Note: Descriptions are shown in the official language in which they were submitted.
~1 7 ~7 6 6 PCT~S94111553
W095/12612 L11 ~1
-- 1--
SUBS~ ul~ DI- AND TRIPEPTIDE INHIBITORS
OF PROTEIN:FARNESYL TRANSFERASE
FIELD OF THE INVENTION
The present invention pertains to a number of
compounds which can be used in the medicinal field to
treat, prophylactically or otherwise, uncontrolled or
abnormal proliferation of ht1mAn tissues. More
specifically, the present invention pertains to a
number of compounds which act to inhibit the farnesyl
transferase enzyme that has been determined to activate
ras proteins which in turn activate cellular divi~ion
and are implicated in cancer and restenosis.
BACKGROUND OF THE INVENTION
Ras protein (or p21) has been ~x~mi n~d extensively
because mutant forms are found in 20~ of most types of
human cancer and greater than 50~ of colon and
pancreatic carc~nnm~s (Gibbs J.B., Cell, 65:l (l99l),
Cartwright T., et al., Chimica Og~i, 10:26 (1992)).
These mutant ras proteins are deficient in the
capability for feedback regulation that is present in
native ras and this deficiency is associated with their
oncogenic action since the ability to stimulate normal
cell division can not be controlled by the normal
endogenous regulatory cofactors. The recent discovery
that the transforming activity of mutant ras is
critically dependent on post-translational
modifications (Gibbs J., et al., Microbiol. Rev.,
~3:171 (1989)) has unveiled an important aspect of ras
function and identified novel prospects for cancer
therapy.
WO95/l2612 PCT~S94/1l553
2-
In addition to cancer, there are other conditions
of uncontrolled cellular proliferation that may be
related to excessive expression and/or function of
native ras proteins. Post surgical vascular restenosis
is such a condition. The use of various surgical
revascularization techniques such as saphenous vein
bypass grafting, endarterectomy and translllm; n~ 1
coronary angioplasty is often accompanied by
complications due to uncontrolled growth of neointimal
tissue, known as restenosis. The biochemical causes of
restenosis are poorly understood and numerous growth
factors and protooncogenes have been implicated
(Naftilan A.J., et al., Hypertension, 13:706 (1989) and
J. Clin. Invest., 83:1419; Gibbons G.H., et al.,
Hypertension, 14:358 (1989); Satoh T., et al., Mollec.
Cell. Biol., 13:3706 (1993)). The fact that ras
proteins are known to be involved in cell division
processes makes them a candidate for intervention in
many situations where cells are dividing uncontrol-
lably. In direct analogy to the inhibition of mutant
ras related cancer, blockade of ras dependant processes
has the potential to reduce or eliminate the
inappropriate tissue proliferation associated with
restenosis, particularly in those instances where
normal ras expression and/or function is exaggerated by
growth stimulatory factors.
Ras functioning is dependent upon the modification
of the proteins in order to associate with the inner
face of plasma membranes. Unlike other membrane-
associated proteins, ras proteins lack conventionaltr~ncm~mhrane or hydrophobic sequences and are
initially synthesized in a cytosol soluble form. Ras
protein membrane association is triggered by a series
of posttranslational processing steps that are signaled
by a carboxyl term;n~l amino acid consensus sequence
that is recognized by protein:farnesyl transferase.
WO 95/12612 2 1 7 ~ 7 6 ~ PCT/US9~/115~i3
.
--3--
This consensus sequence consists of a cysteine residue
located four amino acids from the carboxyl term;
followed by two lipophilic amino acids and the
C-t~rm;n~l residue. The sulfhydryl group of the
cysteine residue is alkylated by farnesyl pyrophosphate
in a reaction that is catalyzed by protein:farnesyl
transferase. Following prenylation, the C-termln~1
three amino acids are cleaved by an endoproteaQe and
the newly exposed alpha-carboxyl group of the
prenylated cysteine is methylated by a methyl
transferase. The enzymatic processing of ras proteins
that begins with farnesylation enables the protein to
associate with the cell membrane. Mutational analysis
of oncogenic ras proteins indicate that these
posttranslational modifications are essential for
transforming activity. Replacement of the consensus
sequence cysteine residue with other amino acids gives
a ras protein that is no longer farnesylated, fails to
migrate to the cell membrane and lacks the ability to
stimulate cell proliferation (Hancock J.F., et al.,
Cell, 57:1617 (1989); Scha~er w.R., et al., Science,
245:379 (1989); Casey P.J., Proc. Natl. Acad. Sci. USA,
86:8323 (1989)).
Recently, protein:farnesyl transferases (PFTs,
also referred to as farnesyl:protein transferaseQ) have
been identified and a specific PFT from rat brain was
purified to homogeneity (Reiss Y., et al., Bioch. Soc.
Trans., 20:487-88 (1992)). The enzyme was charac-
terized as a heterodimer composed of one alpha-subunit
(49 kDa) and one beta-subunit (46 kDa), both of which
are required for catalytic activity. High level
expression of m~mm~lian PFT in a baculovirus system and
purification of the recombinant enzyme in active fonm
has also been accomplished (Chen W.-J., et al.,
J. Biol. Chem., 268:9675 (1993)).
PCT~S9411lS53
WO95/12612
In light of the foregoing, the discovery that the
function of oncogenic ras pro.teins is critically
dependent on their posttranslational processing
provides a means of cancer chemotherapy through
S inhibition of the processing enzymes. The
identification and isolation of a protein:farnesyl
transferase that catalyzes the addition of a farnesyl
group to ras proteins provides a promising target for
such intervention. Recently it has been determined
that prototypical inhibitors of PFT can inhibit ras
processing and reverse cancerous morphology in tumor
cell models (Kohl N.E., et al., Science, 260:1934
(1993); James G.L., et al., Science, 260:1937 (1993);
Garcia A.M., et al., J. Biol. Chem., 268:18415 (1993)).
Thus, it is possible to prevent or delay the onset of
cellular proliferation in cancers that exhibit mutant
ras proteins by blocking PFT. By analogous logic,
inhibition of PFT would provide a potential means for
controlling cellular proliferation associated with
restenosis, especially in those cases wherein the
expression and/or function of native ras is
overstimulated.
PCT Application WO91/16340 discloses cysteine
cont~in;ng tetrapeptide inhibitors of PFT of the
formula CAAX.
European Patent Application 0461869 discloses
cysteine cont~;n;ng tetrapeptide inhibitors of PFT of
the formula Cys-Aaa1-Aaa2-Xaa.
European Patent Application 0520823 discloses
cysteine cont~;n;ng tetrapeptide inhibitors of PFT of
the formula Cys-Xaal-dXaa2-Xaa3.
European Patent Application 0523873 discloses
cysteine cont~;n;ng tetrapeptide inhibitors of PFT of
the formula Cys-Xaa1-Xaa2-Xaa3.
WO95/12612 2 1 7 0 7 6 6 PCT~S94/11553
--5--
European Patent Application 0528486 discloses
cysteine cont~; n; ng tetrapeptide amides inhibitors of
PFT of the formula Cys-Xaa1-Xaa2-Xaa3-NRRl.
European Patent Application 0535730 discloses
pseudotetrapeptide inhibitors of PFT of the following
two formulas:
X R2 o )~_,ZH
J~NH ~H~J~H~OH
HS'~ Y R 0
X R~ o
H R3
HS
European Patent Application 0535731 (US 5,238,922)
discloses esters of pseudotetrapeptide inhibitors of
PFT of the formula:
X R2 o ()~ZH
- H ~ -~ N oR5
~ Y R O
HS
European Patent Application 0482539 discloses
tachykinin antagonists of the formula:
R R3
R
l,Y ~ A` ~ N~R5
0 R~ O
wos5ll26l2 PCT~S9~/11553
--6-
European Patent Application 0457195 discloses
endothelin antagonists of the formula:
~
US 4,022,759 discloses tripeptide antagonists of
luteinizing honmone releasing factor of the formula
A-Rl-Tyr(benzyl)-Ser(benzyl)-R2, wherein one of the
definitions of Rl is His(benzyl).
Compounds disclosed in the above references do not
disclose or suggest a novel combination of structural
variations found in the present invention described
hereinafter. All cited references are hereby
incorporated by reference.
SUMMARY OF THE IN V~N'1'10N
Accordingly, the present invention is a
substituted di- or tripeptide compound of Formula I:
R
/
~rN
N~
~( 'H2) n
A--N R O
R O ~ ) n
2 1 7~766 PCT~Ss~/115~3
W095/12~1~
wherein:
n = l or 2;
A = CoR3, Co2R3, CoNHR3, CSR3, C(S)oR3, C(S)NHR3,
CF3SO2, aryl-SO2, or alkyl-SO2, wherein R3 is
- 5 alkyl, (CH2) m~ cycloalkyl, (CH2) m~ aryl,
( CH2 ) m~ heteroaryl, or (CH2) m ~ alkyl, and m = 0, l,
2, or 3;
R = independently H or Me;
Y - independently H or Me;
Z - independently H or Me;
Rl - H, CO-aryl, (CH2) m ~ aryl, O(CH2) m ~ cycloalkyl,
O(CH2)m-aryl, or O(CH2)m-heteroaryl, wherein m is
as defined above and Rl i8 located at either the
meta or para position;
X = one to four substituents, including H, alkyl, CF3,
F, Cl, Br, I, HO, MeO, NO2, NH2, N(Me) 2' OPO3H2, or
CH2PO3H2;
R2 = NR(CH2)nCo2R3, NR(CH2)nCoNHR3, NR(CH2)nR3,
NR(CH2)n+l0R , NR(CH2)n+lSR ~
NRCH(CoR5)(CH2) n ~ heteroaryl, NRCH(CoR5)(CH2)noR3,
NRCH(CoR5)(CH2)nSR3, or N N- R3
wherein R, R3, and n are as defined above, R4 = H
or R3, and R5 = OH, NH2, oR3, or NHR3; an optical
isomer, diastereomer, or a ph~rm~ceutically
acceptable salt thereof.
The present invention is also directed to the use
of a compound of Formula I, or a pharmaceutically
acceptable salt there~rom, to inhibit the activity of a
protein:farnesyl transferase enzyme as a method for
trea~ing tissue proliferative diseases.
A further embodiment of the present invention is
the use of a pharmaceutical composition including an
e~ective amount of a compound of Formula I as a method
for the treatment of cancer.
PCTIUS94/1 1553
WO 95/12612
8-
A still further embodiment of the present
invention is the use of a pharmaceutical composition
including an effective amount of a compound of
Formula I as a method for the treatment of restenosis.
A still further embodiment of the present
invention i8 a ph~r~ceutical compo~ition for
~mi n; stering an effective amount of a compound of
Fonmula I in unit dosage form in the treatment methods
mentioned above.
A final embodiment of the present invention
pertains to methods for the preparation of compounds of
Formula I by solid phase synthesis, solution phase
synthesis, and simultaneous multiple ~yntheses using a
multiple simultaneous synthesis apparatus.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMæNTS
In the compounds of Formula I, the term "alkyl"
means a straight or branched hydrocarbon radical having
from 1 to 6 carbon atoms and includes, for example,
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl, n-pentyl, n-hexyl, and the like.
The term "cycloalkyl" means a saturated
hydrocarbon ring which contains from 3 to 10 carbon
atoms, for example, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, adamantyl, and the like,
unsubstituted or substituted by an alkyl or aryl group.
The term "aryl" means an aromatic ring which is a
phenyl, 5-fluorenyl, 1-naphthyl or 2-naphthyl group,
unsubstituted or substituted by 1 to 3 substituents
selected from alkyl, O-alkyl and S-alkyl, O-aryl, OH,
SH, F, Cl, Br, I, CF3, NO2, NH2, NHCH3, N(CH3) 2
NHCO alkyl, (CH2)mC02H, (CH2)mCO2-alkyl, (CH2)mSO3H,
(CH2)mPO3H2, (CH2)mPO3(alkYl) 2 ~ ( CH2 ) mS2NH2 ~ and
wossll26l2 2 1 7 0 7 6 6 pcT~ss4lll553
(CH2)mSO2NH-alkyl wherein alkyl is defined a~ above and
m = 0, l, 2, or 3.
The term Uheteroaryl~ means a heteroaromatic ring
which is a 2- or 3-thienyl, 2- or 3-furanyl, 2- or
3-pyrrolyl, 2-, 3- or 4-pyridyl, 2-, 3-, 4-, 5-, 6-,
or 7-indolyl group, unsubstituted or with l or
2 substituents from the group of substituents described
above for aryl.
The following table provides a list of
abbreviations and definitions thereof used in the
present invention.
TABLE OF ABBREVIATIONS
Abbreviation Amino Acid
Ala Alanine
Arg Arginine
Asn Asparagine
Asp Aspartic acid
Cys Cysteine
Glu Glutamic acid
Gln Glutamine
Gly Glycine
His Histidine
Ile Isoleucine
Leu Leucine
Lys Lysine
Met Methionine
Phe Phenylalanine
Pro Proline
Ser Serine
Thr Threonine
Trp Tryptophan
If the optical activity of the amino acid iB other than L(S),
the amino acid or abbreviation i8 preceded by the appropriate
configuration D(R) or D~(RS).
