoxyphenyl, 4-ethoxyphenyl, 4-butoxyphenyl, 4-iso-propylphenyl, 4-
phenoxyphenyl, 4-trifluoromethylphenyl, 4-hydroxymethylphenyl, 3-
methoxyphenyl, 3-hydroxyphenyl, 3-nitrophenyl, 3-fluorophenyl, 3-
chlorophenyl, 3-bromophenyl, 3-phenoxyphenyl, 3-thiomethoxyphenyl, 3-
methylphenyl, 3-trifluoromethylphenyl, 2,3-dichlorophenyl, 2,3-difluorophenyl,
2,4-dichlorophenyl, 2,5-dimethoxyphenyl, 3,4-dichlorophenyl, 3,4-
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 13 --
difluorophenyl, 3,4-methylenedioxyphenyl, 3,4-dimethoxyphenyl, 3,5-
difluorophenyl, 3,5-dichlorophenyl, 3,5-di-(trifluoromethyl)phenyl, 3,5-
dimethoxyphenyl, 2,4-dichlorophenyl, 2,4-difluorophenyl, 2,6-difluorophenyl,
3,4,5-trifluorophenyl, 3,4,5-trimethoxyphenyl, 3,4,5-tri-
(trifluoromethyl)phenyl,
2,4,6-trifluorophenyl, 2,4,6-trimethylphenyl, 2,4,6-tri-
(trifluoromethyl)phenyl,
2,3,5-trifluorophenyl, 2,4,5-trifluorophenyl, 2,5-difluorophenyl, 2-fluoro-3-
trifluoromethylphenyl, 4-fluoro-2-trifluoromethylphenyl, 2-fluoro-4-
trifluoromethylphenyl, 4-benzyloxyphenyl, 2-chloro-6-fluorophenyl, 2-fluoro-6-
chlorophenyl, 2,3,4,5,6-pentafluorophenyl, 2,5-dimethylphenyl, 4-phenylphenyl
and 2-fluoro-3-trifluoromethylphenyl.
When m is one, other preferred R' groups include, by way of example,
adamantyl, benzyl, 2-phenylethyl, 3-phenyl-n-propyl, 4-phenyl-n-butyl, methyl,
ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tent-butyl, n-
pentyl, iso-
valeryl, n-hexyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclopent-
1-
enyl, cyclopent-2-enyl, cyclohex-1-enyl, -CHZ-cyclopropyl, -CH,-cyclobutyl, -
CH,-cyclohexyl, -CHZ-cyclopentyl, -CH~CH~-cyclopropyl, -CH,CH,-cyclobutyl,
-CH,CH,-cyclohexyl, -CH~CH,-cyclopentyl, pyrid-2-yl, pyrid-3-yI, pyrid-4-yl,
fluoropyridyls (including 5-fluoropyrid-3-yl), chloropyridyls (including 5-
chloropyrid-3-yl), thien-2-yl, thien-3-yl, benzothiazol-4-yl, 2-
phenylbenzoxazol-
5-yl, furan-2-yl, benzofuran-2-yl, thionaphthen-2-yl, thionaphthen-3-yl,
thionaphthen-4-yl, 2-chlorothiophen-5-yl, 3-methylisoxazol-5-yl,
2-(thiophenyl)thien-5-yl, 6-methoxythionaphthen-2-yl, 3-phenyl-1,2,4-
thiooxadiazol-S-yl, 2-phenyloxazol-4-yl, indol-3-yl, 1-phenyl-tetraol-S-yl,
allyl,
2-(cyclohexyl)ethyl, (CH3)ZCH=CHCHZCH2CH(CH3)-, ~C(O}CHZ-, thien-2-yl-
methyl, 2-(thien-2-yl)ethyl, 3-(thien-2-yl)-n-propyl, 2-(4-nitrophenyl)ethyl,
2-(4-
methoxyphenyl)ethyl, norboran-2-yl, (4-methoxyphenyl)methyl, (2-
methoxyphenyl)methyl, (3-methoxyphenyl)methyl, (3-hydroxyphenyl)methyl, (4-
hydroxyphenyl)methyl, (4-methoxyphenyl)methyl, (4-methylphenyl)methyl, (4-
fluorophenyl)methyl, (4-fluorophenoxy)methyl, (2,4-dichlorophenoxy)ethyl, (4-
chlorophenyl)methyl, (2-chlorophenyl)methyl, (1-phenyl)ethyl, (1-(p-
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 14 --
chlorophenyl}ethyl, (1-trifluoromethyl)ethyl, (4-methoxyphenyl)ethyl,
CH30C(O)CH,-, benzylthiomethyl, 5-(methoxycarbonyl)-n-pentyl, 3-
(methoxycarbonyl)-n-propyl, indan-2-yl, (2-methylbenzofuran-3-yl),
methoxymethyl, CH3CH=CH-, CH3CH2CH=CH-, (4-chlorophenyl)C(O)CHZ-,
(4-fluorophenyl)C(O)CH~-, (4-methoxyphenyl)C(O)CH,-, 4-(fluorophenyl)-
NHC(O)CHz-, 1-phenyl-n-butyl, (~)ZCHNHC(O)CH,CH,-, (CH3)ZNC(O)CHZ-,
(~)ZCHNHC(O)CHZCHZ-, methylcarbonylmethyl, (2,4-dimethylphenyl)C(O}CH2-
4-methoxyphenyl-C(O)CH,-, phenyl-C(O)CHz-, CH3C(O)N(~)-, ethenyl,
methylthiomethyl, (CH3)3CNHC(O)CH,-, 4-fluorophenyl-C(O)CHZ-,
diphenylmethyl, phenoxymethyl, 3,4-methylenedioxyphenyl-CH,-,
benzo[b]thiophen-3-yl, (CH3)3COC(O)NHCH,-, traps-styryl, HZNC(O)CH~CHZ-,
2-trifluoromethylphenyl-C(O)CH2, ~C(O)NHCH(~)CH~-, mesityl,
CH3CH(=NHOH)CH,-, 4-CH3-~-NHC(O)CHzCHz-, ~C(O)CH(~)CHZ-,
(CH3),CHC(O)NHCH(~)-, CH3CH,OCH~-, CH30C(O)CH(CH3)(CH~)3-, 2,2,2-
trifluoroethyl, 1-(trifluoromethyl)ethyl, 2-CH3-benzofuran-3-yl, 2-(2,4-
dichlorophenoxy)ethyl, ~SO,CH~-, 3-cyclohexyl-n-propyl, CF3CH,CH~CH2- and
N-pyrrolidinyl.
When present, R'- is preferably selected from the group consisting of
alkyl, substituted alkyl, alkenyl, cycloalkyl, aryl, heteroaryl and
heterocyclic.
Each R'-' is preferably (and independently) selected from the group
consisting of hydrogen, alkyl, substituted alkyl, alkenyl, cycloalkyl, aryl,
heteroaryl and heterocyclic.
Particularly preferred RZ and RZ' substituents (when R'-' is other than
hydrogen) include, by way of example, methyl, ethyl, n-propyl, iso-propyl, n-
butyl, iso-butyl, sec-butyl, tert-butyl, -CHZCH(CH,CH3)Z, 2-methyl-n-butyl, 6-
fluoro-n-hexyl, phenyl, benzyl, cyclohexyl, cyclopentyl, cycloheptyl, allyl,
iso-
but-2-enyl, 3-methylpentyl, -CH,-cyclopropyl, -CH,-cyclohexyl, -CHZCHZ-
cyclopropyl, -CHZCHz-cyclohexyl, -CHZ-indol-3-yl, p-(phenyl)phenyl, o-
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/I4007
-- 15 --
fluorophenyl, m-fluorophenyl, p-fluorophenyl, m-methoxyphenyl, p-
methoxyphenyl, phenethyl, benzyl, m-hydroxybenzyl, p-hydroxybenzyl, p-
nitrobenzyl, m-trifluoromethylphenyl, p-(CH3)2NCHZCH,CH20-benzyl, p-
(CH3)3COC(O)CH20-benzyl, p-(HOOCCH~O)-benzyl, 2-aminopyrid-6-yl, p-(N-
morpholino-CH2CH20}-benzyl, -CHzCH2C(O)NH2, -CH2-imidazol-4-yl, -CH,-(3-
tetrahydrofuranyl), -CHZ-thiophen-2-yl, -CHz(1-methyl)cyclopropyl, -CHZ-
thiophen-3-yl, thiophen-3-yl, thiophen-2-yl, -CH,-C(O)O-t-butyl, -CHZ-C(CH3)3,
-CHZCH(CH,CH3)Z, -2-methylcyclopentyl, -cyclohex-2-enyl, -
CH[CH(CH3)2]COOCHj, -CH,CH~N(CH3),, -CH~C(CH3)=CH2, -
CHZCH=CHCH3 (cis and traps), -CH,OH, -CH(OH)CH3, -CH(O-t-butyl)CH3, -
CHZOCH3, -(CHZ)QNH-Boc, -(CHz)4NHZ, -CH,-pyridyl (e.g., 2-pyridyl, 3-
pyridyl and 4-pyridyl), pyridyl (2-pyridyl, 3-pyridyl and 4-pyridyl), -CH2-
naphthyl (e.g., 1-naphthyl and 2-naphthyl), -CH,-(N-morpholino), p-(N-
morpholino-CHzCH20)-benzyl, benzo[b]thiophen-2-yl, 5-
chlorobenzo[b]thiophen-2-yl, 4,5,6,7-tetrahydrobenzo(b]thiophen-2-yl,
benzo[b]thiophen-3-yl, 5-chlorobenzo[b]thiophen-3-yl, benzo[b]thiophen-5-yl, 6-
methoxynaphth-2-yl, -CHZCH~SCH3, thien-2-yl, thien-3-yl, and the like.
Preferably, R'-~ is methyl.
Preferably, R' is selected from the group consisting of hydrogen, alkyl,
substituted alkyl and cycloalkyl. In another preferred embodiment, R' is
alkyl,
substituted alkyl or aryl. More preferably, R' is alkyl.
Particularly preferred R' substituents include, by way of example,
hydrogen, methyl, 2-methypropyl, hexyl, methoxycarbonylmethyl, 3,3-
dimethyl-2-oxobutyl, 4-phenylbutyl, cyclopropylmethyl, 2,2,2-trifluoroethyl,
cyclohexyl, and the like.
When present, R4 is preferably alkyl or substituted alkyl.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 16 --
RS is preferably alkyl; substituted alkyl; phenyl; substituted phenyl, such
as 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyi, 2-chlorophenyl and the
like;
cycloalkyl, such as cyclohexyl and the like; or heteroaryl or heterocyclic,
such as
1-piperdinyl, 2-pyridyl, 2-thiazyl, 2-thienyl and the like.
Preferably, f is 0 or 1. More preferably, f is 0.
When Y is the group -(CHRZ')a NH-, the integer a is preferably 2, 3 or 4,
more preferably 2 or 4, and still more preferably a is equal to 2. In a
preferred
embodiment, Y has the formula -CHRZ'-CH,-NH-, where Rz' is as defined herein,
including the described preferred embodiments.
In one preferred embodiment of this invention, W is a cyclic group of the
formula:
'R6'p ~~ ~~~R7~q
N~ 8
R
O
wherein
each R6 is independently selected from the group consisting of acyl,
acylamino, acyloxy, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy,
alkyl, substituted alkyl, alkynyl, substituted alkynyl, amino, substituted
amino,
aminoacyl, aryl, aryloxy, carboxyl, carboxyalkyl, cyano, cycloalkyl,
substituted
cycloalkyl, halo, heteroaryl, heterocyclic, vitro, thioalkoxy, substituted
thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -
SO-aryl,
-SO-heteroaryl, -SOZ-alkyl, -SO,-substituted alkyl, -SOZ-aryl, and -SO,-
heteroaryl;
CA 02325388 2000-09-21
WO 99/67220 PCTNS99/14007
__ l~ __
each R' is independently selected from the group consisting of acyl,
acylamino, acyloxy, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy,
alkyl, substituted alkyl, alkynyl, substituted alkynyl, amino, substituted
amino,
aminoacyl, aryl, aryloxy, carboxyl, carboxyalkyl, cyano, cycloalkyl,
substituted
cycloalkyl, halo, heteroaryl, heterocyclic, nitro, thioalkoxy, substituted
thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -
SO-aryl,
-SO-heteroaryl, -SO,-alkyl, -SO,-substituted alkyl, -SOZ-aryl, and -S02-
heteroaryl;
R8 is selected from the group consisting of hydrogen, alkyl, substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, aryl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl and heterocyclic;
p is an integer from 0 to 4; q is an integer from 0 to 4.
Preferably, R~ and R' are independently selected from the group
consisting of alkoxy, substituted alkoxy, alkyl, substituted alkyl, amino,
substituted amino. carboxyl, carboxyalkyl, cyano, halo, nitro, thioalkoxy and
substituted thioalkoxy. More preferably, when present, R6 and R' are fluoro.
Rg is preferably selected from the group consisting of hydrogen, alkyl,
substituted alkyl, acyl, aryl, cycloalkyl and substituted cycloalkyl. More
preferably, Rg is selected from the group consisting of hydrogen, alkyl,
substituted alkyl and cycloalkyl.
Particularly preferred R8 substituents include, by way of example,
hydrogen, methyl, 2-methypropyl, hexyl, methoxycarbonylmethyl, 3,3-
dimethyl-2-oxobutyl, 4-phenylbutyl, cyclopropylmethyl, 2,2,2-trifluoroethyl,
cyclohexyl, and the like.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ lg __
In another preferred embodiment of this invention, W is a cyclic group of
the formula:
(R')r
\ /
N
wherein R6, R', and p are as defined herein and r is an integer from 0 to 3.
In still another preferred embodiment of this invention, W is a cyclic
group of the formula:
(R6)P
\ /
N
O
wherein R6 and p are as defined herein.
In yet another preferred embodiment of this invention, W is a cyclic ring
of the formula:
O
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 19 --
wherein R6 and p are as defined herein.
In still another preferred embodiment of this invention, W is a cyclic ring
of the formula:
~Rs)p
R9)9
~Rs)s
'/N
\ R8
O
wherein R6, R8 and p are as defined herein; and
each R9 is independently selected from the group consisting of alkyl,
substituted alkyl, aikenyl, substituted alkenyl, alkynyl, substituted alkynyl,
aryl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl and heterocyclic; and g is an integer from 0 to 2.
When present, R9 is preferably alkyl or substituted alkyl.
In another preferred embodiment of this invention, W is a cyclic ring of
the formula:
~Rs)p
~Rs)s ~Rs)s
~N~
Ra
O
wherein R6, R8, R9, g and p are as defined herein.
In yet another preferred embodiment of this invention, W is a cyclic ring
of the formula:
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 20 --
R9~9 .~~~ R6~P
~R9~9
N~
R8
O
wherein R6, Rg, R9, g and p are as defined herein.
In still another preferred embodiment of this invention, W is a cyclic ring
of the formula:
R8 ~Rs~P
O N \
N~
R$
O
wherein R6, each Rg and p are as defined herein.
In another preferred embodiment of this invention, W is a cyclic ring of
the formula:
18 i ~R6~P
p N \
N~
RB
~R9~9
wherein R6, each R8, R9, g and p are as defined herein.
In another preferred embodiment of this invention, W is a cyclic ring of
the formula:
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 21 --
Rio ~Rs)p
N~
N~Ra
O
wherein R6, Rg and p are as defined herein; and
R'° is selected from the group consisting of alkyl, substituted
alkyl,
alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted amino,
aryl,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
heteroaryl, heterocyclic, thioalkoxy and substituted thioalkoxy.
In another preferred embodiment of this invention, W is a cyclic ring of
the formula:
Rio ~Rs)p
N~
N
E
N-D
wherein R6, R'° and p are as defined herein; and
D-E is selected from the group consisting of alkylene, alkenylene,
substituted alkylene, substituted alkenylene and -N=CH-.
In another preferred embodiment of this invention, W is a cyclic ring of
the formula:
CA 02325388 2000-09-21
WO 99/67220 PC'T/US99/14007
-- 22 --
~Rs~a
~R9~9 Q ~ I
N~
R8
O
wherein R6, R8, R9, g and p are as defined herein; and
Q is oxygen, sulfur, -S(O)- or -S(O}~-.
In another preferred embodiment of this invention, W is a cyclic ring of
the formula:
~Rs~p
~~N
N~
R$
O
wherein R6, R$ and p are as defined herein.
In another preferred embodiment of this invention, W is a cyclic ring of
the formula:
N~
Ra
O
wherein Rg is as defined herein.
CA 02325388 2000-09-21
WO 99!67220 PCT/US99/14007
-- 23 --
In the above formulae, preferably each R6 is independently selected from
the group consisting of alkyl, substituted alkyl, alkoxy and halo; each R' is
independently selected from the group consisting of alkyl, substituted alkyl,
alkoxy and halo; each Rg is independently selected from the group consisting
of
alkyl, substituted alkyl, cycloalkyl and aryl; each R9 is independently
selected
from the group consisting of alkyl, substituted alkyl, cycloalkyl and aryl;
and g,
p, q and r are 0 or 1. More preferably, g, p, q and r are 0.
Compounds of this invention include, by way of example, the following:
H3C CH3 ~
H
HO NCH N~CH3
O CH3 O
i.e., SS-[N'-(2S-hydroxy-3-methylbutyryl)-2S-aminoprop-1-yl]amino-7-
methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one.
The products of this invention include mixtures of R,S enantiomers at any
stereochemical center. Preferably, however, when a chiral product is desired,
the chiral product corresponds to the L-amino acid derivative. In the formulas
set forth herein, a mixture of R,S enantiomers at the stereochemical center is
sometimes indicated by a squiggly line as per convention. Othertimes, no
stereochemical designation is made at the stereochemical center and this also
infers that a mixture of enantiomers is present.
Also included within the scope of this invention are prodrugs of the
compounds of formula I above including acylated forms of alcohols and thiols,
aminals of one or more amines, and the like.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 24 --
DETAILED DESCRIPTION OF THE INVENTION
As above, this invention relates to compounds which inhibit ~i-amyloid
peptide release and/or its synthesis, and, accordingly, have utility in
treating
Alzheimer's disease. However, prior to describing this invention in further
detail, the following terms will first be defined.
Definitions
The term "(3-amyloid peptide" refers to a 39-43 amino acid peptide having
a molecular weight of about 4.2 kD, which peptide is substantially homologous
to the form of the protein described by Glenner, et al.' including mutations
and
post-translational modifications of the normal (3-amyloid peptide. In whatever
form, the (3-amyloid peptide is an approximate 39-43 amino acid fragment of a
large membrane-spanning glycoprotein, referred to as the (3-amyloid precursor
protein (APP). Its 43-amino acid sequence is:
1
Asp Ala Glu Phe Arg His Asp Ser Gly Tyr
~1
Glu Val His His Gln Lys Leu Val Phe Phe
~1
Ala Glu Asp Val Gly Ser Asn Lys Gly Ala
Ile Ile Gly Leu Met Val Gly Gly Val Val
41
Ile Ala Thr (SEQ ID NO: 1 )
or a sequence which is substantially homologous thereto.
"Alkyl" refers to monovalent alkyl groups preferably having from 1 to 20
carbon atoms and more preferably 1 to 6 carbon atoms. This term is exemplified
by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-
hexyl, and the like.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 25 --
"Substituted alkyl" refers to an alkyl group, preferably of from 1 to 10
carbon atoms, having from 1 to 5 substituents,. and preferably 1 to 3
substituents,
selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl,
substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,
acylamino,
acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyacylamino,
cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol,
thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy,
heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -
SO-substituted alkyl, -SO-aryl,-SO-heteroaryl, -SOZ-alkyl, -SOZ-substituted
alkyl,
-SOz-aryl and -SO,-heteroaryl.
"Alkylene" refers to divalent alkylene groups preferably having from 1 to
10 carbon atoms and more preferably 1 to 6 carbon atoms. This term is
exemplified by groups such as methylene (-CHZ-), ethylene (-CHZCHZ ), the
propylene isomers (e.g., -CH,CH~CH,- and -CH(CH3)CH,-) and the like.
"Substituted alkylene" refers to an alkylene group, preferably of from 1 to
10 carbon atoms, having from 1 to 3 substituents selected from the group
consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,
cycloalkoxy, substituted cycloalkoxyl, acyl, acylamino, acyloxy, amino,
substituted amino, aminoacyl, aminoacyloxy, cyano, halogen, hydroxyl,
carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted
thioalkoxy,
aryl, heteroaryl, heterocyclic, heterocyclooxy, nitro -SO-alkyl, -SO-
substituted
alkyl, -SO-aryl, -SO-heteroaryl, -SO,-alkyl, -SOZ-substituted alkyl, -SO~-
aryl,
and -SO,-heteroaryl. Additionally, such substituted alkylene groups include
those where 2 substituents on the alkylene group are fused to form one or more
cycloalkyl, aryl, heterocyclic or heteroaryl groups fused to the alkylene
group.
Preferably such fused cycloalkyl groups contain from 1 to 3 fused ring
structures.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
--26--
"Alkenylene" refers to divalent alkenylene groups preferably having from
2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms. This term is
exemplified by groups such as ethenylene (-CH=CH-), the propenylene isomers
(e.g., -CHZCH=CH- and -C(CH3)=CH-) and the like.
"Substituted alkenylene" refers to an alkenylene group, preferably of from
2 to 10 carbon atoms, having from 1 to 3 substituents selected from the group
consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,
cycloalkoxy, substituted cycloalkoxyl, acyl, acylamino, acyloxy, amino,
substituted amino, aminoacyl, aminoacyloxy, cyano, halogen, hydroxyl,
carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted
thioalkoxy,
aryl, heteroaryl, heterocyclic, heterocyclooxy, nitro -SO-alkyl, -SO-
substituted
alkyl, -SO-aryl, -SO-heteroaryl, -SOZ-alkyl, -SO~-substituted alkyl, -SOZ-
aryl,
and -SO~-heteroaryl. Additionally, such substituted alkylene groups include
those
where 2 substituents on the alkylene group are fused to form one or more
cycloalkyl, aryl, heterocyctic or heteroaryl groups fused to the alkylene
group.
"Alkaryl" refers to -alkylene-aryl groups preferably having from 1 to 8
carbon atoms in the alkylene moiety and from 6 to 10 carbon atoms in the aryl
moiety. Such alkaryl groups are exemplified by benzyl, phenethyi and the like.
"Alkoxy" refers to the group "alkyl-O-" . Preferred alkoxy groups
include, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy,
n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy,
and the like.
"Substituted alkoxy" refers to the group "substituted alkyl-O-" where
substituted alkyl is as defined above.
"Alkylalkoxy" refers to the group "-alkylene-O-alkyl" which includes by
way of example, methylenemethoxy (-CH~OCH3), ethylenemethoxy
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 27 _-
(-CHZCHZOCH3), n-propylene-iso-propoxy (-CH,CHzCH~OCH(CH3)x),
methylene-t-butoxy (-CHZ-O-C(CH3)3) and the like.
"Alkylthioalkoxy" refers to the group "-alkylene-S-alkyl" which includes
by way of example, methylenethiomethoxy (-CHZSCH3), ethylenethiomethoxy
(-CHZCH,SCH3), n-propylene-thio-iso-propoxy (-CHZCH,CHZSCH(CH3)z},
methylenethio-t-butoxy (-CH,SC(CH3)3) and the like.
"Alkenyl" refers to alkenyl groups preferably having from 2 to 10 carbon
atoms and more preferably 2 to 6 carbon atoms and having at least 1 and
preferably from 1-2 sites of alkenyl unsaturation. Preferred alkenyl groups
include ethenyl (-CH=CHZ), n-propenyl (-CH,CH=CHZ}, iso-propenyl (-
C(CH3)=CHZ), and the like.
"Substituted alkenyl" refers to an alkenyl group as defined above having
from 1 to 3 substituents selected from the group consisting of alkoxy,
substituted
alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted
cycloalkoxyl,
acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,
cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol,
thioalkoxy, substituted thioalkoxy, aryl, heteroaryl, heterocyclic,
heterocyclooxy, nitro -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-
heteroaryl,
-SOZ-alkyl, -SO,-substituted alkyl, -SO,-aryl, and -SO~-heteroaryl.
"Alkynyi" refers to alkynyl groups preferably having from 2 to 10 carbon
atoms and more preferably 2 to 6 carbon atoms and having at least l and
preferably from 1-2 sites of alkynyl unsaturation. Preferred alkynyl groups
include ethynyl (-C=CH), propargyl (-CH,C---CH) and the like.
"Substituted alkynyl" refers to an alkynyl group as defined above having
from 1 to 3 substituents selected from the group consisting of alkoxy,
substituted
alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted
cycloalkoxyl,
CA 02325388 2000-09-21
WO 99/67220 PCTNS99/14007
__ 2g __
acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyioxy,
cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol,
thioalkoxy, substituted thioalkoxy, aryl, heteroaryl, heterocyclic,
heterocyclooxy, nitro -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-
heteroaryl,
-S02-alkyl, -SOZ-substituted alkyl, -SOZ-aryl, and -SOZ-heteroaryl.
"Acyl" refers to the groups alkyl-C(O)-, substituted alkyl-C(O)-,
cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)-
and
heterocyclic-C(O)- where alkyl, substituted alkyl, cycloalkyl, substituted
cycloalkyl, aryl, heteroaryl and heterocyclic are as defined herein.
"Acylamino" refers to the group -C(O)NRR where each R is
independently selected from the group consisting of hydrogen, alkyl,
substituted
alkyl, aryl, heteroaryl, heterocyclic and where both R groups are joined to
form
a heterocyclic group, wherein alkyl, substituted alkyl, aryl, heteroaryl and
heterocyclic are as defined herein.
GAmino" refers to the group -NH,.
"Substituted amino" refers to the group -N(R~ where each R is
independently selected from the group consisting of hydrogen, alkyl,
substituted
alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,
cycloalkyl,
substituted cycloalkyl, heteroaryl, heterocyclic and where both R groups are
joined to form a heterocyclic group. When both R groups are hydrogen, -N(R)Z
is an amino group. Examples of substituted amino groups include, by way of
illustration, mono- and di-alkylamino, mono- and di-(substituted alkyl)amino,
mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di-
heterocyclic amino, and unsymmetric di-substituted amines having different
substituents selected from alkyl, substituted alkyl, aryl, heteroaryl and
heterocyclic, and the like.
CA 02325388 2000-09-21
WO 99/67220 PCT/iJS99/14007
-- 29 --
The term "amino-blocking group" or "amino-protecting group" refers to
any group which, when bound to an amino group, prevents undesired reactions
from occurring at the amino group and which may be removed by conventional
chemical and/or enzymatic procedures to reestablish the amino group. Any
known amino-blocking group may be used in this invention. Typically, the
amino-blocking group is selected so as to render the resulting blocked-amino
group unreactive to the particular reagents and reaction conditions employed
in a
subsequent pre-determined chemical reaction or series of reactions. After
completion of the reaction(s), the amino-blocking group is selectively removed
to
regenerate the amino group. Examples of suitable amino-blocking groups
include, by way of illustration, tent-butoxycarbonyl (Boc), benzyloxycarbonyl
(Cbz), acetyl, 1-(1'-adamantyl)-1-methylethoxycarbonyl (Acm), allyloxycarbonyl
(Aloc), benzyloxymethyl (Bom), 2 p-biphenylisopropyloxycarbonyl (Bpoc), tert-
butyldimethylsilyl (Bsi), benzoyl (Bz), benzyl (Bn), 9-fluorenyl-
methyloxycarbonyl (Fmoc), 4-methylbenzyl, 4-methoxybenzyl, 2-
nitrophenylsulfenyl (Nps), 3-nitro-2-pyridinesulfenyl (NPys), trifluoroacetyl
(Tfa}, 2,4,6-trimethoxybenzyl (Tmob), trityl (Trt), and the like. If desired,
amino-blocking groups covalently attached to a solid support may also be
employed.
"Aminoacyl" refers to the group -NRC(O)R where each R is
independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or
heterocyclic
wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as
defined
herein.
"Aminoacyloxy" refers to the group -NRC(O)OR where each R is
independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or
heterocyclic
wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as
defined
herein.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
--30--
"Acyloxy" refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-,
cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)-, aryl-C(O)O-, heteroaryl-
C(O)O-, and heterocyclic-C(O)O- wherein alkyl, substituted alkyl, cycloalkyl,
substituted cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined
herein.
"Aryl" refers to an unsaturated aromatic carbocyclic group of from 6 to
14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed
(fused)
rings (e.g., naphthyl or anthryl). Preferred aryls include phenyl, naphthyl
and
the like.
Unless otherwise constrained by the definition for the aryl substituent,
such aryl groups can optionally be substituted with from 1 to 5 substituents
selected from the group consisting of acyloxy, hydroxy, acyl, alkyl, alkoxy,
alkenyl, alkynyl, substituted alkyl, substituted alkoxy, substituted alkenyl,
substituted alkynyl, amino, substituted amino, aminoacyl, acylamino, alkaryl,
aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl,
heterocyclic, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioaIkoxy,
thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl,
-SO-heteroaryl, -SO~-alkyl, -SO:-substituted alkyl, -SO,-aryl,
-SO=-heteroaryl and trihalomethyl. Preferred substituents include alkyl,
alkoxy,
halo, cyano, nitro, trihalomethyl, and thioalkoxy.
"Aryloxy" refers to the group aryl-O- wherein the aryl group is as defined
above including optionally substituted aryl groups as also defined above.
"Carboxyalkyl" refers to the groups "-C(O)Oalkyl" and "-C(O)O-
substituted alkyl" where alkyl is as defined above.
"Cycloalkyl" refers to cyclic alkyl groups of from 3 to 12 carbon atoms
having a single cyclic ring or multiple condensed rings. Such cycloalkyl
groups
include, by way of example, single ring structures such as cyclopropyl,
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 31 --
cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures
such
as adamantanyl, and the like.
"Substituted cycloalkyl" refers to cycloalkyl groups having from 1 to 5
(preferably 1 to 3) substituents selected from the group consisting of alkoxy,
substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl,
substituted
cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl,
aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl, carboxyl,
carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy,
aryl,
aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy,
hydroxyamino,
alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-
heteroaryl, -
SOZ-alkyl, -SO~-substituted alkyl, -SOZ-aryl, and -SOZ-heteroaryl.
"Cycloalkenyl" refers to cyclic alkenyl groups of from 4 to 8 carbon
atoms having a single cyclic ring and at least one point of internal
unsaturation.
Examples of suitable cycloalkenyl groups include, for instance, cyclobut-2-
enyl,
cyclopent-3-enyl, cyclooct-3-enyl and the like.
"Substituted cycloalkenyl" refers to cycloalkenyl groups having from 1 to
5 substituents selected from the group consisting of alkoxy, substituted
alkoxy,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
acyl,
acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,
oxyacylamino, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto,
thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl,
heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,
-SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SOZ-alkyl, -SOZ-
substituted alkyl, -SO~-aryl, and -SOZ-heteroaryl.
"Halo" or "halogen" refers to fluoro, chloro, bromo and iodo and
preferably is either fluoro or chloro.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
--32--
"Heteroaryl" refers to an aromatic group of from 1 to 15 carbon atoms
and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within at
least
one ring (if there is more than one ring).
Unless otherwise constrained by the definition for the heteroaryl
substituent, such heteroaryl groups can be optionally substituted with I to 5
substituents selected from the group consisting of acyloxy, hydroxy, acyl,
alkyl,
alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy, substituted
alkenyl, substituted alkynyl, amino, substituted amino, aminoacyl, acylamino,
IO alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro,
heteroaryl, heterocyclic, aminoacyloxy, oxyacylamino, thioalkoxy, substituted
thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -
SO-aryl,
-SO-heteroaryl, -SOZ-alkyl, -SOZ-substituted alkyl, -SO,-aryl,
-SO~-heteroaryl and trihalomethyl. Such heteroaryl groups can have a single
ring
(e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or
benzothienyl}. Preferred heteroaryls include pyridyl, pyrrolyl and furyl.
"Heteroaryloxy" refers to the group "-O-heteroaryl".
"Heterocycle" or "heterocyclic" refers to a monovalent saturated or
unsaturated group having a single ring or multiple condensed rings, from 1 to
15
carbon atoms and from I to 4 hetero atoms selected from nitrogen, sulfur or
oxygen within the ring.
Unless otherwise constrained by the definition for the heterocyclic
substituent, such heterocyclic groups can be optionally substituted with 1 to
S
substituents selected from the group consisting of alkoxy, substituted alkoxy,
cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl,
acyl,
acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,
oxyacylamino, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto,
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 33 --
thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,
heteroaryl,
heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, vitro,
-SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SOZ alkyl, -SOZ-
substituted alkyl, -SO,-aryl, and -S02-heteroaryl. Such heterocyclic groups
can
have a single ring or multiple condensed rings. Preferred heterocyclics
include
morpholino, piperidinyl, and the like.
Examples of heterocycles and heteroaryls include, but are not limited to,
pyrrole, furan, imidazole, pyrazole, pyridine, pyrazine, pyrimidine,
pyridazine,
indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline,
quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline,
pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline,
isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine,
imidazoline, piperidine, piperazine, indoline, morpholino, piperidinyl,
tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen containing
heterocycles.
"Heterocyclooxy" refers to the group "-O-heterocycle".
"Keto" or "oxo" refers to the group "=O".
"Oxyacylamino" refers to the group -OC(O)NRR where each R is
independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or
heterocyclic
wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as
defined
herein.
"Thiol" refers to the group -SH.
"Thioalkoxy" refers to the group -S-alkyl.
"Substituted thioalkoxy" refers to the group -S-substituted alkyl.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 34 --
"Thioaryloxy" refers to the group aryl-S- wherein the aryl group is as
defined above including optionally substituted aryl groups also defined above.
"Thioheteroaryloxy" refers to the group heteroaryl-S- wherein the
heteroaryl group is as defined above including optionally substituted aryl
groups
as also defined above.
"Thioketo" refers to the group "=S".
The term "5,7-dihydro-6H-dibenz[b,d]azepin-6-one" refers to a polycyclic
E-caprolactam ring system having the formula:
10 11 12 13
9
d/ 3 2 \b ~ 14
8 4 1\~ 15
.. 7. .,
wherein, for nomenclature purposes, the atoms and bonds are numbered and
lettered, respectively, as shown.
The term "5,6-dihydro-4H-quino[8,1-ab][3]benzazepin-8(9H)-one" refers
to a polycyclic E-caprolactam ring system having the formula:
12 13 1 2
11 ~ 13 3
13b ~ ~ 3a
9a 9 8 N7 4
\6
wherein, for nomenclature purposes, the atoms and bonds are numbered and
lettered, respectively, as shown.
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/14007
-- 35 --
The term "1,3,4,7,I2,I2a-hexahydropyrido[2,1-b][3]benzazepin-6(2H)-
one" refers to a polycyclic E-caprolactam ring system having the formula:
11
11a
12 12a
1
8 7a
6 N~4
3
wherein, for nomenclature purposes, the atoms and bonds are numbered and
5 lettered, respectively, as shown.
The term "4,5,6,7-tetrahydro-3,7-methano-3H-3-benzazonin-2(1H)-one"
refers to a polycyclic e-caprolactam ring system having the formula:
s
11 11a
10 wherein, for nomenclature purposes, the atoms and bonds are numbered and
lettered, respectively, as shown.
As to any of the above groups which contain 1 or more substituents, it is
understood, of course, that such groups do not contain any substitution or
substitution patterns which are sterically impractical and/or synthetically
non-
feasible.
"Pharmaceutically acceptable salt" refers to pharmaceutically acceptable
salts of a compound of formula I which salts are derived from a variety of
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 36 --
organic and inorganic counter ions well known in the art and include, by way
of
example only, sodium, potassium, calcium, magnesium, ammonium,
tetraalkylammonium, and the like; and when the molecule contains a basic
functionality, salts of organic or inorganic acids, such as hydrochloride,
hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like can
be
used as the pharmaceutically acceptable salt.
The term "protecting group" or "blocking group" refers to any group
which when bound to one or more hydroxyl, thiol, carboxyl groups or other
protectable functional group of the compounds which prevents reactions from
occurring at these groups and which protecting group can be removed by
conventional chemical or enzymatic steps to reestablish the unprotected
functional
group. The particular removable blocking group employed is not critical and
preferred removable hydroxyl blocking groups include conventional substituents
such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine,
phenacyl, t-
butyl-diphenylsilyl and any other group that can be introduced chemically onto
a
hydroxyl functionality and later selectively removed either by chemical or
enzymatic methods in mild conditions compatible with the nature of the
product.
Preferred carboxyl protecting groups include esters such as methyl, ethyl,
propyl, t-butyl etc. which can be removed by mild hydrolysis conditions
compatible with the nature of the product.
compound Preparation
When m and n are one, the compounds of formula I are readily prepared
by conventional reductive amination, followed by acylation as illustrated in
Scheme 1.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 3~ __
Scheme 1
(R6)P ._,_ _, (R~)a
B' -H-(CH)a_~ C~ + ~ / \
R2'
N
H2N ~Ra
O 2
1 ) Reductive Amination
2) Deprotection
(Rs)P .' (R')a
z,
N
H N-(CH) H \R$
O
O 3
X" OH Acylation
T R'
(R6)P _ (R')q
0 12, r--
N~ a
X H-(CH)a H O R
TR'
As shown in Scheme 1, a protected aldehyde or ketone ~ (where B' is a
protecting group and RZ' and a are as defined herein) can be coupled with an
amine compound, such as ~ (where R°, R', R8, p and q are as defined
herein), by
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__3g__
conventional reductive amination to provide, after deprotection, intermediate
~.
In Scheme 1, amine ~ is merely representative and those skilled in the art
will
recognize that amino derivatives of any of the other ring systems described
herein
may be employed in this reaction. Typically, this reaction is conducted by
contacting amine ~ with an excess ofl, preferably with 1.1 to 2 equivalents
ofd,
and an excess, preferably 1.1 to 1.5 equivalents, of a reducing agent, such as
sodium cyanoborohydride. Generally, this reaction is conducted in an
essentially
inert diluent, such as methanol, at a temperature ranging from about
0°C to about
50°C, preferably at ambient temperature, for about 0.5 to 3 hours.
Removal of
the amine protecting group using conventional procedures and reagents then
affords intermediate ~.
Intermediate ~ can then be acylated or coupled with a carboxylic acid,
e.g., 4_ (where R', T, X' and X" are as defined herein), to provide compound.
This reaction is typically conducted using conventional coupling reagents and
procedures and at least a stoichiometric amount of intermediate ~ and
carboxylic
acid 4_. For example, well known coupling reagents such as carbodiimides with
or without the use of well known additives such as N-hydroxysuccinimide, 1-
hydroxybenzotriazole, etc. can be used to facilitate coupling. The reaction is
conventionally conducted in an inert aprotic polar diluent such as
dimethylformamide, dichloromethane, chloroform, acetonitrile, tetrahydrofuran
and the like. Alternatively, the acid halide of compound ~ can be employed in
reaction (1) and, when so employed, it is typically employed in the presence
of a
suitable base to scavenge the acid generated during the reaction. Suitable
bases
include, by way of example, triethylamine, diisopropylethylamine, N-
methylmorpholine and the like.
Alternatively, compound ~ can be prepared by reductive amination of a
compound of formula ~:
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
--39--
X, O R2~
N- CH -C
X.. _ H ~ )a_~ ~ 2
R'
TRH 6
where R', RZ', T, X', X" and a are as defined herein, with amine, using
conventional reagents and procedures.
Compounds of formula 1, where n is 1 and m is 0, can similarly be
prepared by reductive amination of an intermediate of formula 7:
R
R~-N-(CH)a_~ C ~
B2 R2,
7
where B'- is a suitable amine protecting group and R' , R'-~ and a are as
defined
herein, with amine 2, followed by deprotection using conventional procedures.
When n is two and both of Y are represented by the formula -(CHRZ')a
NH-, the compounds of formula I can be prepared by further reductive
alkylation
of intermediate ~ with, for example, compound ~ or 7, using conventional
reagents and procedures. Alternatively, intermediate ~ can be reductively
alkylated with protected aldehyde or ketone ~ and the resulting intermediate
deprotected and acylated using procedures similar to those described above.
Compounds in which n is two and one of Y is represented by the formula
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 40 --
-(CHRZ')~ NH- and the other Y is represented by the formula -CHRZ-C(O)NH-
can be prepared by coupling intermediate ~ with a protected carboxylic acid of
formula $:
R2
~ OH
R~' l Z ~ NH
m
O
where R', RZ, Z and m are as defined herein, using conventional coupling
reagents and procedures such as those described above, to afford compounds of
formula L'
Alternatively, compounds of formula I can be prepared by first coupling
an amine, such as _2, with a protected carboxylic acid ~, and then, after
deprotection, reductively alkylating the resulting intermediate ~Q ~as
illustrated in
Scheme 2.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 41 --
Scheme 2
2
~Rs)P _ _ {R')q
B1 _N
OH +
H O
9 H2N~yRs
1 ) Acylation _O 2
2) Deprotection
~R6)p _ ~R')q
RZ ~ /
N
N ~Ra
HZN ~ H O
O
Reductive Alkylation, 6 or 7
or
1 ) Reductive Alkyfation, 1
2) Deprotection
3) Acylation, 4
Compound of Formula I
When n is 2, the aldehyde, ketone or carboxylic acid units may also be
coupled together prior to reaction with an amine using the reductive amination
or
acylation procedures described above, as appropriate. The resulting
intermediate
5 is then coupled to the amine, such as 2, to afford compounds of formula I.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 42 --
Synthesis of Alde~t rde Ketone ands'arboxvlic Acid Starting materials
The aldehyde, ketone and carboxylic acids employed in the above
reactions can be readily prepared by several divergent synthetic routes with
the
particular route selected relative to the ease of compound preparation,
commercial availability of starting materials, whether m is zero or one,
whether n
is one or two, etc.
A. Synthesis of Aldehydes and Ketones
The aldehyde and ketone compounds, e.g. ~, ~ and 7, employed in this
invention can be readily prepared by oxidizing the corresponding alcohol using
conventional oxidizing agents. For example, Swern oxidation of N protected
amino primary alcohols affords the corresponding aldehyde. Typically, this
reaction is conducted by contacting the alcohol with a mixture of oxalyl
chloride
and dimethyl sulfoxide in the presence of a tertiary amine, such as
triethylamine.
Generally, this reaction is conducted in an inert diluent, such as
dichloromethane,
at an intial temperature of about -78°C and then at ambient temperature
for about
0.25 to 2 hours to afford the aldehyde. The alcohols employed in this reaction
are either commercially available or can be prepared using conventional
reagents
and procedures. For example, suitable alcohols can be prepared by reduction of
the corresponding amino acids or amino acid esters using conventional reducing
agents such as lithium aluminum hydride and the like.