PCT~S94/11553
W095112612
10-
Abbreviation Amino Acid (continued)
Tyr Tyrosine
Val Valine
Abbreviation* Modified and Unusual Amino ~çid
Aaa-CO2R An amino acid ester, for examples:
Gly-CO2Bn is Glycine, benzyl ester;
Ser(OBn)-CO2Me is O-Benzyl-serine,
methyl ester.
Aaa-CONHR An amino acid amide, for examples:
Gly-CONHBn is Glycine, N-benzyl
amide; Ser(OBn)-CONHEt is
O-Benzyl-serine, N-ethyl amide;
Tyr(OBn)-CONHCH2CH20Bn is O-Benzyl-
tyrosine, N-(2-(phenylmethoxy)ethyl)
amide.
3Hyp 3-Hydroxyproline
4Hyp 4-Hydroxyproline
Hcy Homocysteine
Nva Norvaline
Nle Norleucine
Orn Ornithine
Bal Beta-alanine (or 3-aminopropionic
acid)
Abu 4-Aminobutyric acid
Ahe 7-Aminoheptanoic acid
Acp 6-Aminocaproic acid
Aoc 8-Aminooctanoic acid
Apn 5-Aminopentanoic acid
Bpa (4-Benzoylphenyl)alanine
Chx 3-Cyclohexylalanine (or
Hexahydrophenylalanine)
Cit Citrulline
If the optical activity of the amino acid i8 other than L(S),
the amino acid or abbreviation i8 preceded by the appropriate
configuration D(R) or DL(RS).
PCT~S94111553
WQ 9s/l26l2 2 1 ~ 0 7 6 6
-11-
Abbrevi~tion Modified and Unusual Amino Acid
(continued)
His(1-Me) l-Methyl-hi~tidine ~or N(~)-Methyl-
histidine)
His(Tr) 1-Triphenylmethyl-histidine (or
N(~-Trityl-histidine)
homoPhe 2-Amino-4-phenylbutanoic acid ~or
Homophenylalanine)
homoTyr 2-Amino-4-(4-hydroxyphenyl)butanoic
acid (or Homotyrosine)
homoTyr(OBn) 2-Amino-4-[4-(phenylmethoxy)phenyl]-
butanoic acid (or O-Benzyl-
homotyrosine)
1-Nal 3-(1'-Naphthyl)alanine
2-Nal 3-(2'-Naphthyl)alanine
Pen Penicillamine
Phe(3-OBn) (3-Benzyloxyphenyl)alanine
Phe(4-Ph) 3-(l,l'Biphen-4-yl)alanine (or
4-Phenyl-phenylalanine)
Pgl Phenylglycine
Pyr 2-Amino-3-(3-pyridyl)-propanoic acid
(or 3-Pyridylalanine)
Ser(OBn) O-Benzyl-serine
Thr(OBn) O-Benzyl-threonine
Tic 1,2,3,4-Tetrahydro-3-i~oquinoline-
carboxylic acid
Tyr(OMe) O-Methyl-tyrosine
Tyr(OEt) O-Ethyl-tyro~ine
Tyr(OBn) O-Benzyl-tyrosine
(~-Me)Tyr(OBn) 2-Amino-3-(4-benzyloxyphenyl)-
2-methyl-propionic acid (or ~-Methyl-
O-benzyl-tyrosine)
(N-Me)Tyr(OBn) N-Methyl-O-benzyl-tyrosine
Trp(For) Nln-Formyltryptophan
If the optical activity of the amino acid i~ other than L(S),
the amino acid or abbreviation i8 preceded by the a~op-iate
configuration D(R) or DL(RS).
PCT/IJS9~/1 1553
WO 9S/12612
2~a~
-12-
Abbreviation Mercapto Acids
Maa Mercaptoacetic acid
Mba 4-Mercaptobutyric acid
Mpa 3-Mercaptopropionic acid
Abbreviation Protectinq Group
Ac Acetyl
Ada 1-A~m~ntyl acetic acid
Adoc A~m~ n tyloxycarbonyl
Bn Benzyl
MeBn 4-Methylbenzyl
Cbz Benzyloxycarbonyl
2-Br-Cbz ortho-Bromobenzyloxycarbonyl
2-Cl-Cbz ortho-Chlorobenzyloxycarbonyl
Bom Benzyloxymethyl
Boc tertiary Butyloxycarbonyl
Dnp 2,4-Dinitrophenyl
For Formyl
Fmoc 9-Fluorenylmethyloxycarbonyl
NO2 Nitro
TMS Trimethylsilyl
Tos 4-Toluenesulfonyl (to~yl)
Tr Triphenylmethyl (trityl)
Abbreviation Solvents and Rea~ents
HOAc Acetic acid
CF3SO2H Trifluoromethanesulfonic acid
DCM Dichloromethane
DCC N,N'-Dicyclohexylcarbodiimide
DIC N,N'-Diisopropylcarbodiimide
DIEA N,N-Diisopropylethylamine
DMAP 4-Dimethylaminopyridine
DMF N,N'-Dimethylfonmamide
EDAC N-Ethyl-N'-Dimethylaminopropyl-
carbodiimide
EtOAc Ethyl acetate
PCT/US94/1 lS53
WO 95112612
~ 21 7~766
-13-
Abbreviation Solvents and Reaqents
Et20 Diethyl ether
HC1 Hydrochloric acid
HF Hydrofluoric acid
HOBT 1-Hydroxybenzotriazole
KOH Potassium hydroxide
MeCN Acetonitrile
MeOH Methanol
NHOS N-Hydroxysuccinimide
NMP N-Methylpyrrolidone
iPrOH iso-Propanol
TBAF Tetra n-Butyl~mmo~-um Fluoride
TFA Trifluoroacetic acid
Abbreviation . Solid Phase Peptide Synthesis Resins
HMP ~esin 4-(Hydroxymethyl)-phenoxymethyl-
polystyrene resin
MBHA Resin Methylbenzhydrylamine resin
PAM Resin 4-(Hydroxymethyl)-
phenylacetamidomethyl-polystyrene
resin
2-Cl-Tr Resin 2-Chlorotrityl-polystyrene resin
NH2-Rink Resin 4-(amino-(2',4'-dimethoxy-
phenyl)methyl)-phenoxymethyl-
polystyrene resin
Abbreviation Biological Reaqents
FPP Farnesyl pyrophosphate
PFT Protein:farnesyl transferase
DTT Dithiothreitol
BSA Bovine serum albumin
PCT~S94/11553
WO95/12612
~ 14-
Abbreviation Miscellaneous
CoR3 1IR3
CoNHR3 1l
CNHR3
CSR3 S
CR3
C ( S ) oR3 s
CoR3
C(S)NHR3 lSI 3
CNHR
CH(CoR5)(CH2)noR3 (CH2)nCNHR3
CH(CoR5)(CH2) n~ heteroaryl o= Ic5
IC-(CH2) n ~ heteroaryl
CH(CoR5)(CH2)noR3 O=C
C- (CH2)noR3
CH(CoR5)(CH2)noR3 o=c5
C- (CH2)nSR3
1l ~
CO(4-Bn-piperazin-1-yl) C-N ~ N-CH2Ph
2 1 7 n PCT~S94/11553
W095/1:612 -15 - u ~ 6 6
Preferred compounds of Formula I consist of
compounds of Formula II below:
N-R
~; ~
R O
A'-N ~ z ~ RZ II
R O ~ "~
~ l
~ R
wherein:
A' ~ Co2R3, CoNHR3, C(S)NHR3, or aryl-S02, wherein R3
is alkyl, (CH2)m-cycloalkyl, (CH2)m-aryl,
(CH2) m~ heteroaryl, and m = O, l, 2, or 3;
R = independently H or Me;
Y - independently H or Me;
Z = independently H or Me;
Rl = (CH2)m-aryl, O(CH2)m-aryl, OPO3H2, or CH2PO3H2,
wherein m is as defined above;
R2 = NR(CH2) 2 oR4 ~ NR(CH2) 2 SR4, NRCH(CoR5)cH2oR3
s 3 ~
NRCH(COR )CH2SR , or , - N ~ N - CH2Ph
wherein R, R3, and n are as defined above, R4 = H
or R3, and R5 = OH, NH2, oR3, or NHR3; an optical
isomer, diastereomer, or a ph~rm~ceutically
acceptable salt thereof.
Other preferred compounds of the present inventio
are those of Formula I as defined above wherein A is
Co2R3 or CoNHR3; or as defined above in Formula I
wherein at least one of Y and Z is Me; or as defined
above in Formula I wherein R2 is (CH2)20R4 or
CH(CoR5)CH2oR3; or as defined above in Formula I
PCT~Ss~/ll553
WO95/12612
~7 a7 66 -16-
wherein A is CoNHR3, R2 is (CH2)2oR4, and at least one
of Y and Z i8 Me.
The most preferred compounds of Fonmula I include
the following:
Cbz-His-Tyr(OBn)-Ser(OBn)-C02Me;
Cbz-His-Tyr(OBn)-Ser(OBn)-CONH2;
Cbz-His-Tyr(OBn)-Ser(OBn)-CONHEt;
Cbz-His-Tyr(OBn)-Ser(OBn);
Cbz-His-Tyr(OBn)-D-Ser(OBn)-CO2Me;
Cbz-D-His-Tyr(OBn)-Ser(OBn)-CONH2;
Cbz-D-His-Tyr(OBn)-Ser(OBn)-CONHEt;
Cbz-D-His-Tyr(OBn)-Ser(OBn)-CO2Me;
Cbz-D-His-Tyr(OBn)-Ser(OBn);
Cbz-His(l-Me)-Tyr(OBn)-Ser(OBn)-CO2Me;
Cbz-His(l-Me)-Tyr(OBn)-Ser(OBn)-CONH2;
Cbz-His(l-Me)-Tyr(OBn)-Ser(OBn)-CONHEt;
Cbz-His(l-Me)-Tyr(OBn)-Ser(OBn);
Cbz-D-His(l-Me)-Tyr(OBn)-Ser(OBn)-C02Me;
Cbz-D-His(l-Me)-Tyr(OBn)-Ser(OBn)-CONH2;
Cbz-D-His~l-Me)-Tyr(OBn)-Ser(OBn)-CONHEt;
Cbz-D-His(l-Me)-Tyr(OBn)-Ser(OBn);
Cbz-His-(~-Me)Tyr(OBn)-Ser(OBn)-CO2Me;
Cbz-His-(~-Me)Tyr(OBn)-Ser(OBn)-CONH2;
Cbz-His-(~-Me)Tyr(OBn)-Ser(OBn)-CONHEt;
Cbz-His-(~-Me)Tyr(OBn)-Ser(OBn);
Cbz-His-D-(~-Me)Tyr(OBn)-Ser(OBn)-CO2Me;
Cbz-His-D-(~-Me)Tyr(OBn)-Ser(OBn)-CONH2;
Cbz-His-D-(~-Me)Tyr(OBn)-Ser(OBn)-CONHEt;
Cbz-His-D-(~-Me)Tyr(OBn)-Ser(OBn);
Cbz-D-His-homoTyr(OBn)-Ser(OBn)-CO2Me;
Cbz-His-Phe(4-Ph)-Ser(OBn)-CO2Me;
Cbz-D-His-Phe(4-Ph)-Ser(OBn)-C02Me;
Cbz-His-Tyr(OBn)-Pyr-C02Me;
Cbz-D-His-Tyr(OBn)-Pyr-CO2Me;
Cbz-His-Tyr(OBn)-CONHCH2CH20Bn;
Cbz-D-His-Tyr(OBn)-CONHCH2CH2OBn;
pcT~ss~llls53
W095/12612
~ 2 1 ~0~66
-17-
Cbz-His-(N-Me)Tyr(OBn)-CONHCH2CH20Bn;
Cbz-D-His-(N-Me)Tyr(OBn)-CONHCH2CH20Bn;
Cbz-His-Tyr(OBn)-CONH(CH2)2Ph;
Cbz-D-His-Tyr(OBn)-CONH(CH2)2Ph;
5 .Cbz-His-Tyr(OBn)-Gly-C02Bn;
Cbz-D-His-Tyr(OBn)-Gly-C02Bn;
Cbz-His-Tyr(OBn)-Gly-CONHBn;
Cbz-D-His-Tyr(OBn)-Gly-CONHBn;
BnNHCO-His-Tyr(OBn)-Ser(OBn)-C02Me;
BnNHCO-His-Tyr(OBn)-Ser(OBn)-CONH2;
BnNHCO-Hi~-Tyr(OBn)-Ser(OBn)-CONHEt;
BnNHCO-His-Tyr(OBn)-Ser(OBn);
BnNHCO-His-Tyr(OBn)-CONHCH2CH20Bn;
BnNHCO-His-Tyr(OBn)-CONHCH2CH2CH20Ph;
BnNHCO-D-His-Tyr(OBn)-Ser(OBn)-CO2Me;
BnNHCO-D-His-Tyr(OBn)-Ser(OBn)-CONH2;
BnNHCO-D-His-Tyr(OBn)-Ser(OBn)-CONHEt;
BnNHCO-D-His-Tyr(OBn)-Ser(OBn);
BnNHCO-D-His-Tyr(OBn)-CONHCH2CH20Bn;
BnNHCO-D-His-Tyr(OBn)-CONHCH2CH2CH20Ph;
Cbz-His-Tyr(OBn)-CON(Me)CH2CH20Bn;
(4-EtOPh)NHCO-D-His-Tyr(OBn)-CONH(CH2)30Ph;
PhCH2CO-D-His-Tyr(OBn)-CONH(CH2)3-(2-MeOPh);
(4-PhOPh)NHCO-D-His-Tyr(OBn)-COHN(CH2)2Ph; and
(4-MePh)S02-D-His-Tyr(OBn)-CO(4-Bn-
piperazin-l-yl).
~ENERAL METHODS FOR THE PREPARATION, EVALUATION
AND USE OF COMPOUNDS OF FORMUhA I
The compounds of Formula I may be prepared by
solid phase peptide synthesis on a peptide synthesizer,
for example, an Applied Biosystems 43lA peptide
3~ synthesizer using activated esters or anhydrides of Boc
or Fmoc protected amino acids, acid chlorides,
WO9Sl12612 PCT~S94/11553
-18-
isocyanates, isothiocyanates, etc., on PAM, MBHA, or
NH2-Rink resins with solution phase modifications to
the carboxyl terminus as appropriate. Methodology for
the solid phase synthesis of peptides is widely known
to those skilled in the art thereof (see, for example:
J.M. Stewart and J.D. Young in Solid Phase Peptide
Synthesis; Pierce Chemical Co.; Rockford, IL (1984);
Fields G.B. and Noble R.L., Int. J. Peptide Protein
~ç~, 35:161-214 (1990)).
Additionally, the compounds of Formula I may also
be prepared by conventional solution peptide synthesis,
substituting ~m; n~s, acid chlorides, isocyanates, etc,
for amino acid derivatives where appropriate. Methods
for solution phase synthesis of peptides are widely
known to those skilled in the art (see, for example,
M. Bodanszky, Principles of Peptide Synthesia,
Springer-Verlag (1984)).
Finally, the compounds of Formula I may be
prepared by simultaneous multiple solid phase syntheses
using an apparatus described by S. H. DeWitt, et al.,
Proc. Natl. Acad. Sci. USA, 90:6909 (1993), and
referred to by the trademark, Diversomer~, both
trA~m~rk and apparatus being owned in whole by the
Warner-~ambert Company. The multiple solid phase
synthesis apparatus is currently the subject of now
ab;tn~on~ US Serial 07/958,383 filed October 8, 1992
and pending continuation-in-part US Serial 08/012,557
filed February 2, 1993.
For example (Scheme I below), Fmoc-D-His-Tyr(OBn)-
CO2-CH2CH2Si(CH3)3 is linked to 2-Cl-Tr resin using a
sterically hindered amine such as DIEA as an HCl
scavenger, the Fmoc protecting group is removed with
piperidine, the resulting free amino term;ntl~ is
acylated with a series of isocyanates, isothiocyanates,
activated esters, acid chlorides and the like, the
TMS-ethyl ester is cleaved with TBAF, the resulting
~ ~ PCT~Ss4/11553
WO95112612 ~ ~ 70~66
.
-19 -
free carboxy term;nl~ is activated with a carbodiimide
reagent such as EDAC, DCC, or DIC, the activated
carboxyl group is reacted with alcohols such as HOBT,
NHOS, or pentachlorophenol to give an activated eQter,
5 the activated ester is reacted with a series of amines
and the reculting array of compounds of Formula I i8
cleaved from the resin by with hot HOAc or by treatment
with TFA at room temperature.
For all three synthetic methods described above
appropriate consideration is given to protection and
deprotection of reactive functional groups and to the
sequence of synthetic steps. Knowledge of the use of
common protecting groups and strategy for the assembly
of complex organic molecules are within the usual realm
of expertise of a practitioner of the art of organic
chemistry (see, for example: T.W. Greene and
P.G.M. Wuts, Protective Groups in Organic Chçmistry,
John Wiley and Sons (l99l); E.J. Corey and X.-M. Cheng,
The Logic of Chemical Synthesis, John Wiley and Sons
(l989)).
The homogeneity and composition of the resulting
compounds is verified by reverse-phase-high pressure
liquid chromatography (RP-HPLC), capillary
electrophoresis, thin layer chromatography (T~C),
proton nuclear magnetic resonance spectrometry (NMR),
amino acid analysis, fast atom bombardment mass
spectrometry (FAB-MS) and electrospray mass
spectrometry (ES-MS).
PCT~S9~/11553
WO95/12612
~7~ 20-
SCHEME I: Multiple Simultaneous Synthesi~ Method
Resin ~ Resin
1. Piperidine
~N~ Cl ~ N Cl
\\ // 2. Eight acylating
N ~ agents N--~
FmocNH ~ ~ OCH2CH2TMS ~ ~ OCH2CH2TMS
O / O
~ ~
BnO BnO
1. TBAF
2. EDAC,
HOBT
3. Five amines,
Resin =
N ~N Cl
H ~ ~ TFA A~ ~ ~ ~N J ~R2
BnO
40 Compounds o~ Formula I /~~~
EnO
pcT~ss~llls53
Wo 95112612 2 ~ 6 6
-21-
The compounds of Formula I are capable of further
forming both pharmaceutically acceptable acid addition
and/or base salts. A11 of these forms are within the
scope of the present invention.
Pharmaceutically acceptable acid addition salts of
the compounds of Formula I include ~alts derived from
nontoxic inorganic acids such as hydrochloric, nitric,
pho~phoric, sulfuric, hydrobromic, hydroiodic,
hyd~ofluoric, phosphorous, and the like, as well as the
salts derived from nontoxic organic acids, such a~
aliphatic mono- and dicarboxylic acids,
phenyl-substituted alkanoic acids, hydroxy alkanoic
acids, ~k~nedioic acids, aromatic acids, aliphatic and
aromatic sulfonic acids, etc. Such salts thus include
sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,
nitrate, phosphate, monohydrogenphosphate,
dihydrogenphosphate, metaphosphate, pyrophosphate,
chloride, bromide, iodide, acetate, trifluoroacetate,
propionate, caprylate, isobutyrate, oxalate, malonate,
succinate, suberate, sebacate, fumarate, maleate,
mandelate, benzoate, chlorobenzoate, methylbenzoate,
dinitrobenzoate, phthalate, benzenesulfonate,
toluenesulfonate, phenylacetate, citrate, lactate,
maleate, tartrate, methanesulfonate, and the like.
Also contemplated are salts of amino acids such as
arginate and the like and gluconate, galacturonate,
n-methyl glucamine (see, for example, Berge S.M.,
et al., "Pharmaceutical Salts," Journal of
Pharmaceutical Science, 66:1-19 (1977)).
The acid addition salts of said basic compounds
are prepared by contacting the free base form with a
sufficient amount of the desired acid to produce the
salt in the conventional manner. Preferably a compound
o~ Formula I can be converted to an acidic salt by
treating with an aqueous solution of the desired acid,
such that the resulting pH is less than 4. The
PCT/US9411 1553
WO 95/12612
b~ -22-
solution can be passed through a C18 cartridge to
absorb the compound, washed with copious amounts of
water, the compound eluted with a polar organic solvent
such as, for example, methanol, acetonitrile, and the
like, and isolated by concentrating under reduced
pressure followed by lyophilization. The free base
form may be regenerated by contacting the salt form
with a base and isolating the free base in the
conventional manner or as above. The free base forms
differ from their respective salt forms somewhat in
certain physical properties such as solubility in polar
solvents, but otherwise the salts are equivalent to
their respective free base for purposes of the present
invention.
Pharmaceutically acceptable base addition salts
are formed with metals or amines, such as alkali and
alkaline earth metals or organic amines. Examples of
metals used as cations are sodium, potassium,
magnesium, calcium, and the like. Examples of suitable
amines are N,N'-dibenzylethylene~'~m;n~,
chloroprocaine, choline, diethanolamine,
dicyclohexylamine, ethylenediamine, N-methylgluc~m;ne,
and procaine (see, for example, Berge S.M., et al.,
"Pharmaceutical Salts", Journal of Pharmaceutical
Science, 66:1-19 (1977)).
The base addition salts of said acidic compounds
are prepared by contacting the free acid form with a
sufficient amount of the desired base to produce the
salt in the conventional manner. Preferably, a
compound of Formula I can be converted to a base salt
by treating with an aqueous solution of the desired
base, such that the resulting pH is greater than 9.
The solution can be passed through a C18 cartridge to
absorb the compound, washed with copious amounts of
water, the compound eluted with a polar organic solvent
such as, for example, methanol, acetonitrile and the
PCT~S94/11553
Wo95112612 2 1 7 0 7 6 6
-23-
like, and i~olated by concentrating under reduced
pressure followed by lyophilization. The free acid
~ form may be regenerated by contacting the salt form
with an acid and isolating the free acid in the
con~entional manner or as above. The free acid forms
differ from their respective salt forms somewhat in
certain physical properties such as solubility in polar
solvents, but otherwise the salts are equivalent to
their respective free acid for purposes of the present
invention.
Certain of the compounds of the present invention
can exist in unsolvated forms as well as solvated
~orms, including hydrated forms. In general, the
solvated forms, including hydrated forms, are
equivalent to unsolvated forms and are intended to be
encompassed within the scope of the present invention.
Certain of the compounds of the present invention
possess one or more chiral centers and each center may
exist in the R(D) or S(L) configuration. The present
invention includes all enantiomeric and epimeric forms
as well as the appropriate mixtures thereo~.
The PFT inhibitory activity of compounds o~
Formula I was assayed in 30 mM potassium phosphate
buffer, pH 7.4, containing 7 mM DTT, 1.2 mM MgC12,
0.1 mM leupeptin, 0.1 mM pepstatin and 0.2 mM
phenylmethylsulfonyl fluoride. Assays were performed
in 96 well plates (Wallec) and employed solutions
composed of varying concentrations of a compound of
Formula I in lO0~ DMS0. Upon addition of both
substrates, radiolabeled ~arnesyl pyrophosphate
(rl-3~], specific activity 15-30 Ci/mmol, final
concentration 0.12 ~M) and (biotinyl)-Ahe-Tyr-Lys-Cys-
Val-Ile-Met peptide (final concentration O.l ~M), the
enzyme reaction was started by addition of 40-fold
purified rat brain farnesyl protein transferase. After
incubation at 37C for 30 minutes, the reaction was
PCT/US9 1/1 1553
WO 95112612
~ 7 ~1 6~
-24-
t~rm;n~ted by diluting the reaction 2.5-fold with a
stop buffer cont~;n;ng 1.5 M magnesium acetate, 0.2 M
H3P04, 0.5~ BSA, and strepavidin beads (Amersham) at a
concentration of 1.3 mg/m~. After allowing the plate
to settle for 30 minutes at room temperature,
radioactivity was quantitated on a microBeta counter
(model 1450, Wallec). Compounds of Formula I show IC50
values of 0.5 nM to 80 ~M (see data table) in this
assay and are thus valuable inhibitors of protein:
farnesyl transferase enzyme which may be used in the
medical treatment of tissue proliferative diseases,
including cancer and restenosis.
IC50 Values for Selected Compounds of
Formula I Against PFT
Example Number IC50 (~M)
1 4.4
4 1.0
5 (3) 2.1
5 (4) 7.3
5 (23) 0.64
5 (27) 30
5 (28) 0.73
5 (30) 73
5 (31) 0.76
5 (35) 66
5 (36) 1.9
5 (46) 1.0
5 (49) 2.9
5 (40) 0.75
5 (52) 1.6
5 (56) 1.1
5 (59) 20
5 (60) 1.4
PCT~S94/11553
WO95/12612
~ 2 1 7~766
-25-
IC50 Values for Selected Compounds of
Formula I Against PFT
Example Number IC50 (~M)
5 (61) 7.2
5 (62) 1.5
5 (63) 1.0
5 (64) 1.7
5 (69) 0.48
5 (79) 3.0
5 (80) 1.6
6 0.42
7 0.26
8 0.074
9 0.27
0.10
11 0.17
12 0.028
13 0.083
16 0.60
17 0.039
18 0.82
19 0.31
21 0.31
22 0.37
23 1.9
PCT/US94/1 1553
WO 95/12612
26-
IC50 Values for Selected Compounds of
Formula I Against PFT (cont'd)
Example Number IC50 (~M)
24 1.0
3.7
28 11
29 3.0
The compounds of the present invention can be
prepared and ~min;stered in a wide variety of oral,
rectal and parenteral dosage forms. Thus, the
compounds of the present invention can be A~mi n; gtered
by injection, that is, intravenously, intramuscularly,
intracutaneously, subcutaneously, intraduodenally, or
intraperitoneally. Also, the compounds of the present
invention can be A~ml n; stered by inhalation, for
example, intranasally. Additionally, the compounds of
the present invention can be administered
transdermally. It will be obvious to those skilled in
the art that the following dosage forms may comprise as
the active component, either a compound of Formula I or
a corresponding phArm~ceutically acceptable salt of a
compound of Formula I.