B. Synthesis of Carboxylic Acids
The carboxylic acids of formula $, where m is 0, can be prepared by
various conventional procedures. For example, reaction of a halo acetic acid ~
(where Z' is a halo group such as chloro or bromo and RZ is as defined herein)
with a primary amine ~ (where R' is as defined herein) provides an amino acid
~ as illustrated in Scheme 3. Alternatively, leaving groups other than halo
may
be employed such as triflate and the like. Additionally, suitable esters of I~
may
be employed in this reaction.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 43 --
O Scheme 3 O
Z, H
OH -I- R'-NH2 _"~' R~~ N OH
R? 11 12 13 Rz
As shown in Scheme 3, a suitable haloacetic acid derivative ~ is reacted
with a primary amine ~2 under conditions which provide for amino acid. This
reaction is described by, for example, Yates, et al.'° and proceeds by
combining
approximately stoichiometric equivalents of haloacetic acid ~ with primary
amine ~ in a suitable inert diluent such as water, dimethylsulfoxide (DMSO)
and
the like. The reaction employs an excess of a suitable base such as sodium
bicarbonate, sodium hydroxide, etc. to scavenge the acid generated by the
reaction. The reaction is preferably conducted at from about 25°C to
about
100°C until reaction completion which typically occurs within 1 to
about 24
hours. This reaction is further described in U.S. Patent No. 3,598.859, which
is
incorporated herein by reference in its entirety. Upon reaction completion, N
substituted amino acid ~ is recovered by conventional methods including
precipitation, chromatography, filtration and the like.
Each of the reagents employed in this reaction (e.g., haloacetic acid ~,
and primary amine j~) are well known in the art with a plurality of each being
commercially available.
In an alternative embodiment, the R' group can be coupled to an alanine
ester {or other suitable amino acid ester) by conventional N arylation. For
example, a stoichiometric equivalent or slight excess of the amino acid ester
can
be dissolved in a suitable diluent such as DMSO and coupled with a halo-R'
compound, Z'-R' where Z' is a halo group such as chloro or bromo and R' is as
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 4ø __
defined above. The reaction is conducted in the presence of an excess of base
such as sodium hydroxide to scavenge the acid generated by the reaction. The
reaction typically proceeds at from 15°C to about 250°C and is
complete in about
1 to 24 hours. Upon reaction completion, N substituted amino acid ester is
recovered by conventional methods including chromatography, filtration and the
like. This ester is then hydrolyzed by conventional methods to provide for
carboxylic acid $, where m is 0.
In still another alternative embodiment, esterified amino acids of formula
$, where m is 0, can be prepared by reductive amination of a suitable pyruvate
ester 14 (where R is typically an alkyl group and RZ is as defined above} with
a
primary amine 12 (where R' is as defined herein) in the manner illustrated in
Scheme 4.
Scheme 4 O
O H
N
OR .i- R~-NH2 - H-~- R~~ OR
R2 , ~ Catalyst
2
14 O 12 15 R
The reaction shown in Scheme 4 is typically conducted by combining
approximately stoichiometric equivalents of pyruvate ester ,~ and amine ~2 in
an
inert diluent such as methanol, ethanol and the like under conditions which
provide for imine formation (not shown). The imine formed is then reduced
under conventional conditions by a suitable reducing agent such as sodium
cyanoborohydride, HZ/palladium on carbon and the like to form the N
substituted
amino acid ester ~. In a particularly preferred embodiment, the reducing agent
is HZ/palladium on carbon which is incorporated into the initial reaction
medium
which permits imine reduction in situ in a one pot procedure to provide for
the N
substituted amino acid ester ~.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 45 --
The reaction is preferably conducted at from about 20°C to about
80°C at
a pressure of from 1 to 10 atmospheres until reaction completion which
typically
occurs within 1 to about 24 hours. Upon reaction completion, N-substituted
amino acid ester ~5 is recovered by conventional methods including
chromatography, filtration and the like. Subsequent hydrolysis of the ester ~5
leads to the corresponding carboxylic acid derivative $, where m is 0.
The carboxylic acids of formula $, where m is 1, can be prepared by
conventional coupling of an acetic acid derivative ~ (where R', T, X' and X"
are
as defined herein) with a primary amine of an esterified amino acid (where R
is
typically an alkyl group and R'- is as defined herein) as illustrated in
Scheme 5.
Scheme 5
O O
X OH + H2N OR
TRH 4 R2 16
X. X.. O
H
R'T N 17
~OR
O
R2
As shown in Scheme S, this reaction merely involves coupling of a
suitable acetic acid derivative 4 with the primary amine of amino acid ester~~
under conditions which provide for the N acetyl derivative ~. This reaction is
conventionally conducted as described for peptide synthesis and synthetic
methods used therein can also be employed to prepare the N acetyl amino acid
esters ~ of this invention. For example, well known coupling reagents such as
carbodiimides with or without the use of well known additives such as N-
hydroxysuccinimide, 1-hydroxybenzotriazole, etc. can be used to facilitate
coupling. The reaction is conventionally conducted in an inert aprotic polar
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 46 --
diluent such as dimethylformamide, dichloromethane, chloroform, acetonitrile,
tetrahydrofuran and the like. Alternatively, the acid halide of compound ~ can
be
employed and, when so employed, it is typically employed in the presence of a
suitable base to scavenge the acid generated during the reaction. Suitable
bases
include, by way of example, triethylamine, diisopropylethylamine, N
methylmorpholine and the like.
The coupling reaction of 4 and ,~ is preferably conducted at from about
0°C to about 60°C until reaction completion which typically
occurs within 1 to
about 24 hours. Upon reaction completion, N acetyl amino acid ester ~ is
recovered by conventional methods including precipitation, chromatography,
filtration and the like or alternatively is hydrolyzed to the corresponding
acid
without purification and/or isolation other than conventional work-up (e.g.,
aqueous extraction, etc.).
Each of the reagents (e.g., acetic acid derivative 4 and amino acid ester
~.ø) are well known in the art with a plurality of each being commercially
available.
Carboxylic acids, such as 4, can also be coupled to amines prepared by
use of polymer supported forms of carbodiimide peptide coupling reagents. A
polymer supported form of EDC, for example, has been described (Tetrahedron
Letters, 34(48), 7685 (1993))1'. Additionally, a new carbodiimide coupling
reagent, PEPC, and its corresponding polymer supported forms have been
discovered and are very useful for the preparation of such compounds.
Poiymers suitable for use in making a polymer supported coupling reagent
are either commercially available or may be prepared by methods well known to
the artisan skilled in the polymer arts. A suitable polymer must possess
pendant
sidechains bearing moieties reactive with the terminal amine of the
carbodiimide.
Such reactive moieties include chloro, brorno, iodo and methanesulfonyl.
CA 02325388 2000-09-21
WO 99/67220 PCTNS99/14007
__ 4? __
Preferably, the reactive moiety is a chloromethyl group. Additionally, the
polymer's backbone must be inert to both the carbodiimide and reaction
conditions under which the ultimate polymer bound coupling reagents will be
used.
Certain hydroxymethylated resins may be converted into chloromethylated
resins useful for the preparation of polymer supported coupling reagents.
Examples of these hydroxylated resins include the 4-hydroxymethylphenyl-
acetamidomethyl resin (Pam Resin) and 4-benzyloxybenzyl alcohol resin (Wang
Resin) available from Advanced Chemtech of Louisville, Kentucky, USA (see
Advanced Chemtech 1993-1994 catalog, page 115). The hydroxymethyl groups
of these resins may be converted into the desired chloromethyl groups by any
of a
number of methods well known to the skilled artisan.
Preferred resins are the chloromethylated styrene/divinylbenzene resins
because of their ready commercial availability. As the name suggests, these
resins are already chloromethylated and require no chemical modification prior
to
use. These resins are commercially known as Merrifield's resins and are
available from Aldrich Chemical Company of Milwaukee, Wisconsin, USA {see
Aldrich 1994-1995 catalog, page 899). Methods for the preparation of PEPC and
its polymer supported forms are outlined in Scheme 6.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 4g
Scheme 6
~NCO O
HzN~N~ ---t /~N~N~\/~N
H H
O
S' CI
O
N~N C N
~LG
P
Functionalized Resin
where O = an inert polymer
and LG = CI, Br. I or OS02CH~
N~N C N~
OP ~ CI '
Such methods are described more fully in U.S. Patent Application Serial
No. 60/019,790'4 filed June 14, 1996 which application is incorporated herein
by
reference in its entirety. Briefly, PEPC is prepared by first reacting ethyl
isocyanate with 1-(3-aminopropyl)pyrrolidine. The resulting urea is treated
with
4-toluenesulfonyl chloride to provide PEPC. The polymer supported form is
prepared by reaction of PEPC with an appropriate resin under standard
conditions to give the desired reagent.
The carboxylic acid coupling reactions employing these reagents are
performed at about ambient to about 45°C, for from about 3 to 120
hours.
Typically, the product may be isolated by washing the reaction with CHCl3 and
concentrating the remaining organics under reduced pressure. As discussed
supra, isolation of products from reactions where a polymer bound reagent has
been used is greatly simplified, requiring only filtration of the reaction
mixture
and then concentration of the filtrate under reduced pressure.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 49 --
Benzodi,gzepines and Relaygd Com~s~nds)
The cyclic compounds and amino-substituted derivatives thereof, such as
~, employed in the reactions described above are either known in the art or
can
be prepared by art-recognized procedures using commercially available starting
materials and reagents.
For example, 5,7-dihydro-6H-dibenz[b,d]azepin-6-one may be prepared
by cyclizing a chloromethyl amide intermediate using the procedures set forth
in
R. F. C. Brown et al., Tetrahedron Letters 1971, 8, 667-670'2 and references
cited therein.
Additionally, the synthesis of a representative cyclic compuond, i.e., a
5,7-dihydro-6H-dibenz[b,d]azepin-6-one, is illustrated in Scheme 7. As will be
readily apparent to those of ordinary skill in the art, the synthetic
procedure
illustrated in Scheme 1 and the reaction conditions described below can be
modified by selecting the appropriate starting materials and reagents to allow
the
preparation of other cyclic amines suitable for use in this invention.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99114007
-- SO --
Scheme 7
~Rs)P ~R7)q
Br Br
1$ CH3 H2N 20
R' q
~Rs)P O Ra ~ )
Br
O Ra
Boc-N
19 CH3 H 21
~Rs)P ~ ~ ~R~)q
CH3 NH
22 Boc
~Rs)P\/1 ~/~R )a
O
23
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 51 __
As shown in Scheme 7, 5,7-dihydro-6H-dibenz[b,d)azepin-6-one
derivatives, 23, wherein R6, R', p and q are as defined above, can be readily
prepared in several steps from a 2-bromotoluene derivative 18 and a 2-
bromoaniline derivative 20. In this synthetic procedure, the 2-bromotoluene
derivative, 18, is first converted into the corresponding 2-
methylphenylboronate
ester, 19. This reaction is typically conducted by treating 18 with about I .0
to
about 2.1 equivalents of an alkyl lithium reagent, preferably sec-butyl
lithium or
tert-butyl lithium, in an inert diluent, such as THF, at a temperature ranging
from
about -80 ° C to about -60 ° C for about 0.25 to about 1 hour.
The resulting
lithium anion is then treated in situ with an excess, preferably 1.5
equivalents, of
a trialkylborate, such as trimethylborate. This reaction is initially
conducted at -
80°C to about -60°C and then allowed to warm to about 0°C
to about 30°C for
about 0.5 to about 3 hours. The resulting meEhyl boronate ester is typically
not
isolated, but is preferably converted in situ into the pinacol ester by
treating the
reaction mixture with an excess, preferably about 2.0 equivalents, of pinacol.
This reaction is typically conducted at ambient temperature for about 12 to
about
24 hours to afford the 2-methylphenylboronate ester, 19, in which both I~
groups
are preferably joined together to form -C(CH3)zC(CH3)z-.
In a separate reaction, the amino group of a 2-bromoaniline derivative,
20, is converted into the N Boc derivative 21 by treating 20 with about 1.0 to
about 1.5 equivalents of di-tert-butyl-dicarbonate. Typically, this reaction
is
conducted at a temperature ranging from 25°C to about 100°C for
about 12 to 48
hours to afford the N Boc-2-bromoaniline derivative 21.
As further illustrated in Scheme 7, the 2-methylphenylboronate ester, 19,
and the N Boc-2-bromoaniline derivative 21 can then be coupled to form the
biphenyl derivative 22. This reaction is typically conducted by contacting 21
with about 1.0 to about I .2 equivalents of 19 and about 1.0 to about 1.2
equivalents of potassium carbonate in the presence of a pallidium catalyst,
preferably tetrakis(triphenylphosphine)pallidium(0). Generally, this coupling
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 52 --
reaction is conducted in a diluent, preferably 20 % water/dioxane, under an
inert
atmosphere at a temperature ranging from about 50°C to about
100°C for about 6
to 24 hours.
Biphenyl derivative 22 is then readily converted into the 5,7-dihydro-6H-
dibenz(b,d]azepin-6-one 23 by carboxylation of the 2-methyl group, followed by
cyclization to form the E-caprolactam. The carboxylation reaction is typically
conducted by contacting 22 with about 2.0 to about 2.5 equivalents of a
suitable
base, such as sec-butyllithium, tent-butyllithium and the like, in an inert
diluent,
such as THF, at a temperature ranging from about -100°C to about -
20°C for
about 0.5 to 6 hours. The resulting dianion is then treated with excess
anhydrous
carbon dioxide to form the carboxylate. Treatment of the carboxylate with
excess hydrogen chloride in a suitable diluent, such as methanol, at a
temperature
ranging from about 25°C to about 100°C then affords the 5,7-
dihydro-6H-
dibenz[b,d]azepin-6-one 23. Various other cyclic compounds can be prepared by
routine modifications of the above described procedures.
Preferred synthetic procedures for aminating a representative compound
are illustrated in Scheme 8. It will be readily apparent to those of ordinary
skill
in the art that the synthetic procedure illustrated in Scheme 8 and the
following
reaction conditions can be modified by selecting the appropriate starting
materials
and reagents to allow the preparation of other amino compounds suitable for
use
in this invention.
CA 02325388 2000-09-21
WO 99/67220 PCTlUS99/14007
-- 53 --
Scheme 8
(R6)p ~ (R')q (R6)P (R')q
i -..
NH
23
O O ci
1 1
(Rs)p ' (R')a (R6)p (R )a
/ \ ~ ~ /
NH
~R8
24 ~ N3 O 28
(Rs)p .~.. (R )q (Rs)Q (R')a
/ \
HON N~Rs
25 O ~ 29
(R7)a (R6)p (R )a
B
26
As shown in Scheme 8, 5,7-dihydro-6H-dibenz[b,d]azepin-6-one, 23, is
optionally N alkylated using conventional reagents and conditions to provide a
7-
CA 02325388 2000-09-21
WO 99/67220 PCTNS99/14007
-- 54 --
alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one derivative, 24. Typically, this
reaction is conducted by first contacting 23 with about 1.0 to 1.5 equivalents
of a
suitable base, such as sodium hydride, sodium bis(trimethysilyl)amide and the
like, in an inert diluent, such as DMF, THF and the like, at a temperature
ranging from about -78°C to about 50°C for about 0.25 to about 6
hours. The
resulting anion is then treated in situ with an excess, preferably about 1.1
to
about 2.0 equivalents, of an alkyl, substituted alkyl, cycloalkyl halide,
etc.,
typically a chloride, bromide or iodide. This reaction is typically conducted
at a
temperature ranging from about 0°C to about 60°C for about 1.0
to about 48
hours to afford the 7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one derivative,
24.
The 7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one 24 is then oximated
by contacting 24 with an excess, preferably with about 1.0 to 1.5 equivalents
of a
suitable base, such as sodium bis(trimethysilyl)amide and the like, in the
presence
of about 1.0 to about 2.0 equivalents of an alkyl nitrite. Suitable alkyl
nitrites for
use in this reaction include, by way of example, butyl nitrite, isoamyl
nitrite and
the like. This reaction is typically conducted in an inert diluent, such as
THF
and the like, at a temperature ranging from about -10°C to about
20°C for about
0.5 to about 6 hours to afford the 7-alkyl-5-oximo-5,7-dihydro-6H-
dibenz[b,d]azepin-6-one derivative 25.
Reduction of 25 using conventional reagents and conditions then affords
the 5-amino-7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one 26. Preferably,
this
reduction reaction is conducted by hydrogenating the oxime 25 in the presence
of
a catalyst, such as Raney nickel. This reaction is typically conducted under
about
200 psi to about 600 psi of hydrogen at a temperature of about 70°C to
about
120°C for about 8 to 48 hours in a diluent, preferably a mixture of
ethanol and
ammonia (about 20:1). Alternatively, in another preferred procedure, the oxime
may be reduced using 10% Pd/C and between about 30 to about 60 psi of
hydrogen at a temperature ranging from about 20°C to about 50°C
for about 4
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 55 --
hours. The resulting 5-amino-7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
26 is generally purified using well known procedures, such as
recrystallization
and/or chromatography.
Alternatively, 5-amino-7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-ones,
26, can be prepared by first forming the 5-iodo derivative 27 of 5,7-dihydro-
6H-
dibenz[b,d]azepin-6-one, 23. This reaction is typically conducted as described
in
A. O. King et a1.13 by treating 23 with an excess, preferably about 1.2 to
about
2.5 equivalents, of trimethylsilyl iodide in the presence of an excess of a
trialkyamine, such as triethylamine, diisopropylethylamine, TMEDA and the
like, at a temperature ranging from about -20°C to about 0°C for
about 3 to 30
minutes and then adding about 1.1 to about 2.0 equivalents of iodine (Iz).
Typically, after addition of the iodide, the reaction is stirred at a
temperature
ranging from about 0°C to about 20°C for about 2 to about 4
hours to afford 5-
iodo-5,7-dihydro-6H-dibenz[b,d]azepin-6-one, 27.
Displacement of iodide from 27 using an alkali metal azide then affords 5-
azido-5,7-dihydro-6H-dibenz[b,d]azepin-6-one, 28. Typically, this reaction is
conducted by contacting 27 with about 1.1 to about 1.5 equivalents of sodium
azide in an inert diluent, such as DMF, at a temperature ranging from about
0°C
to about 50°C for about 12 to about 48 hours.
The azido derivative Z$ is then reduced to the corresponding amino
derivative 29 using conventional procedures and reagents. For example, the
azido group is preferably reduced by contacting 28 with an excess, preferably
with about 3 equivalents, of triphenylphosphine in a diluent, preferably a
mixture
of THF and water. This reduction reaction is typically conducted at a
temperature ranging from about 0°C to about 50°C for about 12 to
48 hours to
afford 5-amino-5,7-dihydro-6H-dibenz[b,d]azepin-6-one, 29.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 56 --
The amino group of 29 is then protected or blocked using a conventional
amino blocking group. Preferably, compound 29 is treated with about 1.0 to
about 1.1 equivalents of di-tert-butyl dicarbonate in the presence of an
excess,
preferably about 2 to about 3 equivalents, of a trialkylamine, such as
triethylamine. This reaction is typically conducted in an inert diluent, such
as
THF, at a temperature ranging from about 0°C to about 50°C for
3 to about 24
hours to provide 5-(N Boc-amino)-5,7-dihydro-6H-dibenz(b,d]azepin-6-one, 30.
Compound 30 is then optionally N alkylated to afford, after de-blocking
of the amino group, a 5-amino-7-alkyl-5,7-dihydro-6H-dibenz(b,d]azepin-6-one,
26. The N alkylation reaction is typically conducted by treating 30 with about
1.0 to 1.5 equivalents of an alkyl halide, a substituted alkyl halide or a
cycloalkyl
halide in the presence of about 1.0 to about 1.5 equivalents of a suitable
base,
such as cesium carbonate and the like. This reaction is generally conducted in
an
inert diluent, such as DMF and the like, at a temperature ranging from about
25°C to about 100°C for about 12 to about 48 hours.
Representative alkyl, substituted alkyl and cycloalkyl halides suitable for
use in this N alkylation reaction include, by way of illustration, 1-iodo-2-
methylpropane, methyl bromoacetate, 1-chloro-3,3-dimethyl-2-butanone, 1-
chloro-4-phenylbutane, bromomethylcyclopropane, 1-bromo-2,2,2-
trifluoroethane, bromocyclohexane, 1-bromohexane and the like.
The N Boc protecting group is then removed using conventional
procedures and reagents to, afford the 5-amino-7-alkyl-5,7-dihydro-6H-
dibenz[b,d]azepin-6-one, 26. This deblocking reaction is typically conducted
by
treating the N Boc compound 30 with anhydrous hydrogen chloride in an inert
diluent, such as 1,4-dioxane, at a temperature ranging from about 0°C
to about
50°C for about 2 to about 8 hours. The resulting 5-amino-7-alkyl-5,7-
dihydro-
6H-dibenz[b,d]azepin-6-one 26 is generally purified using well known
procedures, such as recrystallization and/or chromatography.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 57 __
The 5-amino-7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-ones, 26, can
also be prepared via an azide transfer reaction as illustrated in Scheme 9.
Scheme 9
~R6~P ~ _..- 'R7,q ~R6~P ~ R7~q
NH 23 ~ N~ R8 24
V O
a
'R6/P ~ ~ ~ 7lq ~R6~P W /1 ,=-1 / ~R7~q
~N~ R8 N~ Ra
HzN O N3 OI
26 31
As shown in Scheme 9, 5,7-dihydro-6H-dibenz[b,d]azepin-6-one, 23, is
first N-alkylated as described above using conventional reagents and
conditions to
provide a 7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one derivative, 24.
The 7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one 24 is then reacted
with an azide transfer reagent to afford 5-azido-7-alkyl-5,7-dihydro-6H-
dibenz[b,dJazepin-6-one 31. Typically, this reaction is conducted by first
contacting 24 with an excess, preferably with about 1.0 to 1.5 equivalents of
a
suitable base, such as lithium diisopropylamine and the like, in an inert
diluent
such as THF, at a temperature ranging from about -90°C to about -
60°C for
about 0.25 to about 2.0 hours. The resulting anion is then treated with an
excess,
preferably with about 1.1 to about 1.2 equivalents, of an azide transfer
reagent,
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__sg~
such as 2,4,6-triisopropylbenzenesulfonyl azide (trisyl azide). This reaction
is
typically conducted at a temperature ranging from about -90°C to about -
60°C
for about 0.25 to about 2.0 hours. The reaction mixture is then typically
treated
with an excess of glacial acetic acid and the mixture is allowed to warm to
ambient temperature and then heated at about 35°C to about 50°C
for about 2 to
4 hours to afford the 5-azido-7-alkyl-5,7-dihydro-6H-dibenz(b,d]azepin-6-one
derivative 31. Reduction of 31 as described above using conventional reagents
and conditions then affords the 5-amino-7-alkyl-5,7-dihydro-6H-
dibenz[b,d]azepin-6-one 26.
If desired, the aryl rings of 5-amino-7-alkyl-5,7-dihydro-6H-
dibenz[b,d]azepin-6-ones, 26, and similar or related compounds may be
partially
or fully saturated by treatment with hydrogen in the presence of a
hydrogention
catalyst. Typically, this reaction is conducted by treating 26 with hydrogen
at a
pressure of about 10 to about 100 psi in the presence of a catalyst, such as
rhodium on carbon. This reaction is typically conducted at a temperature
ranging
from about 20°C to about 100°C for about 12 to 96 hours in a
suitable diluent,
such as ethyl acetate/acetic acid (1:1) and the like.
Other methods for preparing intermediates useful in this invention are
described in U.S. Patent Application No. 09/102,726, filed on June 22, 1998
and
in U.S. Patent Application No. /-,-, filed on even date herewith
(Attorney Docket No. 002010-345), both entitled "Polycyclic a-Amino-E-
caprolactams and Related Compounds", the disclosures of which are incorporated
herein by reference in their entirety.
Additionally, the synthesis of various benzapinones and related
compounds are described in Busacca et al., Tetrahedron Lett., 33, 165-168
(1992); Crosisier et al., U.S. Patent No. 4,080,449; J. A. Robl et al.
Tetrahedron Lett., 36(10), 1593-1596 (1995); Flynn et al. J. Med. Chem. 36,
2420-2423 (1993); Orito et al. Tetrahedron, 36, 1017-1021 (1980); Kawase et
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 59 --
al., J. Org. Chem., 54, 3394-3403 (1989); Lowe et al., J. Med. Chem. 37, 3789-
3811 (1994); Robl et al., Bioorg. Med. Chem. Lett., 4, 1789-1794 (1994);
Skiles
et al., Bioorg. Med. Chem. Lett., 3, 773-778 (1993); Grunewald et al., J. Med.
Chem., 39(18), 3539- (1996); Warshawsky et al., Bioorg. Med. Chem. Lett., 6,
957-962 (1996); Ben-Ishai, et aL, Tetrahedron, 43, 439-450 (1987); van Neil et
al, Bioorg. Med. Chem. 5, 1421-1426 (1995); and reference cited therein. These
publications and patents are incorporated herein by reference in their
entirety.
Similarly, various benzodiazepine derivatives suitable for use in this
invention can be prepared using conventional procedures and reagents. For
example, a 2-aminobenzophenone can be readily coupled to a-(isopropylthio)-N-
(benzyloxycarbonyl)glycine by first forming the acid chloride of the glycine
derivative with oxayl chloride, and then coupling the acid chloride with the 2-
aminobenzophenone in the presence of a base, such as 4-methylmorpholine, to
afford the 2-[a-(isopropylthio)-N-(benzyloxycarbonyl)glycinyl]-
aminobenzophenone. Treatment of this compound with ammonia gas in the
presence of an excess, preferably about 1.1 to about 1.5 equivalents, of
mercury
(II) chloride then affords the 2-[N-(a-amino)-N'-(benzyloxycarbonyl)-
glycinyl]aminobenzophenone. This intermediate can then be readily cyclized by
treatment with glacial acetic acid and ammonium acetate to provide the 3-
(benzyloxycarbonyl)amino-2, 3-dihydro-S-phenyl-1H-1,4-benzodiazepin-2-one 1.
Subsequent removal of the Cbz group affords the 3-amino-2,3-dihydro-5-phenyl-
1H-1,4-benzodiazepin-2-one.
Alternatively, 2,3-dihydro-5-phenyl-1H-1,4-benzodiazepin-2-ones can be
readily aminated at the 3-position using conventional azide transfer reactions
followed by reduction of the resulting azido group to form the corresponding
amino group. The conditions for these and related reactions are described in
the
examples set forth below. Additionally, 2,3-dihydro-S-phenyl-1H-1,4-
benzodiazepin-2-ones are readily aIkylated at the 1-position using
conventional
procedures and reagents. For example, this reaction is typically conducted by
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 6p __
first treating the benzodiazepinone with about 1.1 to about 1.5 equivalents of
a
base, such as sodium hydride, potassium tent-butoxide, potassium 1,1,1,3,3,3-
hexamethyldisilazane, cesium carbonate, in an inert diluent, such as DMF. This
reaction is typically conducted at a temperature ranging from about -
78°C to
about 80°C for about 0.5 to about 6 hours. The resulting anion is then
contacted
with an excess, preferably about 1.1 to about 3.0 equivalents, of an alkyl
halide,
typically an alkyl chloride, bromide or iodide. Generally, this reaction is
conducted at a temperature of about 0°C to about 100°C for about
1 to about 48
hours.
Additionally, the 3-amino-2,4-dioxo-2,3,4,5-tetrahydro-1H-1,5-
benzodiazepines employed in this invention are typically prepared by first
coupling malonic acid with a 1,2-phenylenediamine. Conditions for this
reaction
are well known in the art and are described, for example, in PCT Application
WO 96-US8400 960603. Subsequent alkylation and amination using
conventional procedures and reagents affords various 3-amino-1,5-bis(alkyl)-
2,4-
dioxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepines. Such procedures are
described in further detail in the example set forth below.
In the synthesis of compounds of formula I using the synthetic methods
described herein, the starting materials can contain a chiral center (e.g.,
alanine)
and, when a racemic starting material is employed, the resulting product is a
mixture of R,S enantiomers. Alternatively, a chiral isomer of the starting
material can be employed and, if the reaction protocol employed does not
racemize this starting material, a chiral product is obtained. Such reaction
protocols can involve inversion of the chiral center during synthesis.
Accordingly, unless otherwise indicated, the products of this invention are
a mixture of R,S enantiomers. Preferably, however, when a chiral product is
desired, the chiral product corresponds to the L-amino acid derivative.
Alternatively, chiral products can be obtained via purification techniques
which
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 61 --
separates enantiomers from a R,S mixture to provide for one or the other
stereoisomer. Such techniques are well known in the art.
Pharmaceutical Formulations
When employed as pharmaceuticals, the compounds of formula I are
usually administered in the form of pharmaceutical compositions. These
compounds can be administered by a variety of routes including oral, rectal,
transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These
compounds are effective as both injectable and oral compositions. Such
compositions are prepared in a manner well known in the pharmaceutical art and
comprise at least one active compound.
This invention also includes pharmaceutical compositions which contain,
as the active ingredient, one or more of the compounds of formula I above
associated with pharmaceutically acceptable carriers. In making the
compositions
of this invention, the active ingredient is usually mixed with an excipient,
diluted
by an excipient or enclosed within such a carrier which can be in the form of
a
capsule, sachet, paper or other container. When the excipient serves as a
diluent,
it can be a solid, semi-solid, or liquid material, which acts as a vehicle,
carrier or
medium for the active ingredient. Thus, the compositions can be in the form of
tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions,
emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium),
ointments containing, for example, up to 10% by weight of the active compound,
soft and hard gelatin capsules, suppositories, sterile injectable solutions,
and
sterile packaged powders.
In preparing a formulation, it may be necessary to mill the active
compound to provide the appropriate particle size prior to combining with the
other ingredients. If the active compound is substantially insoluble, it
ordinarily
is milled to a particle size of less than 200 mesh. If the active compound is
substantially water soluble, the particle size is normally adjusted by milling
to
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 62 --
provide a substantially uniform distribution in the formulation, e.g. about 40
mesh. '
Some examples of suitable excipients include lactose, dextrose, sucrose,
sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,
tragacanth, gelatin, calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose.
The
formulations can additionally include: lubricating agents such as talc,
magnesium
stearate, and mineral oil; wetting agents; emulsifying and suspending agents;
preserving agents such as methyl- and propylhydroxy-benzoates; sweetening
agents; and flavoring agents. The compositions of the invention can be
formulated so as to provide quick, sustained or delayed release of the active
ingredient after administration to the patient by employing procedures known
in
the art.
The compositions are preferably formulated in a unit dosage form, each
dosage containing from about 5 to about 100 mg, more usually about i0 to about
30 mg, of the active ingredient. The term "unit dosage forms" refers to
physically discrete units suitable as unitary dosages for human subjects and
other
mammals, each unit containing a predetermined quantity of active material
calculated to produce the desired therapeutic effect, in association with a
suitable
pharmaceutical excipient. Preferably, the compound of formula I above is
employed at no more than about 20 weight percent of the pharmaceutical
composition, more preferably no more than about 15 weight percent, with the
balance being pharmaceutically inert carrier(s).
The active compound is effective over a wide dosage range and is
generally administered in a pharmaceutically effective amount. It, will be
understood, however, that the amount of the compound actually administered
will
be determined by a physician, in the light of the relevant circumstances,
including
the condition to be treated, the chosen route of administration, the actual
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 63 --
compound administered, the age, weight, and response of the individual
patient,
the severity of the patient's symptoms, and the like.
For preparing solid compositions such as tablets, the principal active
ingredient is mixed with a pharmaceutical excipient to form a solid
preformulation composition containing a homogeneous mixture of a compound of
the present invention. When referring to these preformulation compositions as
homogeneous, it is meant that the active ingredient is dispersed evenly
throughout the composition so that the composition may be readily subdivided
into equally effective unit dosage forms such as tablets, pills and capsules.
This
solid preformulation is then subdivided into unit dosage forms of the type
described above containing from, for example, 0.1 to about 500 mg of the
active
ingredient of the present invention.
The tablets or pills of the present invention may be coated or otherwise
compounded to provide a dosage form affording the advantage of prolonged
action. For example, the tablet or pill can comprise an inner dosage and an
outer
dosage component, the latter being in the form of an envelope over the former.
The two components can separated by enteric layer which serves to resist
disintegration in the stomach and permit the inner component to pass intact
into
the duodenum or to be delayed in release. A variety of materials can be used
for
such enteric layers or coatings, such materials including a number of
polymeric
acids and mixtures of polymeric acids with such materials as. shellac, cetyl
alcohol, and cellulose acetate.
The liquid forms in which the novel compositions of the present invention
may be incorporated for administration orally or by injection include aqueous
solutions suitably flavored syrups, aqueous or oil suspensions, and flavored
emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or
peanut oil, as well as elixirs and similar pharmaceutical vehicles.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
Compositions for inhalation or insufflation include solutions and
suspensions in pharmaceutically acceptable, aqueous or organic solvents, or
mixtures thereof, and powders. The liquid or solid compositions may contain
suitable pharmaceutically acceptable excipients as described supra. Preferably
the compositions are administered by the oral or nasal respiratory route for
local
or systemic effect. Compositions in preferably pharmaceutically acceptable
solvents may be nebulized by use of inert gases. Nebulized solutions may be
breathed directly from the nebulizing device or the nebulizing device may be
attached to a face masks tent, or intermittent positive pressure breathing
machine.
Solution, suspension, or powder compositions may be administered, preferably
orally or nasally, from devices which deliver the formulation in an
appropriate
manner.
The following formulation examples illustrate the pharmaceutical
compositions of the present invention.
Formulation Examy 1~ a 1
Hard gelatin capsules containing the following ingredients are prepared:
Quantity
Ingredient (mg/capsule)
Active Ingredient 30.0
Starch 305.0
Magnesium stearate 5.0
The above ingredients are mixed and filled into hard gelatin capsules in
340 mg quantities.
Formulation Exam 1~
A tablet formula is prepared using the ingredients below:
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
- 65 -
Quantity
j~~redient (Ingl abletl
Active Ingredient 25.0
Cellulose, microcrystalline 200.0
Colloidal silicon dioxide 10.0
Stearic acid 5.0
. The components are blended and
compressed to force tablets, each
weighing 240 mg.
Formulation Example 3
A dry powder inhaler formulation is prepared containing the following
components:
Ingredient Weight %
Active Ingredient 5
Lactose 95
The active ingredient is mixed with the lactose and the mixture is added to
a dry powder inhaling appliance.
Formulation Exam lie 4
Tablets, each containing 30 mg of active ingredient, are prepared as
follows:
Quantity
I /t
redient bl
ng fmg
a
e~
Active Ingredient 30.0
mg
Starch 45.0
mg
Microcrystalline cellulose 35.0
mg
Polyvinylpyrrolidone
(as 10% solution in sterile water) 4.0 mg
Sodium carboxymethyl starch 4.5 mg
Magnesium stearate 0.5 mg
Talc 1.0 mg
Total 120 mg
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 66 --
The active ingredient, starch and cellulose are passed through a No. 20
mesh U.S. sieve and mixed thoroughly. The solution of polyvinyl-pyrrolidone is
mixed with the resultant powders, which are then passed through a 16 mesh U.S.
sieve. The granules so produced are dried at SO° to 60°C and
passed through a
16 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and
talc, previously passed through a No. 30 mesh U.S. sieve, are then added to
the
granules which, after mixing, are compressed on a tablet machine to yield
tablets
each weighing 150 mg.
Formulation ExamRle 5
Capsules, each containing 40 mg of medicament are made as follows:
Quantity
Ingredient ~m~;/capsule)
Active Ingredient 40.0 mg
Starch 109.0 mg
Magnesium stearate 1.0 m~
Total 150.0 mg
The active ingredient, starch, and magnesium stearate are blended, passed
through a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 150
mg quantities.
Suppositories, each containing 25 mg of active ingredient are made as
follows:
Active Ingredient 25 mg
Saturated fatty acid glycerides to 2,000 mg
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 67 __
The active ingredient is passed through a No. 60 mesh U.S. sieve and
suspended in the saturated fatty acid glycerides previously melted using the
minimum heat necessary. The mixture is then poured into a suppository mold of
nominal 2.0 g capacity and allowed to cool.
Formulation Exam 1~
Suspensions, each containing 50 mg of medicament per 5.0 mL dose are
made as follows:
'en
Active Ingredient 50.0 mg
Xanthan gum 4.0 mg
Sodium carboxymethyl cellulose (11 %)
Microcrystalline cellulose (89%) 50.0 mg
Sucrose 1.75 g
Sodium benzoate 10.0 mg
Flavor and Color q.v.
Purified water to 5.0 mL
The active ingredient, sucrose and xanthan gum are blended, passed through
a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of
the
microcrystalline cellulose and sodium carboxymethyl cellulose in water. The
sodium benzoate, flavor, and color are diluted with some of the water and
added
with stirring. Sufficient water is then added to produce the required volume.
FormulatL~n Example 8
Quantity
Ingredient (mg/capsulel
Active Ingredient 15.0 mg
Starch 407.0 mg
Magnesium stearate 3.0 m~
Total 425.0 mg
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 6g __
The active ingredient, starch, and magnesium stearate are blended, passed
through a No. 20 mesh U.S. sieve, and 611ed into hard gelatin capsules in 560
mg quantities.
Formulation Example 9
A subcutaneous formulation may be prepared as follows:
Ingredient Ouantitv
Active Ingredient 1.0 mg
corn oil 1 mL
(Depending on the solubility of the active ingredient in corn oil, up to about
5.0 mg or more of the active ingredient may be employed in this formulation,
if
desired).
A topical formulation may be prepared as follows:
Ingredient Quantity
Active Ingredient 1-10 g
Emulsifying Wax 30 g
Liquid Paraffin 20 g
White Soft Paraffin to 100 g
The white soft paraffin is heated until molten. The liquid paraffin and
emulsifying wax are incorporated and stirred until dissolved. The active
ingredient is added and stirring is continued until dispersed. The mixture is
then
cooled until solid.
Another preferred formulation employed in the methods of the present
invention employs transdermal delivery devices ("patches"). Such transdermal
patches may be used to provide continuous or discontinuous infusion of the
compounds of the present invention in controlled amounts. The construction and
use of transdermal patches for the delivery of pharmaceutical agents is well
CA 02325388 2000-09-21
WO 99/67220 PCT/LTS99/14007
-- 69 --
known in the art. , ee. e.~., U.S. Patent 5,023,252, issued June 11, 1991,
herein
incorporated by reference. Such patches may be constructed for continuous,
pulsatile, or on demand delivery of pharmaceutical agents.
Frequently, it will be desirable or necessary to introduce the pharmaceutical
composition to the brain, either directly or indirectly. Direct techniques
usually
involve placement of a drug delivery catheter into the host's ventricular
system to
bypass the blood-brain barrier. One such implantable delivery system used for
the transport of biological factors to specific anatomical regions of the body
is
described in U.S. Patent 5,011,472 which is herein incorporated by reference.
Indirect techniques, which are generally preferred, usually involve
formulating the compositions to provide for drug latentiation by the
conversion of
hydrophilic drugs into lipid-soluble drugs. Latentiation is generally achieved
through blocking of the hydroxy, carbonyl, sulfate, and primary amine groups
present on the drug to render the drug more lipid soluble and amenable to
transportation across the blood-brain barrier. Alternatively, the delivery of
hydrophilic drugs may be enhanced by infra-arterial infusion of hypertonic
solutions which can transiently open the blood-brain barrier.
Other suitable formulations for use in the present invention can be found in
Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia,
PA, 17th ed. (1985).
Utility
The compounds and pharmaceutical compositions of the invention are useful
in inhibiting (3-amyloid peptide release and/or its synthesis, and,
accordingly,
have utility in diagnosing and treating Alzheimer's disease in mammals
including
humans.
CA 02325388 2000-09-21
WO 99/67ZZ0 PCT/US99/14007
__ 7p __
As noted above, the compounds described herein are suitable for use in a
variety of drug delivery systems described above. Additionally, in order to
enhance the in vivo serum half life of the administered compound, the
compounds
may be encapsulated, introduced into the lumen of liposomes, prepared as a
colloid, or other conventional techniques may be employed which provide an
extended serum half life of the compounds. A variety of methods are available
for preparing liposomes, as described in, e.g., Szoka, et al., U.S. Patent
Nos.
4,235,871, 4,501,728 and 4,837,028 each of which is incorporated herein by
reference.
The amount of compound administered to the patient will vary depending
upon what is being administered, the purpose of the administration, such as
prophylaxis or therapy, the state of the patient, the manner of
administration, and
the like. In therapeutic applications, compositions are administered to a
patient
already suffering from AD in an amount sufficient to at least partially arrest
further onset of the symptoms of the disease and its complications. An amount
adequate to accomplish this is defined as "therapeutically effective dose."
Amounts effective for this use will depend on the judgment of the attending
clinician depending upon factors such as the degree or severity of AD in the
patient, the age, weight and general condition of the patient, and the like.
Preferably, for use as therapeutics, the compounds described herein are
administered at dosages ranging from about 1 to about 500 mg/kg/day.
In prophylactic applications, compositions are administered to a patient at
risk of developing AD (determined for example by genetic screening or familial
trait) in an amount sufficient to inhibit the onset of symptoms of the
disease. An
amount adequate to accomplish this is defined as "prophylactically effective
dose." Amounts effective for this use will depend on the judgment of the
attending clinician depending upon factors such as the age, weight and general
condition of the patient, and the like. Preferably, for use as prophylactics,
the
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ ~ 1 __
compounds described herein are administered at dosages ranging from about 1 to
about 500 mg/kg/day.
As noted above, the compounds administered to a patient are in the form of
pharmaceutical compositions described above. These compositions may be
sterilized by conventional sterilization techniques, or may be sterile
filtered. The
resulting aqueous solutions may be packaged for use as is, or lyophilized, the
lyophilized preparation being combined with a sterile aqueous carrier prior to
administration. The pH of the compound preparations typically will be between
3 and 11, more preferably from 5 to 9 and most preferably from 7 and 8. It
will
be understood that use of certain of the foregoing excipients, carriers, or
stabilizers will result in the formation of pharmaceutical salts.
The compounds described herein are also suitable for use in the
administration of the compounds to a cell for diagnostic and drug discovery
purposes. Specifically, the compounds may be used in the diagnosis of cells
releasing and/or synthesizing (3-amyloid peptide. In addition the compounds
described herein are useful for the measurement and evaluation of the activity
of
other candidate drugs on the inhibition of the cellular release and/or
synthesis of
(3-amyloid peptide.
The following synthetic and biological examples are offered to illustrate this
invention and are not to be construed in any way as limiting the scope of this
invention.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__
EXAMPLES
In the examples below, the following abbreviations have the following
meanings. If an abbreviation is not defined, it has its generally accepted
meaning.