For preparing pharmaceutical compositions from the
compounds of the present invention, pharmaceutically
acceptable carriers can be either solid or liquid.
Solid form preparations include powders, tablets,
pills, capsules, cachets, suppositories, and
dispersible granules. A solid carrier can be one or
more substances which may also act as diluents,
flavoring agents, binders, preservatives, tablet
disintegrating agents, or an encapsulating material.
pcT~ss~ 553
W095/12612
~ 2 1 70766
-27-
In powders, the carrier is a finely divided solid
which is in a mixture with ~he finely di~ided active
component.
In tablets, the active component is mixed with the
carrier having the necessary binding properties in
suitable proportions and compacted in the shape and
size desired.
The powders and tablets preferably contain from 5
or lO to about 70 percent of the active compound.
Sui~able carriers are magnesium carbonate, magnesium
stearate, talc, sugar, lactose, pectin, dextrin,
starch, gelatin, tragacanth, methylcellulose, sodium
carboxymethylcellulose, a low melting wax, cocoa
butter, and the like. The term "preparation" is
intended to include the formulation of the active
compound with encapsulating material as a carrier
providing a capsule in which the active component with
or without other carriers, is surrounded by a carrier,
which is thus in association with it. Similarly,
cachets and lozenges are included. Tablet~, powders,
capsules, pills, cachets, and lozenges can be used as
solid dosage forms suitable for oral ~m; n; gtration.
For preparing suppositories, a low melting wax,
such as a mixture of fatty acid glycerides or cocoa
butter, is first melted and the active component is
dispersed homogeneously therein, as by stirring. The
molten homogenous mixture i5 then poured into
convenient sized molds, allowed to cool, and thereby to
~olidify.
Liquid form preparations include solutions,
suspensions, and emulsions, for example, water or water
propylene glycol solutions. For parenteral injection
liquid preparations can be formulated in solution in
aqueous polyethylene glycol solution.
Aqueous solutions suitable for oral use can be
prepared by dissolving the active component in water
WO9S112612 PCT~S9~111553
.
1 ~1 6 ~ -28-
and adding suitable colorants, flavors, stabilizing and
thickening agents as desired.
Aqueous suspensions suitable for oral use can be
made by dispersing the finely divided active component
in water with viscous material, such as natural or
synthetic gums, resins, methylcellulose, sodium
carboxymethylcellulose, and other well-known suspending
agents .
Also included are solid form preparations which
are intended to be converted, shortly before use, to
liquid form preparations for oral A~m; n; stration. Such
liquid forms include solutions, suspensions, and
emulsions. These preparations may contain, in addition
to the active component, colorants, flavors,
stabilizers, buffers, artificial and natural
sweeteners, dispersants, thickeners, solubilizing
agents, and the like.
The phAr~Aceutical preparation is preferably in
unit dosage form. In such ~orm the preparation is
subdivided into unit doses contA;n;ng appropriate
quantities of the active component. The unit dosage
form can be a packaged preparation, the package
contA i nl ng discrete quantities of preparation, such as
packeted tablets, capsules, and powders in vials or
ampoules. Also, the unit dosage form can be a
capsules, tablet, cachet, or lozenge itself, or it can
be the appropriate number of any of these in packaged
form.
The quantity of active component in a unit dose
preparation may be varied or adjusted from O.l mg to
lO0 mg preferably 0.5 mg to lO0 mg according to the
particular application and the potency of the active
component. The composition can, if desired, also
contain other compatible therapeutic agents.
In therapeutic use as inhibitors of PFT, the
compounds utilized in the phanmaceutical methods of
PCT/US94/1 1553
WO 95/1~612 _,
~ 6 6
-29-
this invention are administered at the initial dosage
of about 0.01 mg/kg to about 20 mg/kg daily. A daily
dose range of about 0.01 mg/kg to about 10 mg/kg is
preferred. The dosages, however, may be varied
depending upon the requirements of the patient, the
severity of the condition being treated, and the
compound being employed. Determination of the proper
dosage for a particular situation is within the skill
of the art. Generally, treatment is initiated with
smaller dosages which are less than the optimum dose of
the compound. Thereafter, the dosage is increased by
small increments until the optimum effect under the
circumstances is reached. For convenience, the total
daily dosage may be divided and ~m~ n; stered in
port:ions during the day, if desired.
The following nonlimiting examples illustrate the
inventors' preferred methods for preparing the
compounds of the invention. For added clarity, complex
chemical names describing compounds of Formula I are
followed by structural abbreviations, which are shown
in braces, wherein the structural elements are as
defi~ed in the Table of Abbreviations above.
EXAMPLE 1
~-rN-rN-[(Phenylmethoxy)carbonyl]-L-histidyll-
O-(phenylmethyl)-L-tyrosyl]-O-(phenylmethyl)-
L-serinamide ~Cbz-His-Tyr(OBn)-Ser(OBn)-CONH2}
Using an ABI model 431A solid phase peptide
synthesizer, Fmoc-NH2-Rink resin (0.25 mmol scale) was
treated with 20~ piperidine in NMP to afford NH2-Rink
resin. Sequential coupling of Fmoc-protected Ser(OBn)
and Tyr(OBn) (DCC and HOBT in NMP) and Fmoc
deprotection (20~ piperidine in NMP) reactions were run
using a 4-fold excess of reagents in the coupling steps
and traditional resin washing cycles to afford
Tyr(OBn)-Ser(OBn)-CONH2-Rink resin. This dipeptide
PCT~S94/11553
WO95/12612
~lQ16~ ' --
-30-
resin was transferred to an uninstrumented reaction
vessel and treated with a 4-fold excess of Cbz-His,
DCC, and HOBT in DMF, shaking overnight at room
temperature. After removal of excess reagents, the
resulting substituted tripeptide was cleaved from the
resin by treatment with 50~ TFA in DCM at room
temperature for 2.5 hours. Evaporation of solvents and
purification by reversed phase chromatography
(Cl8-column, eluted with a 20-70~ gradient of MeCN in
water (both solvents acidi~ied with 0.l~ TFA) afforded
Cbz-His-Tyr(OBn)-Ser(OBn)-CONH2 as its TFA salt upon
lyophilization. ES-MS: 719 (m+l).
Using analogous methods the following most
preferred compounds of Formula I with carboxamides at
the C-terminus may be prepared:
Cbz-D-His-Tyr(OBn)-Ser(OBn)-CONH2;
Cbz-His(l-Me)-Tyr(OBn)-Ser(OBn)-CONH2;
Cbz-D-His(l-Me)-Tyr(OBn)-Ser(OBn)-CONH2;
Cbz-His-DL-(~-Me)Tyr(OBn)-Ser(OBn)-CONH2;
BnNHCO-His-Tyr(OBn)-Ser(OBn)-CONH2; and
BnNHCO-D-His-Tyr(OBn)-Ser(OBn)-CONH2.
EXAMPLE 2
N-rN-rN-[(Phenylmethoxy)carbonyll-L-histidyll-
O-(phenylmethyl)-L-tyrosyll-O-(phenylmethyl)-L-serine
{Cbz-His-Tyr(OBn)-Ser(OBn)}
Beginning with PAM resin or HMP resin,
Fmoc-Ser(OBn), Fmoc-Tyr(OBn), and Cbz-His are
sequentially coupled using the deprotection and
coupling conditions described in Example l. Cleavage
from the resin is accomplished by treatment with
CF3SO2H for the PAM supported tripeptide or with 50
TFA in DCM for the HMP supported tripeptide.
Chromatography as in Example l provides
Cbz-His-Tyr(OBn)-Ser(OBn) as its TFA salt upon
PCT~S94/11553
W095/12612 2 1 ~ ~66
.
-31-
lyophilization. See also Example 7 for a solution
phase method.
Using analogous methods the following most
preferred compounds of Formula I with a free carboxyl
term; m~ may be prepared:
Cbz-D-His-Tyr(OBn)-Ser(OBn);
Cbz-His(l-Me)-Tyr(OBn)-Ser(OBn);
Cbz-D-His(l-Me)-Tyr(OBn)-Ser(O~n);
Cbz-Hi~-D~-(~-Me)Tyr(OBn)-Ser(OBn3;
BnNHCO-His-Tyr(OBn)-Ser(OBn); and
BnNHCO-D-His-Tyr(OBn)-Ser(OBn).
EXAMPLE 3
Solid phase supported N-~N-~(9H-Fluoren-9-
ylmethoxy)car~onYl]-L-histidyll-O-(phenylmethyl)-
L-tyrosine, 2-trimethylsilylethyl ester {Fmoc-
His(2-Cl-Tr Resin)-Tyr(OBn)-CO2CH2CH2TMS}
Step 1: Boc-Tyr(OBn)-C02CH2CH2TMS
2-Trimethylsilyl ethanol (2.6 g, 22.6 mmol) was
added to a pr~m~ solution of EDAC (4.3 g,
22.6 mmol), DMAP (0.5 g), and Boc-Tyr(OBn)-OH (7.0 g,
18.8 mmol) in dry THF (25 mL). The resulting mixture
was stirred for 18 hours at room temperature. The
solu~ion was diluted with 1:1 EtOAc:Et20 (40 mL),
2~ washed with saturated aqueous NaHC03 (2 x 10 m~) and
with saturated aqueous NaCl (2 x 10 mL), dried (MgSO4),
filtered and concentrated in vacuo to provide an oil
which was further purified by flash chromatography
(SiO2, EtOAc:hexane eluent) to give the pure TM~-ethyl
ester as an oil;
H NMR (HCDCl3): ~ 0.04 (s, 9H), 1.43 (8, 9H), 3.03
(m, 2H), 4.22 (m, 2H), 4.51 (m, lH), 4.95 (m, lH), 5.05
(s, 2H), 6.85-7.48 (m, 9H).
PCT~S94111553
WO95/12612
~7`~76~ --
-32-
Step 2: Tyr(OBn)-CO2CH2CH2TMS
Eighty percent TFA in CH2C12 (20 mL, v/v) was
added to an ice-cooled solution of Boc-Tyr(OBn)-
CO2CH2CH2TMS (14.8 g, 31.4 mmol) in CH2Cl2 (40 mL). The
reQulting mixture was stirred for 1.0 minute before
concentrating in vacuo. The procedure was repeated
once more, and the resulting residue was diluted with
CH2Cl2 and saturated aqueous NaHCO3. The resulting
mixture was filtered through celite. The organic layer
was then separated, washed with saturated aqueous NaCl,
and dried (MgSO4). Filtration and concentration
in vacuo provided an oil which was further purified by
flash chromatography (SiO2, CHCl3:MeOH eluent) to give
the de~ired product;
1H NMR (HCDC13): ~ 0.06 (s, 9H), 1.68 (br ~, 2H),
2.85-3.03 (m, 2H), 3.67 (m, lH), 4.22 (m, 2H), 5.05
(s, 2H), 6.91-7.45 (m, 9H).
Step 3: Fmoc-His(Tr)-Tyr(OBn)-CO2CH2CH2TMS
To a solution of HOBT (2.6 g, 19.3 mmol) in DMF
(10 mL) was added Fmoc-His(Tr) ( 10.0 g, 16.1 mmol)
followed by EDAC (3.7 g, 19.3 mmol). The mixture was
stirred at room temperature for 20 minutes before
adding a solution of Tyr(OBn?-CO2CH2CH2TMS (from Step 2
above, 5.8 g, 16.1 mmol) in DMF (10 mL). The mixture
was stirred overnight at room temperature before
partitioning between a mixture of water and 1:1
Et2O:EtOAc (50 mL). The layers were separated, and the
organic phase was washed with saturated aqueous NaCl
(4 x 20 mL) and dried (MgSO4). Filtration and
concentration in vacuo provided an oil which was
further purified by flash chromatography (SiO2,
CHCl3:MeOH eluent) to give the protected His-Tyr
dipeptide; FAB-MS 974 (m+1).
W095/12612 PCT~S94/11553
2 1 7~66
-33-
Step 4: Fmoc-His-Tyr(OBn)-CO2CH2CH2TMS
Fmoc-His(Tr)-Tyr(OBn)-CO2CH2CH2TMS (from Step 3
above, 5.0 g, 5.1 mmol) was treated with pyridineoHCl
(1.0 g) in MeOH (20 mL). The mixture was allowed to
stir 8 hours at 65 C. The solution was concentrated
in vacuo, and the residue was dissolved in CH2Cl2,
washed with H20 (lx), saturated aqueous NaHC03 (2x),
and dried (MgS04). Filtration and concentration
in vacuo provided an oil which was further purified by
flash chromatography (SiO2, CHCl3:MeOH eluent) to give
Fmoc-His-Tyr(OBn)-CO2CH2CH2STMS as a white solid;
FAB-MS 731 (m+1).