BEMP - 2-tert-butylimino-2-diethylamino-1,3-
dimethylperhydro-1,3,2-diazaphosphorine
Boc - t-butoxycarbonyl
BOP - benzotriazoI-1-yloxy-
tris(dimethylamino)phosphonium
hexafluorophosphate
bd - broad doublet
bs - broad singlet
d - doublet
dd - doublet of doublets
DIC - diisopropylcarbodiimide
DMF - dimethylformamide
DMAP - dimethylaminopyridine
DMSO - dimethylsulfoxide
EDC - ethyl-1-(3-dimethyaminopropyl)carbodiimide
eq. - equivalents
EtOAc - ethyl acetate
g - grams
HOBT - 1-hydroxybenzotriazole hydrate
Hunig's diisopropylethylamine
base -
L - liter
m - multiplet
M - molar
max - maximum
meq - milliequivalent
mg - milligram
mL - milliliter
mm - millimeter
mmol - millimole
MOC - methoxyoxycarbonyl
N - normal
N/A - not available
ng - nanogram
nm - nanometers
OD - optical density
PEPC - 1-(3-(1-pyrrolidinyl)propyl)-3-ethylcarbodiimide
PP-HOBT piperidine-piperidine-1-hydroxybenzotrizole
-
psi - pounds per square inch
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/14007
__ 73 __
- phenyl
q - quartet
quint. - quintet
rpm - rotations per minute
s - singlet
t - triplet
TFA - trifluoroacetic acid
THF - tetrahydrofuran
tlc - thin layer chromatography
~L - microliter
UV - ultra-violet
In the examples below, all temperatures are in degrees Celsius (unless
otherwise indicated). The compounds set forth in the examples below were
prepared using the following general procedures as indicated.
In the following examples and procedures, the term "Aldrich" indicates
that the compound or reagent used in the procedure is commercially available
from Aldrich Chemical Company, Inc., 1001 West Saint Paul Avenue,
Milwaukee, WI 53233 USA; the term "Fluka" indicates that the compound or
reagent is commercially available from Fluka Chemical Corp., 980 South 2nd
Street, Ronkonkoma NY 11779 USA; the term "Lancaster" indicates that the
compound or reagent is commercially available from Lancaster Synthesis, Inc.,
P.O. Box 100 Windham, NH 03087 USA; the term "Sigma" indicates that the
compound or reagent is commercially available from Sigma, P.O. Box 14508, St.
Louis MO 63178 USA; the term "Chemservice" indicates that the compound or
reagent is commercially available from Chemservice Inc., Westchester, PA; the
term "Bachem" indicates that the compound or reagent is commercially available
from Bachem Biosciences Inc., 3700 Horizon Drive, Renaissance at Gulph Mills,
King of Prussia, PA 19406 USA; the term "Maybridge" indicates that the
compound or reagent is commercially available from Maybridge Chemical Co.
Trevillett, Tintagel, Cornwall PL34 OHW United Kingdom; and the term "TCI"
indicates that the compound or reagent is commercially available from TCI
America, 9211 North Harborgate Street, Portland OR 97203; the term "Alfa"
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 74 __
indicates that the compound or reagent is commercially available from Johnson
Matthey Catalog Company, Inc. 30 Bond Street, Ward Hill, MA 01835-0747;
the term "Novabiochem" indicates that the compound or reagent is commercially
available from Calbiochem-Novabiochem Corp. 10933 North Torrey Pines Road,
P.O. Box 12087, La Jolla CA 92039-2087; the term "Oakwood" indicates that
the compound or reagent is commercially available from Oakwood Products,
Inc., 1741 Old Dunbar Rd., West Columbia, SC 29169; the term "Advanced
Chemtech" indicates that the compound or reagent is commercially available
from Advanced Chemtech, Louisville, KY; the term "Pfaltz & Bauer" indicates
that the compound or reagent is commercially available from Pfaltz & Bauer,
Waterbury, CT, USA; and the term "Fluorochem" indicates that the compound
or reagent is commercially available from Fluorochem Ltd. Wesley St. Old
Glossop, Derbyshire SK13 9RY, United Kingdom (for U.S. sales office see
Oakwood Products, Inc. above).
I. Coupling Procedures
The following coupling procedures may be used to prepare compounds of
this invention:
GENERAL PROCEDURE A
To a 1:1 mixture of the corresponding carboxylic acid and the
corresponding amino acid ester or amide in CH,Ch at O°C was added 1.5
equivalents triethylamine, followed by 2.0 equivalents hydroxybenzotriazole
monohydrate and then 1.25 equivalents of ethyl-3-(3-dimethylamino)propyl
carbodiimide~HCl. The reaction mixture was stirred overnight at room
temperature and then transferred to a separatory funnel. The mixture was
washed with water, saturated aqueous NaHC03, 1N HCl and saturated aqueous
NaCI, and then dried over MgSO~. The resulting solution was stripped free of
solvent on a rotary evaporator to~ yield the crude product.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__
GENERAL PROCEDURE B
Second EDC Coupl~g Procedure
A mixture of the corresponding acid (1 eqv), N-1-hydroxybenzotriazole
(1.6 eqv), the corresponding amine (1 eqv), N-methylmorpholine ( 3 eqv) and
dichloromethane (or DMF for insoluble substrates) was cooled in an ice-water
bath and stirred until a clear solution was obtained. EDC (1.3 eqv) was then
added to the reaction mixture. The cooling bath was then allowed to warm to
ambient temperature over 1-2 h and the reaction mixture was stirred overnight.
The reaction mixture was then evaporated to dryness under vacuum. To the
residue was added 20%a aqueous potassium carbonate and the mixture was shaken
throughly and then allowed to stand until the oily product solidified
(overnight if
necessary). The solid product was then collected by filteration, washed
thoroughly with 20% aqueous potassium carbonate, water, 10% HCI, and water
to give the product, usually in pure state. No racemization was observed.
GENERAL PROCEDURE C
Third EDC Coupling Procedure
The carboxylic acid was dissolved in methylene chloride. The
corresponding amino acid ester or amide (1 eq.), N-methylmorpholine (S eq.)
and hydroxybenzotriazole monohydrate (1.2 eq.) were added in sequence. A
cooling bath was applied to the round bottomed flask until the solution
reached
0°C. At that time, 1.2 eq. of 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide
hydrochloride was added. The solution was allowed to stir overnight and come
to room temperature under nitrogen pressure. The reaction mixture was worked
up by washing the organic phase with saturated aqueous sodium carbonate, O.1M
citric acid, and brine before drying with sodium sulfate. The solvents were
then
removed to yield crude product.
CA 02325388 2000-09-21
WO 99167220 PCT/US99/14007
__ 76 __
GENERAL PROCEDURE D
Fourth EDC Coupling Procedure
A round bottom flask was charged with the corresponding carboxylic acid
(1.0 eq.), hydroxybenzotriazole hydrate (1.1 eq.) and the corresponding amine
(1.0 eq.) in THF under nitrogen atmosphere. An appropriate amount (1.1 eq for
free amines and 2.2 eq. for hydrochloride amine salts) of base, such as
Hunig's
base was added to the well stirred mixture followed by EDC (1.1 eq.). After
stirring from 4 to 17 hours at room temperature the solvent was removed at
reduced pressure, the residue taken up in ethyl acetate (or similar solvent)
and
water, washed with saturated aqueous sodium bicarbonate solution, 1 N HCI,
brine, dried over anhydrous sodium sulfate and the solvent removed at reduced
pressure to provide the product.
GENERAL PROCEDURE E
BOP CouDing Procedure
To a stirred solution of N (3,5-difluorophenylacetyl)alanine (2 mmol) in
DMF, cooled in an ice-water bath, was added BOP (2.4 mmol) and N
methylmorpholine (6 mmol). The reaction mixture was stirred for 50 min. and
then a solution of a-amino-y-lactam (2 mmol) in DMF cooled at 0 °C was
added.
The cooling bath was ailowed to warm to ambient temperature over 1-2 h and the
reaction mixture was then stirred overnight. A 20% aqueous potassium
carbonate solution (60 mL) was added and this mixture shaken throughly. No
solid formed. The mixture was then washed with ethyl acetate (150 mL) and
evaporated to dryness under vacuum to give a white solid. Water (50 mL) was
then added and this mixture shaken throughly. The precipitate that formed was
collected by filtration, then washed thoroughly with water, followed by 1 mL
of
diethyl ether to give the product (51 mg, 0.16 mmol, 7.8%).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
GENERAL PROCEDURE F
Cou ling of an Acid Chloride with an Amino Acid Ester
To a stirred solution of (D,L)-alanine isobutyl ester hydrochloride (4.6
mmol) in 5 ml of pyridine was added 4.6 mmol of the acid chloride.
Precipitation occurred immediately. The mixture was stirred for 3.5 h,
dissolved
in 100 mL of diethyl ether, washed with 10% HCl three times, brine once, 20%
potassium carbonate once and brine once. The solution was dried over
magnesium sulfate, filtered, and evaporated to yield the product. Other amino
acid esters may also be employed in this procedure.
GENERAL PROCEDURE G
Counlj~g of a Carboxylic Acid with an Amino Acid Ester
A solution of the carboxylic acid (3.3 mmol) and 1,1'-carbodiimidazole
(CDI) in 20 mL THF was stirred for 2 h. (D,L)-alanine isobutyl ester
hydrochloride (3.6 mmol) was added, followed by 1.5 mL (10.8 mmol) of
triethylamine. The reaction mixture was stirred overnight. The reaction
mixture
was dissolved in 100 mL of diethyl ether, washed with 10% HCl three times,
brine once, 20% potassium carbonate once and brine once. The solution was
dried over magnesium sulfate, filtered, and evaporated to yield the product.
Other amino acid esters may also be employed in this procedure.
GENERAL PROCEDURE H
Fifth EDC Cou~,Lg Procedure
In a round bottom flask was added a carboxylic acid (1.1 eq.) in THF, an
amine hydrochloride (1.0 eq.), 1-hydroxybenzotriazole hydrate (1.1 eq.), N,N-
diisopropylethylamine (2.1 eq.), followed by 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDC) (1.1 eq.). The reaction mixture stirred
at room temperature for 10-20 hours under an atmosphere of nitrogen. The
mixture was diluted with EtOAc and washed with 0.1 M HCI (1 x 10 mL),
saturated NaHC03 (1 x 10 mL), H,O (1 x 10 mL), and brine and dried over
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ ~g __
MgS04. The drying agent was removed by filtration and the filtrate was
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel followed by trituration from EtOAc and hexanes.
GENERAL PROCEDURE I
Sixth EDC Coupling Procedure
To a solution or suspension of the amine or amine hydrochloride (1.0 eq.)
in THF (0.05-0.1 M) under N~ at 0°C was added the carboxylic acid (1.0-
1.1
eq.), hydroxybenzotriazole monohydrate (1.1-1.15 eq.), Hunig's base (1.1 eq.
for free amines and 1.1-2.3 eq. for hydrochloride amine salts), followed by 1-
(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.1-1.15 eq.). The
cooling bath was removed and the mixture allowed to warm to room temperature
for 10-24 hours. The solution or mixture was diluted with EtOAc, in a 3-5
volume multiple of the initial THF volume, and washed with 0.1-1.0 M aq. HCI
(1 or 2x), dilute NaHC03 (I or 2x), and brine (lx). Then, the organic phase
was
dried over either MgSO~ or Na,S04, filtered, concentrated to provide the crude
product, which was either further purified or utilized without further
purification.
GENERAL PROCEDURE J
EEDO Cou~~g Procedure
To a solution of the amine in THF (1.0 eq., 0.05-0.08 M, final molarity)
under N, at room temperature was added the N-t-Boc protected amino acid (1.1
eq., either as a solid or in THF via cannula), followed by EEDQ (Aldrich, I.1
eq.). The pale yellow solution was stirred at room temperature for 16-16.5
hours, then diluted with EtOAc (in a 3-5 volume multiple of the initial THF
volume), and washed with 1M aq. HCl {2x), dilute aq. NaHC03 (2x), and brine
(Ix). The organic phase was dried over either NarS04 or MgS04, filtered, and
concentrated.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 79 __
II. Carboxylic Acids
The following procedures may be used to prepare carboxylic acid
intermediates useful in the present invention:
GENERAL PROCEDURE II-A
Ester Hydrolysis to Free Acid
Ester hydrolysis to the free acid was conducted by conventional methods.
Below are two examples of such conventional de-esterification methods.
Method A: To a carboxylic ester compound in a 1: I mixture of
CH30H/H~O was added 2-5 equivalents of KZC03. The mixture was heated to
50°C for 0.5 to 1.5 hours until tlc showed complete reaction. The
reaction was
cooled to room temperature and the methanol was removed on a rotary
evaporator. The pH of the remaining aqueous solution was adjusted to ~ 2, and
ethyl acetate was added to extract the product. The organic phase was then
washed with saturated aqueous NaCI and dried over MgS04. The solution was
stripped free of solvent on a rotary evaporator to yield the product.
Method B: The amino acid ester was dissolved in dioxane/water (4:1) to
which was added LiOH ( - 2 eq. ) that was dissolved in water such that the
total
solvent after addition was about 2:1 dioxane:water. The reaction mixture was
stirred until reaction completion and the dioxane was removed under reduced
pressure. The residue was dissolved in water and washed with ether. The layers
were separated and the aqueous layer was acidified to pH 2. The aqueous layer
was extracted with ethyl acetate. The ethyl acetate extracts were dried over
Na,SO,, and the solvent was removed under reduced pressure after filtration.
The
residue was purified by conventional methods (e.g., recrystallization).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99l14007
__ g0 __
GENERAL PROCEDURE II-B
Acid Chloride Preparation
3,5-Difluorophenylacetic acid (30 g, 0.174 mol) (Aldrich) was dissolved
in dichloromethane and this solution was cooled to 0°C. DMF (0.5 mL,
catalytic) was added followed by the dropwise addition of oxalyl chloride (18
mL, 0.20 mol) over a S minute period. The reaction was stirred for 3 h and
then
rotoevaporated at reduced pressure to give an oil which was placed on a high
vacuum pump for 1 h to afford 3,5-difluorophenylacetyl chloride as a thin
yellow
oil. Other acid chlorides can be prepared in a similar manner.
GENERAL PROCEDURE II-C
Schotten-Bauman_n_ Procedure
3,5-Difluorophenylacetyl chloride (from General Procedure II-B) was
added dropwise to a 0°C solution of L-alanine (Aldrich) (16.7 g, 0.187
mol) in 2
N sodium hydroxide (215 mL, 0.43 mol). The reaction was stirred for 1 h at
0°C and then overnight at room temperature. The reaction was diluted
with
water (100 mL), then extracted with ethyl acetate (3 x 150 mL). The organic
layer was then washed with brine (200 mL), dried over MgS04, and
rotoevaporated at reduced pressure to a residue. Recrystallization of the
residue
from ethyl acetate/hexanes afforded the desired product (34.5 g, 82% yield).
Other acid chlorides.may be used in this procedure to provide for
intermediates
useful in this invention.
GENERAL PROCEDURE II-D
geductive Amination
To a solution of the arylamine in ethanol in a hydrogenation flask was
added 1 equivalent of the 2-oxocarboxylic acid ester (e.g., pyruvate ester),
followed by 10% palladium on carbon (25 weight percent based on the
arylamine). The reaction was hydrogenated at 20 psi H, on a Parr shaker until
complete reaction was indicated by tlc (30 minutes to 16 hours). The reaction
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ gl __
mixture was then filtered through a pad of Celite 545 (available from Aldrich
Chemical Company, Inc.) and stripped free of solvent on a rotary evaporator.
The crude product residue was then further purified via chromatography.
The following procedures illustrate the synthesis of carboxylic acid
intermediates useful in preparing compounds of this invention:
Example A
Synthesis of N-(Phenylacetyl)-L-alanine
Using General Procedure II-C, the title compound was prepared from
phenylacetyl chloride (Aldrich) and L-alanine (Aldrich) as a solid having a
melting point of 102-104°C.
NMR data was as follows:
'H-nmr (CDC13): 8 = 9.14 (br s, 1H), 7.21-7.40 (m, SH), 6.20 (d, J =
7.0 Hz, 1H), 4.55 (m, 1H), 3.61 (s, 2H), 1.37 (d, J = 7.1 Hz, 3H).
"C-nmr (CDC13): b = 176.0, 171.8, 134.0, 129.4, 127.5, 48.3, 43.2, 17.9.
Example B
Synthesis of N-(3,5-Difluorophenylacetyl)-L-alanine
Using General Procedure II-C, the title compound was prepared from 3,5-
difluorophenylacetyl chloride (General Procedure II-B) and L-alanine
(Aldrich).
NMR data was as follows:
'H-nmr (CD30D): b = 8.32 (br s, 0.3H), 6.71 (m, 2H), 6.60 (m, 1H), 4.74
(br s, 1.7H), 4.16 (m, 1 H), 3.36 (s, 2H), 1.19 (d, J = 7.3 Hz, 3H).
'3C-nmr (CD30D): 8 = 175.9. 172.4, 164.4 (dd, J = 13.0, 245.3 Hz), 141.1,
113.1 (dd, J = 7.8, 17.1 Hz), 102.9 (t, J = 25.7 Hz), 49.5, 42.7, 17.5.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ g2 __
Example C
Synthesis of N-(Cyclopentylacetyl)-L-phenylglycine
Sin A - Prenaration of N-l~vcloRentylace yll-L-~y~elvcine Meths
Following General Procedure A above using cyclopentylacetic acid
(Aldrich) and phenylglycine methyl ester hydrochloride (Novabiochem), the
title
compound was prepared as a solid having a melting point of 83-86°C. The
reaction was monitored by tlc on silica gel (Rf = 0.28 in 25% ethyl
acetate/hexanes) and purification was by recrystalIization from ethyl
acetate/hexanes.
NMR data v~ras as follows:
'H-nmr (CDCl3): S = 7.35 (s, 5H), 6.44 (bd, 1H), 5.6 (d, 1H), 3.72 (s, 3H),
2.24 (bs, 3H), 1.9-1.4 (m, 6H), 1.2-1.05 (m, 2H).
"C-nmr (CDC13): b = 172.3, 171.7, 136.7, 129.0, 128.6, 127.3, 56.2, 52.7,
42.5, 36.9, 32.40, 32.38, 24.8.
C,6HZ~NO3 (MW = 275.35); mass spectroscopy (M+Na) 298.
Sten B - Preparation of N-lCycloRe_~,lylacet3rl)-L-nhenvlelvcine
Following General Procedure II-A above using N-(cyclopentylacetyl)-L-
phenylglycine methyl ester (from Step A), the title compound was prepared as a
solid having a melting point of 155-158°C. The reaction was monitored
by tlc on
silica gel (Rf = 0.18 in 10% methanol/dichloromethane).
NMR data was as follows:
'H-nmr (CDCl3}: 8 = 8.60 (d, J = 7.8 Hz, 1H}, 7.45 (m, 5H0, 5.41 {d, J =
7.2 Hz, 1H), 2.20 (m, 3H), 1.8-1.1 (m, 8H)..
'3C-nmr (CDC13): b = 172.3, 172.0, 137.5, 128.7, 128.1, 127.8, 56.2, 40.9,
36.8, 31.8, 24.5.
C,SH,9N03 (MW = 261.32); mass spectroscopy (M+Na) 284.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ g3 -_
Example D
Synthesis of N-(Cyclopentylacetyl)-L-alanine
Sten A - Preparation of N-lCyclopentylacetyl)-L-alanine Methvl Ester
Following General Procedure A above using cyclopentylacetic acid
(Aldrich) and L-alanine methyl ester hydrochloride (Sigma), the title compound
was prepared as a solid having a melting point of 43-46°C. Purification
was by
recrystallization from ethyl acetate/hexanes.
NMR data was as follows:
'H-nmr (CDC13): b = 6.38 (d, 1H), 4.50 (m, 1H), 3.65 (s, 3H), 2.13 (bs,
3H), 1.80-I.00 (m (includes d at 1.30, 3H), 1 IH).
'3C-nmr (CDC13): b = 173.7, 172.5, 52.1, 47.6, 42.3, 36.8, 32.15, 32.14,
18Ø
C"H,9N03 (MW = 213.28); mass spectroscopy (MH') 214.
Sten B - Preparation of N-(Cvclooen ylacetyly-L-alanine
Following General Procedure II-A above using N-(cyclopentylacetyl)-L-
alanine methyl ester (from Step A), the title compound was prepared. The
reaction
was monitored by tlc on silica gel (Rf = 0.18 in 10%
methanol/dichloromethane).
NMR data was as follows:
' H-nmr (DMSO-db): S = 12.45 (bs, I H), 8.12 (d, J=7.2 Hz, 1 H), 4.24
(quint. J = 7.2 Hz, I H), 2.14 (m, 3H), 1.8-1.4 (m, 6H), 1.29 (d, J = 7.2 Hz,
3H),
1.2-1.0 (m, 3H).
"C-nmr (DMSO-db): b = 174.6, 171.9, 47.3, 41.1, 36.7, 31.8, 24.5, 17.2.
C,oH"N03 (MW = 199.25); mass spectroscopy (MH') N/A.
Example E
Synthesis of N-(Cyclopropylacetyl)-L-alanine
S~en A - Preparation of N-(Cycloprop 1~~)-L-alanine Meth 1
Following General Procedure A above using cyclopropylacetic acid
(Aldrich) and L-alanine methyl ester hydrochloride (Sigma), the title compound
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
_- g4 __
was prepared as an oil. The reaction was monitored by tlc on silica gel (Rf =
0.15
in 25% ethyl acetate/hexanes) and purification was by flash column
chromatography using 25% ethyl acetate/hexanes as the eluant.
NMR data was as follows:
'H-nmr (CDCl3): b = 6.60 (d, 1H), 4.55 (m, 1H), 3.69 (s, 3H), 2.10 (m, 2H),
1.34 (d, 3H), 0.95 (m, 1H), 0.58 (m, 2H), 0.15 (m, 2H).
'3C-nmr (CDC13): b = 173.7, 172.3, 52.3, 47.7, 41.0, 18.2, 6.7, 4.27, 4.22.
C9H,SN03 (MW = 185.22); mass spectroscopy (MH+) N/A.
St B~'- reparation of N-yvclopent Iv acet3rl)-L-alanine
Following General Procedure II-A above using N-(cyclopropylacetyl)-L-
alanine methyl ester (from Step A), the title compound was prepared as an oil.
The
reaction was monitored by tlc on silica gel (Rf = 0.27 in 10%
methanol/dichloromethane).
NMR data was as follows:
H-nmr (DMSO-d6): S = 8.18 (d, 1 H), 4.25 (m, 1 H), 2.08 (m, 2H), 1.30 (d,
3H), 1.00 (m, 1H), 0.50 (m, 2H), 0.19 (m, 2H).
'3C-nmr (DMSO-d6): b = 174.6, 171.7, 47.4, 17.3, 7.6, 4.12, 4.06.
CgH,3N03 (MW = 199.25): mass spectroscopy (MH') N/A.
Example F
Synthesis of N-(Cyclopropylacetyl)-L-phenylglycine
Step A - Preparation of N-f~vcloprop, ly ace~yll-3,~gl~cine Methyl Ester
Following General Procedure A above using cyclopropylacetic acid
(Aldrich) and L-phenylglycine methyl ester, the title compound was prepared as
a
solid having a melting point of 74-76°C. The reaction was monitored by
tlc on
silica gel (Rf = 0.61 in 50% ethyl acetate/hexanes) and purification was by
recrystallization from ethyl acetate/hexanes.
NMR data was as follows:
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ gs __
'H-nmr (CDC13): b = 7.35 (m, SH), 6.97 (bd, J=7.2 Hz, 1 H), 5.59 (d, J=7.8
Hz, 1 H), 3.71 (s, 3H), 2.17 (m, 2H), 1.05-0.95 (m, 1 H), 0.62 (m, 2H), 0.20
(m,
2H).
'3C-nmr (CDCl3): b = 171.9, 174.6, 136.6, 129.0, 128.5, 127.2, 56.1, 52.7,
41.0, 6.9, 4.37, 4.33.
C,4H1,N03 (MW = 247.30); mass spectroscopy (MH+) N/A.
Step B - Preparation of N-(C~cl_ ent lr~acetyj"1-L-phenylglycine
Following General Procedure II-A above using N-(cyclopropylacetyl}-L-
phenylglycine methyl ester (from Step A), the title compound was prepared as a
solid having melting point of 152-157°C. The reaction was monitored by
tlc on
silica gel (Rf = 0.23 in 10% methanol/dichloromethane) and purification was by
recrystallization from ethyl acetate/hexanes.
NMR data was as follows:
'H-nmr (CDCl3): 8 = 8.47 (d, J = 7.69 Hz, 1H), 7.35 (m, SH), 5.34 (d, J =
7.69 Hz. 1 H), 2.10 (m, 2H), 0.90 (m, 1 H), 0.40 (m, 2H), 0.10 (m, 2H).
"C-nmr (CDCl3): 8 = 172.3, 171.8, 137.6, 128.7, 56.2, 7.7, 4Ø
C'~H'SN03 (MW = 233.27}; mass spectroscopy (MH') N/A.
Example H
Synthesis of
N-(2-Biphenyl)-D,L-alanine
2-Aminobiphenyl (2 g, 11.8 mmol, Aldrich), triethylamine ( 1.2 eq.) and
ethyl 2-bromopropionate ( 1.1 eq., Aldrich) were combined and heated to 85
°C
with stirring. After 7 days, the mixture was diluted with chloroform and
washed
with water. The organic portion was dried and concentrated to yield an oil
which
was purified by silica gel chromatography (1:1 CH,CI,/hexanes). The resulting
oil
was dissolved in a 1:2 mixture of water/dioxane (200 mL) and LiOH (2 eq.) was
added. After 2 hours, the mixture was concentrated to yield an oil which was
dissolved in water. The aqueous solution was washed with ether then was
adjusted
to pH 3 with SN HCl and extracted with ethyl acetate. The organic portion was
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ g6 __
dried and concentrated to yield an oil which was purified by silica gel
chromatography (EtOAc) to yield the title compound.
Example I
Synthesis of
N-(Phenyl-furazan-3-yl)-D,L-alanine
Following General Procedure II-D and using 4-phenyl-furazan-3-ylamine
(Maybridge) and ethyl pyruvate (Aldrich), the ethyl ester was prepared.
Following
General Procedure II-A, Method B (LiOH/H,0/dioxane) and using the ethyl ester,
the title compound was prepared.
Example L
Synthesis of
S-(+)-3,5-Ditluoromandelic Acid
Step A - Preparation of Methvl S-lf)-3.5-difluoromandelate
To a solution of 3,5-difluorobenzaldehyde (Aldrich) in CH,CI, (100 mL)
was added ZnCh (6.7 g, 21.1 mmol) to form a slurry. Trimethysilyl cyanide
(21.0
g, 211.2 mmol) dissolved in CH,C1, (100 mL) was slowly added to the slurry at
0°C. The resulting solution was stirred at room temperature for 4 h.
The reaction
mixture was then diluted with water and the organic layer separated. The
combined organic layers were concentrated to a residue. The residue was
dissolved with MeOH (200 mL) at 0°C and anhydrous HCl gas bubbled into
the
solution for I 0 min. After stirring at room temperature for 18 h, the
solution was
concentrated to a solid. The solid was dissolved in CH,C1, / H,O and the
aqueous
portion extracted with CH,CI,. The combined organics were washed with brine,
dried over anhydrous MgSO~ and concentrated to a solid (37.4 g, 87.6%), mp =
77-
78°C.
'H NMR (300 MHz, CDC1;): b = 6.97 (dd, J = 9.6 Hz, J = 1.79 Hz, 2H),
6.74 (dt, J = 8.82, J = 2.28 Hz, 1 H), 5.14 (d, J = 4.64 Hz, 1 H), 3.78 (s, 3
H), 3.54 (d,
J = 5.1 Hz, 1 H).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ g7 __
Step B - Preparation of Methyl S-l+)-3.5-difluoromandelate
Methyl (f)-3,5-difluoromandelate was separated via preparative chiral
HPLC to give a white solid having a melting point of 70-71 °C.
C9HaF,03 (MW = 202.17); mass spectroscopy found (M+NH4a) 220Ø
Anal. calcd for C9HeF,0;: C, 53.47; H, 3.99. Found: C, 53.40; H, 3.89.
Step C - Pret~aration of S-f+)-3.5-Difluoromandelic acid
A solution of methyl S-(+)-3,5-difluoromandelate ( I eq.) in 74% aqueous
THF was cooled to 0 °C and treated with lithium hydroxide. After 40
minutes at 0
°C the reaction was complete by TLC. The contents were transferred to a
separatory funnel and partitioned between CH,CI, and saturated aqueous NaHC03.
The aqueous layer was acidified with 0.5 N NaHSO, and extracted thrice with
ethyl acetate. The combined extracts were washed with brine, dried over
Na~S04,
filtered, and concentrated to a white solid having a melting point of 119-122
°C.
The'H NMR was consistent with known 3,5-difluoromandelic acid.
Example N
Synthesis of
(R)-N,N'-Di-BOC-2-Hydrazinopropionic Acid
Sten A: To (S)-(-)-4-benzyl-2-oxazolidanone (Aldrich) in THF cooled to -
50°C was added n-butyl lithium 1.1 eq. (1.6 M in hexane) dropwise. The
reaction
mixture was allowed to warm to -20°C and then was re-cooled to -
78°C and
propionyl chloride ( 1. I eq) was added in one portion. The reaction mixture
was
allowed to stir an additional 15 min. at -78°C and then was allowed to
warm to
room temperature. The reaction was then quenched with a saturated solution of
sodium bicarbonate and extracted with ethyl acetate. The organic extracts were
washed with water, followed by brine and then dried over sodium sulfate,
filtered
and concentrated to give (S)-(-)-3-propionyl-4-benzyl-2-oxazolidanone.
Step B: To a solution of (S)-(-)-3-propionyl-4-benzyl-2-oxazolidanone in
THF at -78 °C was added LiHMDS ( 1.05 eq.) (Aldrich) dropwise. The
reaction
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/I4007
__ gg __
mixture was allowed to stir at -78 °C for 30 min. and then a precooled
solution of
di-tert-butyl-azodicarboxylate (Aldrich) was added via a cannula. After S min.
2.6
eq. of acetic acid was added. The reaction mixture was then extracted with
dichloromethane and the organic layer was washed with 1 M potassium phosphate.
The organic layer was then dried over sodium sulfate, filtered and
concentrated to
give (S)-(-)-3-[(R)-N,N'-di-BOC-2-hydrazinopropionyl]-4-benzyl-2-
oxazolidanone.
Stern C: To the product from Step B (1.0 g, 2.16 mM) in THF (34 mL) and
water (I3 mL), cooled to 0°C, was added a 30% solution of H,O, (0.734
mL, 6.4
mM) and LiOH (57 mg, 2.37 mM). The reaction mixture was stirred at ambient
temperature for 1.5 h and then quenched vi~ith 1.5 N Na~S03 (15 mL). A
saturated
solution of sodium bicarbonate (48 mL) was added and the mixture was partioned
between dichloromethane and water. The aqueous layer was acidified with 10%
citric acid and extracted into dichloromethane. The organic layer was dried
over
Na,SO,, filtered and concentrated to give the title compound (390 mg, 60%) as
a
colorless glass which was used without further purification.
Example O
Synthesis of
3,5-Difluorophenyl-a-oxoacetic Acid
Step A: Ethyl 3,5-difluorophenyl-a-oxoacetate was prepared from 1-
bromo-3.5-difluorobenzene (Aldrich) according to the procedure described in J.
Org. Chem., 45 (14), 2883-2887 (1980).
Step B: Ethyl 3,5-difluorophenyl-a-oxoacetate was hydrolyzed using
General Procedure II-A (Method B) to afford 3,5-difluorophenyl-a-oxoacetic
acid.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__g9_
Example P
Synthesis of
Cyclopentyl-a-hydroxyacetic Acid
The title compound (CAS No. 6053-71-0) was prepared in two steps from
cyclopentylmethanal (CAS No. 872-53-7, Wiley) using the procedure described by
Gibby, W. A.; Gubler, C. J. Biochemical Medicine 1982, 27, 15-25.
Example Q
Synthesis of N-(3,4-Dichlorophenyl)alanine
Using the procedure set forth in U.S. Patent No. 3,598,859, the disclosure
of which is incorporated herein by reference in its entirety, N-(3,4-
dichlorophenyl)alanine was prepared. Specifically, to a solution of 3,4-
dichloroaniline (1 equivalent) (Aldrich) in isopropanol (about S00 mL per mole
of
3,4-dichloroaniline) is added water (about O.Ob mL per mL of isopropanol) and
2-
chloropropionic acid (2 equivalents) (Aldrich). This mixture is warmed to
40°C
and sodium bicarbonate (0.25 equivalents) is added in successive portions
before
heating under reflux for 4-5 days. After cooling, the reaction mixture is
poured
into water and the unreacted 3,4-dichloroaniline is removed by filtration. The
filtrate is acidified to pH 3-4 with concentrated hydrochloric acid and the
resultant
precipitate is filtered, washed and dried to yield the title compound, m.p. =
148-
149°C.
Example R
Synthesis of N-(3,5-Difluorophenyl)alanine
Using the procedure set forth in U.S. Patent No. 3,598,859 and Example Q
above, N-(3,5-difluorophenyl)alanine was prepared using 3,5-difluoroaniline
(Aldrich) and 2-chloropropionic acid (Aldrich).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 90 --
Example S
Synthesis of
a-Fluoro-3,5-difluorophenylacetic Acid
S»en A - Synthesis of Methyl 3,5-Difluoromandelate
To a solution of 3,5-difluoromandelic acid (Fluorochem) in methanol was
bubbled HC1 gas for 10 minutes. The reaction was refluxed overnight. The
mixture was then concentrated in vacuo and the residue was taken up in ethyl
acetate and washed with saturated NaHCO; and brine. The organic layer was
dried
over Na,SO~, filtered, and concentrated to give the title intermediate as a
white
solid.
C9H8F,0; (MW=202.17); mass spectroscopy 202.
'H NMR (300 MHz, CDCI;): 8 = 7.00 (2H, d, J=6.58 Hz), 6.76 (1H, t,
J=8.86 Hz), 5.16 ( 1 H, d, J=5.29 Hz), 3.81 (3 H, s), 3.54 ( 1 H, d, J=5.39
Hz).
Ste - Synthesis of Methyl a-Fluoro-3,5-difluorophenylacetate
A solution of diethylaminosulfur trifluoride (DAST} (1.1 eq) in methylene
chloride was cooled to 0°C and a pre-cooled solution of methyl 3,5-
difluoromandelate ( 1 eq) in methylene chloride was added. The transfer flask
was
rinsed with a small portion of methylene chloride. After 15 minutes, the
cooling
bath was removed and the reaction mixture was stirred an additional 40 minutes
at
ambient temperature. The mixture was poured over ice and the layers separated.
The organic phase was washed with saturated NaHCO; and brine. The organic
layer was dried over Na,SO~, filtered, and concentrated. The residue was
purified
via HPLC eluting with 7% ethyl acetate/hexanes providing the title
intermediate as
a yellow oil.
C9H,F,0~ (MW=204.16); mass spectroscopy 204.
Anal. calcd for C9H,F;0,: C, 52.95; H, 3.46. Found: C, 52.80; H, 3.73.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 91 --
St~gp C - Synthesis of a-Fluoro-3,5-difluorophenylacetic Acid
Following General Procedure II-A, Method B and using methyl a-fluoro-
3,5-difluorophenylacetate, the title intermediate was prepared as a white
solid
having a melting point of 100-102°C.
CBHSF,O~ (MW = 190.13); mass spectroscopy 190.
Anal. calcd for C8HSF30,: C, 50.54; H, 2.65. Found: C, 50.47; H, 2.79.
Additionally, various other amino acid derivatives suitable for use in this
invention are disclosed in PCT Publication No. WO 98/22430 and WO 98/22493,
both published on May 28, 1998, the disclosures of which are incorporated
herein
by reference in their entirety.
III. Cyclic Com ounds
The following procedures illustrate the synthesis of various cyclic
compound intermediates useful for preparing compounds of this invention:
A. Benz eninone Derivatives and Related Com op ands
GENERAL PROCEDURE 6-A
Alkylation of
1-Amino-1 3 4 5-tetral~ydro-2H-3-benzazepin-2-one
step A: 1-Ethoxycarbonylamino-1,3,4,5-tetrahydro-2H-3-benzazepin-2-
one was prepared according to the procedure of Ben-Ishai et al., Tetrahedron,
1987, ~3, 430.
Step: 1-Ethoxycarbonylamino-1,3,4,5-tetrahydro-2H-3-benzazepin-2-one
(2.0 g, 100 M%) was dissolved in DMF (30 mL) and NaH (95%, 0.17 g, 100M%)
was added in one portion. The reaction mixture was stirred for 1 hour and then
the
appropriate alkyl iodide (300M%) was added and the mixture was stirred for 12
hours. The reaction was poured into water and extracted with ethyl acetate
(3x).
The ethyl acetate extracts were then washed with water (3x) and brine (lx).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 92 --
Treatment with MgSOa, rotoevaporation, and chromotography (30%
EtOAc/hexanes) yielded 1-ethoxycarbonylamino-3-alkyl-1,3,4,5-tetrahydro-2H-3-
benzazepin-2-one in 87% yield.
Step C: 1-Ethoxycarbonylamino-3-alkyl-1,3,4,5-tetrahydro-2H-3-
benzazepin-2-one ( 1.Og, 1 OOM%) was suspended in 30 mL of 30% HBr/HOAc and
heated to 100°C. The reaction mixture was stirred for 5 hours at this
temperature
and then the reaction was cooled and rotoevaporated to yield I-amino-3-alkyl-
1,3,4,5-tetrahydro-2H-3-benzazepin-2-one as the hydrobromide salt (100%
yield).
GENERAL PROCEDURE 6-B
Alkylation of
3-Ami no- I .3.4.5-tetrah~vdro-2H-1-benzazepin-2-one
to : 3-Amino-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one was prepared
from a-tetralone using the methods described in Armstrong et al. Tetrahedron
Letters, 1994, 35, 3239. The following compounds were as prepared by this
procedure for use in the following steps:
5-methyl-3-amino-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (from 4-
methyl-a-tetralone (Aldrich)); and
5,5-dimethyl-3-amino-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (from
4,4-dimethyul-a-tetralone (Aldrich)).
Step B: 3-Amino-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (4.43 g,
100M % ) was suspended in t-butanol (30mL) and BOC-anhydride (7.5 mL,
130M%) was added dropwise. The reaction was stirred for 2 hours and then it
was rotoevaporated to a residue which was chromatographed with 60% ethyl
acetate/hexanes to yield BOC-protected 3-amino-1,3,4,5-tetrahydro-2H-1-
.benzazepin-2-one in 87 % yield.
St~,~p C: BOC-protected 3-amino-1,3,4,5-tetrahydro-2H-1-benzazepin-2-
one (1.5 g, 100M%) was dissolved in DMF (20mL) and NaH (95%, 0.13g,
CA 02325388 2000-09-21
WO 99/67220 PCT/fJS99/14007
-- 93 --
100M % ) was added in one portion. The reaction mixture was stirred for 1 hour
and then the appropriate alkyl iodide (300M % ) was added and stirring was
continued for 12 hours. The reaction was poured into water and extracted with
ethyl acetate (3x). The ethyl acetate extracts were washed with water (3x) and
then brine (lx). Treatment with MgS04, rotoevaporation, and chromotography
(30% EtOAclhexanes) yielded a BOC-protected 3-amino-1-alkyl-1,3,4,5-
tetrahydro-2H-1-benzazepin-2-one in 80% yield.
Step D: The BOC-protected 3-amino-1-alkyl-1,3,4,5-tetrahydro-2H-1-
benzazepin-2-one (l.Og, 100M%) was suspended in 30 mL of l:l
CH~Ch/triflouroacetic acid and the mixture was stirred for 4 hours. The
reaction
was then rotoevaporated to yield the 3-amino-1-alkyl-1,3,4,5-tetrahydro-2H-1-
benzazepin-2-one (100% yield).
Example 6-A
Synthesis of
3-Amino-1,S-dimethyl-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one
St~~ A: 3-Amino-5-methyl-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one
was prepared from 4-methyl-a-tetralone using the methods described in
Armstrong et al. Tetrahedron Letters, 1994, 35, 3239.
Sten BB: 3-Amino-5-methyl-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one
(9.3g 100M%) was dissolved in dioxane (300mL) and the solution was chilled to
0°C. BOC-anhydride (13.89g 130M%) was added and the ice bath was
removed
allowing the solution to come to room temperature and stirring was continued
for
16 hours. The solution was rotory evaporated to remove dioxane to provide an
off white solid. This solid was recrystallized from CHCI~ to yield BOC-
protected 3-amino-5-methyl-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one in 55 %
yield.
CA 02325388 2000-09-21
WO 99/67220 PC'T/US99/14007
-- 94 --
Ste~C: BOC-protected 3-amino-5-methyl-1,3,4,5-tetrahydro-2H-1-
benzazepin-2-one ( 100 M % ) was dissolved in DMF (20mL) and NaH (95 % , 100
M % ) was added in one portion and the reaction mixture was stirred for 1
hour.
Methyl iodide (300 M%) was added and this mixture was stirred for 12 hours.
The reaction was then poured into water and extracted with ethyl acetate (3x)
then backwashed with water (3x) and then brine (lx). Treatment with MgS04,
rotoevaporation, and chromotography (5 % MeOH/CH,C12) yielded BOC-
protected 3-amino-1,5-dimethyl-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one in
75 % yield.
Step D: BOC-protected 3-amino-1,5-dimethyl-1,3,4,5-tetrahydro-2H-1-
benzazepin-2-one ( 100 M % ) was suspended in 30 mL of 1:1
CHZCIZ/triflouroacetic acid. The reaction mixture was stirred for 4 hours. The
reaction was then rotoevaporated to yield 3-amino-1,5-dimethyl-1,3,4,5-
tetrahydro-2H-1-benzazepin-2-one (100% yield).
Example 6-B
Synthesis of
5-(L-Alaninyl)-amino-3,3,7-trimethyl
5,7-dihydro-6H-benz[b]azepin-6-one Hydrochloride
Following the procedure of Example 7-I and using 5-amino-3,3,7-
trimethyl-5,7-dihydro-6H-bent[b]azepin-6-one hydrochloride (Example 6-C), the
title compound was prepared.
Example 6-C
Synthesis of
5-Amino-3,3,7-trimethyl-5,7-dihydro-
6H-benz[b]azepin-6-one Hydrochloride
Sten A: Following General Procedure 5-A and using N-t-Boc-5-amino-
3,3-dimethyl-5,7-dihydro-6H-bent[b]azepin-6-one (General Procedure 6-B,
following by Boc protection) and methyl iodide, N-t-Boc-5-amino-3,3,7-
trimethyl-5,7-dihydro-6H-Benz[b]azepin-6-one was prepared.
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/14007
-- 95 --
Step B: Following General Procedure 8-N and using N-t-Boc-5-amino-
3,3,7-trimethyl-5,7-dihydro-6H-bent[b]azepin-6-one, the title compound was
prepared.
Example 6-D
Synthesis of
3-(S)-Amino-1-methyl-5-oxa-1,3,4,5
tetrahydro-2H-1-benzazepin-2-one
Sit ~p A: 3-(S)-Amino-S-oxa-1,3,4,5-tetrahydro-2H-I-benzazepin-2-one
was prepared from N-Boc-serine (Bachem) and 2-fluoro-1-nitrobenzene (Aldrich)
using the method of R. J. DeVita et al., Bioorganic and Medicinal Chemistry
Lett. 1995, 5(12) 1281-1286.
Following General Procedure 5-A and using the product from
Step A, the title compound was prepared.
Example 6-E
Synthesis of
3-(S)-Amino-1-ethyl-5-oxa-1,3,4,5-
tetrahydro-2H-1-benzazepin-2-one
Step A: 3-(S)-Amino-5-oxa-1,3,4,5-tetrahydro-2H-1-benzazepin-2-one
was prepared from N-Boc-serine (Bachem) and 2-fluoro-1-nitrobenzene (Aldrich)
using the method of R. J. DeVita et al., Bioorganic and Medicinal Chemistry
Lett. 1995, 5(12) 1281-1286.
Ste,~B.~ Following General Procedure S-A and using the product from
Step A, the title compound was prepared.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 96 --
Example 6-F
Synthesis of
3-(S)-Amino-1-methyl-5-thia-1,3,4,5
tetrahydro-2H-1-benzazepin-2-one
The title compound was prepared from N-Boc-cystine (Novabio) and 2-
fluoro-1-nitrobenzene (Aldrich) using the method of R. J. DeVita et al.,
Bioorganic and Medicinal Chemistry Lett. 1995, 5(12) 1281-1286, followed by
General Procedure S-A.
Example 6-G
Synthesis of
7-Amino-1,3,4,7,12,12a-hexahydropyrido[2,1-b][3]
benzazepin-6(2H)-one
Step A - Synthesis of N Chloroace~vl-2-benz3lg,~ rsr~inP
Following General Procedure F and using 2-benzylpyridine, the title
compound was prepared.
Physical data were as follows:
(MW = 251.8); mass spectroscopy (MH+) 252Ø
Step B - Synthesis of 1.3.4.7.12 12a-hexah,~pyrido[,~
blf3]benzaze in-612H1-one
Following General Procedure G and using N chloroacetyl-2-
benzylpiperidine, the title compound was prepared.
Physical data were as follows:
'H-nmr (CDC13): 8 = 1.3-1.9 (6H); 2.42 (t, 1H); 3.08 (m, 2H); 3.47 (m,
1 H); 3.96 (q, 2H); 4.66 (d, 1 H); 7.2 (m, 4H).
(MW = 215.3); mass spectroscopy (MH+) 216.1.
Step C - Synthesis of 7-Oximo-1 _3,,4.7,12 12a-
hexah~R rido[2 1 ~lf~lbenzazepin-6 ~l-one
Following General Procedure A (Step B) and using 1,3,4,7,12,12a-
hexahydropyrido(2,1-b][3]benzazepin-6(2H)-one (from Step B), the title
compound was prepared.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 97 __
Physical data were as follows:
(MW = 244.3); mass spectroscopy (MH+) 245Ø
Step D - Synthesis of 7-Amino-1.3,4.7.12 12a-
hexa ~pyrido(2.1-b~j3lbenzazepin-6l H)-one
Following General Procedure A (Step C) and using 7-oximo-
1,3,4,7,12,12a-hexahydropyrido[2,1-b][3]benzazepin-6(2H)-one (from Step C),
the title compound was prepared.
Physical data were as follows:
'H-nmr (CDC13): 8 = 1.3-1.9 (6H); 2.42 (t, 1 H); 3.08 (m, 2H); 3.47 (m,
1 H); 3.96 (q,2H); 4.66 (d, l H); 7.2 (m,4H).
(MW = 230.3); mass spectroscopy (MH+) 231.1.
Example 6-H
Synthesis of
1-(N'-L-Alaninyl)amino-4,5,6,7-tetrahydro
3,7-methano-3H-3-benzazonin-2(1H)-one
Step A - Synthesis of N Chloroacetyl-3-nhenyl~ eridine
Following General Procedure F and using 3-phenylpyridine hydrochloride
(Aldrich), the title compound was prepared.
Step B - Svnthesis of 4.5.6.7-Tetrahydro-3 7-methano-3H-~-
benzazonin-211 Hl-one
Following General Procedure G and using N chloroacetyl-3-
phenylpiperidine, the title compound was prepared.
Physical data were as follows:
'H-nmr (CDCl3): d = 1.32-1.57 (2H); 2.08 (m, 2H); 2.8I (t, 1H); 3.13
(bs, 1H); 3.37 (m, 2H); 4.36 (m, 2H); 4.50 (d; 1H).
(MW = 201.3); mass spectroscopy (MH+) 202.1.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 9g __
Step C - Synthesis of 1-Oximo-4. 5 6 7-tetrahy~ro-~ 7-methano-3H-
3-Benz, zonin-~(1H)-one
Following General Procedure A {Step B) and using the product from Step
B, the title compound was prepared.
Step D - Synthesis of 1-Amino-4. 5 6 7-terrahydro-'~-7-methano-3H-
3-benzazonin-2~ 1 H)-one
Following General Procedure A (Step C) and using the product from Step
C', the title compound was prepared.
Physical data were as follows:
'H-nmr (CDC13): 8 = 2.86 (t, IH); 3. I7 (bs, 1H); 3.39 (dd, 1H); 4.40 (d,
1H); 4.50 (d, IH); 5.39 (s, 1H).
(MW = 216.3); mass spectroscopy (MH+) 217.4.
Step E - Synthesis of 1-lN'-Boc-L-Ala_ninyl)amino-4 5 6 7-
tetr~hydro-3 .7-methano-~-3-benzazonin-2( 1 H)-one
Following General Procedure D and using N tert-Boc-L-alanine (Aldrich)
and the product from Step D, the title compound was prepared.
Physical data were as follows:
(MW = 387.48); mass spectroscopy (MH+) 388.1.
Step F - Synthesis of 1-(N'-I -Alani~yl)amino-4 5,~ 7-tetrah,
3.7-methano-3H-3-benzazonin-2S 1 H)-one
Following General Procedure E and using the product from Step E, the
title compound was prepared.
Physical data were as follows:
'H-nmr (CDCl3): 8 = 2.85 (t, 1 H); 3.16 (bs, 1 H); 3.40 (dd, 1 H); 3.67 (m,
1 H); 4.3 5 (d, 1 H); 4.56 (d, 1 H); 6.40 (d, 1 H).
(MW = 287.4); mass spectroscopy (MH+) 288.1.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 99 --
B. Dibenzazeninone Derivatives and Related Com o=n unds
GENERAL PROCEDURE 7-A
Preparation of
5-Amino-7-alkyl-5,7-dihydro
6H-dibenz~b.~]azenin-6-one Derivatives
Step A: Following General Procedure 5-A and using 5,7-dihydro-6H-
dibenz[b,d]azepin-6-one and an alkyl halide, the 7-alkyl-5,7-dihydro-6H-
dibenz(b,d]azepin-6-one was prepared.
Step B: The 7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one (1 eq.) was
dissolved in THF and isoamylnitrite (1.2 eq.) was added. The mixture was
cooled to 0°C in an ice bath. NaHMDS (1.1 eq., 1M in THF) was added
dropwise. After stirring for 1 hour or until the reaction was complete, the
mixture was concentrated then acidified with 1N HCl and extracted with EtOAc.
The organic portion was dried and concentrated to yield a crude product which
was purified by silica gel chromatography.
Sten C: The resulting oxime was dissolved in EtOH/NH3 (20:1) and
hydrogenated in a bomb using Raney nickel and hydrogen (500 psi) at
100°C for
hours. The resulting mixture was filtered and concentrated to provide an oil
which was purified by silica gel chromatography to yield the title compound.
GENERAL PROCEDURE 7-B
Preparation of
Fluoro-substituted 5,7-dihydro-6H-
dibenz[b.d]azepin-6-one Derivatjv~e
A modification of the procedure of Robin D. Clark and Jahangir,
Tetrahedron, Vol. 49, No. 7, pp. 1351-1356, 1993'5 was used. Specifically, an
appropriately substituted N-t-Boc-2-amino-2'-methylbiphenyl was dissolved in
THF and cooled to -78°C. s-Butyl lithium (1.3M in cyclohexane, 2.2
eq.) was
added slowly so that the temperature remained below -65°C. The
resulting
CA 02325388 2000-09-21
WO 99/67224 PCT/US99/14007
__ 100 __
mixture was allowed to warm to -25°C and was stirred at that
temperature for 1
hour. The mixture was cooled to -78°C. Dry CO1 was bubbled through the
mixture for 30 seconds. The mixture was allowed to warm to ambient
temperature then was carefully quenched with water. The mixture was
concentrated under reduced pressure then was adjusted to pH 3 with 1N HCI.
The mixture was extracted with EtOAc and the organic portion was dried and
concentrated to yield a crude material. The crude material was dissolved in
methanol and the solution was saturated with HC1. The mixture was heated at
reflux for 12 hours then was allowed to cool. The mixture was concentrated to
provide crude lactam which was purified by chromatography or crystallization.
GENERAL PROCEDURE 7-C
Resolution of
5-Amino-7-methyl-5.7-dij~rdro-6H-diben~[b.d]~,epin-6-one
In a round bottom flask was added the racemic freebase amine (1.0 eq.) in
methanol followed by di p-toluoyl-D-tartaric acid monohydrate (1.0 eq.). The
mixture was concentrated in vacuo to a residue and redissolved in a moderate
volume of methanol and allowed to stir at room temperature open to the
atmosphere (8-72 hours). The solid was removed by filtration. The
enantiomeric excess was determined by chiral HPLC (Chiracel ODR) using 15 %
acetonitrile and 85 % H,O with 0.1 % trifluoroacetic acid and a flow rate of
1.0
mL/min at 35 °C. The resolved di p-toluoyl-D-tartaric salt was then
dissolved in
EtOAc and saturated NaHC03 until pH 9-10 was reached. The layers were
separated and the organic layer was washed again with saturated NaHC03, HBO,
and brine. The organic layer was dried over MgS04 and the drying agent was
removed by filtration. The filtrate was concentrated In vacuo. The free amine
was dissolved in MeOH and HCl (12M, 1.0 eq.) was added. The salt was
concentrated in vacuo and the resulting film was triturated with EtOAc. The
HCl
salt was filtered and rinsed with EtOAc. The ee was determined by chiral
HPLC.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 101 --
Example 7-A
Synthesis of
5-Amino-7-methyl-5,7-dihydro
6H-dibenz[b,d]azepin-6-one Hydrochloride
Step A - Synthesis of 7-Methyl-5.7-dihvdro-6H-dibenzlb dlazenin-6-one
A round bottom flask was charged with sodium hydride (0.295 g, 7.46
mmol) in 9.0 ml of DMF and treated with 5,7-dihydro-6H-dibenz[b,d]azepin-6-
one (1.3 g, 6.22 mmol) (CAS # 20011-90-9, prepared as described in Brown, et.
al., Tetrahedron Letters, No. 8, 667-670, (1971) and references cited
therein).
After stirring at 60°C for 1 h, the solution was treated with methyl
iodide (1.16
ml, 18.6 mmol} and stirring continued for 17 h with the exclusion of light.
After
cooling, the reaction was diluted with CH2Clz/H20, washed with NaHS04
solution, HzO, and dried over NaZS04. Evaporation and flash chromatography
(Si02, CHC13) gave 0.885 g (63 % ) of the title compound as a colorless solid.
NMR data was as follows:
'H-nmr (CDCI3): 6 = 7.62 (d, 2H), 7.26-7.47 (m, 6H), 3.51 (m, 2H), 3.32
(s, 3H).
C,SH,;NO{MW = 223.27); mass spectroscopy (MH+) 223.
Anal. Calcd for C,SH,3N0; C, 80.69 H, 5.87 N, 6.27. Found: C, 80.11 H,
5.95 N, 6.23.
Step B - Synthesis of 7-Methyl-5-oximo-~ 7-dihydro-6H-
dibenz b[~t. )a~gpin-6-one
The compound isolated above (0.700 g, 3.14 mmol) was dissolved in 20 ml
of toluene and treated with butyl nitrite (0.733 ml, 6.28 mmol). The reaction
temperature was lowered to 0°C and the solution was treated with KHMDS
(9.42
ml, 0.5 M) under N, atmosphere. After stirring for 1 h the reaction was
quenched
with a saturated solution of NaHSO~, diluted with CH,CI, and separated. The
organic layer was dried over Na,SO~ and the title compound purified by
chromatography (SiO~, 98:2 CHCl3/MeOH) giving 0.59 g (80 %) as a colorless
solid.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 102 --
C,SH,,N,O, (MW = 252.275); mass spectroscopy (MH+) 252.
Anal. Calcd for C,SH,~N,O~; C, 71.42 H, 4.79 N, 11.10. Found: C, 71.24
H, 4.69 N, 10.87.
Step C - Synthesis of 5-Amino-7-Methyl-5 7-dihvdro-6H-
dibenz[b d~azenin-6-one Hvdrochlor~e
The oxime isolated above (0.99 g, 3.92 mmol) was hydrogenated in a Parr
apparatus at 35 psi over 10 % Pd/C (0.46 g) in 3A ethanol. After 32 h the
reaction
mixture was filtered through a plug of celite, the filtrate evaporated to a
foam and
treated with a saturated solution of HCl (g) in Et,O. The resulting colorless
solid
was filtered, rinsed with cold Et,O and vacuum dried to give 0.66 g (61 %) of
the
title compound.
NMR data was as follows:
'H-nmr (DMSOd6): b = 9.11 (bs, 3H), 7.78-7.41(m, 8H), 4.83 (s, IH), 3.25
(s, 3H).
C,SH,~,N,O. HCl (MW = 274.753); mass spectroscopy (MH+ free base) 238.
Anal. Calcd for C, ~H,~N,O , HCI; C, 65.57 H, 5.50 N, 10.19 Found: C,
65.27 H. 5.67 N, 10.13.
Example 7-B
Synthesis of
(S)- and (R)-5-(L-Alaninyl)-amino-7-methyl
5,7-dihydro-6H-dibenz[b,d]azepin-6-one
Step A - Synthesis of (,S~- and ly-5-lN-Boc-L-Alanin~l amino 7
trlet yl-5.7-dihydro-6H-dihenz~ d~]azepin-6-one
Boc-L-Alanine (0.429 g, 2.26 mmol) (Aldrich) was dissolved in THF and
treated with HOBt hydrate (0.305 g, 2.26 mmol). and 5-amino-7-methyl-5,7-
dihydro-6H-dibenz[b,d]azepin-6-one (0.45 g, 1.89 mmol) (Example 7-A). The
temperature was lowered to 0°C and the reaction mixture treated with
EDC (0.449
g, 2.26 mmol) (Alrich) and stirred 17 hours under N,. The reaction mixture was
evaporated, the residue diluted with EtOAc/H,O, washed 1.0 N HC1, sat. NaHC03,
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 103 --
brine and dried over Na,S04. The diastereomers were separated on a Chiralcel
OD
column using 10% IPA/heptane at 1.5 ml/minute.
Isomer 1: Retention time 3.37 minutes.
NMR data was as follows:
'H-nmr (CDCI,): b = 7.62-7.33 (m, 9H), 5.26 (d, I H), 5.08 (m, 1 H), 4.34
(m, 1H), 3.35 (s, 3H), 1.49 (s, 9H), 1.40 (d, 3H).
Optical Rotation: [a],a = - 96 @ 589 nm (c = 1, MeOH).
C,3H,,N30~ (MW = 409.489); mass spectroscopy (MH+) 409.
Anal. Calcd for C,3H,,N;O~; C, 67.46 H, 6.64 N, 10.26. Found: C, 68.42
H, 7.02 N, 9.81.
Isomer 2: Retention time 6.08 minutes.
NMR data was as follows:
'H-nmr (CDC13): a = 7.74 (bd,1 H), 7.62-7.32 (rn, 8H), 5.28 (d, 1 H), 4.99
(m, 1H), 4.36 (m, 1H), 3.35 (s, 3H), 1.49 (s, 9H), 1.46 (d, 3H).
Optical Rotation: [a],° = 69 @ 589 nm (c = 1, MeOH).
C,3H,,N30, (MW = 409.489); mass spectroscopy (MH+) 409.
Anal. CaIcd for C,,H,,N,O~; C, 67.46 H, 6.64 N, 10.26. Found: C, 67.40
H, 6.62 N, 10.02
Step B - Synthesis of (Sl- and fR~ 5 ~L-Alaniny~,l-amino-7-methyl-
5.7-dih3rdro-6H-dibenzfb.d]azepin-6-one Hydrochloride
The compounds isolated in Part A (each isomer separately) were dissolved
in dioxane and treated with excess HCl (g). After stirring for 17 hours, the
title
compounds were isolated as colorless solids after evaporation and vacuum
drying.
Isomer 1:
C,gH,9N;O~.HC1 (MW = 345.832); mass spectroscopy (MH+ free base)
309.
Optical Rotation: [a],° _ - 55 @ 589 nm (c = 1, MeOH).
Isomer 2:
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- lp4 __
C,8H,9N,O,.HCI (MW = 345.832); mass spectroscopy (MH+ free base)
309.
Optical Rotation: [a],a = 80 @ 589 nm (c = 1, MeOH).
Example 7-C
Synthesis of
(S)- and (R)-5-(L-Valinyl)-amino-7-methyl
5,7-dihydro-6H-dibenz[b,d]azepin-6-one
Step A - Synthesis of lS~ and ( -5~- N-Boc-L-Valin,~, -amino-7-
methvl-5.7-dihydro-6H-dibenzlb.d]aze ip n-6-ong
Boc-L-Valine (0.656 g, 3.02 mmol) (Aldrich) was dissolved in THF and
treated with HOBt hydrate (0.408, 3.02 mmol), Dipea ( 1.05 ml, 6.05 mmol) and
5-
amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one hydrochloride (0.75 g,
2.75 mmol)(Example 7-A). The temperature was lowered to 0°C and the
reaction
mixture treated with EDC (0.601 g, 3.02 mmol)(Alrich) and stirred 17 hours
under
N,. The reaction mixture was evaporated, the residue diluted with EtOAc/H,O,
washed 1.0 N HCI, sat. NaHC03, brine and dried over Na,SO~. The diastereomers
were separated on a Chiralcel OD column using 10% IPA/heptane at 1.5
ml/minute.
Isomer 1: Retention time 3.23 minutes.
Optical Rotation: [a],° _ - 120 @ 589 nm (c = 1, MeOH).
C,SH3,N;0~ (MW = 437.544); mass spectroscopy (MH+) 438
Isomer 2: Retention time 6.64 minutes.
Optical Rotation: [a],° = 50 @ 589 nm (c = 1, MeOH).
C~3H3,N;Oa (MW = 437.544); mass spectroscopy (MH+) 438
Step B - Synthesis of (S)- and (~l-5-lL-Valin~l-amino-7- et ,L
5.7-dihydro-6H-dibenz~b.dlazepin-6-one Hydrochloride
The compounds isolated in Part A (each isomer separately) were dissolved
in dioxane and treated with excess HCl (g). After stirring for 17 hours, the
title
compounds were isolated as colorless solids after evaporation and vacuum
drying.
Isomer 1:
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- I05 --
C,oH,3N30~.HCl (MW = 373.88); mass spectroscopy (MH+ free base) 338.
Optical Rotation: [a],o = - 38 @ 589 nm (c = 1, MeOH).
Isomer 2:
C,oH23N30,.HC1 (MW = 373.88); mass spectroscopy (MH+ free base) 338.
Optical Rotation: [a]ZO = 97 @ 589 nm (c = 1, MeOH).
Example 7-D
Synthesis of
(S)- and (R)-5-(L-tert-Leucine)-amino-7-methyl-
5,7-dihydro-6H-dibenz[b,d]azepin-6-one
Step A - Synthesis of fS - and (R)-5-~V-Boc-L-tent-Leucinyl)-amino-
7-methyl-5.7-dihvdro-6H-dibenz(b.d]azepin-6-one
Boc-L-tert-Leucine (0.698 g, 3.02 mmol) (Fluka) was dissolved in THF and
treated with HOBt hydrate (0.408, 3.02 mmol), Dipea (1.05 ml, 6.05 mmol) and 5-
amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one hydrochloride (0.75 g,
2.75 mmol)(Example 7-A). The temperature was lowered to 0°C and the
reaction
mixture treated with EDC (0.601 g, 3.02 mmol) (Alrich) and stirred 17 hours
under
N,. The reaction mixture was evaporated, the residue diluted with EtOAc/H,O,
washed 1.0 N HCI, sat. NaHCO;, brine and dried over Na,SO.,. The diastereomers
were separated on a Chiralcel OD column using 10% IPA/heptane at 1.5
ml/minute.
Isomer 1: Retention time 3.28minutes.
Optical Rotation: [a],° _ - 128 @ 589 nm (c = 1, MeOH).
C,6H33N,O~ (MW = 451.571 ); mass spectroscopy (MH+) 452
Isomer 2: Retention time 5.52 minutes.
Optical Rotation: [a]zo = 26 @ 589 nm (c = 1, MeOH).
C,6H33N3O4 (MW = 451.571 ); mass spectroscopy (MH+) 452
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 106 --
Step B - ~ynthesis of fS)- and (~l-~L-tent-Leucin,LZamino-7-
m~th3r1-5.7-dihvdro-6H-dibenajb.d]azepin-6-one
I~ydrochloride
The compounds isolated in Part A (each isomer separately) were dissolved
in dioxane and treated with excess HC1 (g). After stirring for 17 hours, the
title
compounds were isolated as colorless solids after evaporation and vacuum
drying.
Isomer 1:
C,,H,SN30,.HCl (MW = 387.91); mass spectroscopy (MH+ free base) 352.
Optical Rotation: [a],o = - 34 @ 589 nm (c = 1, MeOH).
Isomer 2:
C,,H,SN30,.HCl (MW = 387.91 ); mass spectroscopy (MH+ free base) 352.
Optical Rotation: [a],a = 108 @ 589 nm (c = 1, MeOH).
Example 7-E
Synthesis of
5-(N-Boc-Amino)-5,7-dihydro-6H,7H-dibenz[b,d)azepin-6-one
Step A - ~ynthesis of 5-Iodo-5,7-dihydro-6H-dibenz[b-dlazgpin-6-
one
A solution of 5,7-dihydro-6H-dibenz[b,d]azepin-6-one ( 1.0 g, 4.77 mmol)
(Example 7-A) and Et;N ( 2.66 ml. 19.12 mmol) were stirred for 5.0 minutes at -
15°C in CH,Cl,and treated with TMSI (1.36 ml, 9.54 mmol). After
stirring for 15
minutes I, (I .81 g, 7.16 mmol) was added in a single portion and the reaction
allowed to warm to 5-10°C over 3 h. The reaction was quenched with sat.
Na,S03,
diluted with CH,Ch and separated. The organics were washed with Na,S03 and
NaHSO; and dried over MgSOa. After filtration. the organics were concentrated
to
approximately 20 ml and diluted with an additional 20 ml of hexanes. The title
compound was isolated as a tan precipitate by filtration.
CA 02325388 2000-09-21
WO 99/67220 PCTNS99/14007
-- 107 --
Step B - ~~rnthesis of 5-Azido-5,7-dihydro-6H-dibenzjb.dj~,P,~in-6-
The iodide isolate above was dissolved in DMF and treated with 1.2
equivalents of NaN3. After stirring 17 h at 23 °C the mixture was
diluted with
EtOAc/H,O, separated, washed with brine and dried over MgS04. The title
compound was triturated from hot EtOAc as a tan powder.
Step C - Synthesis of 5-lN-Boc-Amine-5.7-dih, dro-6 7H-
dibenzjb.dlazenin-6-one
The azide was dissolved in THF/H,O and stirred at 23 °C for 17 h
in the
presence of 3.0 equivalents of Ph3P. The reaction was diluted with 50
HOAc/toluene, separated, the aqueous layer extracted with toluene and
evaporated
to an oily residue. This was taken to pH 7.0 by the addition of 1 N NaOH, the
resulting HOAc salt was collected and vacuum dried. Finally, the compound was
treated with Boc anhydride ( 1.05 equivalents) and Et3N (2.1 equivalents) in
THF.
After stirring for 5 h at 23 °C the reaction was filtered and the title
compound
isolated as a colorless powder.
Example 7-F
Synthesis of
5-Amino-7-(2-methylpropyl)-5,7-dihydro
6H-dibenz[b,d]azepin-6-one Hydrochloride
Step A - Synthesis of 5-fN-Boc-Aminol-7-l -met yl~opyl -~5 7~-
di~: 6H-dibenzf b.dlazenin-6-one
A solution of 5-(N-Boc-amino)-5,7-dihydro-6H-dibenz[b,d]azepin-
6-one (0.2g, 0.617 mmol) (Example 7-E) in DMF was treated with Cs,CO, (0.22 g,
0.678 mmol) and warmed to 60°C. To the reaction mixture was added 1-
iodo-2-
methylpropane (0.078 ml, 0.678 mmol) and stirring continued for 17 h. After
cooling to 23 °C the mixture was diluted with CH,Ch, washed with
several
portions of brine and dried over Na,SO~. The title compound was purified by
chromatography (SiO,, CHC13/MeOH 9:1 ).
C,3H,8N,03 (MW = 380.41); mass spectroscopy (MH+) 381
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 108 --
Anal. Calcd for C,;H,8N~0;; C, 72.61 H, 7.42 N, 7.36. Found: C, 72.31 H,
7.64 N, 7.17.
Step B - Synthesis of 5-Amino-7-l2-methvlnroRyll-5.7-dihydro-6H-
dibenzl[b.d)azepin-6-one Hvdrochloride
The compound isolated in Part A was deprotected in dioxane saturated with
gaseous HCI. The title compound was isolated as a slightly colored solid after
evaporation and vacuum drying.
Example 7-G
Synthesis of
5-Amino-7-(methoxyacetyl)-5,7-dihydro
6H-dibenz(b,d]azepin-6-one Hydrachloride
Step A- S nthesi~ of 5-lN-Boc-Amino)-~methox,~yl_ -L
dihvdro-6H-dibenz(b.~]azepin-6-one
A solution of 5-(N-Boc-amino)-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
(1.03, 3.08 mmol) (Example 7-E) in DMF was treated with Cs,CO; (1.10 g, 3.39
mmol) and warmed to 60°C. To the reaction mixture was added bromomethyl
acetate (0.321 ml, 3.39 mmol) (Aldrich) and stirring continued for 17 h. After
cooling to 23 °C the mixture was diluted with CH,C1,, washed with
several
portions of brine and dried over Na,SO~. The title compound was purified by
chromatography (SiO,, CHC13).
C"H,~N,OS (MW = 396.44); mass spectroscopy (MH+) 397
Anal. Calcd for C"H,~N,OS; C, 66.65 H, 6.10 N, 7.07. Found: C, 66.28 H,
5.72 N, 6.50.
Step B - Synthesis of ~-Amino-7lmethox.~rac~yll-5.7-dih~dro-6H-
dibenzfb d,~azepin-6-one Hydrochloride
The compound isolated in Part A was deprotected in dioxane saturated with
gaseous HCI. The title compound was isolated as a colorless solid after
evaporation and vacuum drying.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 109 --
C"H,6N,03 HCI (MW = 332.78); mass spectroscopy (MH+ free base) 297.
Example 7-H
Synthesis of
5-Amino-7-(3,3-dimethyl-2-butanonyl)
5,7-dihydro-6H-dibenz[b,d]azepin-6-one Hydrochloride
Step A- Synthesis of 5-lN-Boc-Amino)-7-L'i.3-dimetl~rl-bu non~l~
5.7-dihydro-6H-dibenz(~.dlazenin-6-one
A solution of 5-(N-Boc-amino)-5,7-dihydro-6H-dibenz[b,dJazepin-6-one
(0.2 g, 0.617 mmol) (Example 7-E) in DMF was treated with Cs,C03 (0.3 g, 0.925
mmol) and warmed to 60°C. To the reaction mixture was added 1-chloro-
3,3-
dimethyl-2-butanone (0.096 ml, 0.74 mmol) (Aldrich) and stirring continued for
17
h. After cooling to 23 °C, the mixture was diluted with CH,CI,, washed
with
several portions of brine and dried over Na,SO~. The title compound was
isolated
as a colorless solid.
C,SH;oN,Oa (MW = 422.522); mass spectroscopy (MH+) 423
Step B - ~vnthesis of 5-Amino-7-1~.3-dimethvl-2-butanonyll-5.7-
dihvdro-6H-dibenz[b.d]aze~n-6-one Hydrochloride
The compound isolated in Part A was deprotected in dioxane saturated with
gaseous HC1. The title compound was isolated as a colorless solid after
evaporation and vacuum drying.
Example 7-I
Synthesis of
L-Alaninyl-5-amino-7-methyl-5,7-dihydro-
6H-dibenz[b,d]azepin-6-one Hydrochloride
Step A: Following General Procedure D and using N-t-Boc-L-alanine and
5-amino-7-methyl-5,7-dihydro-6H-dibenz[b,dJazepin-6-one, N-t-Boc-L-alaninyl-S-
amino-7-methyl-5,7-dihydro-6H-dibenz[b,dJazepin-6-one was prepared.
Sten B: Following General Procedure 8-N and using the N-t-Boc-L-
alaninyl-5-amino-7-methyl-5,7-dihydro-6H-dibenz(b,dJazepin-6-one, the title
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 110 --
compound was prepared. Other substituted N-t-Boc-L-alaninyl-5-amino-7-methyl-
5,7-dihydro-6H-dibenz[b,d]azepin-6-ones can also be prepared by this
procedure.
Example 7-J
Synthesis of
L-Valinyl-5-amino-7-methyl-5,7-dihydro-
6H-dibenz(b,d)azepin-6-one Hydrochloride
S_ten A: Following General Procedure D and using N-t-Boc-L-valine and
5-amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one, N-t-Boc-L-valinyl-S-
amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one was prepared.
Ste~B ~. Following General Procedure 8-N and using the N-t-Boc-L-
valinyl-~-amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one, the title
compound was prepared. Other substituted N-t-Boc-L-valinyl-5-amino-7-methyl-
5,7-dihydro-6H-dibenz[b,d]azepin-6-ones can also be prepared by this
procedure.
Example 7-K
Synthesis of
5-Amino-7-phenbutyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
Following General Procedure 7-A and using 5,7-dihydro-6H-
dibenz[b,d]azepin-6-one (prepared as described in Brown, et. al., Tetrahedron
rs, No. 8, 667-670, ( 1971 ) and references cited therein) and I -chloro-4-
phenylbutane (Aldrich), the title compound was prepared.
Example 7-L
Synthesis of
5-Amino-7-cyclopropymethyl-~,7-dihydro
6H-dibenz[b,d]azepin-6-one
Following General Procedure 7-A and using ~,7-dihydro-6H-
dibenz[b,d]azepin-6-one (prepared as described in Brown, et. al., Tetrahedron
tte , No. 8, 667-670, ( I 971 ) and references cited therein) and
(bromomethyl)cyclopropane (Aldrich), the title compound was prepared.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 111 --
Example 7-M
Synthesis of
5-Amino-7-(2',2',2'-trifluoroethyl)-5,7-dihydro
6H-dibenz[b,d]azepin-6-one
Following General Procedure 7-A and using 5,7-dihydro-6H-
dibenz[b,d)azepin-6-one (prepared as described in Brown, et. al., Tetrahedron
L er , No. 8, 667-670, ( 1971 ) and references cited therein) and 1-bromo-
2,2,2-
trifluoroethane (Aldrich), the title compound was prepared.
Example 7-N
Synthesis of
5-Amino-7-cyclohexyl-5,7-dihydro
6H-dibenz[b,d)azepin-6-one
Following General Procedure 7-A and using 5,7-dihydro-6H-
dibenz[b,d)azepin-6-one (prepared as described in Brown, et. al., Tetrahedron
Letters, No. 8, 667-670, ( 1971 ) and references cited therein) and
bromocyclohexane (Aldrich), the title compound was prepared.
Example 7-O
Synthesis of
5-(L-Alaninyl)amino-9-fluoro-7-methyl
5,7-dihydro-6H-dibenz[b,d]azepin-6-one Hydrochloride
Sten 1:1: 2-Bromo-5-fluorotoluene was stirred in THF at -78C. s-BuLi (1.05
eq., 1.3 M in cyclohexane) was slowly added and the mixture was stirred for 45
minutes. Trimethylborate ( 1.5 eq) was added and the mixture was allowed to
warm to ambient temperature. After stirring for 1 hour, pinacol (2 eq.) was
added.
The mixture was stirred for 16 hours then was concentrated under reduced
pressure. The resulting residue was slurried in CH,CI, and filtered through
Celite.
The filtrate was concentrated to yield an oil which was purified by
chromatography
on deactivated silica gel (Et3N) to yield the arylboronate ester.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 112 --
Sl~~,n 2: 2-Bromoaniline (1 eq.) and di-t-butyl-dicarbonate (1.1 eq.) were
stirred at 80°C for 20 hours. The resulting mixture was allowed to cool
and was
directly distilled using house vacuum to provide N-t-Boc-2-bromoaniline.
N-t-Boc-2-bromoaniline (Step 2, 1 eq.), the arylboronate ester
{Step 1, 1.1 eq.), K,C03 (1.1 eq.) and
tetrakis(triphenylphosphine)palladium(0)
(0.02 eq} were stirred in 20% water/dioxane under nitrogen. The solution was
heated at reflux for 10 hours. The mixture was allowed to cool then was
concentrated. The resulting residue was partitioned between water and
chloroform.
The organic portion was dried and concentrated to yield an oil which was
purified
by silica gel chromatography using 1:1 CH,CI,/hexanes.
Ste~4: Following General Procedure 7-B and using the substituted
biphenyl from step 3, the 9-fluoro-5,7-dihydro-6H-dibenz(b,d]azepin-6-one was
prepared.
Stgp 5: 9-Fluoro-5,7-dihydro-6H-dibenz[b,dJazepin-6-one (1 eq., Step 4),
cesium carbonate ( 1.1 eq., Aldrich) and methyl iodide ( 1.1 eq., Aldrich)
were
stirred in dry DMF at ambient temperature for 16 hours. The mixture was
concentrated under reduced pressure to provide a residue which was partitioned
between EtOAc and water. The organic portion was dried and concentrated to
yield an oil which was purified by silica gel chromatography to 9-fluoro-7-
methyl-
5,7-dihydro-6H-dibenz[b,d]azepin-6-one.
ten 6: Following General Procedure 7-A, Step B and 9-fluoro-7-methyl-
5,7-dihydro-6H-dibenz[b,d]azepin-6-one from Step 5, 5-amino-9-fluoro-7-methyl-
5,7-dihydro-6H-dibenz[b,d]azepin-6-one was prepared.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- I I3 --
Step Following the procedure of Example 7-I and using 5-amino-9-
fluoro-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one from Step 6, the title
compound was prepared.
Example 7-P
Synthesis of
5-(L-Alaninyl)amino-13-fluoro-7-methyl
5,7-dihydro-6H-dibenz[b,d]azepin-6-one Hydrochloride
Following the procedure of Example 7-O and using 2-bromo-4-
fluoroaniline (Step 2, Lancaster) and o-tolylboronic acid (Step ~, Aldrich),
the title
compound was prepared.
Example 7-Q
Synthesis of
5-(L-Aianinyl)amino-10-fluoro-7-methyl
5,7-dihydro-6H-dibenz[b,d]azepin-6-one Hydrochloride
Following the procedure of Example 7-O and using 2-bromo-4-
fluorotoluene (Step 1 ), the title compound was prepared.
Example 7-R
Synthesis of
5-(L-Alanyl)-amino-7-cyclopropylmethyl
5,7-dihydro-6H-dibenz[b,d]azepin-6-one Hydrochloride
Following the procedure of Example 7-I and using 5-amino-7-
cyclopropylmethyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one (Example 7-L}, the
title compound was prepared.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 114 --
Example 7-S
Synthesis of
5-(L-Alaninyl)amino-7-phenbutyl
5,7-dihydro-6H-dibenz[b,d]azepin-6-one Hydrochloride
Following the procedure of Example 7-I and using ~-amino-7-phenbutyl-
5,7-dihydro-6H-dibenz[b,dJazepin-6-one (Example 7-K), the title compound was
prepared.
Example 7-T
Synthesis of
5-(L-Valinyl)amino-7-cyclopropylmethyl
5,7-dihydro-6H-dibenz[b,d]azepin-6-one Hydrochloride
Following the procedure of Example 7-J and using 5-amino-7-
cyclopropylmethyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one (Example 7-L), the
title compound was prepared.
Example 7-U
Synthesis of
5-(L-Valinyl)amino-7-phenbutyl
5,7-dihydro-6H-dibenz[b,dJazepin-6-one Hydrochloride
Following the procedure of Example 7-J and using ~-amino-7-phenbutyl-
5,7-dihydro-6H-dibenz[b,d]azepin-6-one (Example 7-U), the title compound was
prepared.
Example 7-V
Synthesis of
5-(L-Valinyl)amino-7-hexyl-5,7-dihydro
6H-dibenz(b,d]azepin-6-one Hydrochloride
Sten A: Following General Procedure 7-A and using 5,7-dihydro-6H-
dibenz[b,d]azepin-6-one (prepared as described in Brown, et. al., Tetrahg~lron
Letters, No. 8, 667-670, ( 1971 ) and references cited therein) and 1-
bromohexane
(Aldrich), 5-amino-7-hexyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one was
prepared.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 115 --
Std B: Following the procedure of Example 7-J and using 5-amino-7-
hexyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one, the title compound was prepared.
Example 7-W
Synthesis of
5-(L-Valinyl)amino-10-tluoro-7-methyl
5,7-dihydro-6H-dibenz[b,d]azepin-6-one Hydrochloride
Following the procedure of Example 7-J and using 5-amino-10-fluoro-7-
methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one (as prepared in Example 7ld~l-
Q),
the title compound was prepared.
Example 7-X
Synthesis of
5-(L-Valinyl)amino-I3-fluoro-7-methyl
5,7-dihydro-6H-dibenz[b,dJazepin-6-one Hydrochloride
Following the procedure of Example 7-J and using the 5-amino-13-fluoro-
7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one (as prepared in Example 7-P),
the title compound was prepared.
Example 7-Y
Synthesis of
5-(L-Valinyl)amino-13-tluoro-7-methyl
5,7-dihydro-bH-dibenz(b,d(azepin-6-one Hydrochloride
Following the procedure of Example 7-J and using the 5-amino-9-fluoro-7-
methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one (as prepared in Example 7-O},
the
title compound was prepared.
Example 7-Z
Synthesis of
(5-Amino-7-methyl-1,2,3,4,5,7-hexahydro
6H-dicyclohexyl[b,dJazepin-6-one
The 5-amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
hydrochloride (Example 7-A) was dissolved in a 1:1 mixture of EtOAc/HOAc. 5%
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 116 --
Rh/C was added and the mixture was stirred at 60°C under 60 psi of
hydrogen.
After 3 days, the mixture was filtered and the filtrate was concentrated to
provide
an oil which was purified by SCX-cation exchange chromatography to yield the
title compound.
Example 7-AA
Synthesis of
5-(S)-Amino-7-methyl-5,7-dihydro
6H-dibenz[b,d]azepin-6-one Hydrochloride
Following General Procedure 7-C using racemic S-amino-7-methyl-5,7-
dihydro-6H-dibenz[b,d]azepin-6-one (1.0 eq.) and di p-toluoyl-D-tartaric acid
monohydrate (1.0 eq.) in methanol, the title compound was prepared as a solid.
The product was collected by filtration. Enantiomeric excess was determined by
chiral HPLC.
Desired enantiomer 1: retention time of 9.97 minutes.
Undesired enantiomer 2: retention time of 8.62 minutes.
NMR data was as follows:
'H-nmr (CDCI;): S = 9.39 (s, 2H), 7.75-7.42 (m, 8H), 4.80 (s, 1H), 3.30 (s,
3H).
C,SH,SC1N,0 (MW = 274.75); mass spectroscopy (MH') 239.1.
Anal Calcd for C,SH,SC1N,0;; C, 65.57; H, 5.50; N, 10.20; Found: C,
65.51, H, 5.61; N, 10.01.
Example 7-AB
Synthesis of
9-Amino-5, 6-dihydro-4H-quino[8,1-ab] [3]benzazepin-8 (9H)-one
Hydrochlororide
Step A - Synthesis of 8-Phenxlquinoline
A degassed solution of 8-bromoquinoline (1.0 g, 4.81 mmol) (Aldrich) in
dioxane (50 mL)/H20 (10 mL) was treated with phenylboronic acic (0.64 g, 5.29
mmol) (Aldrich), Pd(Ph;P)4 (0.050 g, 0.04 mmol) and K,CO; (0.73 g, 5.29
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 117 --
mmol). After refluxing for 4 h under a N, atmosphere the reaction was allowed
to cool, diluted with EtOAc and separated. After drying over NazS04 and SiO,
chromatography (95:5 Hexanes/EtOAc) the titled compoltnd was isolated as a
colorless oil.
Physical data were as follows:
'H-nmr (CDC13): 8 = 8.97 (d, 1H), 8.22 (dd, 1H), 7.87-7.39 (m, 9H).
C,SH"N (MW = 205); mass spectroscopy (MH+ ) 206.
Step B - Synthesis of 8-Pher~,yl-1.2.3.4-tetrah dv ropu'inoline
The product from Step A (0.99 g, 4.82 mmol) was hydrogenated according
to the procedure described by Honel, M., et. al., J.C.S. Perkin I, (1980),
1933-1938.
Physical data were as follows:
'H-nmr (CDCl3): S = 7.46 (m, 3H), 7.38 (m, 2H), 6.98 (m, 2H), 6.70 (m,
1H), 3.27 (t, 2H), 2.86 (t, 2H), 1.96 (m, 2H).
C,SH,SN (MW = 209); mass spectroscopy (MH+ ) 210.
Step C - Synthesis of 1-Chlorometh~acet~rl-8-phenyl-1.2.3.4-
tetrahydroquinoline
The product from Step B (1.0 g, 4.78 mmol) was dissolved in CH,C1, (20
mL)/ H,O (20 mL) and treated with NaHCO; (0.602 g, 7.18 mmol) followed by
chloroacetyl chloride (0.478 ml, 5.26 mmol). After stirring for 17 h at
23°C, the
reaction was diluted with CH,C1,, washed with saturated NaHC03, dried over
Na,SOa and purified by SiO, chromatography (CHCl3/Hexanes 9:1 ). The product
was isolated as a colorless solid.
Physical data were as follows:
C"H,6C1N0 (MW = 286.77); mass spectroscopy (MH+ ) 287.
Anal. Calcd for C"H,6C1N0; C, 71.45 H, 5.64 N, 4.90. Found: C, 71.63 H,
5.60 N, 4.87.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 118 --
Step D - Synthesis of 5.6-Dih, d~ ro-4H-auino~8.1-ablf3lbenzaze~in-
~9H1-one
The product from Step C (0.89 g, 3.11 mmol) was mixed thoroughly with
AlCl3 (0.87 g, 6.54 mmol) at 23°C and the mixture heated neat at
100°C for 5-7
minutes. After vigorous gas evolution, the molten mixture was allowed to cool
and
extracted with several portions of CH,C1,lNaHC03 (sat). The combined organic
layers were dried over Na,SO~ and the title compound was purified by
chromatography (SiO,, CHC13/hexanes 9:1 ), yielding a colorless oil which
solidified upon standing.
Physical data were as follows:
C"H,SNO (MW = 249.312); mass spectroscopy (MH+ ) 250.