Step 5: Fmoc-His(2-Cl-Tr Resin)-Tyr(OBn)-CO2CH2CH2TMS
To a suspension of Fmoc-His-Tyr(OBn)- C02CH2CH2TM~
(from Step 4 above, 5.3 g, 7.3 mmol) in CHC13 (20 mL)
was added 2-chloroltrityl chloride resin (Novabiochem)
(7.1 g) followed by DIEA (0.96 g, 7.4 mmol). The
resulting mixture was subjected to brief sonication to
disperse the resin and then agitated on a shaker for
2.0 hours. The modified resin was collected by
filtration, washed with DMF (2x), MeOH (2x), CHCl2
(2x), and dried in vacuo for 18 hours to yield 10.5 g
(loading corresponds to approximately 1 mmol/g resin).
EXAMPLE 4
N-r3-PhenoxYpropyll-O-(phenylmethyl)-N~-rN-
r r (phenylmethyl)aminolcarbonyll-L-histidyl]-L-
tyrosinamide {BnNHCO-His-Tyr(OBn)-CONH(CH2)30Ph}
Fmoc-His(2-Cl-Tr-Resin)-Tyr(OBn)-C02CH2CH2TMS
(from Example 3 above, 2.0 g) was suspended in 20
piperidine in DMF. The resulting suspension was
subjected to sonication for 10 minutes and then
agitated by shaking for 30 minutes. The resin was
filtered and washed with DMF (3x). The resin was again
subjected to the same reaction conditions for an
PCT~S94111553
W095/12612
?~1~766
-34-
additional 20 minutes. The resin was filtered and
washed with DMF (4x) and CHCl3 (3x) to provide
His(2-Cl-Tr-Resin)-Tyr(OBn)-C02CH2CH2TMS which was
suQpended in DCM (lO mL), agitated by shaking for
30 minutes, treated with benzyl isocyanate (l.l g,
8.0 mmol), and agitated for an additional 30 minutes.
The resin was filtered, washed with DCM (3x),
resuspended in DCM, and the benzyl isocyanate treatment
was repeated. The resin was filtered and washed with
DMF (2x) and CHCl3 (5x) to give BnNHCO-His(2-Cl-Tr-
Resin)-Tyr(OBn)-C02CH2CH2TMS which was next suspended
in a mixture of 4:3 dioxane:MeOH (14 mL) and treated
with l.O M TBAF in THF (2.0 mL, 2.0 mmol). The
suspension was agitated by shaking for 18 hours,
filtered, washed sequentially with a 2:l mixture of
dioxane and lO~ citric acid (3x lO mL), dioxane:MeOH
(3x lO mL), dioxane (3x lO mL), and CHCl3 (3x lO mL) to
provide BnNHCO-His(2-Cl-Tr-Resin)-Tyr(OBn). The
BnNHCO-His(2-Cl-Tr-Resin)-Tyr(OBn) was suspended in DMF
(lO mL) and treated with a carbodiimide coupling
reagent such as DIC (0.2 g, l.6 mmol) and HOBT (0.22 g,
l.6 mmol). The resulting mixtuEe was stirred
30 minutes and 3-phenoxypropylamine (0.24 g, 1.6 mmol)
was added. The resulting mixture was shaken 18 hours
before filtering the resin and washing with DMF (3x)
and CHCl3 (3x). The resin was suspended in DMF ~lO mL)
and the carbodiimide/HOBT/3-phenoxypropylamine coupling
reaction was repeated. After 18 hours, the resin was
filtered and washed with lO mL each of MeOH (2x), DCM
(3x), DMF (2x), MeOH (2x), and CHCl3 (2x) to give
BnNHCO-His(2-Cl-Tr-Resin)-Tyr(OBn)-CONH(C~2)30Ph. The
highly substiuted dipeptide was cleaved from the resin
by treatment with 40~ TFA in DCM, shaking for l hour at
room temperature. The supernate, cont~in;ng the free
dipeptide, was filtered away from the resin and the
resin was washed with DCM (6x). The combined supernate
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and washings were concentrated in vacuo to pro~ide
BnNHCO-His-Tyr(OBn)-CONH(CH2)30Ph TFA. The product was
partitioned between water and DCM, and both layers were
treated dropwise with saturated aqueous NaHCO3 until
the aqueous layer r~m~;ned basic. The layers were
separated, and the organic phase was washed with
saturated aqueous NaCl and dried (MgSO4). Filtration
and concentration yielded BnNHCO-His-Tyr(OBn)-
CONH(CH2)30Ph; ES-M~ 675 (m+1).
EXAMPLE 5
Mul~iple, Simultaneous Solid Phase Synthe~is
The method described in Example 4 may be employed
in simultaneous multiple syntheses using the Diversomer
apparatus described by S.H. DeWitt, et al., Proc. Natl.
Acad. Sci. USA, 90:6909 (1993). Fmoc-D-His(2-Cl-
Tr Resin)-Tyr(OBn)-CO2iPr, prepared according to from
Example 3 by substituting Fmoc-D-His(Tr) for Fmoc-
His(Tr) in Step 3, (100-200 mg) is placed in each of
40 ~as dispersion tubes, and the tubes are placed in
the Diversomer apparatus. The sequential deprotection
and coupling reactions described in Example 4 are
followed, employing the following acylating agents and
~m; neS in various combinations:
Acylatinq aqents Amines
1) benzyl isocyanate 1) 3-pheno~y~lu~ylamine
2) p-toluenesulfonyl chloride 2) 2-(phenylmethoxy)ethyl
amine
3) cyclohexyl isocyanate 3) 2-[(phenylmethyl)-
thio]-ethylamine
4) phenyl isocyanate 4) 4-phenylbutylamine
5) i-propyl isocyanate 5) 3-(2-methoxyphenyl)-
propylamine
6) n-butyl isocyanate 6) 1-benzyl piperazine
7) 4-chlorophenyl isocyanate 7) o-benzyl-hy~o~ylamine
8) l-napthyl isocyanate 8) methionine methyl ester
9) 3-methoxypropyl isocyanate 9) benzylamine
10) 4 ethoxyphenyl isocyanate 10) 2-phenylethylamine
11) 2~phenethyl isocyante
PCTrUS9~/11553
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~7~ 36-
12) 3-phenylpropionyl chloride
14) phenylacetyl chloride
15) 4-phenoxyphenyl isocyanate
16) benzyl chloroformate
17) (trans)-2-phenylcyclopropyl isocyanate
18) l-adamantyl chloroformate
Array 1. Following cleavage from the resin and work-up
as described in Example 4, the following substituted
dipeptides (1-40) o~ Formula I are prepared:
1. PhNHCO-D-His-Tyr(OBn)-CONHCH2CH20Bn
2. PhNHCO-D-His-Tyr(OBn)-CONHCH2CH2SBn BS-~S 677 (m+l)
3. PhNHCO-D-His-Tyr(OBn)-CONHCH2CH2CH20Ph BS-MS 661 (m+1)
4. BuNHCO-D-His-Tyr(OBn)-CONH(CH2)4Ph ES-NS 639 (m+1)
5. BuNHCO-D-His-Tyr(OBn)-CO(4-Bn-piperazin-
l-yl) BS-MS 665 (m)
6. (4-MePh)SO2-D His-Tyr(OBn)-CONHCH2CH2Ph BS-MS 666 (m+l)
7. (4-MePh)S02-D-His-Tyr(OBn)-CONHCH2CH2SBn BS-MS 712 (m+l)
8. CF3S02-D-His-Tyr(OBn)-CONH-Met-C02Me
9 CF3S02-D-His-Tyr(OBn)-CONHCH2CH2CH2-(2-MeO-Ph)
10. CF3S02-D-His-Tyr(OBn)-CO(4-Bn-piperazin-1-yl)
11 BnNHCO-D-His-Tyr(OBn)CONHCH2cH2Ph
12. MeO(CH2~3NHCO-D-His-Tyr(OBn)CON~ORn
13. MeO(CH2)3NHCO-D-His-Tyr(OBn)CONHCH2CH2CH20Ph
14. MeO(CH2)3NHCO-D-His-Tyr(OBn)CONH(CH2)3-
(2-MeO-Ph)
15. BnNHCO-D-His-Tyr(OBn)CONHCH2Ph BS-MS 631 (m+1)
16. (4-ClPh)NHCO-D-His-Tyr(OBn)CONHCH2CH20Bn BS-MS 695 (m+1)
17. l-Napthyl-NHCO-D-His-Tyr(OBn)C~N~ORn ES-~S 683 (m+1)
18. 1-Napthyl-NHCO-D-His-Tyr(OBn)CONH-Met-C02Me ES-MS 723 (m+l)
19. (4-ClPh)NHCO-D-His-Tyr(OBn)CONH(CH2)4Ph ES-MS 693 (m+l)
20. (4-ClPh)NHCO-D-His-Tyr(OBn)CONHCH2Ph ES-MS 651 (m+1)
21. BnOCO-D-His-Tyr(OBn)CONHCH2CH20Bn ES-MS 676 (m+l)
22. 1-adamantyl-OCO-D-His-Tyr(OBn)CONHCH2CH2SBn
23. BnOCO-D-His-Tyr(OBn)CONHCH2CH2CH20Ph ES-MS 676 (m+1)
24. 1-adamantyl-OCO-D-His-
Tyr(OBn)CONHCH2CH2CH2CH2Ph
25. BnOCO-D-His-Tyr(OBn)CO(4-Bn-piperazin-
1-yl) ES-MS 700 (m)
26. PhCH2CO-D-His-Tyr(OBn)CONHCH2CH2Ph ES-MS 630 (m+1)
27. PhCH2CH2NHCO-D-His-Tyr(OBn)CONHCH2CH2SBn BS-MS 705 (m+1)
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28. PhCH2CH2NHCO-D-His-Tyr(OBn)CONH-Met-
CO2Me . ES-MS 701 (m+l)
29. PhCH2CH2NHCO-D-His-Tyr(OBn)CONH(CH2)3-
(2-MeO-Ph) ES-MS 703 (m+1)
30. PhCH2CO-D-His-Tyr(OBn)CO(4-Bn-piperazin-
1-yl) ES-MS 684 (m)
31. (t-2-Ph-c-propyl)-NHCO-D-His-
Tyr(OBn)CONHCH2CH2Ph ES-MS 671 (m+l)
32. (t-2-Ph-c-propyl)-NHCO-D-His-
Tyr(OBn)CONUoRn ES-MS 673 (m+l)
33. c-hexyl-NHCO-D-His-Tyr(OBn)
CONH(CH2)3Oph ES-MS 666 (m+1)
34. c-hexyl-NHCO-D-His-Tyr(OBn)CONH(CH2)3-
(2-MeO-Ph) ES-MS 681 (m+l)
35. c-hexyl-NHCO-D-His-Tyr(OBn)CONHCH2Ph ES-MS 623 (m+l)
36. PhCH2CH2CO-D-His-Tyr(OBn)CONHCH2CH2OBn ES-MS 674 (m+1)
37. PhCH2CH2CO-D-His-Tyr(OBn)C~N~ORn ES-MS 646 (m+l)
38. (CH3)2CHNHCO-D-His-Tyr(OBn)CONH-Met-
CO2Me ES-MS 639 (m+1)
39. PhCH2CH2CO-D-His-Tyr(OBn)CoNH(cH2)3ph ES-MS 672 (m+l)
40. (CH3)2CHNHCO-D-His-Tyr(OBn)CONHCH2Ph ES-MS 583 (m+l)
Array 2. Following cleavage from the resin and work-up
as described in Example 4, the following substi~uted
dipeptides (41-80) of Formula I are prepared:
41. n-BuNHCO-D-HiR-Tyr(OBn)-CONHCH2CH2OBn ES-MS 641 (m+1)
42. n-BuNHCO-D-HiR-Tyr(OBn)-CONHCH2CH2SBn ES-MS 657 (mll)
43. n-BuNHCO-D-His-Tyr(OBn)-CONHCH2CH2CH2OPh ES-MS 641 (m+1)
44. Ph~HCO-D-His-Tyr(OBn)-CONHCH2CH2CH2CH2Ph ES-MS 659 (m+1)
45. PhNHCO-D-His-Tyr(OBn)-CO(4-Bn-piperazin-l-yl)ES-MS 685 (m)
46. (4-PhOPh)NHCO-D-His-Tyr(OBn)-CONHCH2CH2Ph ES-MS 723 (m+l)
47. (4-PhOPh)NHCO-D-His-Tyr(OBn)-CONHCH2CH2SBn ES-MS 769 (m+1)
48. (4-MePh)SO2-D-His-Tyr(OBn)-CONH-Met-CO2Me ES-MS 708 (m+1)
49. (4-MePh)SO2-D-His-Tyr(OBn)-CONHCH2CH2CH2-
(2-MeO-Ph)
50. (4-MePh)SO2-D-His-Tyr(OBn)-CO(4-Bn-piperazin-
1-yl) ES-MS 720 (m)
51. MeO(CH2)3NHCO-D-His-Tyr(OBn)CONHCH2CH2Ph
52. BnNHCO-D-His-Tyr(oBn)cnN~oR~ ES-MS 647 (m+1)
53. BnNHCO-D-His-Tyr(OBn)CONHCH2CH2CH2OPh ES-~S 675 (m+l)
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54. BnNHCO-D-His-Tyr(OBn)CONH(CH2)3-