Anal. Calcd for C"H,SNO; C, 81.90 H, 6.06 N, 5.62. Found: C. 81.75 H,
6.11 N, 5.86.
Step E - Synthesis of 9-Oximo-5.6-Dihydro-4H-quinoj8.1-
abl(3]benzaze in n-81~~ one
The product from Step D (0.490 g, 1.97 mmol) was dissolved in THF and
butyl nitrite (0.46 mL, 3.93 mmol) and treated with KHMDS (0.5 M, 4.52 mL,
2.26 mmol) at 0°C. After stirring for 1 h, the reaction was quenched
with cold 1 N
HC1, extracted with EtOAc, the combined organic layers dried over Na,SO, and
the product purified by SiO, chromatography (CHC1;/MeOH, 99: I ). The title
compound was isolated as a colorless solid.
Physical data were as follows: ,
C"H,~N,O, (MW = 278.3); mass spectroscopy (MH+ ) 279.
Anal. Calcd for C"H"N,O, Ø3317 mol H,O; C, 71.82 H, 5.19 N, 9.85.
Found: C, 71.85 H, 5.09 N, 9.59.
Step F - Synthesis of 9-Amino-5.6-Dih~rdro-4H-quinoj_8.1-
ab](3)benzazeyn-8,19H -one
The product from Step E (0.360 g, 1.29 mmol) was hydrogenated over
Ra/Ni (0.05 g) in EtOH (50 mL)/ NH3 (anhydrous) (5.0 mL) at 100°C and
500 psi
for 10 h. The catalyst was removed by filtration and the resulting filtrate
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 119 --
chromatographed over SiO~ (CHCI3/MeOH, 98:2) yielding the titled compound as
a colorless oil which solidified upon standing.
Physical data were as follows:
C"H,6N~0 (MW = 264.326); mass spectroscopy (MH+ ) 266.
Anal. Calcd for C"H,bN,O; C, 77.25 H, 6.10 N, 10.60. Found: C, 77.23 H,
6.15 N, 10.49.
Example 7-AC
Synthesis of
9-(N'-L-Alaninyl)amino-5,6-dihydro
4H-quino[8,1-ab][3]benzazepin-8(9H)-one Hydrochloride
Step A - Synthesis of 9-(N'-Boc-L-Alaninyl)amino-5 6-Dihydro-4H-
quino[8.1-abl[3]benzaze in-8 9Hl-one
Following General Procedure D and using N-Boc-Alanine (Aldrich)
and 9-amino-5,6-dihydro-4H-quino[8,1-ab][3]benzazepin-8(9H)-one (from
Example 7-AC), the title compound was prepared.
Physical data were as follows:
C,SH,9N3O4 (MW = 435.521 ); mass spectroscopy (MH + ) 436.
Anal. Calcd for C~;H,9N304 Ø4102 mol H,O; C, 67.79 H, 6.79 N, 9.49;
Found: C, 67.83 H, 6.91 N, 9.29.
Step B - Synthesis of 9-(N'-L- lanin~)amino-5.6-dihydro-4H-
quino[8.1-ab][~~ben?az~in-$(9H)-one H3rdrochloride
Following General Procedure E and using the product from Step A, the
title compound was prepared.
Physical data were as follows:
CzoHz~N30~.HC1 (MW = 371.6); mass spectroscopy (MH+ free base )
335.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 120 --
C. Benzodiazenine Derivatives and Related Com on unds
GENERAL PROCEDURE 8-A
N-1-Methvlation of Benzodiaze i~ nes
A solution of benzodiazepine (1 eq.) in DMF (0.1 M concentration) at
0°C
was treated with potassium tert-butoxide ( 1.0 eq., 1.0 M solution in THF).
After
stirring for 30 minutes at 0°C, iodomethane (l.3 eq.) was added and
stirring
continued for 25 minutes. The mixture was diluted with methylene chloride and
washed with water and brine. The organic phase was dried over Na,SOa,
filtered,
and concentrated. The crude product was then either purified by trituration
with
1:1 ether/hexanes or chromatographed via HPLC using ethyl acetate/hexanes as
the
eluent.
GENERAL PROCEDURE 8-B
Cbz Removal Procedure
A flask was charged with the Cbz-protected 3-aminobenzodiazepine (1 eq.).
To this was added HBr (34 eq.; 30% solution in acetic acid}. Within 20 minutes
all
of the starting material dissolves. The reaction was stirred for 5 hours at
ambient
temperature. Ether was added to the orange solution causing the HBr~amine salt
to
precipitate. The mixture was decanted. This process of adding ether and
decanting
was repeated thrice in an effort to remove acetic acid and benzyl bromide.
Toluene
was added and the mixture concentrated in vacuo. This step was also repeated.
The HBr salt was partitioned between ethyl acetate and 1 M K~CO;. The aqueous
layer was back-extracted with ethyl acetate. The combined organics were washed
with brine, dried over Na,SO~, filtered, and concentrated.
GENERAL PROCEDURE 8-C
BcZc Removal Procedure
A solution of Boc-protected amine ( 1 eq.) in methylene chloride (0.15 M
concentration} was cooled to 0°C and treated with trifluoroacetic acid
(30 eq.).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 121 --
After 10 minutes at 0°C, the cooling bath was removed and stirring
continued at
ambient for 20 minutes to 1 hour. The mixture was concentrated in vacuo to
remove excess trifluoroacetic acid. The residue was dissolved in methylene
chloride and washed with saturated aqueous NaHC03 or 1 M K.,C03 and brine.
The organic layer was dried over Na,SOa, filtered, and concentrated.
GENERAL PROCEDURE 8-D
Azide Transfer Reaction Using KHMDS
The azido derivative was prepared using the procedure described in John
W. Butcher et al., Tet. Lett., 37, 6685-6688 ( 1996).
GENERAL PROCEDURE 8-E
Azide Transfer Reaction L1s',_ng LDA
To a solution of diisopropylamine ( 1.1 eq.) in 1 mL of dry THF cooled to -
78 °C was added n-butyl lithium ( 1.6M in hexane) ( 1.1 eq.) dropwise
maintaing the
reaction temperature at -78 °C. The reaction mixture was stirred for 30
min. at -
78°C and then the lactam (0.471 mM) was added dropwise as a solution in
1 mL of
dry THF. The reaction mixture was stirred at -78°C for 30 min. and then
a pre-
cooled solution of trisyl azide ( 1.2 eq.) was added as a solution in 1 mL of
dry
THF. The reaction mixture was stirred at -78 °C for 20 min. and then
quenched
with acetic acid (4.0 eq.). The reaction mixture was then stirred at
40°C for 2 hrs.
The reaction was then poured into EtOAc and washed with water, sodium
bicarbonate and brine, and then dried over sodium sulfate, filtered and
concentrated. The residue was purified by LC 2000 chromatography.
GENERAL PROCEDURE 8-F
Azido Groun Reduction
The azido group was reduced to the corresponding primary amine using the
procedure described in John W. Butcher et al., Tet. Lett., 37, 6685-6688
(1996).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 122 --
GENERAL PROCEDURE 8-G
N-Alkylation of Amides or Lactams
Using Sodium Hydride or Potassium tent-Butoxide
To a slurry of sodium hydride or potassium tert-butoxide ( 1.1 eq) in I S mL
of dry DMF was added the appropriate amide (0.0042 moles) as a solution in I 0
mL of DMF. The alkyl iodide was then added and a thick slurry resulted. The
reaction became less thick as time elapsed and when complete by TLC the
reaction had become homogeneous. The reaction mixture was poured over ice and
extracted into ethyl acetate. The organic layer was washed with water,
followed by
brine. The organic layer was then dried over sodium sulfate, filtered and
concentrated under reduced pressure. The residue was purified by HPLC (LC
2000), eluting with an ethyl acetate/hexane system.
GENERAL PROCEDURE 8-H
N-Alkylation of
Amides or Lactams Using KHMDS
To the appropriate amide or lactam in THF cooled to -78°C was
added
KHMDS dropwise and the reaction mixture was stirred for 30 min. at -78
°C. The
alkyl iodide was then added dropwise while maintaining the temperature at -
70°C.
The cooling bath was then removed and reaction was allowed to warm to room
temperature and stirring was continued for 2 hours. The reaction mixture was
then
poured over ice and extracted into ethyl acetate. The organic extracts were
washed
with water, followed by brine. The organic layer was then dried over sodium
sulfate, filtered and concentrated under reduced pressure. The residue was
purified
by HPLC (LC 2000), eluting with an ethyl acetate/hexane system.
GENERAL PROCEDURE 8-I
N-Al~cvlation of Amides or Lactams Using Cesium Carbo~~g
To a solution of the amide or lactam in DMF was added cesium carbonate
(1.05 eq) and an alkyl iodide (1.1 eq). The mixture was allowed to stir
overnight at
room temperature and then the reaction mixture was dilluted with ethyl acetate
and
CA 02325388 2000-09-21
WO 99167220 PCT/US99/14007
- 123 -
washed with water; followed by brine. The organic layer was dried over sodium
sulfate, filtered and concentrated under reduced pressure. The residue was
purified
by HPLC (LC 2000), eluting with an ethyl acetate/hexane system.
GENERAL PROCEDURE 8-J
BOC Removal Procedure
To an N-Boc protected compound was added CH,Ch/TFA (4:1) at room
temperature. The reaction mixture was stirred at room temperature for 3 hours
and
then concentrated. The residue was extracted into dichloromethane and washed
with water, saturated sodium bicarbonate, dried over Na,SO~, filtered and
concentrated to give the free amine.
GENERAL PROCEDURE 8-K
Azide Transfer Procedure
This azide transfer procedure is a modification of the procedure described
in Evans, D. A.; Britton, T. C.; Ellman, J. A.; Dorow, R. L. J. Am. Chem. Soc.
1990, 112, 4011-4030. To a solution of the lactam substrate ( 1.0 eq.) in THF
(~0.1
M) under N, at -78 °C was added a solution of KN(TMS), (1.1 eq. of
0.5 M in
Toluene, Aldrich) dropwise over a period of 2-10 minutes. A slight exotherm
was
often observed by an internal thermometer, and the resulting solution was
stirred
for 5-1~ minutes, while re-cooling to -78°C. Then, trisyl azide (1.1-
1.5 eq., CAS
No. 36982-84-0, prepared as described by references in the Evans reference
above)
in THF (~0.5 M), either precooled to -78°C or at room temperature, was
added via
cannuia over a period of 0.5-5 minutes. Again, a slight exotherm was generally
noted. The resulting solution was stirred for from 5-10 minutes, while re-
cooling
to -78°C. Then, AcOH (4.5-4.6 eq., glacial) was added, the cooling bath
removed
and the mixture allowed to warm to room temperature with stirring for 12-16
hours. The mixture was diluted with EtOAc, in a 2-5 volume multiple of the
initial
THF volume, and washed with dilute aq. NaHC03 (1-2x), 0.1-1.0 M aq. HCl (0-
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 124 --
2x), and brine ( 1 x). The organic phase was then dried over MgS04, filtered,
concentrated to provide the crude product.
GENERAL PROCEDURE 8-L
Azide Reduction to an Amine
A mixture of the azide in absolute EtOH (0.03-0.07 M) and 10% Pd/C
(~1/3 by weight of the azide) was shaken in a Parr apparatus under H, (35-45
psi)
at room temperature for 3-6 hours. The catalyst was removed by filtration
through
a plug of Celite, rinsing with absolute EtOH, and the filtrate concentrated to
provide the crude amine product.
GENERAL PROCEDURE 8-M
Amide Alkvlation Using Cesium Carbonate
This procedure is a modification of the procedure described in Claremon,
D. A.; et al, PCT Application: WO 96-US8400 960603. To a mixture of 2,4-
dioxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine (CAS No. 49799-48-6) in DMF
(1.0 eq., 0.7 M) under N, at room temperature was added Cs,C03 (2.2 eq.) and
the
appropriate alkyl halide (2.2 eq.). The mixture was stirred at room
temperature for
5.~-16 hours. The mixture was partitioned between EtOAc and sat. NaHCO;. The
aqueous layer was extracted with EtOAc (1-2x) and the combined EtOAc extracts
were dried over Na,SOa, filtered, and concentrated to provide the crude
product.
GENERAL PROCEDURE 8-N
BOC Removal Procedure
A stream of anhydrous HCl gas was passed through a stirred solution of the
N-t-Boc protected amino acid in 1,4-dioxane (0.03-0.09 M), chilled in a ice
bath to
~10°C under N,, for 10-15 minutes. The solution was capped, the cooling
bath
removed, and the solution was allowed to warm to room temperature with
stirring
for 2-8 hours, monitoring by TLC for the consumption of starting material. The
solution was concentrated (and in some instances dissolved in CH,CI, then re-
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 125 --
concentrated and placed in vacuum oven at 60-70°C to remove most of the
residual dioxane) and used without further purification.
Example 8-A
Synthesis of
3-Amino-1,3-dihydro-5-(1-piperidinyl)-2H-1,4-benzodiazepin-2-one
Step A - Preparation of 1.2-Dihydro-3H-1-methvl-S-( 1-niberidinvll-
1.4-benzodiazenin-2-one
A solution of phosphorous pentachloride (1.2 eq) in methylene chloride
was added dropwise to a solution of 1-methyl-1,2,3,4-tetrahydro-3H-1,4-
benzodiazepin-2,5-dione (Showell, G. A.; Bourrain, S.; Neduvelil, J. G.;
Fletcher,
S. R.; Baker, R.; Watt, A. P.; Fletcher, A. E.; Freedman, S. B.; Kemp, J. A.;
Marshall, G. R.; Patel, S.; Smith, A. J.; Matassa, V. G. J. Med Chem. 1994,
37,
719.) in methylene chloride. The resultant yellowish-orange solution was
stirred at
ambient temperature for 2.5 hours; the solvent was removed in vacuo. The
orange
residue was redissolved in methylene chloride, cooled to 0 °C, and
treated with a
solution of piperidine (2 eq) and triethylamine (2 eq) in methylene chloride.
The
cooling bath was removed and the reaction stirred for 18 hours. The reaction
mixture was washed with saturated aqueous NaHC03 (back-extracted with
methylene chloride) and brine. The organic phase was dried over Na,SO,, f
ltered,
and concentrated. The residue was purified via HPLC eluting with a gradient of
4
to 10% methanol/methylene chloride affording the title intermediate as a
yellow
solid having a melting point of 103-105 °C.
C~sH~9N30 (MW 257.37); mass spectroscopy 257.
Anal. Calcd for C,SH,9N30: C, 70.01; H, 7.44; N, 16.33. Found: C, 69.94;
H, 7.58; N, 16.23.
Step B - Preparation of 1.2-Dih~ro-3H-1-methyl-3-oximido-S-(1-
eridinyj)-1.4-benzodiazenin-2-one
Potassium tert-butoxide (2.5 eq) was added in two portions to a -
20°C
solution of 1,2-dihydro-3H-1-methyl-5-(1-piperidinyl)-1,4-benzodiazepin-2-one
(1
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/14007
-- 126 --
eq) in toluene. After stirnng at - 20°C for 20 min, isoamyl nitrite
(1.2 eq.;
Aldrich) was added to the red reaction mixture. The reaction was stirred at -
20 °C
for 5 hours at which time the reaction was done by TLC. The cooling bath was
removed and the reaction quenched with 0.5 M citric acid. After stirnng for 10
minutes, diethyl ether was added. The suspension was stirred at ambient
temperature overnight then filtered washing with ether. The resultant cream
colored solid had a melting point of 197-200°C.
'H NMR data of the E/Z isomers was as follows:
'H NMR (300 MHz, CDC1,): 8 = 7.64 (1H, bs), 7.48 (2H, d, J=7.4 Hz),
7.35-7.20 (6H, m), 6.75 (1H, bs), 3.8-3.2 (8H, m), 3.46 (3H, s), 3.42 (3H, s),
1.90-
1.40 ( 12H, m).
C,SH,gN.,O, (MW = 286.37); mass spectroscopy 286.
Step C - P~gnaration of 1.2-dihydro-3H-1-met~vl-3-(O-
Lethvlaminocarbon~)oximido]-~1-pi eridin~ -
benzodiazenin-2-one
A mixture of I,2-dihydro-3H-1-methyl-3-oximido-5-(1-piperidinyl)-1,4-
benzodiazepin-2-one ( I eq) in THF was treated with ethyl isocyanate ( 1.7 eq)
and
triethylamine (0.6 eq). The mixture was heated to 64°C for 4 hours. The
mixture
was concentrated and the residue purified by HPLC eluting with 5%
methanol/methylene chloride.
'H NMR data of the E/Z isomers was as follows:
'H NMR (300 MHz, CDC13): 8 = 7.50 (2H, dd, J=8.4, 1.5 Hz), 7.35-7.22
(6H, m), 6.42 ( 1 H, bt), 6.20 ( 1 H, bt), 3.7-3.4 (8H, m), 3.46 (3H, s), 3.44
(3H, s),
3.25 (4H, m), 1.9-1.4 (12H, m), 1.12 (3H, t, J=6.3 Hz), 1.10 (3H, t, J=6.3
Hz).
C,eH,,N503 (MW = 357.46); mass spectroscopy 357.
Step D - Preparation of 3-Amino-1 3-dihvdro-2H-1-methy_,[~ 1
eridinyll-1.4-benzodiazepin-2-one
The 1,2-dihydro-3H-1-methyl-3-[O-(ethylaminocarbonyl)oximido]-5-(1-
piperidinyl)-1,4-benzodiazepin-2-one (1 eq) was hydrogenated in methanol over
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 127 --
5% palladium on carbon (0.15 eq) at 43 psi for 3.25 hours. The reaction was
filtered through celite and concentrated in vacuo. The residue was taken up in
methylene chloride and filtez~ed a second time through celite. The filtrate
was
concentrated and the resultant foam was used immediately.
Example 8-B
Synthesis of
3-(L-Alaninyl)-amino-2,3-dihydro
1-methyl-5-phenyl-1H-1,4-benzodiazepin-2-one
Step A - Preparation of f~-3-amino-1 3-di'~~vdro-1-meth~phen ~~1-
2H-1.4-benzodiazenin-2-one,~,(,j S)-7.7-dimeth,
oxobicvclof2.2. l lhentane-1-methanesulfonate
The title intermediate was prepared according to Reider, P. J.; Davis, P.;
Hughes, D. L.; Grabowski, E. J. J. J. Org. Chem. 1987, 52, 955 using 3-amino-
1,3-
dihydt~o-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one (Bock M. G.; DiPardo, R.
M.; Evans, B. E.; Rittle, K. E.; Veber, D. F.; Freidinger, R. M.; Hirshfield,
J.;
Springer, J. P. J. Org. Chem. 1987, ~2, 3232.) as the starting material.
Step B - Preparation of 3=j 1~'-(tert-Butvlcar~amalg]-L-alaninvll-
ar~inQ-~ 3-di~vdro-1-meth~S~vl-1H-1 4-
benzodiazepin-2-one
(S)-3-Amino-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one,
(1S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-methanesulfonate was free based
by partitioning between methylene chloride and 1 M potassium carbonate. The
free
amine was then coupled with N-Boc-alanine following General Procedure D.
C~QH,8N~0~ (MW = 436.56); mass spectroscopy 436.
Anal. Calc. for C,~HZgN~Oa: C, 66.03: H, 6.47; N, 12.84. Found: C, 65.79;
H, 6.68; N, 12.80.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 128 --
Step C - Preparation of 3-(L-Alaniny~~-amino-2,3-dihxdro-1-methyl-
5-phenyl- I H-~,,4-benzodiazenin-2-one
Following General Procedure 8-C using 3-[N'-(tent-butylcarbamate)-L-
alaninyl]-amino-2,3-dihydro-1-methyl-S-phenyl-1H-1,4-benzodiazepin-2-one, the
title compound was prepared as a white foam.
Anal. Calc. for C,9H,9N~0,: C, 69.21; H, 6.64; N, 15.37. Found: C, 70.11;
H, 6.85; N, 15.01.
Example 8-C
Synthesis of
3-(L-Alaninyl)-amino-7-chloro-2,3-dihydro
1-methyl-5-phenyl-1H-1,4-henzodiazepin-2-one
Step A - Preparation of 3-lBenzvlox,Ycarbonyl)-amino-7-chloro-2.3-
dihvdro-1-methyl-5-tzhenvl-1 H-I .4-benzodiazenin-2-one
A solution of 3-(benzyloxycarbonyl)-amino-7-chloro-2,3-dihydro-5-
phenyl-1H-1,4-Benzodiazepin-2-one (1 eq; Neosystem) in DMF was cooled to
0°C
and treated with potassium tert-butoxide (1 eq; 1.OM solution in THF). The
resultant yellow solution was stirred at 0°C for 30 minutes then
quenched with
methyl iodide ( 1.3 eq). After stirring an addition 25 minutes the reaction
was
diluted with methylene chloride and washed with water and brine. The organic
phase was dried over Na,SO;, filtered, and concentrated. The residue was
purified
via HPLC chromatography eluting with a gradient of 2030% ethyl
acetate/hexanes.
C,,,H,°C1N303 (MW = 433.92); mass spectroscopy 433.
Anal. calcd for C,aH,°C1N303: C, 66.44; H, 4.65; N, 9.68. Found:
C,
66.16; H, 4.50; N, 9.46.
Step B - Preparation of 3-Amino-7-chloro-I.3-dihydro-1-meth,
phenyl-2H-1,4-benzodiazepin-2-one
Following General Procedure 8-B using 3-(benzyloxycarbonyl)-amino-7-
chloro-2,3-dihydro-I-methyl-5-phenyl-1H-1,4-benzodiazepin-2-one, the title
intermediate was prepared as a white foam which was used immediately in Step
C.
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/I4007
-- 129 --
Step C - Preparation of 3-(N'-tert-Butvlcarbamate -L-al ni y~-
amino-7-chloro-1.3-dihydro-1-methyl-5-phenyl-2H-1.4-
benzodiazenin-2-one
Following General Procedure D using N-Boc-L-alanine and 3-amino-7-
chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one, the title
intermediate was prepared as a white foam.
C,4H,SC1N404 (MW = 471.18); mass spectroscopy 471
Anal. calcd for C,4HZgC1N~0~: C, 61.21; H, 5.78; N, 11.90. Found: C,
61.24; H, 5.59; N, 11.67.
Step D - ~enaration of 3-j -Alanir~yllamino-7-chloro-1 3-dihydro-1
m~th~phenvl-2H-1.4-benzodiazenin-2-one
Following General Procedure 8-C using 3-[N'-tert-butylcarbamate)-L-
alaninylJ-amino-7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-
one, the title intenmediate was prepared as a white foam. The crude material
was
used immediately.
Example 8-D
Synthesis of
3-(L-Alaninyl)amino-7-brorno-2,3-dihydro
1-methyl-5-(2-fluorophenyl)-1H-1,4-benzodiazepin-2-one
Step A - Preparation of 3-(Benzyloxycarbonyl)-amino-7-bromo-2,3-
dihydro-1-methyl-5-(2-fluorophenyl)-1 H-1,4-
benzodiazepin-2-one
Following General Procedure 8-A using 3-(benzyloxycarbonyl)-amino-7-
bromo-2,3-dihydro-5-(2-fluorophenyl)-1H-1,4-benzodiazepin-2-one (Neosystem),
the title intermediate was prepared as a white foam.
C,4H~9BrFN3O3 (MW = 496.36); mass spectroscopy 497.
Anal. calcd for C,4H~9BrFN3O3: C, 58.08; H, 3.86; N, 8.47. Found: C,
57.90; H, 4.15; N, 8.20.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 130 --
Step B - Prerzaration of 3-Amino-7-bromo-1.3-dihydro-1-methxl-5-
(2-fluoro~henyl)-2H-1.4-benzodiazepin-2-one
Following General Procedure 8-B using 3-(benzyloxycarbonyl)-amino-7-
bromo-2,3-dihydro-1-methyl-5-(2-fluorophenyl)-1H-1,4-benzodiazepin-2-one, the
title intermediate was prepared as a white foam which was used immediately in
Step C.
Step C - Preparation of 3-(~1' ~tert-Bu~ylcarbamate)-L-alaninyll~
amino-7-bromo-1.3-dihydro-I -methv~2-fluor~,phenvll-
2H-1.4-benzodiazenin-2-one
Following General Procedure D using N-Boc-L-alanine (Novo) and 3-
amino-7-bromo-I,3-dihydro-I -methyl-5-(2-fluorophenyl)-2H-1,4-benzodiazepin-
2-one, the title intermediate was prepared as a white foam.
CzaHzeBrFN404 (MW = 533.12); mass spectroscopy 533.2.
Anal. calcd for C,~H,6BrFNa04: C, 54.04; H, 4.91; N, 10.50. Found: C,
53.75; H, 4.92; N, 10.41.
Step D - Preparation of 3-lL-Alanin~l)-amino-7-bromo-I.3siih, dro-
1-methyl-5-(2-fluoro~aheny,~"1-2H-1.4-benzodiazepin-2-one
Following General Procedure 8-C using 3-[N'-(tert-butylcarbamate)-L-
alaninylJ-amino-7-bromo- I ,3-dihydro- I -methyl-5-(2-fluorophenyl)-2H-1,4-
benzodiazepin-2-one, the title intermediate was prepared as a white foam. The
crude material was used immediately.
Example 8-E
Synthesis of
3-(N'-Methyl-L-alaninyl)-amino-2,3-dihydro-
1-methyl-S-phenyl-1H-1,4-benzodiazepin-2-one
Step A - Preparation of 3- 1[~'-ltert-Butvlcarbamate)-N'-methyl-j~
alaninyl]-amino-2.3-dihydro-I-met~~5=phe~vl-I H-1.4-
benzodiazenin-2-one
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 131 --
Following General Procedure D and using (S)-3-amino-1,3-dihydro-1-
methyl-5-phenyl-2H-1,4-benzodiazepin-2-one (Example 8-B) and N-tert-Boc-N-
methyl-alanine (Sigma), the title intermediate was obtained as a white solid.
C25H30N404 (MW = 450.2); mass spectroscopy (M+1) 451.2.
Anal. calcd for C,SH3pN~O4: C, 66.65; H, 6.71; N, 12.44. Found: C, 66.66;
H, 6.89; N, 12.21.
Step A - Preparation of 3-lN'-Methvl-L-alaniny~-ammo-2 3-dihydro-
1-met yl-S-~envl-1H-1 4-benzodiazepin-2-one
Following General Procedure 8-C and using 3-[N'-(tert-butylcarbamate)-
N'-methyl-L-alaninyl]-amino-2.3-dihydro-1-methyl-5-phenyl-1 H-1,4-
benzodiazepin-2-one, the title intermediate was prepared as a white foam.
CzoHZ,N40z (MW =350.46); mass spectroscopy (M+1) 351.4.
Anal. calcd for C,oH,~N~O,: C, 68.55; H, 6.33; N, 15.99. Found, C, 68.36;
H, 6.20; N, 15.79.
Example 8-F
Synthesis of
3-(L-Alaniny!)amino-7-chloro-2,3-dihydro
1-methyl-5-(2-chlorophenyl)-1H-1,4-benzodiazepin-2-one
Step A - Preparation of 3-(Benzyloxycarbo~yll-amino-7-chloro-2 3-
dihvdro-1-methyl-5-(2-chloro~X~, -1
benzodiazenin-2-one
Following General Procedure 8-A using 3-(benzyloxycarbonyl)-amino-7-
chlpro-2,3-dihydro-5-(2-chlorophenyl)-1H-1,4-benzodiazepin-2-one (Neosystem),
the title intermediate was prepared as a white solid having a melting point of
232-
233°C.
C,4Hi9Cl,N3O3 (MW = 468.36); mass spectroscopy 468.
'H NMR (300 MHz, CDCl3): 8 = 7.67 ( 1 H, m), 7.52 ( 1 H, dd, J=2.4,
8.7 Hz), 7.42-7.26 (9H, m), 7.07 ( 1 H, d, J=2.4 Hz), 6.70 ( I H, d, J=8.3
Hz), 5.3 5
( 1 H, d, J=8.4 Hz), 5.14 (2H, ABq, J=19.6 Hz), 3.47 (3 H; s).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99II4007
-- 132 -
'3C NMR (75 MHz, CDC13): b = 166.66, 165.65, 155.72, 140.52, 136.99,
136.0, 132.87, 131.99, 131.47, 131.40, 131.38, 131.16, 130.54, 130.06, 128.45,
128.08, 128.03, 127.72, 127.22, 123.28, 122.01, 68.95, 67.02, 35.32.
Step B - P_~~paration of 3-Amino-7-chloro-1.3-dihyd y-1-methyl-5-
(2-chlorophen,1.r_l)-2H-1.4-benzodiazenin-2-one
Following General Procedure 8-B using 3-(benzyloxycarbonyl)-amino-7-
chloro-2,3-dihydro-1-methyl-5-(2-chlorophenyl)-1H-1,4-benzodiazepin-2-one, the
title intermediate was prepared as a white foam which was used immediately in
Step C.
Step C - Preparation of 3-J]V'-Lert-Butylcarbamate~- -al ni 11-
amino-7-chloro-1.3-dihvdro-1-meth-S,S2-chlorophenyjl-
2H-1.4-benzodiazepin-2-one
Following General Procedure D using N-Boc-L-alanine and 3-amino-7-
chloro-1,3-dihydro-1-methyl-5-(2-chlorophenyl)-2H-1,4-benzodiazepin-2-one, the
title intermediate was prepared as a white foam.
C,qH,6CI~N~O4 (MW = 505.44); mass spectroscopy 505.2.
Step D - Preparation of 3-(1 -Alaninvl)-amino-7-chloro-1.3-dihydro-
1-meth~(2-chloro~y~)-2H-1 4-benzodiaze~~~ 2-one
Following General Procedure 8-C using 3-[N'-(tert-butylcarbamate)-L-
alaninyl]-amino-7-chloro-1,3-dihydro-1-methyl-5-(2-chlorophenyl)-2H-1,4-
benzodiazepin-2-one, the title intermediate was prepared as a white foam. The
crude material was used immediately.
Example 8-G
Synthesis of
3-(L-Alaninyl)amino-5-cyclohexyl-2,3-dihydro
1-methyl-IH-1,4-Benzodiazepin-2-one
Step A - Per paration of 3~Ben oxycarbonyll-amino-5-cvlclohexyl-
2 3-di~,vdro-1-methyl-1H-1 4-benzodiazP~' - -one
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 133 --
Following General Procedure 8-A using 3-(benzyloxycarbonyl)-amino-5-
cyclohexyl-2,3-dihydro-1H-1,4-benzodiazepin-2-one (Neosystem), the title
intermediate was prepared as a white solid having a melting point of 205-
206°C.
C~qH"N3O; (MW = 405.54); mass spectroscopy 405.
' H NMR (300 MHz, CDC13): 8 = 7.54 ( 1 H, d, J=7.9 Hz), 7.48 ( 1 H, d, J=7.7
Hz), 7.36-7.26 (7H, m), 6.54 (1H, d, J= 8.3 Hz), 5.15 (1H, d, J=8.0 Hz), 5.09
(2H,
ABq, J=17.1 Hz), 3.39 (3H, s), 2.77 (1H, m), 2.01 (IH, bd, J=13.6 Hz), 1.85
(1H,
bd, J=12.4 Hz), 1.68-1.49 (4H, m), 1.34-1.02 (4H, m).
Step B - Prepa_ration of 3-Amino-S-cyclohexyl-1.3-dih~dro-1-methyl-
2H-1.4-benzodiazenin-2-one
Following General Procedure 8-B using 3-(benzyloxycarbonyl)-amino-5-
cyclohexyl-2,3-dihydro-1-methyl-1H-1,4-benzodiazepin-2-one, the title
intermediate was prepared as a white foam which was used immediately in Step
C.
C,6H,,N,O (MW+H = 272.1763); mass spectroscopy 272.1766
Step C - Preparation of 3-(N'-(tert-Butylcarbamatel-L-alaninYll-
a~nino-5-cy,~Iohexyl- I .3-dih_vdro- I -meth'rl-2H-1.4-
benzodiazepin-2-one
Following General Procedure D using N-Boc-L-alanine and 3-amino-S-
cyclohexyl-1,3-dihydro-I-methyl-2H-1,4-benzodiazepin-2-one, the title
intermediate was prepared as a white foam.
C,4H3~Na0, (MW = 442.62); mass spectroscopy (M+H) 443.2.
Step D - Preparation of 3-(,L-Alaninxl)amino-5-cyclohexxl-1.3-
~lih3rdro- I -methy l-2 H-1.4-benzodiaze~in-2-one
Following General Procedure 8-C using 3-[N'-(tert-butylcarbamate)-L-
alaninyl]-amino-5-cyclohexyl-1,3-dihydro-1-methy(-ZH-1,4-benzodiazepin-2-one,
the title intermediate was prepared as a white foam. The crude material was
used
immediately.
C,9H,6N~0, (M+H = 343.2136); mass spectroscopy found 343.2139.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 134 -
Example 8-H
Synthesis of
3-(L-Alaninyl)amino-2,3-dihydro-1-methyl-
7-nitro-5-phenyl-1H-1,4-benzodiazepin-2-one
Step A - Pr~aration of 2~a-Isopropylthio)-N'-
(benzvloxvcarbonvl)-elvcinvll-amino-5-nitrobenzonhenone
A solution of a-(isopropylthio)-N-(benzyloxycarbonyl)glycine (1 eq;
prepared according to Zoller, V.; Ben-Ishai, D. Tetrahedron 1975, 31, 863.) in
dry
THF was cooled to 0 °C and treated with oxalyl chloride (1 eq.) and 3
drops of
DMF. After stirring for 15 minutes at 0°C, the cooling bath was
removed and
stirring continued at ambient temperature for 40 minutes. The solution was
recooled to 0°C. A solution of 2-amino-5-nitrobenzophenone (0.9 eq.;
Acros) and
4-methylmorpholine (2.0 eq.) in dry THF was added via cannulation to the acid
chloride. The cooling bath was removed and the reaction stirred at ambient for
S
hours. The reaction was diluted with methylene chloride and washed with 0.5 M
citric acid, saturated aqueous NaHC03, and brine. The organic phase was dried
over Na,SO,,, filtered, and concentrated. The residue was purified via
preparative
LC2000 eluting with a gradient of 1520% ethyl acetate/hexanes giving an off
white foam.
C,6H,SN3O6S (MW = 507.61 ); mass spectroscopy found 507.9.
Anal. calcd for C,6H,SN;O6S: C, 61.53; H, 4.96; N, 8.28. Found: C, 61.70;
H, 4.99; N, 8.22.
Step B - Preparation of 2-[~1- a-Amino,)-N' ~benzylo~ycarbo 11-
El3rcin~)-amino-5-nitrobenzophenone
Ammonia gas was bubbled into a solution 2-[N-(a-isopropylthio)-N'-
(benzyloxycarbonyl}-glycinyl]-amino-S-nitrobenzophenone (1 eq) in THF at
0°C.
After 35 minutes mercury(II) chloride (1.1 eq) was added. The ice bath was
removed and ammonia gas was continued to bubble through the suspension for 4
hours. The bubbler was removed and the reaction continued to stir for 16
hours.
The mixture was filtered through celite washing with THF. The filtrate was
CA 02325388 2000-09-21
WO 99/67220 PCTIUS99/14007 _ _ ,
-- 135 --
concentrated in vacuo. The crude solid was used in step C without further
purification.
Step C - p~gparation of 3-(Benzyloxvcarbony~-amino-2.3-dihX, 0-7
nitro-5-p, m3r1-1H-1.4-benzodiazepin-2-one
2-[N-(a-Amino)-N'-(benzyloxycarbonyl)-glycinyl]-amino-S-
nitrobenzophenone (1 eq) was treated with glacial acetic acid and ammonium
acetate (4.7 eq). The suspension was stirred at ambient temperature for 21
hours.
After concentrating the reaction in vacuo, the residue was partitioned between
ethyl acetate and 1 N NaOH. The aqueous layer was back-extracted with ethyl
acetate. The combined organics were washed with brine, dried over Na,S04,
filtered, and concentrated. The residue was purified via flash chromatography
eluting with a gradient of 2--3% isopropyl alcohol/methylene chloride.
C,3H,gN405 (MW = 430.45); mass spectroscopy found (M+H) 431.2.
Anal. calcd for C,3H,8Na05: C, 64.18; H, 4.22; N, 13.02. Found: C, 64.39;
H, 4.30; N, 13.07.
Step D - Preparation of 3-lBenzyloxycarbonyll-amino-2.3-dih d
methXl-7-nitro-5-phen,~-1 H-1.4-be~zodiazepin-2-one
Following General Procedure 8-A and using 3-(benzyloxycarbonyl)-amino-
2,3-dihydro-7-nitro-5-phenyl-IH-1,4-benzodiazepin-2-one, the title
intermediate
was prepared as a yellow foam.
C,4H,°N4O5 (MW = 444.48); mass spectroscopy found (M+H) 445.2.
Anai. calcd for C,4H,°N405: C, 64.86; H, 4.54; N, 12.60. Found: C,
65.07;
H, 4.55; N, 12.46.
Step E - ~g;paration of 3-Amino-1.3-dihydro-1-methyl-7-nitro-5-
phen 1-y 2H-1.4-benzodiazenin-2-one
Following General Procedure 8-B and using 3-{benzyloxycarbonyl)-amino-
2,3-dihydro-1-methyl-7-nitro-5-phenyl-1H-1,4-benzodiazepin-2-one, the title
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007 _
-- 136 --
intermediate was prepared as a yellow foam which was used immediately in Step
F.
Step F - Preparation of 3-[N'-(tert-Butylcarbamate, -L-alaninyh'
amino-2.3-dihvdro- I -methyl-7-nitro-5-nheny l-1 H-1.4-
benzodiazepin-2-one
Following General Procedure D using N-Boc-L-alanine and 3-amino-1,3-
dihydro-I-methyl-7-nitro-5-phenyl-2H-1,4-benzodiazepin-2-one, the title
intermediate was prepared as a yellow solid.
C,4H,,N506 (MW = 481.56); mass spectroscopy found (M+H) 482.3.
Anal. calcd for C,~HZ,N506: C, 59.88; H, 5.61; N, 14.55. Found: C, b0.22;
H, 5.75; N, 13.91.
Step G - Preparation of 3-(L-Alani~rll-amino-2.~-di~ydro-1-meth ~~1
7-nitro-5-phenyl-I H-1.4-benzodiazepin-2-one
Following General Procedure 8-C using 3-[N'-(tert-butylcarbamate)-L-
alaninyt ]-amino-2,3-dihydro-1-methyl-7-nitro-5-phenyl-1 H-1,4-benzodiazepin-2-
one, the title intermediate was prepared as a yellow foam. The crude material
was
used immediately.
Example 8-I
Synthesis of
3-(L-Alaninyl)amino-2,3-dihydro-1-methyl
5-(2-fluorophenyl)-1 H-1,4-benzodiazepin-2-one
Step A - Preparation of 3-Amino-1.3-dih~rdro-1-methyl-5-(2-
fluoronhenyl)-2H-1.4-benzodiazg~n-2-one
A flask was charged with 3-(benzyloxycarbonyl)-amino-7-bromo-2,3-
dihydro-I-methyl-5-(2-fluorophenyl)-IH-1,4-benzodiazepin-2-one (1 eq.; Example
8-D, Step A) and 10% palladium on carbon. Methanol was added, and the flask
was placed under a balloon of H,. The reaction was stirred for 21 hours. The
mixture was filtered through celite washing with methanol. The filtrate was
concentrated to a white solid.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 137 --
C,6H,4FN30 (MW = 283.33); mass spectroscopy found (M+H) 284.1.
Step B - Preparation of 3-[~l~grt-Butylcarbamate)-L-alaninyh~
amino-1.3-dihydro-1-meth,~,(2-fluorophen~)-2H-1.4-
benzodiazenin-2-one
Following General Procedure D using N-Boc-L-alanine and 3-amino-1,3-
dihydro-i-methyl-S-(2-fluorophenyl)-2H-1,4-benzodiazepin-2-one, the title
intermediate was prepared as a white solid.
C2aHnFNaGa (MW = 454.50}; mass spectroscopy found (M+H) 455.4.
Anal. calcd for Cz4Hz,FN404: C, 63.44; H, 5.95; N, 12.33. Found: C,
63.64; H, 6.08; N, 12.16.
Step C - Preparation of 3-lL-Alaninvl)-amino-7-bromo-I .3-dih
I-ethyl-5-l2-fluorop enyjl-2H-1.4-benzodiaze~ain-2-one
Following General Procedure 8-C using 3-[N'-(tert-butylcarbamate)-L-
alaninyl]-amino-1,3-dihydro-1-methyl-5-(2-fluorophenyl)-2H-1,4-benzodiazepin-
2-one, the title intermediate was prepared as a white foam. The crude material
was
used immediately.
Example 8-J
Synthesis of
3-(L-Alaninyl)-amino-2,3-dihydro
1-methyl-5-(3-fluorophenyl)-1H-1,4-benzodiazepin-2-one
Step A - Preparation of 2-Amino 3'-fluorobenzophenone
A solution of 3-bromofluorobenzene (1 eq.) in THF was cooled to -
78°C
under nitrogen and treated with tert-butyllithium (2.05 eq., I .6 M solution
in
pentane) at a rate of 40 ml/h. The internal temperature did not rise above -
74°C.
The orange solution was stirred at -78°C for 30 minutes prior to the
addition of
anthranilonitrile (0.6 eq.) as a solution in THF. The reaction was warmed to
0°C
and stirred for 2 hours. 3N HCl was added to the mixture and stirnng continued
for 30 minutes. The reaction was diluted with ethyl acetate and the layers
were
separated. The aqueous layer was back-extracted thrice with ethyl acetate. The
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 138 --
combined extracts were washed with brine, dried over Na~SO~, filtered, and
concentrated. The residue was purified via HPLC eluting with 93:7
hexanes/ethyl
acetate.
C,3H,oFNO (MW = 215.24); mass spectroscopy found (M+H) 216.3.
' H NMR (300 MHz, CDC13) d 7.44-7.19 (6H, m), 6.74 ( 1 H, d, J=8.0 Hz),
6.61 ( 1 H, dd, J=0.94, 7.9 Hz), 6.10 (2H, bs).
Step B - Preparation of 2-j~1- a-Iso~pylthi~-N'-
jbenzyloxycarbonyl)-glycin~~-amino-3'-
ftuorobenzo henone
A solution of a-(isopropylthio)-N-(benzyloxycarbonyl)glycine ( 1 eq;
prepared according to Zoller, V.; Ben-Ishai, D. Tetrahedron 1975, 31, 863.) in
dry
THF was cooled to 0°C and treated with oxalyl chloride (1 eq.) and 3
drops of
DMF. After stirring for 15 minutes at 0°C, the cooling bath was
removed and
stirring continued at ambient temperature for 40 minutes. The solution was
recooled to 0°C. A solution of 2-amino-3'-8uorobenzophenone (0.9 eq.)
and 4-
methylmorpholine (2.0 eq.) in dry THF was added via cannulation to the acid
chloride. The cooling bath was removed and the reaction stirred at ambient for
S
hours. The reaction was diluted with methylene chloride and washed with 0.5 M
citric acid, saturated aqueous NaHCO;, and brine. The organic phase was dried
over Na,SO~, filtered, and concentrated. The residue was purified via
preparative
LC2000 eluting with a gradient of 15-20% ethyl acetate/hexanes giving an off
white foam.