(2-MeO-Ph) ES-MS 689 (m+l)
55. MeO(CH2)3NHCO-D-His-Tyr(OBn)CONHCH2Ph
56. 1-napthyl-NHCO-D-His-Tyr(OBn)CONHCH2CH2OBn ES-MS 711 (m+l)
57. (4-ClPh)NHCO-D-His-Tyr(oBn)coNu~Rn
58. (4-ClPh)NHCO-D-His-Tyr(OBn)CONH-
Met-CO2Me BS-MS 707 (m+l)
59. 1-napthyl-NHCO-D-His-
Tyr(OBn)CONHCH2CH2CH2CH2Ph BS-MS 709 (m+1)
60. 1-napthyl-NHCO-D-His-Tyr(OBn)CONHCH2Ph
61. (4-EtOPh)NHCO-D-Hi 8 -
Tyr(OBn)CONHCH2CH2OBn ES-MS 705 (m+l)
62. BnOCO-D-His-Tyr(OBn)CONHCH2CH2SBn
63. (4-BtOPh)NHCO-His-
Tyr(OBn)CONHCH2CH2CH2OPh ES-MS 705 (m+l)
64. BnOCO-D-His-Tyr(OBn)CONHCH2CH2CH2CH2Ph
65. (4-EtOPh)NHCO-D-His-Tyr(OBn)CO(4-Bn-
piperazin-1-yl) ES-MS 729 (m)
66. PhCH2CO-D-His-Tyr(OBn)CONHCH2CH2Ph ES-MS 659 (m+1)
67. PhCH2CO-D-His-Tyr(OBn)CONHCH2CH2SBn ES-MS 676 (m+l)
68. PhCH2CO-D-His-Tyr(OBn)CONH-Met-CO2Me
69. PhCH2CO-D-His-Tyr(OBn)CONH(CH2)3-
(2-MeO-Ph) ES-MS 674 (m+l)
70. PhCH2CH2NHCO-D-His-Tyr(OBn)CO(4-Bn-
piperazin-1-yl) ES-MS 713 (m)
71. c-hexyl-NHCO-D-His-
Tyr(OBn)CONHCH2CH2Ph BS-NS 637 (m+l)
72. c-hexyl-NHCO-D-His-Tyr(oBn)CoN~nRn ES-NS 639 (m+l)
73. (t-2-Ph-c-propyl)-NHCO-D-His-
Tyr(OBn)CONH(CH2)30Ph ES-MS 701 (m+l)
74. (t-2-Ph-c-propyl)-NHCO-D-His-
Tyr(OBn)CONH(CH2)3-(2-MeO-Ph)
75. (t-2-Ph-c-propyl)-NHCO-D-His-
Tyr(OBn)CONHCH2Ph ES-NS 657 (m+l)
76. (CH3)2CUNUCO-D-His-
Tyr(OBn)CONHCH2CH2OBn ES-NS 627 (m+l)
77- (cH3)2cH~Hco-D-His-Tyr(oBn)c~N~oRn ES-MS 599 (m+l)
78. PhCH2CH2CO-D-His-Tyr(OBn)CONH-Met-CO2Me ES-MS 686 ~m+l)
79. (CH3)2CHNHCO-D-His-
Tyr(OBn)CONHCH2CH2CH2CH2Ph ES-~S 625 (m+l)
80. PhCH2CH2CO-D-His-Tyr(OBn)CONHCH2Ph ES-~S 630 (m+l)
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EXAMPLE 6
N-rN-rN- r (Phenylmethoxy)carbonyll-L-histidyl]-
O-(phenylmethyl)-L-tyrosyll-O-(phenylmethyl)-L-qerine,
methyl ester {Cbz-His-Tyr(OBn)-Ser(OBn)-CO2Me}
Step 1: Boc-Tyr(OBn)-Ser(OBn)-CO2Me
To a solution of Boc-Tyr(OBn) (1.88 g, 6.50 mmol)
in EtOAc ~30 mL) at 0C was added HOBT hydrate (1.19 g,
7.80 mmol) followed by DCC (1.61 g, 7.80 mmol). A
solution of Ser(OBn)-CO2Me TFA (2.1 g, 6.50 mmol) in
EtOAc (20 mL) was added followed by Et3N (1.09 mL,
7.80 mmol). The mixture was allowed to warm to room
temperature and stirred overnight. The mixture was
filtered, diluted with EtOAc, and washed twice with
saturated aqueous NaHC03, brine, dried over MgSO4, and
concentrated. Flash chromatography (40~ EtOAc/hexane)
gave 2.67 g (73~) of the title compound as a white
solid, mp 81-84C.
Step 2: Tyr(OBn)-Ser(OBn)-CO2Me TFA
Boc-Tyr(OBn)-Ser~OBn)-C02Me (from Step 1 above,
2.64 g, 4.69 mmol) was dissolved in CH2C12 (15 mL),
cooled to 0C and TFA (5 mL) was added. The ~olution
was warmed to room temperature and stirred for 4 hours.
The solution was concentrated, taken up in CH2Cl2 and
reconcentrated twice. The resulting oil was triturated
with ether to provide 2.7 g of the title compound as a
white solid.
Step 3: Cbz-His-Tyr(OBn)-Ser(OBn)-CO2Me
To a solution of Cbz-His (1.00 g, 3.47 mmol) in
DMF (15 mL) at 0C was added HOBT (0.64 g, 4.16 mmol)
and DCC (0.86 g, 4.16 mmol). Tyr(OBn)-Ser(OBn)-
CO2Me TFA (from Step 2 above, 2.0 g, 3.47 mmol) was
added followed by Et3N (0.58 mL, 4.16 mmol). The
mixture was allowed to warm to room temperature and
stirred overnight. The mixture was filtered and the
PCT~S94tllS53
WO95/12612
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filtrate was diluted with CHC13, washed twice with
saturated aqueous NaHCO3, brine, dried over MgSO4, and
concentrated. Flash chromatography (2-5~ MeOH/CHCl3)
gave 2.14 g of the title compound as a white solid,
mp 175-176C; FAB-MS 734 (m~1);
Anal. Calc. for C4lH43N58
C, 67.11; H, 5.91; N, 9.54;
Found: C, 66.96; H, 6.01; N, 9.41.
EXAMPLE 7
N-[N-rN-~(Phenylmethoxy)carbonyl]-L-histidyll-
O-(phenylmethyl)-L-tyrosyll-O-(phenylmethyl)-L-serine
{Cbz-His-Tyr(OBn)-Ser(OBn)}
To a solution of Cbz-His-Tyr(OBn)-Ser(OBn)-CO2Me
(from Example 6 above, 2.02 g, 2.75 mmol) in THF
(50 m~) and MeOH (15 mL) at 0C was added 0.lN LiOH
(30.3 mL, 3.03 mmol). The solution was stirred for
6 hours at 0C, then concentrated. Water was added and
the pH was adjusted to 4-5 with lN HCl. The mixture
was filtered, and the solid was collected and dried to
provide 1.55 g (78~) of the title compound as a white
solid, mp 187-192C; ES-MS 720 (m+1);
Anal. Calc. for C40H4lN58 1 5H2O:
C, 64.33; H, 5.94; N, 9.38;
Found: C, 64.29; H, 5.73; N, 9.15.
EXAMP~E 8
N-rN-[N-[(Phenylmethoxy)carbonyl]-D-histidyll-
O-(phenylmethyl)-L-tyrosyl]-O-(phenylmethyl)-L-serine,
methyl ester ~Cbz-D-His-Tyr(OBn)-Ser(OBn)-CO2Me}
Step 1: Tyr(OBn)-Ser(OBn)-CO2Me HCl
A solution of Boc-Tyr(OBn)-Ser(OBn)-CO2Me (from
Bxample 6, Step 1 above, 9.90 g, 17.6 mmol) in EtOAc
was cooled to 0C. Anhydrous HCl gas was bubbled
through the cold solution for 5 minutes. The solution
was allowed to warm to room temperature and stirred
PCT/US94/11553
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~ 21 70766
-41-
overnight. The solution was concentrated, taken up in
EtOAc and reconcentrated to provide 8.75 g of the title
compound as a foam; CI-MS 463 (m+1).
Step 2: Cbz-D-His(Tr)-Tyr(OBn)-Ser(OBn)-CO2Me
According to Example 6, Step 3, by substituting
Cbz-D-His(Tr) for Cbz-His and Tyr(OBn)-Ser(OBn)-
CO2Me HCl for Tyr(OBn)-Ser(OBn)-CO2Me TFA, the title
compound was obtained as a white solid, mp 78-88C;
FAB-MS 976 (m+1).
Step 3: Cbz-D-His-Tyr(OBn)-Ser(OBn)-CO2Me
A solution of Cbz-D-His(Tr)-Tyr(OBn)-Ser(OBn)-
CO2Me (from Step 2 above, 0.27 g, 0.28 mmol) in
HOAc:H2O (4:1, 2 mL) was stirred at 80C for 5 minutes,
then cooled to room temperature. The solution was
partitioned between EtOAc and saturated aqueous NaHCO3.
The organic layer was washed with brine, dried (MgSO4),
and concentrated. Flash chromatography (2-5~
MeOH:CHC13) yielded 0.10 g of the title compound as a
foam; FAB-MS 734 (m+l).
EXAMPLE 9
N- rN- rN- r (Phenylmethoxy)carbonyll-L-histidyll-O-
(phenylmethyl)-L-tyrosyll-O-(phenylmethyl)-D-~erine,
methyl ester {Cbz-His-Tyr(OBn)-D-Ser(OBn)-CO2Me}
According to Example 6, by substituting
D-Ser(OBn)-CO2Me TFA for Ser(OBn)-CO2Me-TFA in Step 1,
the title compound was obtained, mp 168-170C;
FAB-MS 734 (m+l).
PCT~S94/11553
WO9S/12612
z~3~ 42-
EXAMPLE 10
N-r~-Methyl-N-rN- r (phenylmethoxy)carbonyll-L-hi tidyll-
O-(phenylmethyl)-DL-tyrosyll-O-(phenylmethyl)-L-serine.
methyl ester {Cbz-His-DL-(~-Me)Tyr(OBn)-Ser(OBn)-CO
According to Example 6, by substituting Boc-DL-
(~-Me)Tyr(OBn) for Boc-Tyr(OBn) in Step 1, the title
compound was obtained; FAB-MS 748 (m+l).
EXAMPLE 11
N-Ethyl-N~-[N- rN- r (phenylmethoxy)carbonyl]-L-histidyll-
O-(phenylmethyl)-L-tyrosyll-O-(phenylmethyl)-h-
serinamide {Cbz-His-Tyr(OBn)-Ser(OBn)-CONHEt}
According to Example 6, by substituting
Boc-Tyr(OBn)-Ser(OBn)-CONHEt for Boc-Tyr(OBn)-
Ser(OBn)-CO2Me in Step 2, the title compound was
obtained, mp 182-188C; FAB-MS 747 (m+l).
EXAMPLE 12
N-Ethyl-N~- rN- rN- r (Phenylmethoxy)carbonyll-D-histidyll-
O-(phenylmethyl)-L-tyrosyll-O-(phenylmethyl)-
L-serinamide {Cbz-D-His-Tyr(OBn)-Ser(OBn)-CONHEt}
According to Example 6, by substituting Boc-
Tyr(OBn)-Ser(OBn)-CONHEt for Boc-Tyr(OBn)-Ser(OBn)-
CO2Me in Step 2 and Cbz-D-His for Cbz-His in Step 3,
the title compound was obtained, mp 193-196C;
ES-MS 747 (m+l).
EXAMPLE 13
N-rN-rl-Methyl-N- r (phenylmethoxy)carbonylll-L-
histidyll-
O-(phenylmethyl)-L-tyrosyll-O-(phenylmethyl)-L-Serine~
methyl ester {Cbz-His(l-Me)-Tyr(OBn)-Ser(OBn)-CO2Me}
Step 1: Cbz-His(l-Me)
Benzyl chloroformate (0.24 mL, 1.7 mmol) was added
dropwise to a slurry of l-methyl-~-histidine (0.25 g,
WO9S/12612 P~T~S94111553
~ 21 70766
1.5 mmol) in THF (5 mL) and saturated aqueous NaHCO3
(5 mL) at 0C. The mixture was allowed to warm to room
temperature and stirred overnight. The mixture was
concentrated and diluted with H2O, washed with ether,
and the pH adjusted to 6-7 with lN HCl. The mixture
was concentrated, then diluted with CHCl3 (150 mL) and
MeOH (15 mL), and stirred for 1 hour. The mixture was
dried (MgSO4) and concentrated to pro~ide 0.48 g of the
title compound which was used without further
purification.
Step 2: Cbz-His(1-Me)-Tyr(OBn)-Ser(OBn)-CO2Me
To a slurry of Cbz-His(1-Me) (from Step 1 abo~e,
0.36 g, 1.2 mmol), Tyr(OBn)-Ser(OBn)-CO2Me HCl (from
Example 8, Step 1 above, 0.60 g, 1.2 mmol), DCC
(0.30 g, 1.4 mmol), and HOBT (0.19 g, 1.4 mmol) in
CH2Cl2 was added Et3N (0.17 mL, 1.2 mmol) and the
mixture was stirred overnight at room temperature. The
mixture was diluted with CHCl3, washed with saturated
aqueous NaHCO3, brine, dried (MgSO4), and concentrated.