C,6H,SN~O~S (MW = 480.60); mass spectroscopy found (M+NH4') 498.3.
'H NMR (300 MHz, CDCl3) d 11.39 (1H, s), 8.59 (IH, d, J=6.0 Hz), 7.63-
7.55 (2H, m), 7.48-7.27 (9H, m), 7.14 ( 1 H, dt, J=1.2, 8.4 Hz), 5.94 ( 1 H,
d, J=7.2
Hz), 5.58 ( 1 H, d, J=8.7 Hz), 5.17 (2H, ABq, J=14.7 Hz), 3.25 ( 1 H, sep,
J=6.6 Hz),
1.44 (3H, d, J=6.0 Hz), 1.28 (3H, d, J=6.6 Hz).
CA 02325388 2000-09-21
WO 99/67220 PCT/U599/14007
-- 139 --
Step C - Preparation of 2-(~-,~a-Amino)-~1'-lbenzyloxycarbo~yl)
gl~invl]-amino-3'-fluorobenz~phenone
Ammonia gas was bubbled into a solution 2-(N-(a-isopropylthio)-N'-
(benzyloxycarbonyl)-glycinyl}-amino-3'-fluorobenzophenone (1 eq) in THF at
0°C. After 35 minutes mercury(II) chloride (1.1 eq) was added. The ice
bath was
removed and ammonia gas was continued to bubble through the suspension for 4
hours. The bubbler was removed and the reaction continued to stir for 16
hours.
The mixture was filtered through celite washing with THF. The filtrate was
concentrated in vacuo. The crude solid was used in step D without further
purification.
Step D - Preparation of 3-lBenzvloxycarbon~l-amino-2.3-dihydro-5=
(_3-fluoronher~vl l-1 H-1 _4-benzodi azepin-2-one
2-[N-(a-Amino)-N'-(benzyloxycarbonyl)-glycinyl]-amino-3'-
fluorobenzophenone ( I eq) was treated with glacial acetic acid and ammonium
acetate (4.7 eq). The suspension was stirred at ambient temperature for 21
hours.
After concentrating the reaction in vacuo, the residue was partitioned between
ethyl acetate and 1 N NaOH. The aqueous layer was back-extracted with ethyl
acetate. The combined organics were washed with brine, dried over Na,SO.,,
filtered, and concentrated. The residue was purified via flash chromatography
eluting with a gradient of 2~3% isopropyl alcohol/methylene chloride.
C,3H,gFN303 (MW = 403.44); mass spectroscopy found (M+H) 404.4.
Anal. calcd for C,3H,gFN30;~O.SH~O: C, 66.98; H, 4.64; N, 10.18. Found:
C, 67.20; H, 4.64; N, 9.77.
Step E - Plgparation of 3-(Benzvlox card rbon~rl)-amino-2 3-dih~!~ro-1-
meth 1-~5-(3-fluor eny~)-1H-1 4-benzodiaze~in-2-one
Following General Procedure 8-A and using 3-(benzyioxycarbonyl)-amino-
2,3-dihydro-5-(3-fluorophenyl)-IH-1,4-benzodiazepin-2-one, the title
intermediate
was prepared as a yellow foam.
C,4H,oFN30, (MW = 417.47); mass spectroscopy found (M+H) 418.3.
CA 02325388 2000-09-21
WO 99/67220 PC"T/US99/14007
-- 140 --
Anal. calcd for C,4H,oFN303: C, 69.06; H, 4.83; N, 10.07. Found: C,
69.33; H, 4.95; N, 9.82.
Step F - Preparation of 3-Amino-1.3-dihydro-1-methyl-5-f3-
.fluoropheny~,l-2H-1.4-benzodiazenin-2-one
Following General Procedure 8-B and using 3-(benzyloxycarbonyl)-amino-
2,3-dihydro-1-methyl-S-(3-fluorophenyl)-1H-1,4-benzodiazepin-2-one, the title
intermediate was prepared as a yellow foam which was used immediately in Step
G.
Step G - Preparation of 3-j~'_ltert-Butylcarbamate)-L-alaninvll-
amino-2,3-dit,~,ydro-1-methyj-5-l3-fluorophen~l-1 H-1.4-
benzodiazepin-2-one
Following General Procedure D using N-Boc-L-alanine and 3-amino-1,3-
dihydro-1-methyl-5-(3-fluorophenyl)-2H-1,4-benzodiazepin-2-one, the title
intermediate was prepared as a yellow solid.
C,~H,,FN~O~ (MW = 454.50); mass spectroscopy found (M+H) 455.3.
Anal. calcd for C,~H,,FN,04: C, 63.42; H, 5.99; N, 12.33. Found: C,
63.34; H, 6.01; N, 12.08.
Step H - Preparation of 3-(L-Alanin~)-amino-2.3-dihvdro-1-methvl-
5-~(3-fluoropheny,~l-1 H-1.4-benzodiazepjn-2-one
Following General Procedure 8-C using 3-[N'-(tent-butylcarbamate)-L-
alaninyl]-amino-2,3-dihydro-1-methyl-5-(3-fluorophenyl)-1 H-1,4-benzodiazepin-
2-one, the title intermediate was prepared as a yellow foam. The crude
material
was used immediately.
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/14007 _
-- 141 --
Example 8-K
Synthesis of
3-(L-Alaninyl)amino-2,3-dihydro-1-methyl
5-(4-fluorophenyl)-1H-1,4-benzodiazepin-2-one
Step A - Preparation of 2-Amino-4'-fluorobenzo hn enone
A solution of 4-bromofluorobenzene (1 eq.) in THF was cooled to -
78°C
under nitrogen and treated with tert-butyllithium (2.05 eq., 1.6 M solution in
pentane) at a rate of 40 ml/h. The internal temperature did not rise above -
74°C.
The orange solution was stirred at -78°C for 30 minutes prior to the
addition of
anthranilonitrile (0.6 eq.) as a solution in THF. The reaction was warmed to
0°C
and stirred for 2 hours. 3N HCl was added to the mixture and stirring
continued
for 30 minutes. The reaction was diluted with ethyl acetate and the layers
were
separated. The aqueous layer was back-extracted thrice with ethyl acetate. The
combined extracts were washed with brine, dried over Na,SO~, filtered, and
concentrated. The residue was purified via HPLC eluting with 93:7
hexanes/ethyl
acetate.
C,3H,oFNO (MW = 215.24); mass spectroscopy found (M+H) 216.3.
Anal. calcd for C,3H,oFNO: C, 72.55; H, 4.68; N, 6.51. Found: C, 72.80;
H, 4.51; N, 6.74.
Step B - Preparation of 2-(N-(a-Isogrop,ylthio -
(benzyloxvcarbonyl_l-gl~j~1]-amino-4'-
fluorobenzo henone
A solution of a-(isopropylthio)-N-(benzyloxycarbonyl)glycine (1 eq;
prepared according to Zoller, V.; Ben-Ishai, D. Tetrahedron 1975, 31, 863.) in
dry
THF was cooled to 0°C and treated with oxalyl chloride (1 eq.) and 3
drops of
DMF. After stirring for I S minutes at 0°C, the cooling bath was
removed and
stirring continued at ambient temperature for 40 minutes. The solution was
recooled to 0°C. A solution of 2-amino-4'-fluorobenzophenone (0.9 eq.)
and 4-
methylmorpholine (2.0 eq.) in dry THF was added via cannulation to the acid
chloride. The cooling bath was removed and the reaction stirred at ambient for
5
hours. The reaction was diluted with methylene chloride and washed with 0.5 M
CA 02325388 2000-09-21
WO 99/67220 PCTIUS99/14007
-- 142 --
citric acid, saturated aqueous NaHC03, and brine. The organic phase was dried
over Na2S04, filtered, and concentrated. The residue was purified via
preparative
LC2000 eluting with a gradient of 1520% ethyl acetate/hexanes giving an off
white foam.
C,6H~SN~04S (MW = 480.60); mass spectroscopy found (M+NH4+) 498.2.
'H NMR (300 MHz, CDC13) d 11.28 (1H, s), 8.56 (1H, d, J=8.4 Hz), 7.78-
7.73 (2H, m), 7.61-7.53 (2H, m), 7.36-7.32 (SH, m). 7.20-7.14 (3H, m), 5.98
(1H,
d, J=7.5 Hz), 5.57 ( 1 H, d, J=7.8 Hz), 5.16 (2H, ABq, J=14.7 Hz}, 3.25 ( 1 H,
sep,
J=6.0 Hz), 1.43 (3H, d, J=6.3 Hz), 1.27 (3H, d, J=6.6 Hz).
Step C - Preparation of 2-[~(a-Amino)-N'-lbenz~ c~xjl
glvcin~]-amino-4'-fluorobenzo hep none
Ammonia gas was bubbled into a solution 2-[N-(a-isopropylthio)-N'-
(benzyloxycarbonyl)-glycinyl]-amino-3'-fluorobenzophenone (1 eq) in THF at
0°C. After 35 minutes mercury(II) chloride (1.1 eq) was added. The ice
bath was
removed and ammonia gas was continued to bubble through the suspension for 4
hours. The bubbler was removed and the reaction continued to stir for 16
hours.
The mixture was filtered through celite washing with THF. The filtrate was
concentrated in vacuo. The crude solid was used in step D without further
purification.
Step D - Preparation of 3-f~3enzvloxycarbonXl_lamino-2.3-did dv ro=5=
f 4-fl uoronhen~l-1 H-1.4-benzodiazenin-2-one
2-[N-(a-Amino)-N'-(benzyloxycarbonyi)-glycinyl]-amino-4'-
fluorobenzophenone ( 1 eq) was treated with glacial acetic acid and ammonium
acetate (4.7 eq). The suspension was stirred at ambient temperature for 21
hours.
After concentrating the reaction in vacuo, the residue was partitioned between
ethyl acetate and 1 N NaOH. The aqueous layer was back-extracted with ethyl
acetate. The combined organics were washed with brine, dried over Na,SO.,,
filtered, and concentrated. The residue was purified via flash chromatography
eluting with a gradient of 2--3% isopropyl alcohol/methylene chloride.
CA 02325388 2000-09-21
WO 99167220 PCT/US99114007
-- 143 --
Cz3H~gFN3O3 (MW = 403.44); mass spectroscopy found (M+H) 404.4.
Anal. calcd for C,3H,8FN303~1.25H~0: C, 64.85; H, 4.85. Found: C,
64.80; H, 4.55.
Step E - Preparation of 3-lBenzvloxycarbonvll-amino-2.3-dihydro-1-1-
methvl-5-l4-fluoropher~ Iv_)-1 H-~ 4-benzodiazxpin-2-one
Following General Procedure 8-A and using 3-(benzyloxycarbonyl)-amino-
2,3-dihydro-5-(4-fluorophenyl)-1H-1,4-benzodiazepin-2-one, the title
intermediate
was prepared as a yellow foam.
C,4H,oFN303 (MW = 417.47); mass spectroscopy found (M+H) 418.2.
Anal. calcd for C~4H,oFN303: C, 69.06; H, 4.83; N, 10.07. Found: C,
69.35; H, 4.93; N, 9.97.
Step F - Prgparation of 3-Amino-1 3-dihydro-1-methyl-5-l4-
fluoronheny~l-2H-1,4-benzodiaze~in-2-one
Following General Procedure 8-B and using 3-(benzyloxycarbonyl)-amino-
2,3-dihydro-1-methyl-5-(4-fluorophenyl)-1H-1,4-benzodiazepin-2-one, the title
intermediate was prepared as a yellow foam which was used immediately in Step
G.
Step G - Preparation of 3-j~I=-(tert-Butylcarb~rr~at~- -alani~,~l-
amino-2.3-dihxdro-1-methyl-5-{4-fluoro~henyl)-1 H-1_ 4
benzodiazenin-2-one
Following General Procedure D using N-Boc-L-alanine and 3-amino-1,3-
dihydro-1-methyl-5-(4-fluorophenyl)-2H-1,4-benzodiazepin-2-one, the title
intermediate was prepared as a yellow solid.
C,4H,,FN404 (MW = 454.50); mass spectroscopy found (M+H) 455.4.
Anal. calcd for C,.,HZ,FN40~~ 1.SH,0: C, 59.86; H, 6.28; N, 11.64. Found:
C, 60.04; H, 5.62; N, 11.27.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ lq.rl __
Step H - Preparation of 3-lL-Alanyll-amino-2.3-d~vdro-1-methxl-
S-l4-fluorophen~l-1 H-1.4-benzodiazepin-2-one
Following General Procedure 8-C using 3-(N'-(tert-butylcarbamate)-L-
alaninylJ-amino-2,3-dihydro-1-methyl-5-(4-fluorophenyl)-1 H-1,4-benzodiazepin-
2-one, the title intermediate was prepared as a yellow foam. The crude
material
was used immediately.
Example 8-L
Synthesis of
3-(N'-L-Alaniny!)amino-2,3-dihydro-1-isobutyl
5-phenyl-1H-1,4-benzodiazepin-2-one
Step A: 1,3-Dihydro-~-phenyl-2H-1,4-benzodiazepin-2-one (prepared
according to the procedure of M. G. Bock et al., J. Org. Chem. 1987, 52, 3232-
3239) was alkylated with isobutyl iodide using General Procedure 8-G to afford
1,3-dihydro-1-isobutyl-5-phenyl-2H-1,4-benzodiazepin-2-one.
Ste~B: Following General Procedures 8-D and 8-F and using the product
from Step A, 3-amino-1,3-dihydro-1-isobutyl-5-phenyl-2H-1,4-benzodiazepin-2-
one was prepared.
Step C: The product from Step B and N-Boc-L-alanine (Sigma) were
coupled using General Procedure D, followed by removal of the Boc group using
General Procedure 8-J, to afford 3-(N'-L-alaninyl)amino-1,3-dihydro-1-isobutyl-
5-
phenyl-2H-1,4-benzodiazepin-2-one.
By substituting isopropyl iodide, n-propyl iodide, cyclopropylmethyl iodide
and ethyl iodide for isobutyl iodide in Step A above, the following additional
intermediates were prepared:
3-(N'-L-alaninyl)amino-1,3-dihydro-1-isopropyl-5-phenyl-2H-1,4-
benzodiazepin-2-one
CA 02325388 2000-09-21
WO 99167220 PCT/US99/14007
-- 145 --
3-(N'-L-alaninyl)amino-1,3-dihydro-1-propyl-5-phenyl-2H-1,4-
benzodiazepin-2-one
3-(N'-L-alaninyl)amino-1,3-dihydro-1-cyclopropylmethyl-5-phenyl-2H-
1,4-benzodiazepin-2-one
3-(N'-L-alaninyl)amino-1,3-dihydro- I -ethyl-5-phenyl-2H-1,4-
benzodiazepin-2-one.
Example 8-M
Synthesis of
3-(N'-L-Alaninyl)amino-1-methyl-5-phenyl
1,3,4,5-tetrahydro-2H-1,5-benzodiazepin-2-one
Sten A: 1,3,4,5-Tetrahydro-5-phenyl-2H-1,5-benzodiazepin-2-one (CAS
No. 32900-17-7) was methylated using General Procedure 8-I to afford 1-methyl-
5-phenyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepin-2-one.
Step B: Following General Procedures 8-E and 8-F and using the product
from Step A, 3-amino-1-methyl-5-phenyl-1,3,4,5-tetrahydro-2H-1,5-
benzodiazepin-2-one was prepared.
St~C ~. The product from Step B and N-Boc-L-alanine (Sigma) were
coupled using General Procedure D, followed by removal of the Boc group using
General Procedure 8-N, to afford 3-(N'-L-alaninyl)amino-1-methyl-S-phenyl-
1,3,4,5-tetrahydro-2H-1,5-benzodiazepin-2-one.
Example 8-N
Synthesis of
3-(N'-L-Alaninyl)amino-2,4-dioxo-1-methyl-5-phenyl
2,3,4,5-tetrahydro-1 H-1,5-benzodiazepine
3-Amino-2,4-dioxo-1-methyl-5-phenyl-2,3,4,5-tetrahydro-1 H-1,5-
benzodiazepine (CAS No. 131604-75-6) was coupled with N-Boc-L-alanine
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 146 -
(Sigma) using General Procedure D, followed by removal of the Boc group using
General Procedure 8-N, to afford the title compound.
Example 8-O
Synthesis of
3-((R)-Hydrazinopropionyl)amino-2,3-dihydro-
1-methyl-5-phenyl)-1H-1,4-benzodiazepin-2-one
3-Amino-2,3-dihydro-1-methyl-5-phenyl-1H-1,4-benzodiazepin-2-one was
coupled to (R)-N,N'-di-BOC-2-hydrazinopropionic acid (Example N) using
General Procedure D. Removal of the Boc group using General Procedure 5-B
afforded the title compound.
Example 8-P
Synthesis of
3-Amino-2,4-dioxo-1,5-bis-(1-methylethyl)-
2,3,4,5-tetrahyd ro-1 H-1,5-benzodiazepine
Step A: - Synthesis of 2,4-Dioxo-2,3,4,5-tetrahydro-1H-1,5-
benzodiazepine
2,4-Dioxo-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine (CAS No. 49799-48-
6) was prepared from 1,2-phenylenediamine (Aldrich) and malonic acid (Aldrich)
using the procedure of Claremon, D. A.; et al, PCT Application: WO 96-US8400
960603.
Ste : - Synthesis of 2,4-Dioxo-1,5-bis-(1-methylethyl)-2,3,4,5-
tetrahydro-1 H-1,5-benzodiazepine
2,4-Dioxo-1,5-bis-( 1-methylethyl)-2,3,4,5-tetrahydro-1 H-1,5-
benzodiazepine (CAS No. 113021-84-4) was prepared following General
Procedure 8-M using the product from Step A and 2-iodopropane (Aldrich).
Purification was by flash chromatography eluting with EtOAc/hexanes (3:7
gradient to 1:1 ), then recrystalization from EtOAc/hexanes.
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/14007
-- 147 --
to ~ - Synthesis of 3-Azido-2,4-dioxo-1,5-bis-(1-methylethyl)
2,3,4,5-tetrahydro-1 H-1,5-benzodiazepine
Following General Procedure 8-K using the product from Step B, 3-azido-
2,4-dioxo-I,5-bis-(1-methylethyl)-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine
(CAS
No. 186490-50-6) was prepared as a white solid. The product was purified by
flash chromatography eluting with hexanes/EtOAc (4:1 ) to provide a separable
23:1 mixture of pseudo-axial/pseudo-equatorial azides. The pure pseudo-axial
azide was used in the next step.
Step D: - Synthesis of 3-Amino-2,4-dioxo-1,5-bis-(1-methylethyl)
2,3,4,5-tetrahydro-1H-1,5-benzodiazepine
Following General Procedure 8-L using the product from Step C, 3-amino-
2,4-dioxo-I,5-bis-{1-methylethyl}-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine
(CAS
No. 186490-51-7) was prepared as a white solid. Purification was by flash
chromatography eluting with CH,CI,/MeOH (98:2 gradient to 95:5). The isolated
pseudo-axial amine atropisomer was completely converted to the pseudo-
equatorial
amine atropisomer by heating in toluene to 100-105 °C for 15 minutes,
and the
pseudo-equatorial amine atropisomer was used in the next step. The isomers
were
distinguished by'H-NMR in CDCI3. Selected'H-NMR (CDC1,): Pseudo-axial
amine 4.40 (s, 1 H); Pseudo-equatorial amine 3.96 (s. 1 H).
Example 8-Q
Synthesis of
3-(R-2-Thienylglycinyl)amino-2,4-dioxo
I,5-bis-(1-methylethyl)-2,3,4,5-tetrahydro
IH-1,5-benzodiazepine Hydrochloride
to ~ - Synthesis of N-(t-Butoxycarbonyl)-R-2-thienylglycine
N-(t-Butoxycarbonyl)-R-2-thienylglycine (CAS No. 74462-03-1) was
prepared from L-a-(2-thienyl)glycine (Sigma) by the procedure described in
Bodansky, M. et aI; The Practice ofPeptide Synthesis; Springer Verlag; 1994,
p.
17.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007 _ _.,
-- 148 --
SteR B: - Synthesis of 3-[N'-(t-Butoxycarbonyl)-R-2-
thienylglycinyl]-amino-2,4-dioxo-1,5-bis-(1-methylethyl)-
2,3,4,5-tetrahydro-1H-1,5-benzodiazepine
Following General Procedure J above using the product from Example 8-P
and the product from Step A above, 3-[N'-(t-butoxycarbonyl)-R-2-
thienylglycinyl]-amino-2,4-dioxo-1,5-bis-( I -methylethyl)-2,3,4,5-tetrahydro-
1 H-
I ,5-benzodiazepine was prepared as a white foam. Purif canon was by flash
chromatography eluting with CH,CI,/EtOAc (9:1 gradient to 5:1).
Synthesis of 3-(R-2-Thienylglycinyl)amino-2,4-dioxo-1,5-
bis-(I-methylethyl)-2,3,4,5-tetrahydro-1H-1,5-
benzodiazepine Hydrochloride
Following General Procedure 8-N above using the product from Step B, the
title compound was prepared as a white solid.
Example 8-R
Synthesis of
3-(L-Alaninyl)-amino-2,4-dioxo-I,5-bis-methyl
2,3,4,5-tetrahydro-1H-1,5-benzodiazepine Hydrochloride
a A: - Synthesis of 2,4-Dioxo-1,5-bis-methyl-2,3,4,5-tetrahydro-
1 H-I,5-benzodiazepine
2,4-Dioxo-1,5-bis-methyl-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine (CAS
No. 23954-54-3) was prepared following General Procedure 8-M using the product
from Example 8-P, Step A and iodomethane (Aldrich). The white solid product
precipitated during partial concentration of the reaction after work-up, and
was
isolated by filtration.
Ste~B: - Synthesis of 3-Azido-2,4-dioxo-1,5-bis-methyl-2,3,4,5-
tetrahydro-1H-1,5-benzodiazepine
For this substrate, General Procedure 8-K was modified in the following
manner. Initially the product from Step A was suspended (not a solution) in
THF
at -78°C, and following addition of the KN(TMS), solution, this
suspension was
allowed to warm to -35 °C over a period of 12 minutes, during which the
CA 02325388 2000-09-21
WO 99/67220 PCT/US99114007
-- 149 --
suspension became a solution, and was re-cooled~to -78°C; then treated
as
described in the General Procedure. 3-Azido-2,4-dioxo-1,5-bis-methyl-2,3,4,5-
tetrahydro-1H-I,5-benzodiazepine was purified by flash chromatography eluting
with CHC13/EtOAc (7: I ), then trituration from hot CHC13 with hexanes and
cooled
to -23°C. The product was isolated as a white solid.
SteRC: - Synthesis of 3-Amino-2,4-dioxo-1,5-bis-methyl-2,3,4,5-
tetrahydro-1H-1,5-benzodiazepine
Following General Procedure 8-L using the product from Step B, 3-amino-
2,4-dioxo-I,5-bis-methyl-2,3,4,5-tetrahydro-IH-I,5-benzodiazepine was prepared
as a white solid. The crude product was used without further purification.
Ste;~D: - Synthesis of 3-[N'-(t-Butoxycarbonyl)-L-alaninyl]-
amino-2,4-dioxo-1,5-bis-methyl-2,3,4,5-tetrahydro-1H-
1,5-benzodiazepine
Following General Procedure I above using N-Boc-L-alanine
(Novabiochem) and the product from Step C, 3-[N'-(t-butoxycarbonyl)-L-
alaninyl ]-amino-2,4-dioxo- I ,~-bis-methyl-2,3,4,5-tetrahydro- I H-1,5-
benzodiazepine was prepared as a white foam. Purification was by flash
chromatography eluting with CH,CI,/EtOAc (2:1 gradient to 1:1 ).
Step E: - Synthesis of 3-(L-alaninyl)-amino-2,4-dioxo-1,5-bis-
methyl-2,3,4,5-tetrahydro-1 H-1,5-benzodiazepine
Hydrochloride
Following General Procedure 8-N above using the product from Step D, the
title compound was prepared as an off white amorphous solid.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 150 --
Example 8-S
Synthesis of
3-(L-Alaninyl)amino-2,4-dioxo-1,5-bis-(2-methylpropyl)-
2,3,4,5-tetrahydro-1H-1,5-benzodiazepine Hydrochloride
,$ten A: - Synthesis of 2,4-Dioxo-1,5-bis-(2-methylpropyl)-2,3,4,5-
tetrahydro-1H-1,5-benzodiazepine
2,4-Dioxo-1,5-bis-(2-methylpropyl)-2,3,4,5-tetrahydro-1 H-I ,5-
benzodiazepine was prepared following General Procedure 8-M using the product
from Example 8-P, Step A and 1-iodo-2-methylpropane (Aldrich). Purification
was by flash chromatography eluting with EtOAc/hexanes (3:7 gradient to 1: I
),
then recrystalization from EtOAc/hexanes.
SteRB: - Synthesis of 3-Azido-2,4-dioxo-1,5-bis-(2-methylpropyl)
2,3,4,5-tetrahydro-1H-1,5-benzodiazepine
Following General Procedure 8-K (a precipitate formed during the addition
of the KN(TMS),, but dissolved upon addition of the trisyl azide) using the
product
from Step A, 3-azido-2,4-dioxo-1,5-bis-(2-methylpropyl)-2,3,4,5-tetrahydro-1H-
1,5-benzodiazepine was prepared as a white solid. The product was purified by
flash chromatography eluting with hexanes/EtOAc (4: I ) and a second flash
chromatography eluting with CH,Ch/hexanes/EtOAc ( 10:10:1 gradient to 8:6:1 ).
Step C: - Synthesis of 3-Amino-2,4-dioxo-1,5-bis-(2-methylpropyl)-
2,3,4,5-tetrahydro-1H-1,5-benzodiazepine
Following General Procedure 8-L using the product from Step B, 3-amino
2,4-dioxo-1,5-bis-(2-methylpropyl)-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine
was
prepared as a white solid. Purification was by flash chromatography eluting
with
CH,C1,/MeOH (98:2 gradient to 95:5, with 5% NH3 in the MeOH).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007 _
-- I51 --
Sten D:D: - Synthesis of 3-[N'-(t-Butoxycarbonyl)-L-alaninyl]-
amino-2,4-dioxo-1,5-bis-(2-methylpropyl)-2,3,4,5-
tetrahydro-1H-1,5-benzodiazepine
Following General Procedure I above using N-Boc-L-alanine
(Novabiochem) and the product from Step C, 3-[N'-(t-butoxycarbonyl)-L-
alaninyl]-amino-2,4-dioxo-1,5-bis-(2-methylpropyl)-2,3,4,5-tetrahydro-1 H-1,5-
benzodiazepine was prepared as a white foam. Purification was by flash
chromatography eluting with CH,CI,/EtOAc (3:1 gradient to 3:2).
Step E: - Synthesis of 3-(L-Alaninyl)-amino-2,4-dioxo-1,5-bis-(2-
methylpropyl)-2,3,4,5-tetrahydro-1H-I,5-benzodiazepine
Hydrochloride
Following General Procedure 8-N above using the product from Step D, the
title compound was prepared as an amorphous white solid.
Example 8-T
Synthesis of
3-(S-Phenylglycinyl)amino-2,4-dioxo-1,5-bis
(2-methylpropyl)-2,3,4,5-tetrahydro-
1H-I,5-benzodiazepine Hydrochloride
Stgp A: - Synthesis of 3-(N'-(t-Butoxycarbonyl)-S-phenylglycinyl]-
amino-2,4-dioxo-1,5-bis-(2-methylpropyl)-2,3,4,5-
tetrahydro-I H-1,5-benzodiazepine
Following General Procedure J above using the product from Example 8-S,
Step C and the Boc-L-phenylglycine (Novabiochem, CAS No. 2900-27-8), 3-[N'-
(t-butoxycarbonyl)-S-phenylglycinyl)-amino-2,4-dioxo-1,5-bis-(2-methylpropyl)-
2,3,4,5-tetrahydro-1H-1,5-benzodiazepine was prepared as a white foam.
Purification was by flash chromatography eluting with CH,CI,/EtOAc (9:1
gradient to 5:1 ).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007 _ .
-- 152 --
ten B: - Synthesis of 3-(S-Phenylglycinyl)-amino-2,4-dioxo-1,5-
bis-(2-methylpropyl)-2,3,4,5-tetrahydro-1 H-1,5-
benzodiazepine Hydrochloride
Following General Procedure 8-N above using the product from Step A, 3-
(S-phenylglycinyl)-amino-2,4-dioxo-1,5-bis-(2-methylpropyl)-2,3,4,5-tetrahydro-
1H-1,5-benzodiazepine hydrochloride was prepared as an off white solid.
Example 8-U
Synthesis of
3-(L-Alaninyl)amino-2,4-dioxo-1,5-bis-(cyclopropylmethyl)
2,3,4,5-tetrahydro-1H-1,5-benzodiazepine Hydrochloride
Step A: - Synthesis of 2,4-Dioxo-1,5-bis-(cyclopropylmethyl)-
2,3,4,5-tetrahydro-1 H-1,5-benzodiazepine
2,4-Dioxo-1,5-bis-(cyclopropylmethyl)-2,3,4,5-tetrahydro-1 H-1,5-
benzodiazepine was prepared following General Procedure 8-M using the product
from Example 8-P, Step A, and (bromomethyl)cyclopropane (Lancaster).
Purification was by flash chromatography eluting with EtOAc/hexanes (3:7
gradient to straight EtOAc), then recrystalization from EtOAc/hexanes.
Synthesis of 3-Azido-2,4-dioxo-1,5-bis-
(cyclopropylmethyl)-2,3,4,5-tetrahydro-1H-1,5-
benzodiazepine
For this substrate General Procedure 8-K was modified in the following
manner. Initially the product from Step A was suspended (not a solution) in
THF
at -78°C, and following addition of the KN(TMS)~ solution, this
suspension was
allowed to warm to -30°C, during which the suspension became a
solution, and
was re-cooled to -78°C. Upon re-cooling to -78°C a precipitate
began to form,
therefore the reaction flask containing the mixture was partially raised above
the
cooling bath until the internal temperature rose to -50°C; then the
trisyl azide
solution was added. The cooling bath was removed and the mixture allowed to
warm to -20°C whereupon the mixture had become a nearly homogenous
solution,
and the AcOH was added. Then, treated as described in the general procedure. 3-
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 153 --
Azido-2,4-dioxo-1,5-bis-(cyclopropylmethyl)-2,3,4, 5-tetrahydro-1 H-1,5-
benzodiazepine was purified by trituration with hot to room temperature EtOAc,
followed by recrystalization from hot to -23 °C CHC13/EtOAc/EtOH (5:5:1
) and
isolated as a white solid.
Step C: - Synthesis of 3-Amino-2,4-dioxo-1,5-bis-
(cyclopropylmethyl)-2,3,4,5-tetrahydro-1H-1,5-
benzodiazepine
Following General Procedure 8-L using the product from Step B, 3-amino-
2,4-dioxo-1,5-bis-(cyclopropy!methyl)-2,3,4,5-tetrahydro-1 H-1,5-
benzodiazepine
was prepared as a white solid. Purification was by flash chromatography
eluting
with CH,CI,/MeOH (98:2 gradient to 95:5, with S% NH3 in the MeOH) followed
by recrystalization from warm CH~CI,/hexanes ( 1:1 ) to -23 °C.
Sten D: - Synthesis of 3-[N'-(t-Butoxycarbonyl)-L-aianinyl]-
amino-2,4-dioxo-1,5-bis-(cyclopropylmethyl)-2,3,4,5-
tetrahydro-1 H-1,5-benzodiazepine
Following General Procedure I above using N-Boc-L-alanine
(Novabiochem) and the product from Step C, 3-(N'-(t-butoxycarbonyl)-L-
alaninyl]-amino-2,4-dioxo-1.~-bis-(cyclopropy!methyl)-2,3,4,5-tetrahydro-1 H-
1,5-
benzodiazepine was prepared as a white foam. Purification was by flash
chromatography eluting with CH,CI,/EtOAc (3:1 gradient to 2:1 ).
Ste~E: - Synthesis of 3-(L-Alaninyl)-amino-2,4-dioxo-1,5-bis-
(cyclopropyimethyl}-2,3,4,5-tetrahydro-1H-1,5-
benzodiazepine Hydrochloride
Following General Procedure 8-N above using the product from Step D, the
title compound was prepared as an off white solid.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 154 --
Example 8-V
Synthesis of
3-(L-Alaninyl)-amino-2,4-dioxo-1,5-bis-(2,2-dimethylpropyl~
2,3,4,5-tetrahydro-1H-1,5-benzodiazepine Hydrochloride
Step A: - Synthesis of 2,4-Dioxo-1,5-bis-(2,2-dimethylpropyl)-
2,3,4,5-tetrahydro-1H-1,5-benzodiazepine
To a stirred suspension of the product from Example 8-P, Step A (1.0 eq.,
17.08 g) in DMSO (500 mL) at room temperature was added neopentyl iodide
(43.01 g, 2.24 eq., Aldrich) and Cs,C03 (72.65 g, 2.3 eq., Aldrich). The
resulting
mixture was heated to 75°C for 30 minutes, then additional Cs,C03
(31.59 g, 1.0
eq.) was added and the mixture rapidly stirred at 75°C for 6 hours. The
mixture
was allowed to cool and H,O (500 mL) and EtOAc (1000 mL) were added. The
phases were partitioned and the organic phase washed with HBO ( 1 x500 mL), 1
M
aq. HCl (2x500 mL), and brine ( 1 x500 mL). Then, the organic phase was dried
over MgSO:,, filtered, concentrated, and purified by flash chromatography
eluting
with hexanes/EtOAc (3:2 gradient to 2:3) to provide 2,4-dioxo-1,5-bis-(2,2-
dimethylpropyl)-2,3,4,5-tetrahydro-1H-1~,5-benzodiazepine as a white solid.
Step B: - Synthesis of 3-Azido-2,4-dioxo-1,5-bis-(2,2-
dimethylpropyl)-2,3,4,5-tetrahydro-1H-I,5-
benzodiazepine
Following General Procedure 8-K using the product from Step A, 3-azido-
2,4-dioxo-1,5-bis-(2,2-dimethylpropyl)-2,3,4,5-tetrahydro-1 H-1,5-
benzodiazepine
was prepared as a white solid. The product was purified by flash
chromatography
eluting with hexanes/CH,C1~/EtOAc ( 10:5:1 gradient to 5:5:1 ) to provide a
separable 13:1 mixture of pseudo-axial/pseudo-equatorial azides. The pure
pseudo-axial azide was used in the next step. Selected'H-NMR (CDC13): Pseudo-
axial azide 5.12 (s, 1 H); Pseudo-equatorial azide 4.03 (s, 1 H).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007 _.
-- 155 --
Step C: - Synthesis of 3-Amino-2,4-dioxo-1,5-bis-(2,2-
dimethylpropyl)-2,3,4,5-tetrahydro-1H-1,5-
benzodiazepine
Following General Procedure 8-L using the product from Step B, 3-amino-
2,4-di oxo-1,5-bis-(2,2-dimethy lpropyl)-2,3,4, 5-tetrahydro-1 H-1, 5-
benzodiazepine
was prepared as a white solid. Purification was by flash chromatography
eluting
with CH,CI,/MeOH (98:2 gradient to 95:5, with 5% NH3 in the MeOH). The
isolated white solid product was identified as a ~-4:1 mixture of pseudo-axial
and
pseudo-equatorial amines atropisomers by'H-NMR. The mixture was heated in
toluene to 100 °C for 20 minutes, then re-concentrated to provide the
pure pseudo-
equatorial amine atropisomer, as a white solid, and this was for the next
step.
Selected'H-NMR (CDCl3): Pseudo-axial amine 4.59 (s, 1H); Pseudo-equatorial
amine 4.03 (s, 1 H).
step ~: - Synthesis of 3-[N'-(t-Butoxycarbonyl)-L-alaninyl]-
amino-2,4-dioxo-1,5-bis-(2,2-dimethylpropyl)-2,3,4,5-
tetrahydro-1 H-1,5-benzodiazepine
Following General Procedure I above using N-Boc-L-alanine
(Novabiochem) and the product from Step C, 3-[N'-(t-butoxycarbonyl)-L-
alaninyl]-amino-2,4-dioxo-1,5-bis-(2,2-dimethylpropyl)-2,3,4,5-tetrahydro-1 H-
1,5-
benzodiazepine was prepared as a white foam. Purification was by flash
chromatography eluting with CH,C1,/EtOAc (4:1 gradient to 5:2).
to E~ - Synthesis of 3-(L-Alaninyl)-amino-2,4-dioxo-1,5-bis-(2,2-
dimethylpropyl)-2,3,4,5-tetrahydro-1H-1,5-
benzodiazepine Hydrochloride
Following General Procedure 8-N above using the product from Step D, the
title compound was prepared as an off white solid.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 156 --
Example 8-W
Synthesis of
3-(L-Alaninyi)amino-2,4-dioxo-1,5-bis-phenyl
2,3,4,5-tetrahydro-1H-1,5-benzodiazepine hydrochloride
Sten A: - Synthesis of 2,4-Dioxo-1,5-bis-phenyl-2,3,4,5-tetrahydro-
1H-1,5-benzodiazepine
This procedure is a modification of the procedure described in Chan, D. M.
T. Tetrahedron Lett. 1996, 37, 9013-9016. A mixture of the product from
Example 8-P, Step A (1.0 eq., 7.50 g), Ph3Bi (2.2 eq., 41.26 g, Aldrich),
Cu(OAc),
(2.0 eq., 15.48 g, Aldrich), Et,N (2.0 eq., 8.62 g) in CHzCI, { 100 mL) was
stirred
under N, at room temperature for 6 days (monitoring by TLC). The solids were
removed by filtration through a plug of Celite rinsing with CH,Ch/MeOH (3x75
mL). The filtrate was concentrated, dissolved in hot CH,C12/MeOH (9:1 ) and
filtered through a large plug of silica gel eluting with CH,CI,/MeOH (9:1,
2L).
The filtrate was concentrated and the residue purified by flash chromatography
eluting with straight CH,CI, gradient to CH,CI,/MeOH (9:1). 2,4-Dioxo-1,5-bis-
phenyl-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine crystallized during
concentration
of the fractions containing the product, and was isolated by filtration as a
white
solid.
Step B: - Synthesis of 3-Azido-2,4-dioxo-1,5-bis-phenyl-2,3,4,5-
tetrahydro-1H-1,5-benzodiazepine
For this substrate, General Procedure 8-K was modified in the following
manner. Initially the product from Step A was suspended (not a solution) in
THF
at -70°C, and following addition of the KN(TMS), solution, this
suspension was
allowed to warm to -20°C over a period of 10 minutes, during which the
suspension became a solution, and was re-cooled to -70°C; then treated
as
described in the general procedure. The title compound was purified by
trituration
with hot CHCl3/hexanes (1:1) to yield 3-azido-2,4-dioxo-1,5-bis-phenyl-2,3,4,5-
tetrahydro-1H-1,5-benzodiazepine as a white solid.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 157 --
Sten C:C: - Synthesis of 3-Amino-2,4-dioxo-1,5-bis-phenyl-2,3,4,5-
tetrahydro-1H-1,5-benzodiazepine
Following General Procedure 8-L using the product from Step B, 3-amino-
2,4-dioxo-I,5-bis-phenyl-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine was prepared
as a white solid. Purification was by flash chromatography eluting with
CH,CI,/MeOH (98:2 gradient to 95:5, with 5% NH3 in the MeOH).
Step D: - Synthesis of 3-[N'-(t-Butoxycarbonyl)-L-alaninyl)-
amino-2,4-dioxo-1,5-bis-phenyl-2,3,4,5-tetrahydro-1H-
1,5-benzodiazepine
Following General Procedure I above using N-Boc-L-alanine
(Novabiochem) and the product from Step C, 3-[N'-(t-butoxycarbonyl)-L
alaniny 1]-amino-2,4-dioxo-1, 5-bis-pheny I-2, 3,4, 5-tetrahydro-1 H-1,5-
benzodiazepine was prepared as a white foam. Purification was by flash
chromatography eluting with CH,CI,/EtOAc (4:1 gradient to 3:1).
Ste ~ - Synthesis of 3-(L-Alaninyl)-amino-2,4-dioxo-1,5-bis-
phenyl-2,3,4,5-tetrahydro-1 H-1,5-benzodiazepine
Hydrochloride
Following General Procedure 8-N above using the product from Step D, the
title compound was prepared as a white amorphous solid.
Example 8-X
Synthesis of
3-Amino-2,3-dihydro-1-methyl-5-phenyl-1H 1,4-benzodiazepin-2-one
Following the method of R. G. Sherrill et al., J. Org. Chem.,1995, 60, 730-
734 and using glacial acetic acid and HBr gas, the title compound was
prepared.
CA 02325388 2000-09-21
WO 99/67220 PC"T/US99/14007
-- 158 --
Example 8-Y
Synthesis of
3-(L-Valinyl)-amino-2,3-dihydro-1-methyl
5-phenyl-1H-1,4-benzodiazepin-2-one
Step A - Synthesis of 3-(N'-(tent-Butylcarbamate)-L-valinyl]-
amino-2,3-dihydro-1-methyl-5-phenyl-1H-1,4-
benzodiazepin-2-one
(S)-3-Amino-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one,
(1S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-methanesulfonate (Example 8-B,
Step A) was free based by partitioning between methylene chloride and 1 M
potassium carbonate. The free amine was then coupled with N-Boc-valine
following General Procedure D to give the title compound.
C,6H3,N40a (MW 464.62); mass spectroscopy 464.3.
Anal. Calcd for C~6H3,Na0~: C, 67.22; H, 6.94; N, 12.06. Found: C,
67.29; H, 6.79; N, 11.20.
Step - Synthesis of 3-(L-valinyl)-amino-2,3-dihydro-1-methyl-5-
phenyl-1 H-1,4-benzodiazepin-Z-one
Following General Procedure 8-C and using 3-[N'-(tert-butylcarbamate)-L-
alaninyl]-amino-2,3-dihydro-1-methyl-5-phenyl-1H-1,4-benzodiazepine-2-one, the
title compound was prepared as a white foam.
C,,H,;NdO, (MW 363.48); mass spectroscopy (M+H) 364.2.
Example 8-Z
Synthesis of
3-(L-tert-Leucinyl)-amino-2,3-dihydro-1-methyl
5-phenyl-1 H-1,4-benzodiazepin-2-one
to - Synthesis of 3-(N'-{tert-Butylcarbamate)-L-tert-leucinyl]-
amino-2,3-dihydro-1-methyl-5-phenyl-1H-1,4-
benzodiazepin-2-one
(S)-3-amino-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one,
(1S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-methanesulfonate (Example 8-B,
Step A) was free based by partitioning between methylene chloride and 1 M
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 159 --
potassium carbonate. The free amine was then coupled with N-Boc-tert-leucine
following General Procedure D to give the title compound.
C,,H35N404 (MW 479.66); mass spectroscopy 479
Step - Synthesis of 3-(L-tert-Leucinyl)-amino-2,3-dihydro-1-
methyl-5-phenyl-1H-1,4-benzodiazepin-2-one
Following General Procedure 8-C and using 3-[N'-(tert-butylcarbamate)-L-
tert-leucinyl]-amino-2,3-dihydro-1-methyl-5-phenyl-1 H-1,4-benzodiazepine-2-
one, the title compound was prepared as a white foam.
Anal. Calcd for C"H,aN~O, ~ O.SH,O: C, 68.19; H, 7.02; N, 14.40. Found:
C, 68.24; H, 7.00; N, 14.00.
Example 8-AA
Synthesis of
3-(L-Alaninyl)-amino-2,3-dihydro-1,5-dimethyl
1H-1,4-benzodiazepine
2,3-Dihydro-1,5-dimethyl-IH-1,4-benzodiazepine was prepared following
General Procedures 8-I (using methyl iodide), 8-D and 8-F. Coupling of this
intermediate with Boc-L-alanine (Novo) using General Procedure D, followed by
deprotection using General Procedure 5-B afforded the title compound which was
used without further purification.
Example 8-AB
Synthesis of
3-(L-3-Thienylglycinyl)amino-2,4-dioxo
1,5-bis-(2,2-dimethylpropyl)-2,3,4,5-tetrahydro
1H-1,5-benzodiazepine
Step - Synthesis of N-(t-Butoxycarbonyl)-L-3-thienylglyeine
N-(t-Butoxycarbonyl)-L-3-thienylglycine was prepared from L-a-(3-
thienyl)glycine (Sigma) by the procedure described in Bodansky, M. et al; The
Practice of Peptide Synthesis; Springer Verlag; 1994, p. 17.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 160 __
Step B: - Synthesis of 3-[N'-(t-Butoxycarbonyl)-L-3-
thienylglycinyl]-amino-2,4-dioxo-1,5-bis-(2,2-
dimethylpropyl)-2,3,4,5-tetrahydro-1H-1,5-
benzodiazepine
Following General Procedure D above using the product from Example 8-
V, Step C and the product from Step A above, 3-[N'-(t-butoxycarbonyl)-L-3-
thienylglycinyl]-amino-2,4-dioxo-1,5-bis-(2,2-dimethylpropyl)-2,3,4,5-
tetrahydro-
1H-1,5-benzodiazepine was prepared.
Step C: - Synthesis of 3-(L-3-Thienylglycinyl)amino-2,4-dioxo-1,5-
bis-(2,2-dimethylpropyl)-2,3,4,5-tetrahydro-1H-1,5-
benzodiazepine
Following General Procedure 8-N above using the product from Step B, the
title compound was prepared.
Using the following procedures, the following additional intermediates can
be prepared for use in this invention.
GENERAL PROCEDURE C-H
The intermediates shown in Table C-t were synthesized in parallel in using
the following procedure:
to : To a solution of 3-(tert-butoxycarbonyl)amino-2,3-dihydro-5-
phenyl-IH-1,4-benzodiazepin-2-one (CA No. 125:33692: 100 mg, 0.28 mmol) in 1
mL of anhydrous DMF was added 600 uL of a solution of 0.5 M potassium
bis(trimethylsilyl)amide (0.30 mmol) in toluene. Neat alkyl halide (0.56 mmol;
as
indicated in Table C-1 ) was added immediately in one portion and the reaction
mixture was left undisturbed overnight. When an alkyl chloride was used, 1
equivalent of sodium iodide was added to the reaction mixture. After
concentration under reduced pressure, the crude reaction residue was
partitioned
between methylene chloride (2 mL) and aqueous saturated bicarbonate (2 mL) and
then passed through a 5 g Extralut QE cartridge (EM Science; Gibbstown, NJ)
using 10 mL of methylene chloride. The resulting filtrate was concentrated
under
CA 02325388 2000-09-21
WO 99/67220 PC'T/US99/14007
-- 161 --
reduced pressure and the crude product was further purified using automated
semi-
preparative HPLC (YMC 20 X SO mm Silica column; gradient elution; 0-5 % (S.5
min.), 5-20 % (3.5 min.), 20-100 % (2 min.), 100% (4 min.) ethyl
acetate/methylene chloride, flow rate of 25 mL/min.). Product provided the
expected M+1 peak by IEX MS and were carried on without further purification
and characterization.
Std B: The product obtained from Step A was dissolved in 5 mL of a 15
TFA/methylene chloride solution and allowed to stand undisturbed for 16 h.
After concentration under reduced pressure, the TFA salt was dissolved in
methanol and loaded directly onto a 1 g SCX column. The column was washed 3
X with 2 mL portions of methanol and the product was eluted from the column
using 6 mL of 2.0 M solution of ammonia/methanol. After concentration under
reduced pressure, the product were characterized by IEX MS and carried on
without further purification.
Step C:_ To the crude product obtained from Step B (1.05 equiv.) was
added sequentially a 0.3 mM stock solution of HOBt~H,O (1.05 equiv.) in DMF, a
0.3 mM stock solution ofN-t-BOC-L-alanine (1.0 equiv.) in THF and 0.3 mM
stock solution of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (1.05 equiv.)
in
THF. After standing undisturbed for 24 h, the reaction mixture was
concentrated
and the residue redissolved in 2 mL of a 10% methanol/methylene chloride
solution. This solution was then filtered through a pre-washed (methanol) 1 g
SCX
(Varian Sample Preparation) column using an additional 8 mL of the same
solvent.
For Example C-V a I g Si column (Varian Sample Preparation) was used). The
filtrate was concentrated under a stream of nitrogen to approximately 1/3 its
original volume and then passed over a plug (500 mg) of AG 1-8x anion exchange
resin (BioRad; Hercules, California; Columns were pre-washed with 1N NaOH,
water and methanol) using an additional 10 mL of methanol. The resulting
filtrate
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
- 162 --
was concentrated under reduced pressure and the crude product was carned on
without further purification after characterization by IEX MS.
Step D: The crude product obtained from Step C was dissolved in 5 mL of
a 15 % TFA/methylene chloride solution and allowed to stand undisturbed for 16
h. After concentration under reduced pressure, the TFA salt was dissolved in
methanol and loaded directly onto a 1 g SCX column. The column was washed 3
X with 2 mL portions of methanol and the product were eluted from the column
using 6 mL of 2.0 M solution of ammonia/methanol. After concentration under
reduced pressure, the product were characterized by IEX MS and carried on
without further purification. The intermediates prepared by this method are
shown
in Table C-A.
TABLE C-A
Intermediates
Ex. Alkyl Halide Intermediate MS
1
C-A 3-Fluorobenzyl bromide3-(L-alaninyl)amino-5-phenyl-2,3-431.1
(Aldrich) dihydro- I -(3-fluorobenzyl)-1
H-
1,4-benzodiazepin-2-one
C-B Benzyl bromide 3-(L-alaninyl)amino-S-phenyl-2,3-513.2
(Aldrich) dihydro-1-(benzyl)-1
H-1,4-
benzodiazepin-2-one
C-C tert-Butylbenzyl bromide3-(L-alaninyl)amino-5-phenyl-2,3-469.2
(Aldrich) dihydro-1-(4-tert-butylbenzyl)-
1 H-1,4-benzodiazepin-2-one
C-D 2-Bromoethylcyclohexane3-(L-alaninyl)amino-5-phenyl-2,3-433.2
(Fairfield) dihydro-1-(2-cyclohexylethyl)-1
H-
1,4-benzodiazepin-2-one
C-E 1-Bromo-3,3-dimethylbutane3-(L-alaninyl)amino-S-phenyl-2,3-407.2
(Wiley) dihydro-1-(3,3-dimethylbutyl)-1
H-
I,4-benzodiaze in-2-one
C-F Methyl alpha- 3-(L-alaninyl)amino-5-phenyl-2,3-471.2
bromophenylacetate dihydro-1-(1-methoxycarbonyl-1-
(Aldrich) phenylmethyl)-1 H-1,4-
benzodiaze in-2-one
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 163 --
Ex. Alkyl Halide Intermediate MS
~
C-G I-Bromo-2-ethylbutane3-(L-alaninyl)amino-5-phenyl-2,3-407.2
(Aldrich) dihydro- I -(2-ethylbutyl
)-1 H-1,4-
benzodiazepin-2-one
C-H Bromomethylcyclohexane3-(L-alaninyl)amino-5-phenyl-2,3-419.2
(Aldrich) dihydro-I-(cyclohexylmethyl}-I
H-
1,4-benzodiazepin-2-one
C-I 2-(Bromoethyl)benzene3-(L-alaninyl)amino-5-phenyl-2,3-427.2
(Aldrich) dihydro-I -(2-phenylethyl)-1
H-1,4-
benzodiazepin-2-one
C-J 3-(Bromopropyl)benzene3-(L-alaninyl)amino-5-phenyl-2,3-441.2
(K and K Laboratories)dihydro-1-(3-phenylpropyl)-IH-
1,4-benzodiazepin-2-one
C-K N-(2-Bromoethyl)phthalimide3-(L-alaninyl)amino-5-phenyl-2,3-496.2
(Al drich) dihydro- I -(2-(N-
phthalimidyl)ethyl)- I
H-1,4-
benzodiazepin-2-one
C-L 2-Phenylbenzyl bromide3-(L-alaninyl)amino-5-phenyl-2,3-489.2
(Aldrich) dihydro-I-(2-biphenylmethyl)-1
H-
1,4-benzodiazepin-2-one
C-M Tetrahydrofurfuryl 3-(L-alaninyl)amino-5-phenyl-2,3-407.2
bromide
(Lancaster) dihydro- I -((2-
tetrahydrofuranyI)methyl}-1
H- I ,4-
benzodiazepin-2-one
C-N 2-Bromomethyl-1,4- 3-(L-alaninyl)amino-5-phenyl-2,3-471.2
benzodioxane dihydro- I -(2-( 1,4-
(Acros ) benzodioxanyl)methyl)-1H-1,4-
benzodiazepin-2-one
C-O 3-Bromomethyl-5- 3-(L-alaninyl)amino-S-phenyl-2,3-503.1
chlorobenzo[b]thiophenedihydro-I-((3-(5-chlorobenzo[b]
(Maybridge) thienyl))methyl}-1 H-1,4-
benzodiazepin-2-one
C-P I-Bromopinacolone 3-(L-alaninyl)amino-5-phenyl-2,3-421.1
(Lancaster) dihydro- I -(3,3-dimethyl-2-oxo-
propyl)-1 H-1,4-benzodiazepin-2-
one
C-Q 5-(Bromomethyl)benzo-3-(L-alaninyl)amino-5-phenyl-2,3-455.2
furazan dihydro-I-(5-
(Maybridge) benzofurazanyl methyl)-1
H- I ,4-
benzodiaze in-2-one
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/14007
__ 1(~4 --
Ex. Alkyl Halide Intermediate MS
~
C-R 3-Phenoxypropyl bromide3-(L-alaninyl)amino-5-phenyl-2,3-457.2
(Aldrich) dihydro-1-(3-phenoxypropyl)-1
H-
1,4-benzodiazepin-2-one
C-S 6-(Bromomethyl)-2- 3-(L-alaninyl)amino-5-phenyl-2,3-533.2
(trifluoromethyl)quinolinedihydro-1-(6-(2-
(Maybridge) trifluoromethylquinolinyl)methyl)-
1 H-1,4-benzodiazepin-2-one
C-T 1-Bromo-2-methylbutane3-(L-alaninyl)amino-5-phenyl-2,3-393.2
(Aldrich) dihydro-1-(2-methylbutyl)-1
H-
1,4-benzodiazepin-2-one
C-U Ethyl bromide 3-(L-alaninyl)amino-5-phenyl-2,3-351.2
(Aldrich) dihydro-1-(ethyl )-1 H-1,4-
benzodiazepin-2-one
C-V 3-Picolyl chloride 3-(L-alaninyl)amino-5-phenyl-2,3-414.1
hydrochloride dihydro-1-(3-pyridylmethyl)-1H-
(Aldrich) 1,4-benzodiazepin-2-one
C-W 1-(2-Chloroacetyl)indoline3-(L-alaninyl)amino-5-phenyl-2,3-482.2
(Maybridge) dihydro-1-(2-oxo-2-(N-
indolinyl)ethyl)-1 H-1,4-
benzodiazepin-2-one
C-Y 4-(Chloromethyl)-3,5-3-(L-alaninyl)amino-S-phenyl-2,3-432.2
dimethylisoxazole dihydro-1-((4-(3,5-
(Aldrich) dimethyl)isoxazolyl)methyl)-1
H-
1,4-benzodiazepin-2-one.
C-Z 2-Bromoethyl methyl 3-(L-alaninyl)amino-5-phenyl-2,3-381.2
ether
(Aldrich) dihydro-1-(2-methoxyethyl}-1
H-
1,4-benzodiazepin-2-one
Example C-AA
Synthesis of
(S)-3-(L-phenylglycinyl)amino
2,3-dihydro-1-methyl-5-phenyl-1H-1,4-benzodiazepin-2-one
Sten A: Synthesis of (S)-3-(N'-(tert-Butoxycarbonyl)-L-
phenylglycinyi)amino-2,3-dihydro-1-methyl-5-
phenyl-1H-1,4-benzodiazepin-2-one
To a solution of triethyl amine (519 uL, 3.8 mmol) and (S)-3-amino-5-
phenyl-2-oxo-1,4-benzodiazepine (1.0 g, 3.8 mmol) (prepared according to the
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/14007
-- 165 --
procedure of M. G. Bock et al., J. Org. Chem. 1987, 52, 3232-3239) in 100 mL
of
anhydrous methylene chloride at -20°C was added N-Boc-L-phenylglycine
fluoride (Carpino et al, J. Org. Chem. 1991, 56, 2611-2614) in one portion.
The
reaction mixture was stirred for 15 min. and quenched with saturated aqueous
bicarbonate ( 10 mL). The layers were seperated, the organic layer washed
sequentially with saturated aqueous bicarbonate, water and brine and then
dried
over sodium sulfate. Purification of the crude product using silica gel
chromatography (10-50% ethyl acetate / hexane) gave 1.3 g (69%) of a
hygroscopic white foam.
NMR data was as follows:
'H NMR (300 MHz, CDCI;): b = 1.35 (br s, 9H), 3.41 (s, 3H), 5.30-5.45
(m, 2H), 5.75-5.95 (m, 1H), 7.15-7.75 (m, 15H).
IR (CDC13): 1709.7, 1676.6, 1489, 1166.3 cm'.
IEX MS (M+1); 498Ø
Ste~B:, Synthesis of (S)-3-(L-phenylglycinyl)amino-2,3-
dihydro-1-methyl-5-phenyl-1H-1,4-
benzodiazepin-2-one
(S)-3-(N'-(tert-Butoxycarbonyl)-L-phenylglycinyl)amino-2,3-dihydro-1-
methyl-5-phenyl-1H-1,4-benzodiazepin-2-one (1.27 g, 2.55 mmol) was added to
50 mL of a stirring solution of I 5 % TFA in methylene chloride in one
portion.
After stirring 1 h, the reaction mixture was concentrated under reduced
pressure
and the residue dissolved in 100 mL of methylene chloride. This solution was
washed twice with saturated sodium bicarbonate, once with brine and then dried
over sodium sulfate. Purification of the crude product using silica gel column
chromatography (5-10% methanol/methylene chloride) gave 743 mg (73%) of a
very light green foam:
NMR data was as follows:
'H NMR (CDCl3): S = 2.05 (br s, 1 H), 3.45 (s, 3 H), 5.51 (d, J = 8.39 Hz,
1 H), 7.15-7.70 (m, 14 H), 8.60 (d, J = 830 Hz, 1 H).
IR (CDC13): 1673.3, 1601.1, 1506.1 cm'.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
1(~ __
IEX MS (M+1 ): 399.2.
Example C-AB
Synthesis of
3-(L-Alaninyl)amino-2,3-dihydro-1
(2-oxo-2-phenylethyl)-5-phenyl-1H-1,4-benzodiazepin-2-one
Step Synthesis of 3-(Benzoxycarbonyl)amino-2,3-dihydro-1-
(2-oxo-2-phenylethyl)-5-phenyl-1H-1,4-benzodiazepin-2-
one
To a solution of 3-(Benzoxycarbonyl)amino-2,3-dihydro-5-phenyl-1 H-1,4-
benzodiazepin-2-one (Rock, M. G. et al, Tetrahedron Lett. 1987, 28, 939; 4.0
g,
10.4 mmol} in 40 mL of anhydrous DMF at 0°C was added potassium tert-
butoxide ( 1.5 l g, 13.5 mmol) in one portion. The reaction mixture was
stirred 20
min. and a-bromoacetophenone (Lancaster; Windham, NH; 2.9 g, 14.6 mmol)
was added. The reaction mixture was warmed to roam temperature over 30 min.
and then diluted with 100 mL of water and 200 mL of methylene chloride. The
layers were separated. The organic layer was extracted with water and dried
over
sodium sulfate. Purification of the crude product by silica gel column
chromatography (0-S% ethyl acetate/methylene chloride) gave 4.2 g (81%) of an
off white foam.
NMR data was as follows:
'H NMR (300 MHz, CDC13): b = S.I6 (s, 2 H), 5.34 (s, 2 H), 5.50 (d, J =
8.33 Hz, 1 H), 6.70 (d, J = 8.28 Hz, 1 H), 7.20-7.70 (m, 12 H), 7.91 (d, J =
7.54 Hz,
2 H).
IR (CHCI;): 1706.04, 1685.3, 1505.9, 1489.1, 1450.3, 1244.7 cm '.
IEX MS (M+1 ): 504.3.
Ste~B: Synthesis of 3-Amino-2,3-dihydro-1-{2-oxo-2-
phenylethyl)-5-phenyl-1H-1,4-benzodiazepin-2-one
A solution of 3-(Benzoxycarbonyl)amino-2,3-dihydro-1-(2-oxo-2-
phenylethyl)-5-phenyl-1H-1,4-benzodiazepin-2-one (3.7 g, 7.36 mmol) in 100 mL
of anhydrous methylene chloride was cooled to 0°C under nitrogen. A
stream of
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/14007
-- 167 --
anhydrous HBr gas was then bubbled through this solution for 1 h. The bubbler
was removed and the reaction was warmed to room temperature under nitrogen.
After stirnng 1 h the reaction was concentrated under vacuum and the residue
was
redissolved in 20 mL of methylene chloride. The crude HBr salt of the product
was precipitated from solution using 300 mL of anhydrous ether and collected
by
filtration as a light yellow solid. After washing with ether , the solid was
dissolved
in methylene chloride and saturated sodium bicarbonate. The layers were
separated and the organic layer was extracted with saturated sodium
bicarbonate.
The combined aqueous layers were then back extracted twice with methylene
chloride. The combined organic layers were extracted once with water and dried
over sodium sulfate. After concentration under vacuum, 2.27 g of the product
was
obtained as an orange foam which was carried on without further purification.
NMR data was as follows:
'H NMR (300 MHz, CDCl3): 8 = 2.60 (br s, 2 H), 4.72 (s, 1 H), 5.34 (s, 2
H), 7.10-7.70 (m, 12 H), 7.91 (d, J = 7.60 Hz, 2 H).
IEX MS (M+1): 370.2
Sten C: Synthesis of 3-(N'-(tert-Butoxycarbonyl)-L-
alaninyl}amino-2,3-dihydro-1-(2-oxo-2-phenylethyl)-5-
phenyl-1H-1,4-benzodiazepin-2-one
To a solution of HOBt-H,O (697 mg, 5.16 mmol), N,N-
diisopropylethylamine (900 uL, 5.16 mmol) and N-t-BOC-L-alanine (975 mg, 5.16
mmol) in 20 mL of anhydrous THF at 0°C was added 1-(3-
dimethylaminopropyl)-
3-ethyl carbodiimide hydrochloride (EDCI; 986 mg, 5.16 mmol) in one portion.
After stirring 5 min., a solution of 3-amino-2,3-dihydro-1-(2-oxo-2-
phenylethyl)-S-
phenyl-1H-i,4-benzodiazepin-2-one (2.0 g, 5.43 mmol) in 20 mL of anhydrous
THF was added via syringe and the reaction mixture was warmed to room
temperature and stirred overnight. The reaction mixture was diluted with 200
mL
methylene chloride, extracted sequentially with 10 % citric acid, saturated
sodium
bicarbonate, water and brine and then dried over sodium sulfate. Purification
of
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007 _ .
-- 168 --
the crude product using silica gel chromatography (10%-30% ethyl
acetate/methylene chloride) gave 2.59 g (93%) of a white foam.
NMR data was as follows:
'H NMR (300 MHz, CDC13): 8 = 1.30-1.60 (m, 12 H), 4.35 (br s, 1 H),
5.00-5.50 (m, 3 H), 5.65-5.70 (m, 1 H), 7.15-7.65 (m, 12 H), 7.70-7.80 (m, 1
H),
7.85-7.95 (m, 1 H).
IR (CHC13): 1705.8, 1678.8, 1488.7, 1450.2, 1230.4, 1164.4 cni'.
IEX MS (M+1 ): 541.2.
Stgp D: Synthesis of 3-(L-Alaninyl)amino-2,3-dihydro-1-(2-oxo-
2-phenylethyl)-5-phenyl-1H-1,4-benzodiazepin-2-one
3-(N'-(tert-Butoxycarbonyl)-L-alaninyl)amino-2,3-dihydro-1-(2-oxo-2-
phenylethyl)-5-phenyl-1H-1,4-benzodiazepin-2-one (2.5 g, 4.63 mmol) was added
to 100 mL of a stirring solution of 15 % TFA/methylene chloride in one
portion.
After stirring 2 h, the reaction mixture was concentrated under reduced
pressure
and the residue was dissolved in 150 mL of methylene chloride. This solution
was
washed twice with saturated sodium bicarbonate, once with brine and then dried
over sodium sulfate. Purification of the crude product using silica gel column
chromatography (1-10% methanol/methylene chloride) gave 1.91 g (94%) of the
title compound as a white foam.
NMR data was as follows:
'H NMR (300 MHz, CDC1;): 8 = 1.30-1.50 (m, 3 H), 1.80-2.20 (br s, 2 H},
3.55-3.75 (m, 1 H), 5.20-5.45 (m, 2 H), 5.67 (t, J = 7.48 Hz, 1 H), 7.20-7.65
(m, 12
H), 7.90 (d, J = 7.7 Hz, 2 H), 8.80 (dd, J, = 25.09 Hz, J, = 8.33 Hz, 1 H).
EX MS (M+1 ): 441.2.
Example C-AC
Synthesis of
3-(L-Alaninyl)amino-2,3-dihydro-1
(4,4,4-trifluorobutyl)-5-phenyl-1H-1,4-benzodiazepin-2-one
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 169 --
Sten A: Synthesis of 3-(Benzoxycarbonyl)amino-2,3-dihydro-1-
(4,4,4-trifluorobutyl)-5-phenyl-1 H-1,4-benzodiazepin-2-
one
To a solution of 3-(benzoxycarbonyl)amino-2,3-dihydro-S-phenyl-1H-1,4-
benzodiazepin-2-one (3.7 g, 9.61 mmol) in 40 mL of anhydrous DMF at 0°C
was
added potassium tert-butoxide (1.6 g, 14.4 mmol) in one portion. The reaction
mixture was stirred 20 min. and 4,4,4-trifluoro-1-bromobutane (Lancaster;
Windham, NH; 2.6 g, 13.4 mmol) was added. The reaction mixture was warmed
to room temperature over 30 min. and then diluted with 100 mL of water and 200
mL of methyiene chloride. The layers were separated. The organic layer was
extracted with water and dried over sodium sulfate. Purification of the crude
product by silica gel column chromatography (0-3 % ethyl acetate / methylene
chloride) gave 1.52 g (32 %) of an off white foam.
NMR data was as follows:
'H NMR (300 MHz, CDC13): 8 = 1.50-2.10 (m, 4 H), 3.70-3.90 (m, 1 H),
4.35-4.55 (m, 1 H), 5.15 (s, 2 H), 5.33 (d, J = 8.47 Hz, 1 H), 6.67 (d, J =
8.40 Hz, I
H), 7.2-7.70 (m, 14 H).
IR (CHCI3): 1720.4, 1683.0, 1604.8, 1505.5, 1451.1, 1323.9, 1254.5,
1148.4 cm'.
IEX MS (M+1 ): 496.3.
SteRB: Synthesis of 3-Amino-2,3-dihydro-1-(4,4,4-
trifluorobutyl)-5-phenyl-1H-1,4-benzodiazepin-2-one
A solution of 3-(benzoxycarbonyl)amino-2,3-dihydro-1-(4,4,4-
trifluorobutyl)-S-phenyl-1H-1,4-benzodiazepin-2-one (1.42 g, 2.87 mmol) in 50
mL of anhydrous methylene chloride was cooled to 0°C under nitrogen. A
stream
of anhydrous HBr gas was slowly bubbled through the solution for 1 h. The
bubbler was removed and the reaction was warmed to room temperature under
nitrogen. After stirring for 1 h, the reaction was concentrated under vacuum
and
the residue was redissolved in 10 mL of methylene chloride. The crude HBr salt
of
the product was precipitated from solution using 90 mL of anhydrous ether and
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/I4007 _.
-- 170 --
collected by filtration. After washing with ether, the HBr salt was dissolved
in
methylene chloride and saturated sodium bicarbonate. The layers were separated
and the organic layer was extracted with saturated sodium bicarbonate. The
combined aqueous layers were then back extracted twice with methylene
chloride.
The combined organic layers were extracted once with water and dried over
sodium sulfate. After concentration under vacuum, 1.06 g ( 100%) of the
product
was obtained as a white foam which was carried on without further
purification.
NMR data was as follows:
'H NMR (300 MHz, CDCl3): b = 1.60-2.10 (m, 4 H), 2.76 (br s, 2 H), 3.75-
3.85 (m. 1 H), 4.40-4.60 (m, 2 H), 7.20-7.70 (m, 9 H).
IEX MS (M+1): 362.1.
Sten C:C: Synthesis of 3-(N'-(tent-Butoxycarbonyl)-L-
alaninyl)amino-2,3-dihydro-1-(4,4,4-trifluorobutyl)-5-
phenyl-1 H-1,4-benzodiazepin-2-one
To a solution of HOBt-H,O (373 mg, 2.76 mmol), N,N-
diisopropylethylamine (481 uL, 2.76 mmol) and N-t-BOC-L-alanine (522 mg, 2.76
mmol) in 10 mL of anhydrous THF at 0°C was added 1-(3-
dimethylaminopropyl)-
3-ethyl carbodiimide hydrochloride (EDCI; 527 mg, 2.76 mmol) in one portion.
After stirring ~ min., a solution of 3-amino-2,3-dihydro-1-(4,4,4-
trifluorobutyl)-5-
phenyl-1 H-1,4-benzodiazepin-2-one ( 1.05 g, 2.91 mmol) in 10 mL of anhydrous
THF was added via syringe and the reaction mixture was warmed to room
temperature and stirred overnight. The reaction mixture was diluted with 100
mL
methylene chloride, extracted sequentially with 10% citric acid, saturated
sodium
bicarbonate, water and brine and then dried over sodium sulfate. Purification
of
the crude product using silica gel chromatography ( 10%-30% ethyl
acetate/methylene chloride) gave 1.28 g (83%) of a white foam.
NMR data was as follows:
'H NMR (300 MHz, CDC13): 8 = 1.40-2.10 (m, 16 H), 3.70-3.85 (m, 1 H),
4.30-4.55 (m, 2 H), 5.10 (br s, 1 H), 5.45-5.55 (m, 1 H), 7.25-7.80 (m, 10 H).
CA 02325388 2000-09-21
WO 99167220 PCT/US99/14007 _ .
-- 171 --
IR (CDC13): 1676.6, 1605.2, 1488.6, 1450.9, 1393.2, 1338.7, 1324.9,
1253.8, 1150.4 cm''.
IEX MS (M+1): 533.1.
Step D: Synthesis of 3-(L-Alaniny!)amino-2,3-dihydro-1-(4,4,4-
trifluorobutyl)-5-phenyl-1H-1,4-benzodiazepin-2-one
3-(N'-(tert-Butoxycarbonyl)-L-alaniny!)amino-2,3-dihydro-1-(4,4,4-
trifluorobutyl)-5-phenyl-1H-1,4-benzodiazepin-2-one (I.21 g, 2.27 mmol) was
added to 50 mL of a stirring solution of 15 % TFA / methyIene chloride in one
portion. After stirnng 2 h, the reaction mixture was concentrated under
reduced
pressure and the residue was dissolved in 100 mL of methylene chloride. This
solution was washed twice with saturated sodium bicarbonate, once with brine
and
then dried over sodium sulfate. Purification of the crude product using silica
gel
column chromatography (1-5% methanol / methylene chloride) gave 670 mg
(68%) of a light pink foam.
NMR data was as follows:
'H NMR (300 MHz, CDC13): b = 1.43 (t, J = 7.0 Hz, 3 H), 1.60-2.20 (m, 7
H), 3.60-3.85 (m, 2 H), 4.35-4.55 (m, 1 H), 5.51 (dd, J, = 8.36 Hz, J, = 2.48
Hz, 1
H), 7.20-7.70 (m, 9 H), 8.80 (dd, J, = 27.73 Hz, 3, = 8.34 Hz, 1 H).
IEX MS (M+I ): 433.2.
Example C-AE
Synthesis of
3-[(L-Alaniny!)amino]-2,3-dihydro-1-methyl
5-(2-pyridyl)-1 H-1,4-benzodiazepin-2-one
Step A: Synthesis of 3-Amino-2,3-dihydro-I-methyl-5-(2-
pyridyl)-1H-1,4-benzodiazepin-2-one
The title compound was synthesized as described in Synth. Commun.,
26(4), 721-727 (1996).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 172 --
Step B: Synthesis of 3-[(N-tert-Butoxycarbonyl-L-
alaninyl)amino]-2,3-dihydro-1-methyl-5-(2-pyridyl)-1H-
1,4-benzodiazepin-2-one
A solution of L-Boc-alanine ( 1.74 g, 9.20 mmol), HOBt monohydrate ( 1.24
g, 9.20 mmol}, diisopropylethylamime ( 1.6 mL, 9.20 mmol) and CHZC12 (30 mL)
was purged with nitrogen and cooled in an ice bath. To the cold solution was
added 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (1.76 g, 9.20
mmol) followed by a solution of 3-amino-2,3-dihydro-1-methyl-5-(2-pyridyl)-1H-
1,4-benzodiazepin-2-one (2.45 g, 9.20 mmol) dissolved in CH,CI, (15 mL). The
cold bath was removed and the solution stirred overnight at room temperature.
The
reaction mixture was extracted with HBO, 0.1 N aq. citric acid, S% aq. NaHCO;,
and brine. The remaining CH,CI, solution was dried (MgS04) and concentrated to
a tan foam. The title compound was crystallized from CH,CI,/EtOAc to give 3.47
g (86% yield) of white crystals, mp. 228-229°C.
Anal. Calcd for C~3H,,NSO~: C, 63.14; H, 6.22; N, 16.01. Found: C,
63.25; H, 6.15; N, 15.95. MS (FD') 437 m/z.
Step C: Synthesis of 3-[(L-Alaninyl)amino]-2,3-dihydro-1-
methyl-5-(2-pyridyl)-1 H-1,4-benzodiazepin-2-one
A solution of 3-[(N-tert-butoxycarbonyl-L-alaninyl)amino]-2,3-dihydro-1-
methyl-S-(2-pyridyl)-1H-1,4-benzodiazepin-2-one (3.42 g, 7.82 mmol) in CH,C1,
(90 mL) was cooled in an ice bath and treated with TFA (13.2 mL, 172 mmol).
The cold bath was removed and the solution stirred at room temperature for
four
hours. The reaction mixture was washed with 1 M aq. K,C03 and the aqueous
back-extracted with CH,CI,. The combined extracts were washed with H,O, dried
(MgSO~) and concentrated to obtain 1.75 g (66% yield) of the title compound as
an
off white foam. MS (IS') 338 (m/e).
'HNMR (CDC13): b = 8.76-8.86 (1H, m), 8.63 (1H, m}, 8.17 (1H, m), 7.82
(2H, m), 7.60 ( 1 H, m), 7.41 (3H, m), 5.60 ( 1 H, m), 3.63 ( 1 H, m), 3.49
(3H, s), 1.66
(2H, broad), 1.45 (3H, m}.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 173 --
Example C-AF
Synthesis of
3-[(L-Alaninyl)amino]-2,3-dihydro-1-(2-N,N-diethylaminoethyl)
5-(2-pyridyl)-1H-1,4-benzodiazepin-2-one
Step Synthesis of 3-Amino-2,3-dihydro-1-(2-N,N-
diethylaminoethyl)-5-(2-pyridyl)-1H-1,4-benzodiazepin-
2-one
The title compound was synthesized as described in Synth. Commun.,
26(4), 721-727 (1996).
Synthesis of 3-[(N-tent-Butoxycarbonyl-L-
alaninyl)amino]-2,3-dihydro-1-(2-N,N-
diethylaminoethyl)-5-(2-pyridyl)-1H-1,4-benzodiazepin-
2-one
A solution of L-Boc-alanine (1.80 g, 9.50 mmol), HOBt monohydrate (1.28
g, 9.50 mmol), diisopropylethylamime (1.65 mL, 9.50 mmol) and CH,Ch (40 mL)
was purged with nitrogen and cooled in an ice bath. To the cold solution was
added 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (1.82 g, 9.50
mmol) followed by a solution of 3-amino-2,3-dihydro-1-(2-N,N-
diethylaminoethyl)-5-(2-pyridyl)-1H-1,4-benzodiazepin-2-one (3.34 g, 9.50
mmol)
dissolved in CH,Ch (25 mL). The cold bath was removed and the solution stirred
overnight at room temperature. The reaction mixture was extracted with H,O, 5%
aq. NaHC03, and brine. The remaining CH,CI, solution was dried (MgS04) and
concentrated to a tan foam. The title compound was isolated via column
chromatography (2% MeOH/CH,CI, to 10% MeOH/CH,C1,) to give 3.53 g (71%
yield) of yellow foam.
MS (FD') 522 (m/z).
' HNMR (CDC13): b = 8.62 ( 1 H, d), 8.11 ( 1 H, m), 7.80 (2H, m), 7.59 (2H,
m), 7.32-7.45 (2H, m), 5.54 ( I H, m), 5.02-5.18 ( 1 H, m), 4.3 8 ( 1 H, m),
4.20 ( 1 H,
m), 3.83 (1H, m), 2.62 (2H, t), 2.44 (4H, m), 1.40-1.56 (12H, m), 0.88 (6H,
m).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 174 -
Sten C: Synthesis of 3-[(L-Alaninyl)amino]-2,3-dihydro-1-(2-
N,N-diethylaminoethyl)- 5-(2-pyridyl~lH-1,4-
benzodiazepin-2-one
The title compound was synthesized using the procedure described in
Example C-AE, Step C. A solution of 3-[(N-tent-butoxycarbonyl-L-
alaninyl)amino]-2,3-dihydro-1-(2-N,N-diethylaminoethyl)-S-(2-pyridyl)-1 H-1,4-
benzodiazepin-2-one (3.52 g, 6.73 mmol) was treated with TFA ( 11.4 mL, 148
mmol) to give 2.61 g (92% yield) the title compound as a light yellow foam.
MS (IS+) 423 (m/e).
' HNMR (CDCI3): b = 8.78-8.93 ( 1 H, m), 8.62 ( 1 H,d); 8.11 ( 1 H, m), 7.80
(2H, m), 7.5 8 (2H, m), 7.39 (2H, m), 5.5 8 ( 1 H, m), 4.22 ( 1 H, m), 3.88 (
1 H, m),
3.61 ( 1 H, m), 2.67 (2H, t), 2.49 (4H, m), 1.73 (2H, broad), 1.42 (3H; m),
0.91 (6H,
m).
Example C-AG
Synthesis of
3-[(L-Alaninyl)amino]-2,3-dihydro-1-(3,3-dimethyl-2-oxobutyl)
5-(2-pyridyl)-1 H-1,4-benzodiazepin-2-one
Sten A: Synthesis of 3-Amino-2,3-dihydro-1-(3,3-dimethyl-2-
oxobutyl)-5-(2-pyridyl)-1H-1,4-benzodiazepin-2-one
The title compound was synthesized as described in Synth. Commun. ,
26(4), 721-727 (1996).
Step Synthesis of 3-[(N-tert-Butoxycarbonyl-L-
alaninyl)amino]-2,3-dihydro-1-(3,3-dimethyl-2-
oxobutyl)- 5-(2-pyridyl)-1H-1,4-benzodiazepin-2-one
A solution of L-Boc-alanine (1.57 g, 8.33 mmol), HOBt monohydrate (1.13
g, 8.33 mmol), diisopropylethylamime (1.45 mL, 8.33 mmol) and CHZCI, (40 mL)
was purged with nitrogen and cooled in an ice bath. To the cold solution was
added I-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (1.60 g, 8.33
mmol) followed by a solution of 3-amino-2,3-dihydro-1-(3,3-dimethyl-2-
oxobutyl)-5-(2-pyridyl)-IH-1,4-benzodiazepin-2-one (2.92 g, 8.33 mmol)
dissolved in CH,CI, (25 mL). The cold bath was removed and the solution
stirred
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 175 --
overnight at room temperature. The reaction mixture was extracted with H,O,
0.1
N aq. citric acid, S% aq. NaHC03, and brine. The remaining CHZC12 solution was
dried (MgS04) and concentrated to a yellow foam. The title compound was
isolated via column chromatography (20% EtOAc/hexanes to 60%
EtOAc/hexanes) to give 4.19 g (96% yield} of light yellow foam.
MS (FD+) 521 (m/z).
' HNMR (CDC13): S = 8.65 ( 1 H, t), 8.17 ( 1 H, t), 7.90 ( 1 H, t), 7.71-7.85
( 1 H,
m), 7.54 ( 1 H, m), 7.44 ( 1 H, t), 7.3 7 ( 1 H, d), 7.24-7.32 ( 1 H, m), 7.14
( 1 H, m), 5.67
( 1 H, dd), S .18 ( 1 H, broad), 4.93-5.07 ( 1 H, m), 4.50-4.64 ( 1 H, m), 4.3
8 ( 1 H,
broad), 1.42-1.51 (12H, m), 1.26 (9H, d).
St~C: Synthesis of 3-[(L-Alaninyl)amino]-2,3-dihydro-1-(3,3-
dimethyl-2-oxobutyl)-5-(2-pyridyl)-1 H-1,4-
benzodiazepin-2-one
The title compound was synthesized using the procedure described in
Example C-AE, Step C. A solution of 3-[(N-tert-butoxycarbonyl-L-
alaninyl)amino]-2,3-dihydro-1-(3,3-dimethyl-2-oxobutyl)-S-(2-pyridyl)-1 H-1,4-
benzodiazepin-2-one (4.18 g, 8.01 mmol) was treated with TFA ( 13.6 mL, 176
mmol) to give 3.14 g (93% yield) the title compound as an off white foam.
MS (IS') 422 (m/e).
' HNMR (CDCl3) b 8.85-8.99 ( 1 H, m), 8.68 ( 1 H, d), 8.20 ( 1 H, t), 7.87 ( 1
H,
t), 7. 5 8 ( 1 H, t), 7.42 ( 2 H, m), 7.30 ( 1 H, t), 7.17 ( 1 H, d), 5.72 ( 1
H, m), 5.08 ( 1 H,
d), 4.60 ( 1 H, d), 3.66 ( 1 H, m), 1.47 (3 H, m), 1.28 (9H, m).
Example C-AH
Synthesis of
3-[(L-Alaninyl)amino]-2,3-dihydro-1-methyl
5-(2-thiazyl)-1H-1,4-benzodiazepin-2-one
Step A: Synthesis of 3-Amino-2,3-dihydro-1-methyl-5-(2-thiazyl)-
1H-1,4-benzodiazepin-2-one
The title compound was synthesized in a manner similar to the procedure
described in Synth. Commun., 26(4), 721-727 ( 1996), starting with 2-(2-
CA 02325388 2000-09-21
WO 99/67220 PGT/ITS99/14007
-- 176 --
aminobenzoyl)thiazole (prepared as described in Tetrahedron, 51(3), 773-786,
(1995)).
MS (IS+) 273 (m/e).
'HNMR (CDC13): 8 = 7.83-7.94 (2H, m), 7.61 (1H, t), 7.50 (1H, d), 7.34
(2H, m), 4.60 ( 1 H, s), 3.46 (3H, s), 1.97 (2H, broad).
Step B: Synthesis of 3-[(N-tert-Butoxycarbonyl-L-
alaninyl)amino]-2,3-dihydro-1-methyl-5-(2-thiazyl}-1H-
1,4-benzodiazepin-2-one
A solution of L-Boc-alanine ( 1.85 g, 9.77 mmol), HOBt monohydrate ( 1.32
g, 9.77 mmol), diisopropylethylamime (1.70 mL, 9.77 mmol) and CH~CIz (30 mL)
was purged with nitrogen and cooled in an ice bath. To the cold solution was
added 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride ( 1.87 g,
9.77
mmol) followed by a solution of 3-amino-2,3-dihydro-1-methyl-5-(2-thiazyl)-1H-
1,4-benzodiazepin-2-one (2.66 g, 9.77 mmol) dissolved in CH,Ch (20 mL). The
cold bath was removed and the solution stirred overnight at room temperature.
The
reaction mixture was extracted with H,O, 0.1 N aq. citric acid, 5% aq. NaHC03,
and brine. The remaining CH,CI, solution was dried (MgS04) and concentrated to
a light yellow foam. The title compound was crystallized from EtOAc/hexane to
give 3.22 g (74% yield) of white crystals, mp. 196-197°C. Anal. Calcd
for
CZ,H,SNSO~S: C, 56.87; H, 5.68; N, 15.79. Found: C, 56.74; H, 5.75; N, 15.55.
MS (IS') 444 m/e.
Ste~C: Synthesis of 3-[(L-Alaninyl)amino]-2,3-dihydro-1-
methyl-5-(2-thiazyl)-1H-1,4-benzodiazepin-2-one
The title compound was synthesized using the procedure described in
Example C-AE, Step C.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007 _..
__ 177 -_
Example C-AI
Synthesis of
3-((L-Alaninyl)amino]-2,3-dihydro-1-methyl-
5-(thiophen-2-yl)-1 H-1,4-benzodiazepin-2-one
Step Synthesis of 3-Amino-2,3-dihydro-1-methyl-5-(2-
thiophen-2-y1)-1H-1,4-benzodiazepin-2-one
The title compound was synthesized in a manner similar to the procedure
described in Synth. Commun., 26(4), 721-727 (1996), starting with 2-(2-
aminobenzoyl)thiophene (prepared as described in Collect. Czech. Chem.
Commun., 34(2), 468-478, (1969)).
MS (IS') 272 (m/e).
' HNMR (CDCl3): a = 7.68 ( 1 H, d), 7.60 ( 1 H, t), 7.48 ( 1 H, m}, 7.3 5 (2H,
d),
7.28 ( 1 H, m), 7.15 ( 1 H, d), 7.05 ( 1 H, d), 4.50 ( I H, broad), 3.45 (3H,
s), 2.26 (2H,
broad).