Flash chromatography (1~ MeOH:CHCl3) provided the title
compound as a white solid, mp 161.5-163.5C; FA~3-MS 748
(m+1).
EXAMPLB 14
N-~M-rl-Methyl-N- r (phenylmethoxy)carbonyll-D-histidyll-
O-(phenylmethyl)-L-tyrosyl]-O-(~henylmethyl)-L-serine.
methyl ester {Cbz-D-His(l-Me)-Tyr(OBn)-Ser(OBn)-CO2Me}
According to Example 13, by substituting 1-methyl-
D-histidine for 1-methyl-L-histidine, the title
compound was obtained; FAB-MS 748 (m+1).
WO9S/12612 PCT~S94/11553
.
44-
EXAMPLE 15
N-[L-2-Amino-N - rN - r ( Phenylmethoxy)carbonyll-D-
histidyll-4-r4-(phenylmethoxy)phenyllbutanoyll-0-
(phenylmethyl-L-serine methyl ester {Cbz-D-His-
homoTyr(OBn)-Ser(OBn)-CO2Me}
According to Example 6, by substituting Boc-
homoTyr(OBn) for Boc-Tyr(OBn) in Step 1, and
substituting Cbz-D-His for Cbz-His in Step 3, the title
compound was obtained; ES-MS 748 (m+l).
EXAMPLE 16
N-r4-Phenyl-N-rN- r (phenylmethoxy)carbonyll-L-histidyll-
L-phenylalanyll-O-(phenylmethyl)-L-serine methyl ester
{Cbz-His-Phe(4-Ph)-Ser(OBn)-CO2Me}
According to Example 6, by substituting Boc-
Phe(4-Ph) for Boc-Tyr(OBn) in Step 1, the title
compound was obtained, mp 184-187C; FAB-MS 704 (m+l).
EXAMPLE 17
N- ro- (Phenylmethyl)-NrN- r r (Phenylmethyl)aminol-
carbonyll-L-histidyll-L-tyrosyll-O-(phenylmethyl)-
serine, methyl ester {BnNHCO-His-Tyr(OBn)-Ser(OBn)-
C02Me }
Step 1: Fmoc-His(Tr)-Tyr(OBn)-Ser(OBn)-C02Me
According to Example 13, Step 2, by substituting
Fmoc-His(Tr) for Cbz-His(l-Me), the title compound was
obtained, mp 82-92C.
Step 2: His(Tr)-Tyr(OBn)-Ser(OBn)-C02Me
Piperidine (4.0 mL) was added to a slurry of
Fmoc-His(Tr)-Tyr(OBn)-Ser(OBn)-CO2Me (from Step 1
above, 1.85 g, 1.74 mmol) in CH2C12 (20 mL). The
solution was stirred for 2 hours at room temperature,
then concentrated. The residue was taken up in EtOAc
(150 mL), washed with water (3 x 50 mL), dried (MgS04),
and concentrated. The resulting oil was triturated
W O 95/12612 2 PCTrUS94/11553
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with Et2O/hexane. Flash chromatography of the residue
(2~ MeOH/CHC13) gave 1.03 g o~ the title compound as a
foam, mp 61.5-70C; ES-MS 843 (m+1).
Ste~ 3: BnNHCO-His(Tr)-Tyr(OBn~-Ser(OBn)-CO2Me
Benzyl isocyanate (0.053 mL, 0.43 mmol) was added
in one portion to a solution of His(Tr)-Tyr(OBn)-
Ser(OBn)-C02Me (~rom Step 2 above, 0.33 g, 0.39 mmol)
in EtOAc (5 mL). The resulting slurry was stirred for
3 hours at room temperature, then concentrated to yield
the title compound (0.4 g), which was used without
fur~her purification.
Step 4: BnNHCO-His-Tyr(OBn)-Ser(OBn)-CO2Me
According to Example 8, by substituting BnNHCO-
His(Tr)-Tyr(OBn)-Ser(OBn)-CO2Me for Cbz-His(Tr)-
Tyr(OBn)-Ser(OBn)-CO2Me, the title compound was
obtained, mp 19 6.5- 199 C; ES-MS 733 (m+1).
EXAMPLE 18
N-rN-rN-(1-Oxo-3-phenylpropvl)-L-histidyll-O-
(phenYlmethyl)-L-tyrosyl]-O-(phenylmethyl)-L-serine
methyl ester {PhCH2CH2CO-His-Tyr(OBn)-Ser(OBn)-C02Me}
Step 1: PhCH2CH2CO-His(Tr)-Tyr(OBn)-Ser(OBn)-CO2Me
To a cooled (0C) solution of His(Tr)-Tyr(OBn)-
Ser(OBn)-CO2Me (from Example 17, Step 2 above, 0.33 g,
0.39 mmol) in THF (5 mL) was added Et3N (O. 06 mL,
0.43 mmol) followed by phenylpropionyl chloride
(0.064 mL, 0.43 mmol). The resulting slurry was
brought to room temperature and stirred overnight. The
mixture was partitioned between EtOAc and saturated
aqueous NaHCO3. The organic layer was washed with
brine, dried (MgSO4), and concentrated to yield the
title compound as a solid which was u~ed without
~urther purification.
wossll26l2 pcT~s9~ ss3
.
~1 31 ~ -46-
Step 2: PhCH2CH2CO-His-Tyr(OBn)-Ser(OBn)-CO2Me
According to Example 8, Step 3, by substituting
PhCH2CH2CO-His(Tr)-Tyr(OBn)-Ser(OBn)-CO2Me for Cbz-D-
His(Tr)-Tyr(OBn)-Ser(OBn)-C02Me, the title compound was
obtA;ne~, mp 193-196.5C; ES-MS 732 (m+l).
EXAMPLE 19
~-rN-~(Phenylmethoxy)carbonyll-L-histidyl]-
O-(~henylmethyl)-N-r2-(phenylmethoxy)ethyll-
~-tyrosinamide {Cbz-His-Tyr(OBn)-CONHCH2CH20Bn}
Step 1: Cbz-His-Tyr(OBn)-CO2Me
According to Example 6, Step 3, by substituting
Tyr(OBn)-CO2Me TFA for Tyr(OBn)-Ser(OBn)-CO2Me TFA, the
title compound was obtained as a white powder,
mp 145-148C; CI-MS 557 (m+l).
Step 2: Cbz-His-Tyr(OBn)
According to Example 7, by substituting Cbz-His-
Tyr(OBn)-CO2Me ~or Cbz-His-Tyr(OBn)-Ser(OBn)-CO2Me, the
title compound was obtained as a white powder,
mp 79-92C; CI-MS 543 (m+l).
Step 3: Cbz-His -Tyr(OBn)-CONHCH2CH20Bn
To a solution of Cbz-His-Tyr(OBn) (from Step 2
above, 0.43 g, O.79 mmol) in DMF (4 mL) at 0C was
added HOBT (0.15 g, 0.95 mmol) and DCC (0.20 g,
0.95 mmol). A solution of 2-(phenylmethoxy)ethylamine
(0.12 g, 0.79 mmol) in DMF (1 mL) was then added. The
mixture was allowed to warm to room temperature and
stirred overnight. The mixture was filtered, diluted
with CHCl3, washed twice with saturated aqueous NaHC03,
washed with brine, dried over MgSO4, and concentrated.
Flash chromatography (3-5~ MeOH/CHC13) afforded 0.34 g
(63~) of the title compound as a white solid,
mp 136-150C; FAB-MS 676 (m+l);
WO 95/12612 2 1 7 0 l 6 6 PCTIUS94/11553
-47-
Anal. Calc. :Eor C39H41N506:
C, 69.32; H, 6.12; N, 10.36;
Found: C, 69.43; H, 6.24; N, 10.45.
EXAMPLE 20
~- rN- r (Phenylmethoxy) carbonyl]-D-histidyl]-N-
r 2-(phenylmethoxy)ethyll-O-(phenylmethyl)-L-
tyrosin~nide {cbz-D-His-Tyr(oBn)-cor~HcH2~H2c:)Bn}
According to Example 6, by substituting 2- (phenyl-
methoxy)ethylamine for Ser(OBn) TFA and omitting Et3N
in Step 1 and by substituting Cbz-D-His for Cbz-His in
Step 3, the title compound was prepared, mp 161-165C;
FAB-MS 676 (m+1).
EXAMPLE 21
~a~ rN-Methyl-Nr(phenylmethoxy)carbonyl]-D-histidyll-N-
r2-~phenylmethoxy)ethyll-0-(phenylmethyl)-L-
tyrosinamide {Cbz-D-His-(N-Me)Tyr(OBn)-CONHCH2CH20Bn}
According to Example 20, by substituting Boc-
(N-Me)Tyr(OBn) for Boc-Tyr(OBn), the title compound was
obtained, mp 64-78C; ES-MS 690 (m+l).
EXAMPBE 22
~a-rcY-Methyl-N-rN-~(~henylmethoxy)carbonyll-
D-histidyll-N-r2-(phenylmethoxy)ethyll-O-(phenyl-
methyl)-L-tyrosin~mide {Cbz-D-His-(cY-Me)Tyr(QBn)-
CONHCH2CH2OBn}
According to Example 20, by substituting Boc-
(cY-Me)Tyr(OBn) for Boc-Tyr(OBn), the title compound was
obtained, mp 66-78C; ES-MS 690 (m+1).
W095/12612 PCT~S91111553
EXAMPLE 23
N-(2-Phenylethyl~-N~-[N- r (phenylmethoxy)carbonyll-
T-histidyll-O-(phenylmethyl)-L-tyrosinamide
{Cbz-His-Tyr(OBn)-CONHCH2CH2Ph}
According to Example 19, Step 3, by substituting
2-phenylethylamine for 2-(phenylmethoxy)ethylamine, the
title compound was obtained as a white solid,
mp 188-189.5C; FAB-MS 646 (m+1).
EXAMPLE 24
N-rN-rN-r(Phenylmethoxy)carbonyll-L-histidyll-
O-(phenylmethyl)-L-tyrosyll-3-(3-pyridinyl)-L-alanine,
methyl ester {Cbz-His-Tyr(OBn)-Pyr-CO2Me}
According to Example 19, Step 3, by substituting
Pyr-CO2Me for 2-(phenylmethoxy)ethylamine, the title
compound was obtained as a white solid, mp 180-182.5C
(dec); FAB-MS 705 (m+1).
EXAMPLE 25
(S.R)-N- r 2-(4-Benzyloxy-phenyl)-1-(3-phenoxy-
propylcarbamoyl)-ethyll-2-r3-(4-ethoxy-phenyl)-
ureidol-3-(3H-imidazol-4-yl)-propionamide
{(4-EtOPh)NHCO-D-His-Tyr(OBn)-CONH(CH2)30Ph}
Step 1. Boc-Tyr(OBn)-CONH(CH2)30Ph
2-(Phenylmethoxy)ethylamine (0.81 g, 5.4 mmol) was
added to a premixed solution of EDAC (1.2 g, 6.5 mmol),
HOBT (0.87 g, 6.5 mmol), and Boc-Tyr(OBn)-OH (2.0 g,
5.4 mmol) in dry DMF (15 mL). The resulting mixture
was stirred for 18 hours at room temperature. The
solution was diluted with 1:1 EtOAc:Et20 (40 mL),
washed with saturated aqueous NaCl (4 x 10 mL), dried
(MgSO4), filtered, and concentrated in vacuo to provide
a solid which was further purified by trituration with
h~n~ to give the pure product, mp 145-146-C.
WO95/12612 PCT~S94/11553
21~0766
-49-
Step 2. Tyr(OLn)-CONH(CH2)3OPh
Dry HCl gas was bubbled into an ice cold ~olution
of Boc-Tyr(OBn)-CONH(CH2)3OPh (from Step l above,
2.0 g, 3.9 mmol) in MeOH (15 mL) for 4 minutes. The
resulting mixture was stirred for l hour at 0 C and
then allowed to warm to room temperature and stir
l hour. The solution was concentrated in vacuo to
provide a solid which was triturated with ether to
provide Tyr(OBn)-CONH(CH2)3OPh HCl; CI-MS 405 (m+l).
The title compound was suspended in CHCl3, cooled in an
ice bath, and NH3 gas was bubbled through the mixture
for 2 minutes. The NH4Cl was filtered off, and the
supernate was concentrated in vacuo to yield the free
base of the title compound which was used in the next
step without further purification.
Step 3. Fmoc-D-His(Tr)-Tyr(OBn)-CONH(CH2)3OPh
To a solution of HOBT (0.48 g, 3.5 mmol) in DMF
(10 m~) was added Fmoc-D-His(Tr)-CO2H (2.0 g, 3.2 mmol)
followed by EDAC (0.67 g, 3.5 mmol). The mixture was
stirred at room temperature for 20 minutes before
adding a solution of Tyr(OBn)-CONH(CH2)3OPh (from
Step 2 above, 1.4 g, 3.2 mmol) in DMF (l0 mL). The
mixture was stirred overnight at room temperature
before partitioniny between a mixture of water (20 mL)
and l:l Et2O:EtOAc (50 mL). The layers were separated,
and the organic phase was washed with saturated aqueous
NaCl (4x 20 mL) and dried (MgSO4). Filtration and
concentration in vacuo provided an oil which was
further purified by flash chromatography (SiO2, CHCl3:
MeOH eluent) to give the protected His-Tyr dipeptide;
FAB-MS 1006 (m).