Ste~B: Synthesis of 3-[(N-tert-Butoxycarbonyl-L-
alaninyl)amino]-2,3-dihydro-1-methyl-5-(thiophen-2-yl)-
1H-1,4-benzodiazepin-2-one
The title compound was synthesized in a manner similar to the procedure
described in Example C-AH, Step B.
MS (IS') 443 (m/e).
'HNMR (CDC13): 8 = 7.69 (IH, d), 7.61 (2H, m), 7.48 (1H, d}, 7.27-7.42
(2H, m), 7.18 ( 1 H, m}, 7.05 ( 1 H, m), 5.5 I ( 1 H, d), 5.13 ( 1 H, broad),
4.36 ( I H,
broad), 3.44 (3H, s), 1.38-1.57 (12H, m).
Ste~C: Synthesis of 3-[(L-Alaninyl)amino]-2,3-dihydro-1-
methyl-5-(thiophen-2-yl)-1H-1,4-benzodiazepin-2-one
The title compound was synthesized in a manner similar to the procedure
described in Example C-AE, Step C.
MS (IS') 343 (m/e).
'HNMR (CDCl3): 8 = 8.55 (1H, d), 7.68 (1H, d), 7.59 (IH, m), 7.48 (1H,
d), 7.3 6 ( 1 H, d), 7.3 I ( 1 H, d), 7.16 ( 1 H, m), 7.04 ( 1 H, t), 5 . 54 (
1 H, d), 3 . 5 8 ( 1 H,
m), 3.45 (3H, s), 1.41 (3H, d).
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 178 -_
Example 1
Synthesis of
5-[N'-(2S-hydroxy-3-methylbutyryl)-2S-aminopropyl]amino
7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
Step A - Synthesis of N Boc-L-Alaninal
Methylene chloride (50 mL) was added to a flask equipped with an addition
funnel and cooled to -78°C before adding 1.6 mL oxalyl chloride (18.3
mmol, 2.4
eq). This was stirred for 5 minutes, at which time 1.8 mL dimethyl sulfoxide
(25.4
mmol, 3.3 eq) dissolved in 20 mL CH,C12 was added over ~ minutes. This was
stirred for an additional S minutes before adding 1.4 g N-Boc-L-alaninol (7.8
mmol, 1.0 eq) (Aldrich) in 20 mL CHZCI,. The reaction mixture was stirred for
S
minutes before 6.5 mL triethylamine (46.6 mmol, 6.0 eq) was added and the
reaction warmed to room temperature for 10 minutes. The reaction was quenched
with 200 mL 0.1 N aqueous HCI, and this was extracted with 2 x 100 mL CHZCI,.
The combined organic layers were washed with 100 mL saturated NaHC03
followed by 100 mL saturated NaCI, then dried over Na,SO~ and removed in
vacuo. The remaining oil was passed through a silica gel plug with ethyl
acetate
(EtOAc). The solvent was removed in vacuo to yield 1.14 g (85%) of the desired
product.
NMR data was as follows:
'H NMR (300 MHz, CDCl3): b = 9.56 (s, 1H, CHO), 5.06 (bs, 1H, NH),
4.24 (bq, 1H, CH), 1.45 (s, 9H.. C(CH3)3), 1.33 (d, 3H, J= 7.2 Hz, CHCH3).
M+ (ionspray) Calcd for CgH,s N03 173.2, Obs. 174.2 (M + 1 H).
Step B - ~~nthesis of 5-fN'-pert-butvloxvc rbnnvll-2S-
To a solution of 620 mg of 5-amino-7-methyl-5,7-dihydro-6H-
dibenz[b,d]azepin-6-one (2.6 mmol, 1.0 eq) (from Example 7-A) in 50 mL
methanol was added 653 mg of N Boc-L-alaninal (3.8 mmol, 1.4 eq) (from Step
A}, followed by 74 mg of sodium cyanoborohydride (1.2 mmol, 0.5 eq). This was
stirred at room temperature for 1 hour after which the solvent was removed.
The
CA 02325388 2000-09-21
WO 99/67220 PC"T/US99/14007
- I79 --
resulting solid was dissolved in 50 mL methyIene chloride and washed with 50
mL
aqueous saturated NaHC03. The aqueous layer was re-extracted with 50 mL
methylene chloride. The combined organic solvents were dried over Na2S04 and
removed in vacuo. The crude product was purified by flash chromatography ( 1:
I
hexanes:ethyl acetate) to yield 309 mg (30%) of the desired product.
NMR data was as follows:
'H NMR (300 MHz, CDCI3): b = 7.64-7.31 (m, 8H, aromatic Hs), 4.04 (bs,
1H, CHN), 3.73 (bs, 1H, CHCH3), 3.34 (s, 3H, NCH3), 2.78 (dd, 1H, J= 4.9, 12.0
Hz, CHHN), 2.48 (dd, 1 H, J= 6.8, 12.0 Hz, CHHN), 1.43 (s, 9H, CCH3), 1.17 (d,
3H, J= 6.8 Hz, CHCH3).
M+ (ionspray) Calcd for C23Hz9N3O3 395.5, Obs. 396.1 (M + 1H}.
Step C - Synthesis of 5-f S-amino rn oov],Lamino-7-meth. 1
dihydro-6H-dibenz(b.d]~enin-6-one
HCl (g) was bubbled through a solution of 317 mg of 5-[N'-(tert-
butyloxycarbonyl)-2S-aminopropyl]amino-7-methyl-5,7-dihydro-6H-
dibenz[b,d]azepin-6-one (0.8 mmol) (from Step B) in 30 mL dioxane for 15
minutes. This was stirred at room temperature for 17 hours at which time the
solvent was removed in vacuo to yield 248 mg (94 %) of a white solid. This was
used without further purification.
NMR data was as follows:
'H NMR (300 MHz, CD30D) 7.73-7.45 (m, 8H, aromatic Hs), 3.76 (m, 1H,
CHCH,), 3.38 (m, SH, NCH3 and CH,N), 1.46 (d, 3H, J= 6.4 Hz, CHCH3).
M+ (ionspray) Calcd for C,BHZ,N,O 295.4, Obs. 296.4 (M + 1H).
Step D - Synthesis of 5-fN'
To a solution of 240 mg 5-(2S-aminopropyl)-amino-7-methyl-5,7-dihydro-
6H-dibenz[b,d]azepin-6-one (0.8 mmol, 1.0 eq} (from Step C) in 25 mL 4:1
tetrahydrofuran (THF): dimethylformamide (DMF) was added 125 mg of 2S-2-
hydroxy-3-methylbutyric acid ( 1.1 mmol, 1.3 eq), 184 mg of N-ethyl-N'-(3-
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 180 --
dimethylaminopropyl)-carbodiimide (1.0 mmol, 1.2 eq), 139 mg 1-
hydroxybenzotriazole hydrate (0.9 mmol, 1.1 eq), and 0.5 mL of
diisopropylethylamine (2.9 mmol, 3.5 eq). The reaction mixture was stirred at
room temperature for 20 hours. The solvent was removed and the remaining oil
diluted with 30 mL CH,CIz and washed with 25 mL 0.1 N aqueous HCI. The
aqueous layer was back extracted with 25 mL CHZCIz. The combined orgainc
layers were dried over NazS04 and removed in vacuo. The crude product was
purified by flash column chromatography (ethyl acetate) to yield 38 mg (12 %)
of
the desired product as a white solid.
NMR data was as follows:
'H NMR (300 MHz, CDCl3): b = 7.62-7.37 (m, 8H, aromatic Hs), 6.67 (d,
1 H, J = 9.0 Hz, NHCO) 4.61 (m, 1 H, CHCH3), 4.40 (bs, 1 H, CHNH), 4.13 (d, 1
H,
J= 3.8 Hz, CHOH), 3.38 (s, 3H, NCH3), 2.95 (m, 2H, CH,NH), 2.10 (m, 1H,
CH(CH3)z), 1.26 (d, 3H, J= 6.4 Hz, CHCH3), 1.06 (d, 3H, J= 6.8 Hz, CH(CH3)z),
0.93 (d, 3H, J= 6.8 Hz, CH(CH3)z).
M' (ionspray) Calcd for Cz,H,9N303 395.5, Obs. 396.1 (M + 1 H).
Anal. Calcd for C,3H,9N3O3 C, 69.85; H, 7.39; N, 10.62. Found: C, 69.63;
H, 7.44; N, 10.32.
Additionally, using the procedures described herein as well as art
recognized starting materials, reagents and procedures, compounds of formula I
can be prepared wherein the portion of the compound having the formula -NH-W
is selected from the following structures (where, when undefined, X, Y and Z
represent aryl ring substitutes):
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/14007 _
-- 181 --
rr'
1Y~/IV'R ' IV If N~R ;'N
0
H O (-I O H O
R= H, Et
R'
1
X \ ~ F '/,
I
~~ N N.R ~~ N N.R
H O H O
X=SO~, R'=H
X=O, R'=F
F
~~N~N~ ~R
H O H O
R
N/ \ l N~ \ l
N N ~ .'~--_... ~N~R
O R
O
R' = Me, isopropyl, cyclohexyl
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- I82 --
HN I ~ HN
HN I \ HN
N~ N~ N
O O O N
O ~1
HN ~ ~ HN ~ ~ HN
~ HN
O N O N_ ~ O N~~ N
O i
,1
HN I ~ HN I ~ HN I ~ HN
N'~ N
O O O N O N
~ 1
HN ( ~ HN I ~ HN ~ ~ HN
O N'~% O N~ O N~
O
HN I ~ HN I ~ HN I ~ HN
O N~ O N~ O N~ N~
O
,1
HN O I ~ HN O.I \ HN O I \ HN O w
O N O N O N N
O
HN S I ~ HN S I ~ HN S ( \ HN S w
I
O N O N O N N
O
b
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 183 --
HN ~ ~ HN ~ w
N~ N
O ~ O
HN ~ ~ HN
N~ N
O ~ O
HN ~ ~ HN
N~ N
O ~ O
HN ~ ~ HN
N~ N
O ~ O
HN ~ ~ HN
N~ N
O ~ O
O O
HN ~ ~ HN
O O N\
~S S
HN-( ~ ~ HN I w
N O N
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 1 g4 __
HN O ~ ~ HN O ~ \ HN O ~ ~ HN O
I
i
O N O N O N O N
~1
HN S ~ ~ HN S I ~ HN S ~ ~ HN S w
N
O O N O N N
O
HN O I ~ HN O I ~ HN O I ~ HN O
O N O N O N N
O
b
I S .. I S ~ I
HN ~ ~ HN I ~ HN I ~ HN I
O N O N O N O Iv
~ 1
HN I ~ HN ~ ~ HN I ~ HN
O N_~X O N_~CX O NIX O NIX
~ 1
b
HN I
N
O
X
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 185 --
HN O ~ ~ HN O
N N
O ~ O
HN S I ~ HN S ~
N N
O ~ O
HN O I ~ HN O
N N
O ~ O
S S
HN I ~ HN
N N
O ~ O
HN I ~ HN
O N v~X O N ~X
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 186 --
r ~ r
1
HN N ~ HN N HN N HN N
O O
O O
X X X
r ~ / ~
I ;V I ~1 I ~,. !
HN ~ HN ~ HN ~ HN
O O O O
l ~ r
IN~IN I~ I~,r
HN HN ~ HN ~ HN
O O O O
r ~ , ~ , ~
IN~IN I~,T (y
HN HN ~ HN ~ HN
0 o O O b
, ~, ~, ~,
HN N ~ HN N HN N HN
O O~ D
O O ~~
r ~, ~, ~r
HN N ~ HN ~ N HN ~ HN
O O ~ O
O
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 187 -_
I N I N ~ I N
HN HN I HN
O O ~ O
X X X
~ / ~
N I ~1 ~ I N
HN HN I F~~1
0 0 0
Y I
Z
I N i ~~ ~ i N
HN HN I HN
O O ~ O
I
~ / ~
HN N HN
N ~ HN N
O O I ~ O
I
N I N ~ 1 N
HN HN I HN
O O ~ O ,
~ / ~
N
HN N HN N ~ HN
O O I i O
I
CA 02325388 2000-09-21
WO 99/67220 PCT/US99J14007
__ 188 -
/ \ / \ / \ / \
HN N~H HN N\ HN N~/ HN N
0 0 0 o
,\ /~\ /\ /\
N N
HN~('N~H HN N~ HN ~/ HN
O O O O b
/ \ / \ / \ / \
r r ~ ) )
HN N~H HN N~ HN N~/ HN N
0 ob
/\ /\ /\ /\
N
HN N~H HN ~'T~ HN N~/ HN
O O O
/ \ / \ / \ / \
HN N~H HN N~ HN N~/ HN N
O O O O
/ \ / \ / \ / \
HN ~ H HN ~ HN ~/ HN
O O O O
CA 02325388 2000-09-21
WO 99/67220 PCTNS99/14007
-- 189 --
/ \ / \ / \
I N I N I N ~ I
HN ~ HN ~ HN ~(
O O I ~ O
X X ~ X
/ \ /'\ / \
i
HN N HN N HN N ~
O O I ~ O
/ \ ~.\ Y / \
I N t
HN ~( HN ~ HN
O O I ~ O
i
/ \ / \ / \
i
i N i N I N ~
HN ~ HN ~ HN
O O I ~ O
i
/ \ / \ / \
i
i N I ~ I ~ ~ I
HN ~ HN ~ HN
O O I ~ O
i
/ \ / \ / \
i N I N I N i I
HN 1( HN ~( HN 1(
O OIL O
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 190 --
HN~ HN~ HN~ HN'~ HN~ HN
0 or o
HN~ HN~ HN~ HN ~ HN~ HN
HN N_ HN~~HN , I~1 HN'~ HN~ HN O N
l
HN~~HN~~HN~ HN~HN~ HN~ o
HN'~ HN'~ ~ HN'~ HN~ HN~ HN'
O ~p O ~ O ~O O ~'O O ?'O O ~'S
'J
HN~~ONU ~N SNO ' HN~ HN~' 1
O ~-S ~O
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 191 --
HN~ HN~ HN~'1 HN~ HN~ HN
~ T
/ -OH ~N, OMe Me0 O t-But-O 0
~ NMe,
HN N NN N HN N HN N HN N I '~I
HN
p b' p b p , O o O
HN'~~ HN'~ HN ~ HN ~ HN'
CJ
HN N HN N HN N
O S O EtN O HN N HN N
NEt O ~SO~ O ~SO~
HN N HN N HN N HN N HN N
O NEt O ~ O ~ O ~ O
NEt N SO, S
Et O'-
HN N HN N ~ N HN N HN N
O ~SO~ O HN O O O S
~S
S S
O
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 192 --
S~ S~ S~ S~ S~ S~
HN N HN N HN N HN ~. N HN ~ N HN N
O ~ O r O ~ O ~ O
Si Si Si S~ Si S~
i ) f ) I > I ) I ) I )
HN I~ HN N HN N HN N HN N HN N
O O ~ O b O b O ~ p
s, s, s, s, s, s,
I ) i ) I ~ ~ ~ ~ )
HN N HN ~- N HN N HN N HN N HN N
O ~ O ~ O ~ O O ~ n
Si Si Si Si Si Si
I ) I ) i ) I ) I 7 ~ ~ )
HN O N~HN O N~ N O N HN O N HN O N HN p N O
Si Si Si Si Si Si
HN N
HN~,-N OHN~N HN~-N HN~-N HN N
O ~O O ~ O ~O O ~O O p O S
Si S~ Si Si S~ S~
HN N HN N HN N HN N HN N HN N
O ~O O ~O O ~S O ~ O ~O O
O
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/14007
- 193 --
S~ S~ S~ S~ S~ S~
i ~ y ~ ,~ ) iI ) I ~ I
HN N HN N HN N HN ~ ~-N HN N HN N
0 ~ o N o '~' o l o 0
/ _OH ~ OMe
Me0 O t-But-O O
NMe~
Si Si Si Si Si Si
I ' I ~ I~ ~ I~ ) , ) ,
HN N HN N HN N HN N HN N HN N
O ~ O ~ O ~ O ~ O ~ O
S~ S_/ S~ S~ S~
HN N HN N HN'~-N HN N HN N
O ~ O O O O
S~ S~ S~ S, S~
HN N HN N HN N
HN N HN N
O S pEtN O
NEt O ~SO~ O ~SO,
S~ S~ .S~ S~ S~
HN N HN N HN N HN N HN N
O NEt O ~ O ~ O ~ O
NEt N SO~ S
Et O'-'
S~ S~ S~ S~ S~
I ) I > ( ) I ) I
HN N HN N HN N HN N HN N
O~SO~ O~ O~S O~ O~S
O~ ~JS
CA 02325388 2000-09-21
WO 99!67220 PCT/US99/14007
-- 194 --
r=N ,.N ~N rN ~=N rN
S~ S~ S~ S~ S~ S~
HN N, HN ~ HN N HN N HN N HN N
p O O r O ~ O ~ O
~=N i=N rN ~N ~N rN
Si Si Si Si Si Si
I ~ I > I ) I )
HN l~ HIV N HN N HN N HN N HN N
O O ~ O b p b p ~ o
i=N rN r=N r-N r'N ~N
S~ S~ S~ Si S~ Si
HN N HN N HN N HN N HN N HN N
p b p ~ O ~ O O ~ p
rN r-N r-N rN rN rN
Si S~ Si S~ Si Si
i ~ I ~ I ) I ~ I ~ I
HN N HN N HN N HN N HN N HN N
O b O ,'O O ~ O O O O
r-N r-N ~N rN - r-N
S ~ S ~ S ~ S ~ S ~ S ~
I ) I >
HN N HN N O HN N HN N HN N HN N
O ~O O ~ O O O O O O O S
r-N ~N rN ~N r-N ~N
S i S i S i S i S i S i
I ) I > I ~ I ) I ) I )
HN N HN N HN N HN N HN N HN :~f
O ~p O ~ O ~S O ~ O O O
O
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 195 --
=N r-N r-N ,=N r-N r-N
S~ S~ S~ S, S~ S~
I ) i ~ 1 ~ I ~ I 7
HN N HN N HN N HN N HN N HN N
O N O ~ O j O O
OH ~ ~ OMe Me0 O t-But-O O
NMe
r- N r- N r- N r- N ~= N r
S~ S~ S~ S~ S~ S N
HN N HN N i I I I
HN N HN N HN ~~- N HN N
O ~ O O O O
NMe,
r-N r-N ~N - r=N rN
S~ S~ S~ S~ S~
HN N I ) I
HN N HN N HN N HN N
O ~ O O O O
~=N ~N r-N r-N rN
S ~ S ~ S ~ S ~ S ~
HN N HN N HN N HN N HN N
O S O EtN O NEt O ~SO~ O ~SO~
r'N rN r=N ~N ~N
S~ Si S~ Si Si
I ) I ) I ) I ) I >
HN N HN N HN N HN N HN N
O NEt O ~NEt O ~ O ~SO, O
N - S
Et O
r-N ~=N ~N r-N rN
S~ S~ S~ S~ S~
I ) I ~ I ) ~ ) I >
HN N HN N HN N HN N HN N
p~SO~ O~ O~S O~ O~S
O, S
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 196 --
rN ,_N rN ~N r-N ~=N
Oi Oi Oi Oi Oi Oi
I > I ) I > I > I > I )
HN O N~ HN O ~ HN O ~ HN O N~", HN O N~HN O N
r-N r=N rN ~N i=N r-N
O, Oi Oi O~ Oi Oi
HN n- ~ I ~ I ~ I T ) I ) I )
I~ HN N HN N HN ~N HN N HN N
p p~ p~ pb pb ~bb
i=N r=N
Oi Oi p N ~ N ~ N Or-N
HN N HN N HN N HN N HN N HN N
O b Ob O O O ~ Ob
r-N r-N ~N r-N r-N r-N
Oi Oi Oi Oi Oi Oi
1 ) I > I ) I ) I ) I )
HN N HN N HN N HN N HN N HN N
O ~ O ~ O ~ O O O O
r-N r=N ~=N r=N r=N r-N
Oi Oi Oi O~ Oi Oi
HN N HN N HN N HN N HN N HN N
O ~OO ~ O ~O O O O O O S
rN ~N r=N r-N r-N r-N
Oi Oi Oi Oi Oi Oi
) ~ ) I ) I ~ I ~ I
HN N HN N HN N HN N HN N HN N
O~CO O~ O~S O~O O
O S O
O
CA 02325388 2000-09-21
WO 99/67220 PGT/US99/14007
-- 197 --
~=N rN rrN
0 N ~ N rN O~ O~
Oi Oi
HN N HN N HN N HIV N HN N HN N
°~ ° N ° ~ ° j ° ~ °
OH ~ ~ OMe Me0 O t-But-O O
NMe2
~=N ~=N r-N rN ~N r=N
Oi Oi Oi Oi Oi Oi
HN N HN N HN N HN N HN N HN N
O b O b O ~ ° ~ ° ~ °
r-N r-N r N r-~N r N
Oi Oi Oi Oi
I > I ) i ) I ) I
HN N HN N HN N HN N HN N
O ~ O O O O
r-N rN r=N i=N rN
Oi Oi Oi
I > I > I ) I ) I 7
HN N HN N HN N HN N HN N
O S O EtN ° NEt ° ~S02 O ~SO,
rN r-N r N r-N rN
Oi Oi Oi Oi Oi
t ' I ' I ~ I ~
HN N HN N HN N HN N HN N
O NEt O ~ O ~ O ~ O
NEt N SO~ S
Et O
,_N r-N ,.N rN rN
O~ O~ O~ O~ O~
HN N HN N HN N HN N HN N~
O ~SO~ O~ O~S O~ ° (,S
O-, ~.. ~S
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 198 -
~ O Oi O Oi Oi
HN~-N, HN~-N' HN~Nr HN~-N~ HN~-N~ HIY~-N
o, o. o, o, o, o,
i ) i ~ i ~ , ~ , ~
HN N HN N HN ~-N HN ~,-N HN N HN N
o H o b ° ~ ° ~ ° ~ °
o~ o~ o~ o~ o~ o~
HN N HN ~-N HN ~ N HN ~-N HN N HN N
o b o ~ o ~ o o ~ o
~N
Oi Oi Oi Oi Oi Oi
I ~ I ~ i ~ I ~ I ~ I
HN N HN N HN ~-N HN N HN N HN N
o ~ o ~p o '~ o 0 0 0
rN
Oi Oi Oi Oi Oi Oi
I ) I > i ) ~ ) ~ ) ~
HN N HN N O HN N HN N HN N HN N
O ~O O ~ O ~O O ~O O O O S
Oi Oi Oi Oi Oi O
HN N HN ~'"N HN ~-N HN N HN N HN ''I
O~CO O O O'~S O~O O
~.-S ~O
O
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 199 __
O~ O~ O~ O
t ~ p ~ I ~ I 7 I ~ I
HN ON HN ONE HN ON~HN O ~ HN ON HN ON
/ 'OH ~ N OMe
Me0 O t-But-O O
NMe,
Oi Oi Oi Oi Oi Oi
I ~ i ) I ~ I ~ I ~
HN O ~ HN O N \ HN N HN N HN N HN N
b pb p~ p~ p
o, o, o, o, o,
I ' I ) I ) I ) I )
HN N HN ~ N HN N HN N HN N
O .~ O O O O
Oi Oi O~ Oi Oi
HN ) I I ~ I ~ I
N HN N HN N HN ~'T~ HN ~'
O S OEtN O NEt O ~SO~ O ~SO~
Oi Oi Oi Oi Oi
I ' I ) I ) I ) I
HN O N NEt HN O N HN O N HN O N HN O N
~NEt ~ ~SO,
N - S
Et O,
Oi Oi Oi O~ Oi
HN N HN N HN N HN N HN N
O~SO~ O~ O~S O~ O~S
O~ S
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 2pp __
HN N HN N HN N HN N HN N
O / ~ O / IV O /-.N O / _ ~ O /
N
HN N H HN N I ~ I ~ I
O / N O , O HN O ~ / S HIS O NHS HN ~'~O
N~ O
J a .J
HN N HN N HN N HN N HN
O ~NH ON H O~O O~S «~S
HN N HN N HN N HN N
HN ~- N
O i ~ O i N O i , O i ~ O i
~N N
N
HN N HN N HN N HN N Her
O N O N O ~ 4~ O
~,NH N ,,O , NfJ
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 201 --
HN ~N HN'~N HN ~ HIY'~ HIT
~-, ~O rN rN
O ~N O (tYJ O N.J O N~.b O ,.b
J
HN N HN N HN N HN N HN N
-O ~ O
O N.~ O NJ O N. S O N O N. SS
HN N HN N HN N HN N HN N
O O ~ O
O N.,O ~ \ N \ N ' N '
L.O L$ LO ESN
HN N HN N HN N HN N HN N
O ~ N O ~N O N N O O
~N~ N, ~ LNG ~ \ ~ \
O S
HN N HN N HN N HN N HN N HN N
O ~ O ~ O O~_ O~_ O
Nr N' ,- N, O N S ~ \
/O NHS
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 202 --
S i S i S i S i S i
HN N HN N HN'~ N HN N HN N
O / \ O / N O / ,N O / \ O /
N
S ~ S i S i S i S i
HN N HN N
I ) H HN N HN N HN N
O S
° ~' ° ~~ ° ~~ ° J ° J
s~ s~ s~ s~
HN N I ~
HN N HN N HN N HN N
°~NH °~ H °~O °~S ° S
S i S i S i S ~ S i
I ~ ~ >
HN N HN N HN N HN N HN N
O i' O ~ N O ~ , ° , O i N
W ~ N , N~ ~ N
S i S i S i S i S i S i
HN N HN N HN ~ N HN N HN N HN N
ON~ °N~ ON O O O,
° ~S ~,NH NCO N.~S ,,,ICJ
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 203 --
S i S i S i S i S ~
HN N HN N HN N HN N I N
O O ~N) O N ~ O N b HN O ?=N
rt -N ~ J N . O
S i S i S i S i S ~
HN N I , I
O r,0 HN O Nr0 HN Nr N HN N~ HN ?=v
NJ NJ O N. S O N. O O N. S
S i S i S i S i S i
)
HN N HN N HN N I N
O ~ ~' HN ~' HN ~-- N
N..O O O O
LD ~S L N
O S
S i S i S i S i S i
I > I ) I ~ I ~
HN N HN N HN N HN N HN N
O ~ N O '~' N O ~' N O O
N, ~ ~N~ I \ I \
N O S
S i S i S i S ~ S i S i
HN N HN N HN N HN N HN N HN '~
ON ON O~ O~ O ) O
NCO N.-~S ~ N~ O Nr~\S
CA 02325388 2000-09-21
WO 99/67220 PG"T/US99/14007
__ 2p4 __
rN /=N rN r-N r-N
S i S i S i S i S i
I > I ) ~ ) ~ ) I )
HN N HN N HN N HN N HN N
O / \ O / N O / ,N O / \ O /
N
r-IV r-N ~N rN ,rN
S i S i S i S i S ~
HN N H HN N HN N HN N
O / N O / O O / S O ?-S HN O N~-O
NJ NJ
r-N r-N ~N i=N r-N
S i S i S i S i S i
~ > I ) ~ ) ~ )
HN N HN N HN N HN N HN N
O ~NH O N ~, NH O ~O O ~S O N JS
i=N rN r=N ~N r-N
S i S i S i S i S i
> I > ~ ) I ) I )
HN N HN N HN N HN N HN N
O ~ ~ O ~ N O ~ , O ~ 1 O ~ N
,.. ~ ~ N ,N ~ 1V
r-N ~N r-N ~N r-N r-N
S i S i S i S i S i S i
I ) I > I > I > ~ ) ~ >
HN N HN N HN N HN N HN N HN N
ON ON ON O O O ,
,O ~S ~.NH N ~O N ~S , NO
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 205 --
r-N ~N r=N /=N r-N
S i S i S i S i S i
HN N ( ) I ) I ) I
O / N HIV N N HN r0 IV ~=N N 1V?= N
O , O 1V~ H O N, b H O N. O
N
/=N ~N r-N r-N i=N
S i S i S i S i S i
N I ) ~ ) ~ )
HN O N O HN O NCO HN O NrN HN O N~ HN O N~
J N~ N. S N. O N. S
r=N ~N ~N r=N r=N
S i S i S i S i S i
I ) I ) ( ) ~ )
HN N HN N HN N HN N HN N
O ~ O O O
N O ~\ ~\ ~ ~ N N
O S O
r-N r=N r=N rN r-N
S i S i S i S i S i
I ) ~ ) I ) I
HN N HN N HN N HN N HN N
O ~ N O ~N O N N O O
~N~ N J 'N~ I \ I \
O S
r-N ~N r-N ~N r-N r-N
S i S i S i S i S i S i
)
HN N HN N HN N HN N HN N HN N
O N-C O N-C O O~_ O~_ O
.. N O N'S I
NCO N,,S
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007 _
__ 2p6 __
r-N rN /=N /=N ~N
Oi Oi Oi Oi Oi
HN N HN N N HN N HN N HN N
O ~ ~ O ~ ~ O ~ ~N O ~_~ O
N
/= N r= N i= N r- N r N
Oi Oi O~ Oi Oi
HN N H HN N HN N HN N I
O / N O / ~ O / S ~ ?,-S HN ~ Nr O
NJ
rN /=N ~N r=N r-N
Oi Oi Oi Oi Oi
HN N HN N HN N HN N HN N
O ~NH O N H O ~O O ~S O N
r- N
r-N ~N rN ,=N
O~ Oi Oi O~ Oi
> I > I ) I ) i
HN N HN N HN N HN N HN N
O i ~ O / N O / , O / , O / N
N ~ ~ N
N
r-N ~N r-N ~N r=N /=N
Oi Oi Oi Oi O,
) I ) I ) l )
HN N HN N HN N HN N HN N HN N
ON~ ON~ ON~ O~_ O~_ O_
~,S ~,NH N JO N .rS NO
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 207 --
/=N r-N f=N ~N r=N
Oi Oi Oi Oi Oi
HN N N HN N HN N HN N HN N
0~~~ O N Or-O O~-N OrN
N , N,~ N.b N,O
N
r'N /=N r=N r-N r--N
Oi Oi Oi Oi Oi
I ~ I > I > i ) I )
HN N r HN NrO HN N~-N HN N~ HN N~
O J O N~ O N, S O N. O O N, S
r=N r=N r=N ,=N ~I~1
Oi Oi Oi Oi Oi
~ i )
HN N HN N HN N HN N HN N
O O O ~ O
L.\ L\ L\ N ~\ N
O S O ~S
r-N r-N r-N r-N r-N
Oi Oi Oi Oi Oi
I ) l ) I ) I ) I )
HN N HN N HN N HN N HN N
O ~ N O ~N O N N O O
~N~ NJ LN) I \ I \
O S
r-N ~N r=N r-N r-N r-N
Oi Oi Oi O, Oi O,
I ) I > I > I ) I > ~ )
HN N HN N HN N HN N HN N HN N
O ~ O , O~ O~ O
NCO NHS ~ Nr-\O N- S ~ \
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 208 --
Oi Oi Oi Oi Oi
I > I > I ) I ) I )
HN N HN N N HN N HN N HN N
O / ~ O /-~ O /=N O / ~ O /
N
Oi Oi Oi Oi Oi
HN N H HN N HN N HN N HN N
/ ~ / ~ ~ ~-O
o y o y o y o J o NJ
o~ o~ o~ o-~ o~
HN N HN N HN N HN N HN N
O ~NH O N~,NH O ~p O ~s O NHS
Oi Oi Oi Oi Oi
HN N HN N HN N HN N HN N
O i ~ O / N O ~ , O ~ ~ O ~ N
W ~ N ,N ~ N
Oi pi Oi Oi Oi Oi
i ) i ~ I ~ I > I ~ I
HN N HN N HN N HN N HN N HN N
ON ON ON O O O
',O ~,S ~NH N ,,O N ~S , NO
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
__ 2p9 __
Oi Oi Oi Oi Oi
I >
HN N N HN N HN ~-N HN N HN N
O ~ a p ~ j O J O N b O I~1
N N
Oi Oi Oi Oi Oi
HN N HN N HN ~-N HN N HN N
O~O O~-O O~N O?=v O
N~ N~ NYS N. O N. S
Oi Oi Oi O, Oi
HN N HN N HN N HN N HN N
O ~ O
O N ''O ON \ ON \
~O ~S N N N N
~O ~S
Oi Oi Oi Oi Oi
HN N I , ~ , ~ , I
HN N HN N HN N HN N
O / N O ~N O ~''N O O
~N'' N J LN'~ ~ \ ~ \
O S
r- N
Oi Oi Oi Oi Oi Oi
HN N HN N HN N HN N HN N HN ~-N
O ~ O ~ O ~ O~ O~ O
N.rO NaS ~' N.O N- S
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 210 --
Example 2
Cellular Screen for the Detection of Inhibitors of (3-Amyloid Production
A compound of formula I above was assayed for its ability to inhibit ~3-
amyloid production in a cell line possessing the Swedish mutation. This
screening
assay employed cells (K293 = human kidney cell line) which were stably
transfected with the gene for amyloid precursor protein 751 (APP751 )
containing
the double mutation Lys65,Met65, to Asn65,Leu~s, (APP751 numbering) in the
manner described in International Patent Application Publication No. 94/105698
and Citron et al.'6. This mutation is commonly called the Swedish mutation and
the cells, designated as "293 751 SWE", were plated in Corning 96-well plates
at
2-4 x l0a cells per well in Dulbecco's minimal essential media (Sigma, St.
Louis.
MO} plus 10% fetal bovine serum. Cell number is important in order to achieve
[3-
amyloid ELISA results within the linear range of the assay (~-0.2 to 2.5 ng
per mL).
Following overnight incubation at 37°C in an incubator
equilibrated with
10% carbon dioxide, media were removed and replaced with 200 ~L of a
compound of formula I (drug) containing media per well for a two hour
pretreatment period and cells were incubated as above. Drug stocks were
prepared
in i 00% dimethyl sulfoxide such that at the final drug concentration used in
the
treatment, the concentration of dimethyl sulfoxide did not exceed 0.5% and, in
fact,
usually equaled 0.1 %.
At the end of the pretreatment period, the media were again removed and
replaced with fresh drug containing media as above and cells were incubated
for an
additional two hours. After treatment, plates were centrifuged in a Beckman
GPR
at 1200 rpm for five minutes at room temperature to pellet cellular debris
from the
conditioned media. From each well, I00 uL of conditioned media or appropriate
dilutions thereof were transferred into an ELISA plate precoated with antibody
266
[P. Seubert, Nature (1992) 359:325-327] against amino acids 13-28 of (3-
amyloid
peptide as described in International Patent Application Publication No.
94/105698
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 211 --
and stored at 4°C overnight. An ELISA assay employing labelled antibody
3D6
[P. Seubert, Nature (1992) 359:325-327]" against amino acids 1-5 of [i-amyloid
peptide was run the next day to measure the amount of ~i-amyloid peptide
produced.
Cytotoxic effects of the compounds were measured by a modif cation of the
method of Hansen, et al.'$. To the cells remaining in the tissue culture plate
was
added 25 ,uL of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (Sigma, St. Louis, MO) stock solution (5 mg/mL) to a final concentration
of 1 mg/mL. Cells were incubated at 37°C for one hour, and cellular
activity was
stopped by the addition of an equal volume of MTT lysis buffer (20% w/v sodium
dodecylsulfate in SO% dimethylformamide, pH 4.7). Complete extraction was
achieved by overnight shaking at room temperature. The difference in the
ODs6zM,
and the ODbso~m was measured in a Molecular Device's UVma~ microplate reader
as
an indicator of the cellular viability.
The results of the [3-amyloid peptide ELISA were fit to a standard curve
and expressed as ng/mL [3-amyloid peptide. In order to normalize for
cytotoxicity,
these results were divided by the MTT results and expressed as a percentage of
the
results from a drug free control. All results are the mean and standard
deviation of
at least six replicate assays.
The test compound was assayed for [i-amyloid peptide production
inhibition activity in cells using this assay. The results of this assay
demonstrate
that the compounds of formula I inhibit ~3-amyloid peptide production by at
least
30% as compared to the control when employed at 10 ug/mL.
CA 02325388 2000-09-21
WO 99/67220 PCT/US99/14007
-- 212 --
Example 3
In Vivo Suppression of (3-Amyloid Release and/or Synthesis
This example illustrates how the compounds of this invention could be
tested for in vivo suppression of [i-amyloid release and/or synthesis. For
these
experiments, 3 to 4 month old PDAPP mice are used [Games et al., (1995) Nature
373:523-527].'9 Depending upon which compound is being tested, the compound
is usually formulated at between 1 and 10 mg/mL. Because of the low solubility
factors of the compounds, they may be formulated with various vehicles, such
as
corn oil (Safeway, South San Francisco. CA); 10% ethanol in corn oil; 2-
hydroxypropyl-(3-cyclodextrin (Research Biochemicals International, Natick
MA);
and carboxy-methyl-cellulose (Sigma Chemical Co., St. Louis MO).
The mice are dosed subcutaneously with a 26 gauge needle and 3 hours
later the animals are euthanized via CO, narcosis and blood is taken by
cardiac
puncture using a 1 cc 25G 5/8" tuberculin syringe/needle coated with solution
of
0.5 M EDTA, pH 8Ø The blood is placed in a Becton-Dickinson vacutainer tube
containing EDTA and spun down for 1 S minutes at 1500 xg at 5 °C. The
brains of
the mice are then removed and the cortex and hippocampus are dissected out and
placed on ice.
1. Brain Assax
To prepare hippocampal and cortical tissue for enzyme-linked
immunosorbent assays (ELISAs) each brain region is homogenized in 10 volumes
of ice cold guanidine buffer (5.0 M guanidine-HC1, 50 mM Tris-HC1, pH 8.0)
using a Kontes motorized pestle (Fisher, Pittsburgh PA). The homogenates are
gently rocked on a rotating platform for three to four hours at room
temperature
and stored at -20°C prior to quantitation of (3-amyloid.
The brain homogenates are diluted 1:10 with ice-cold casein buffer [0.25%
casein, phosphate buffered saline (PBS), 0.05% sodium azide, 20 pg/ml
aprotinin,
CA 02325388 2000-09-21
WO 99/67220 ~ PCT/US99/14007 _
-- 213 --
mM EDTA, pH 8.0, 10 pg/ml leupeptin], thereby reducing the final
concentration of guanidine to 0.5 M, before centrifugation at 16,000 xg for 20
minutes at 4°C. Samples are further diluted, if necessary, to achieve
an optimal
range for the ELISA measurements by the addition of casein buffer with 0.5 M
guanidine hydrochloride added. The ~3-amyloid standards (1-40 or 1-42 amino
acids) were prepared such that the final composition equaled 0.5 M guanidine
in
the presence of 0.1% bovine serum albumin (BSA).
The total ~3-amyloid sandwich ELISA, quantitating both ~3-amyloid (aa 1-
40) and (3-amyloid (aa 1-42) consists of two monoclonal antibodies (mAb) to ~i-
amyloid. The capture antibody, 266 [P. Seubert, Nature ( 1992) 359:325-327],
is
specific to amino acids 13 - 28 of ~3-amyloid. The antibody 3D6 [Johnson-Wood
et
al., PNAS USA (1997) 94:1550-1555],'-° which is specific to amino acids
1 - 5 of ~i-
amyloid, is biotinylated and served as the reporter antibody in the assay. The
3D6
biotinylation procedure employs the manufacturer's (Pierce, Rockford IL)
protocol
for NHS-biotin labeling of immunoglobulins except that 100 mM sodium
bicarbonate, pH 8.5 buffer is used. The 3D6 antibody does not recognize
secreted
amyloid precursor protein (APP) or full-length APP but detects only (3-amyloid
species with an amino terminal aspartic acid. The assay has a lower limit of
sensitivity of ~50 pg/ml ( I I pM) and shows no cross-reactivity to the
endogenous
murine ~i-amyloid peptide at concentrations up to 1 ng/ml.
The configuration of the sandwich ELISA quantitating the level of (3-
amyloid (aa l-42) employs the mAb 21FI2 [Johnson-Wood et al., PNAS USA
(1997) 94:1550-1555] (which recognizes amino acids 33-42 of [i-amyloid) as the
capture antibody. Biotinylated 3D6 is also the reporter antibody in this assay
which has a lower limit of sensitivity of 125 pg/ml (28 pM).
The 266 and 21 F 12 capture mAbs are coated at 10 p,g/ml into 96 well
immunoassay plates (Costar, Cambidge MA) overnight at room temperature. The
CA 02325388 2000-09-21
WO 99/67220 PCT/IJS99/14007 _ .
-- 214 --
plates are then aspirated and blocked with 0.25% human serum albumin in PBS
buffer for at least 1 hour at room temperature, then stored desiccated at
4°C until
use. The plates are rehydrated with wash buffer (Tris-buffered saline, 0.05%
Tween 20) prior to use. The samples and standards are added to the plates and
incubated overnight at 4°C. The plates are washed Z 3 times with wash
buffer
between each step of the assay. The biotinylated 3D6, diluted to 0.5 pg/ml in
casein incubation buffer (0.25% casein, PBS, 0.05% Tween 20, pH 7.4) is
incubated in the well for 1 hour at room temperature. Avidin-HRP (Vector,
Burlingame CA) diluted 1:4000 in casein incubation buffer is added to the
wells
for 1 hour at room temperature. The colorimetric substrate, Slow TMB-ELISA
(Pierce, Cambridge MA), is added and allowed to react for 15 minutes, after
which
the enzymatic reaction is stopped with addition of 2 N H,SO~. Reaction product
is
quantified using a Molecular Devices Vmax (Molecular Devices, Menlo Park CA)
measuring the difference in absorbance at 450 nm and 650 nm.
2. Blood Assav
The EDTA plasma is diluted 1:1 in specimen diluent (0.2 gm/1 sodium
phosphate ~ H,O (monobasic), 2.16 gm/1 sodium phosphate ~ 7H,0 (dibasic),
O.~gm/l thimerosal. 8.5 gm/1 sodium chloride, 0.5 ml Triton X-405, 6.0 g/1
globulin-free bovine serum albumin; and water). The samples and standards in
specimen diluent are assayed using the total ~3-amyloid assay (266 capture/3D6
reporter) described above for the brain assay except the specimen diluent was
used
instead of the casein diluents described.
Formulations other than those described above can also be used for oral
delivery and intravenous delivery to a mammal. For oral delivery, the compound
can be mixed with either 100% com oil or, alternatively, in a solution
comtaining
80% corn oil, 19.5% oleic acid and 0.5% labrafil. The compound can be mixed
with the above solutions in concentrations ranging from 1 mg/mL to 10 mg/mL.
The compound in solution is preferably administered orally to the mammal at a
CA 02325388 2000-09-21
WO 99/67220 PCT/I1S99/14007
-- 215 --
dose volume of 5 mL/kg of body weight. For IV delivery, the compound is
preferably mixed with a solution of 3% ethanol, 3% solutol HS-15 and 94%
saline.
The compound is preferably mixed with the above solution in concentrations
ranging from 0.25 mg/mL to 5 mg/mL. The compound in solution is preferably
administered by IV to the mammal at a dose volume of 2 mL/kg of body weight.
From the foregoing description, various modifications and changes in the
composition and method will occur to those skilled in the art. All such
modifications coming within the scope of the appended claims are intended to
be
included therein.