Step 4. D-His(Tr)-Tyr(OBn)-CONH(CH2)3OPh
Fmoc-D-His(Tr)-Tyr(OBn)-CONH(CH2)30Ph (from Step 3
above, 1.O g, 0.99 mmol) in CH2C12 (5 mL) was treated
PCT~S94/1l553
Wo9SI12612
so- '
with piperidine (0.18 g, 2.1 mmol). The resulting
mixture was stirred 2 hours before concentrating
in vacuo and purifying the resulting oil by flash
chromatography (SiO2, CHC13:MeOH eluent) to give
(4-EtOPh)NHCO-D-His(Tr)-Tyr(OBn)-CONH(CH2)30Ph;
ES-MS 784 (m+l).
Step 5. (4-EtOPh)NHCO-D-His(Tr)-Tyr(OBn)-CONH(CH2L30Ph
D-His(Tr)-Tyr(OBn)-CONH(CH2)30Ph (from Step 4
above, 0.55 g, 0.7 mmol) in CH2Cl2 (5 mL) was treated
with 4-ethoxyphenyl isocyanate (0.1 g, 0.7 mmol). The
resulting mixture was stirred 1 hour at room
temperature. Concentrated in vacuo. The resulting oil
was purified by flash chromatography (SiO2, CHC13:MeOH
eluent) to give (4-EtOPh)NHCO-D-His(Tr)-Tyr(OBn)-
CONH(CH2)30Ph; ES-MS 947 (m+1).
Step 6. (4-EtOPh)NHCO-D-His-Tyr(OBn)-CONH(CH2L30Ph
(4-EtOPh)NHCO-D-His(Tr)-Tyr(OBn)-CONH(CH2)30Ph
(from Step 5 above, 0.5 g, 0.52 mmol) in MeOH (5 mL)
was treated with Pyridine HCl (catalytic). The
resulting mixture was stirred at 65 C for 6 hours.
Concentrated in vacuo to obtain an oil which was
purified by by flash chromatography (SiO2, CHCl3:MeOH
eluent) to give (4-EtO-Ph)NHCO-D-His-Tyr(OBn)-
CONH(CH2)30Ph, mp 185-187 C; ES-MS 705 (m+1).
EXAMPLE 26
(S.R)-N-{2-(4-Benzyloxy-phenyl~-1-r3-(2-methoxy-
phenyl)-propylcarbamoyll-ethyl}-3-(3H-imidazol-4-yl)-
2-phenylacetylamino-propionamide {PhCH2CO-D-His-
Tyr(OBn)- CONH(CH2L3(2-MeOPh)}
Step 1. Boc-Tyr(OBn)-CONH(CH2)3(2-MeOPh)
According to Example 25, Step 1, by substituting
3-(2-methoxyphenyl) propyl amine for 2-(phenylmethoxy)-
PCT/US94/1 1553
WO 95/12612 ~ 1 ~ O ~ ~ 6
-51-
ethylamine, the title compound was obtained as a white
solid, mp 125-126.5-C.
.
Stel? 2. Tyr(OBn)-CONH(CH2)3(2-MeOPh)
According to Example 25, Step 2, by substituting
Boc-Tyr(OBn)-CONH(CH2)3(2-MeOPh) for Boc-Tyr(OBn)-
CONH(CH2)30Ph, the title compound was obtained as a
white solid; CI-MS 419 (m+1).
Step 3. Fmoc-D-His(Tr)-Tyr(OBn)-CQNH(CH2)3(2-MeOPh)
According to Example 25, Step 3, by substituting
Tyr~OBn)-CONH(CH2) 3 ( 2-MeOPh) for Tyr(OBn)-
CONH(CH2)30Ph, the title compound was obtained as a
foam; ES-MS 1020 (m).
Step 4. D-His(Tr)-Tyr(OBn)-CONH(CH2)3(2-MeOPh)
According to Example 25, Step 4, by substituting
Fmoc-D-His(Tr)-Tyr(OBn)-CONH(CH2)3(2-MeOPh) for Fmoc-D-
His(Tr)-Tyr(OBn)-CONH(CH2)30Ph, the title compound was
obt~;ne~ as a white foam; ES-MS 798 (m+1).
Step 5. PhCH2CO-D-His(Tr)-Tyr(OBn)-CONH(CH2) 3 (2-MeOPh)
To a solution of D-His(Tr)-Tyr(OBn)-
CONH(CH2)3(2-MeOPh) (0.4 g, 0.5 mmol) in CH2Cl2 (5 mL)
was added N-methyl morpholine (0.05 g, 0.5 mmol)
followed by phenyl acetyl chloride (0.08 g, 0.5 mmol).
The resulting mixture was stirred 2 hours at room
temperature. Diluted with DCM and washed with
saturated aqueous NaHC03, saturated aqueous NaCl, and
dried (MgSO4). Purified by flash chromatography (SiO2,
CHCl3:MeOH eluent). The title compound was obtained as
a foam; FAB-MS 916 (m).
Step 6. PhCH2CO-D-His-Tyr(OBn)-CONH(CH2) 3 (2-MeOPh)
According to Example 25, Step 6, by substituting
PhCH2CO-D-His(Tr)-Tyr(OBn)-CONH(CH2) 3 (2-MeOPh) for
WO9S/12612 PCT~S94111S53
.
52-
(4-EtOPh)NHCO-D-His(Tr)-Tyr(OBn)-CONH(CH2)30Ph, the
title compound was obtained as a white foam; ES-MS 674
(m+1).
EXAMPLE 27
(S,R)-N-r2-(4-Benzyloxy-phenyl)-1-phenethylcarbamoyl-
ethyl]-3-(3H-imidazol-4-yl)-2-r3-(4-phenoxy-phenyl)-
ureidol-propionamide {(4-PhOPh)NHCO-D-His-Tyr(OBn)-
CoNH(cH2L2ph}
Step 1. Boc-Tyr(OBn)CONH(CH2)2Ph
According to Example 25, Step 1, by substituting
phenethylamine for 2-(phenylmethoxy)ethylamine, the
title compound was obtained as a white solid; CI-MS 475
(m+1).
Step 2. Tyr(OBn)-cONH(cH2L2Ph
According to Example 25, Step 2, by substituting
BocTyr(OBn)-CONH(CH2)2Ph for BocTyr(OBn)-CONH(CH2)30Ph,
the title compound was obtained as a white solid;
CI-MS 375 (m+1).
Step 3. Fmoc-D-His(Tr)-Tyr(OBn?-CONH(CH2L2Ph
According to Example 25, Step 3, by substituting
Tyr(OBn)-CONH(CH2)2Ph for Tyr(OBn)-CONH(CH2)30Ph, the
title compound was obtained as a foam; ES-MS 977 (m+1).
Step 4. Fmoc-D-His-Tyr(OBn)-CONH(CH2L2Ph
Fmoc-D-His(Tr)-Tyr(OBn)-CONH(CH2)2Ph (1.2 g,
1.6 mmol) in MeOH (5.mL) was treated with Pyridine HCl
(catalytic). The resulting mixture was stirred at 65 C
overnight and concentrated in vacuo to obtain an oil
which was purified by by flash chromatography (SiO2,
CHC13:MeOH eluent) to give a white solid; ES-MS 734
(m+1).
PCT~S94111553
W09S/12612
~t~ 66
-53-
Step 5. (4-phoph)NHco-D-His-Tyr(oBn)-coNH(cH2L2ph
Fmoc-D-His-Tyr(OBn)-CONH(CH2)2Ph (0.6 g, O.8 mmol)
in CH2C12 (5 mL) was treated with piperdine (0.14 g,
1.6 mmol). The resulting mixture was stirred 2 hours
- 5 before concentrating in vacuo and purifying the
resulting by flash chromatography (SiO2, CHC13:MeOH
eluent) to give D-His-Tyr(OBn)-CONH(CH2)2Ph. The foam
was ~issolved in CH2Cl2 (5 mL) and treated with
4-phenoxyphenyl isocyanate (0.05 g, 0.23 mmol). The
resulting mixture was stirred 1 hour at room
temperature, concentrated in vacuo, and purified the
re~u:Lting oil by flash chromatography (SiO2, CHCl3:MeOH
eluent) to obtain (4-PhOPh)NHCO-D-His-Tyr(OBn)-
CONH(CH2)2Ph as a foam; ES-MS 723 (m+1).
EXAMPLE 28
(S,R)-N-rl-(4-Benzyloxy-benzyl)-2-(4-benzyl-piperazin-
1-yl)-2-oxo-ethyl]-3-(3H-imidazol-4-yl)-2-(toluene-
4-sulfonylamino)-propionamide {(4-MePh)SO2-D-His-
Tyr(OBn)-CO(4-Bn-piperazin-1-yl) HCl}
Step 1. Boc-Tyr(OBn)-CO(4-Bn-piperazin-1-yl)
According to Example 25, Step 1, by substituting
l-benzylpiperizine for 2-(phenylmethoxy)ethylamine, the
title compound was obtained as a white solid;
CI-MS 530 (m+l).
Step 2. Tyr(OBn)-CO(4-Bn-piperazin-1-yl)
According to Example 25, Step 2, by substituting
Boc-Tyr(OBn)-CO(4-Bn-piperazin-1-yl) for Boc-Tyr(OBn)-
CONH(CH2)30Ph, the title compound was obtained as a
white solid; CI-MS 430 (m+l).
WO95/12612 PCT~S9~111553
-54-
Step 3. Fmoc-D-His(Tr)-Tyr(OBn)-CO(4-Bn-pi~erazin-
l-yl)
According to Example 25, Step 3, by substituting
Tyr(OBn)-CO(4-Bn-piperazin-1-yl) ~or Tyr(OBn)-
CONH(CH2)30Ph, the title compound was obt~;ne~ as a
foam; ES-MS 1032 (m+1).
Step 4. (4-MePh)SO2-D-His(Tr)-Tyr(OBn)-CO(4-Bn-
piperazin-1-yl)
Fmoc-D-His-Tyr(OBn)-CONH(CH2)30Ph, (0.7 g,
0.69 mmol) in CH2C12 (5 mL) was treated with piperidine
(0.14 g, 1.6 mmol). The resulting mixture was stirred
2 hours be~ore concentrating in vacuo and purifying the
resulting oil by flash chromatography (SiO2, CHC13:MeOH
eluent) to give D-His-Tyr(OBn)-CONH(CH2)2Ph. The ~oam
was dissolved in CH2C12 (5 mL) and treated with
pyridine (0.05 g, 0.63 mmol), followed by
4-toluenesulfonyl chloride (0.12 g, 0.63 mmol). The
resulting mixture was stirred 3 hours at room
temperature, concentrated in vacuo, and purified the
resulting oil by flash chromatography (SiO2, CHC13:MeOH
eluent) to obtain (4-MePh)SO2-D-His(Tr)-Tyr(OBn)-
CO(4-Bn-piperazin-1-yl); ES-MS 963 (m).
5 Step 5. (4-MePh)SO2-D-His-Tyr(OBn)-CO(4-Bn-piperazin-
1-yl) HCl
(4-MePh)SO2-D-His(Tr)-Tyr(OBn)-CO(4-Bn-piperazin-
l-yl) (0.21 g, 0.22 mmol) was treated with 80~ aqueous
HCl (3 mL) and heated to 80 C for 5 minutes. The
mixture was cooled and diluted with water (5 mL). The
solid was ~iltered of~, and the supernate was
concentrated in vacuo to provide an oil. The oil was
dissolved in water (15 mL), frozen, and lyophilized to
provide (4-MePh)SO2-D-His-Tyr(OBn)-CO(4-Bn-piperazin-
l-yl) HCl; ES-MS 720 (m).
W095/12612 2 ~ 7 0 7 6 6 PCT~S94/11553
.
-55-
EXAMPLE 29
N- r (Phenylmethoxy)carbonyll-L-histidyl]-O-
(~henylmethyl)-N-methyl-N-[2-(phenylmethoxY)ethyll-
L-tyrosinamide {Cbz-His-Tyr(OBn)-CON(Me)CH2CH20Bn}
According to Example 19, Step 3, by substituting
N-methyl-N-[2-(phenylmethoxy)ethyl]amine for
2-(phenylmethoxy)ethylamine, the title compound was
prepared; FAB-MS 690 (m+1).
The present invention may be embodied in other
spec:ific forms without departing from its ~pirit or
essential characteristics. The described embodiments
are to be considered in all respects only as
illustrative and not restrictive. The scope of the
invention is, therefore, indicated by the appended
claims rather than by the foregoing description. All
changes which come within the me~nlng and range of
equivalency of the claims are to be embraced within
their scope.
,
