Note: Descriptions are shown in the official language in which they were submitted.
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8-HYDROCARBYL SUBSTITUTED BEN~ODIZOC~JE DERlVAl'rVES, THED~ PREPARAT~ON
AND THElR USE AS PROTEIN KINASE C ('PKC) MODULAlORS
~ 5 Priority of Invention
This application claims priority from U.S. Provisional
Application Number 60/017,532, filed May 10, 1996.
Background of the Invention
Protein kinases serve a regulatory function which is crucial for all
aspects of cellular development, differentiation and transformation. One of the
largest gene families of non-receptor serine-threonine protein kinases is protein
kinase C (PKC). Since the discovery of PKC more than a decade ago by
Ni.~hi7l-k~ and coworkers (Kikkawa et al., J. Biol. Chem., 257, 13341 (1982)),
and its identification as a major receptor for phorbol esters (Ashendel et al.,
Cancer Res.~ 43, 4333 (1983)), a multitude of physiological sign~ling
mech~ni~m~ have been ascribed to this enzyme. The intense interest in PKC
stems from its unique ability to be activated in vitro by diacylglycerol (and its
phorbol ester mimetics), an effector whose formation is coupled to phospholipid
turnover by the action of growth and differentiation factors.
The PKC gene family consists presently of 11 genes which are
divided into four subgroups: 1) classical PKCa"B" ~2(~1 and ~2 are alternately
spliced forms of the same gene) and y, 2) novel PKC~ , and ~, 3) atypical
PKC~ and I and 4) PKC~l.PKC~ resembles the novel PKC isoforms but
differs by having a putative transmembrane domain (reviewed in Blobe et al.,
Cancer Metast. Rev., 13, 411 (1994)); Hug et al., Biochem J., 291, 329 (1993);
Kikkawa et al., Ann. Rev. Biochem, 58, 31 (1989)) (Figure 1). The a,~"~ and
~ isoforms are Ca2+, phospholipid- and diacylglycerol-dependent and represent
the classical isoforms of PKC, whereas the other isoforms are activated by
phospholipid and diacylglycerol but are not dependent on Ca2+. All isoforms
encompass 5 variable (Vl-V5) regions and the a,~ and ~ isoforms contain four
(C 1 -C4) structural domains which are highly conserved. All isoforms except
PKCa"B, and ~ lack the C2 domain, and the ~, ~ and I isoforms also lack one of
,
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two cysteine-rich zinc finger domains in C 1 to which diacylglycerol binds. The
C1 domain also contains the pseudo substrate sequence which is highly
conserved among all isoforms, and which serves an autoregulatory function by
blocking the substrate-binding site to produce an inactive conformation of the
5 enzyme (House et al. Science. ~, 1726 (1987)).
Because of these structural features, diverse PKC isoforms are
thought to have highly specialized roles in signal transduction in response to
physiological stimuli (Ni~hi71lk~ Cancer, 10, 1892 (1989)), as well as in
neoplastic transformation and differentiation (Glazer, Protein Kinase C. J. F.
10 Kuo, ed., Oxford U. Press (1994) at pages 171-198).
From a pharmacological perspective, PKC has served as a focal
point for the design of anticancer drugs (Gescher, Brit. J. Cancer~ 66, 10
(1992)). Antisense expression of either the PKCa cDNA (Ahmad et al.,
Neurosur~ery, 35, 904 (1994)) or a phosphorothioate oligodeoxynucleotide (S-
15 oligo) for PKCa has shown the efficacy of targeting PKC to inhibit theproliferation of A549 lung carcinoma cells (Dean et al., J. Biol. Chem.. 269,
16416 (1994)) and U-87 glioblastoma cells. Similar studies have not been
conducted with breast tumors, but historical and preliminary data suggest that
PKC is a logical molecular target by which to inhibit tumor growth and/or
induce apoptosis. However, it is not clear which isoforms are most crucial for
tumor proliferation and what role different PKC isoforms play in such critical
cellular processes as cell proliferation and apoptosis. Nonetheless, it is
reasonable to conclude that isoform selective, non-tumor promoting modulators
of PKC that cause downregulation may find use in cancer treatment through the
initiation of cancer cell death through apoptosis. Selective cancer cell killingmay be achieved either through the targeting of those isoforms found to be
overexpressed in the cancer cells, or through the synergistic interaction of a
cytotoxic drug like 1-,B-D-arabinofuranosylcytosine with an appropliate PKC-
based signaling interceptor.
Teleocidin was first isolated from the mycelia of Streptomyces
mediocidicus as a mixture of highly toxic compounds by T~k~h~hi et al., Bull.
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Agr. Chem. Soc. Japan~ 24, 647 (1960). The structure of one of these
metabolites was assigned by Hirata as shown by Figure 2, formula 1. The
lyngbyatoxin series can be obtained together with the teleocidin B group from
Streptomyces mediocidicus as disclosed by S. Sakai et al., Tetrahedron Lett.~ 27,
5 5219 (1986). Therefore, as depicted in ~igure 2, they were named as teleocidinA-1 (2a) and A-2 (2b) by Sakai. Indolactarn V (3, ILV), which contains the
basic ring structure of the teleocidins, is the simplest member of the family, and
is produced in large quantities by actinomycetes strain NA34-17 (Figure 2).
Investigations with 1 2-O-tetradecanoylphorbol- 1 3-acetate (TPA)
10 have provided considerable information on tumor promotion. In the two stage
model of skin carcinogenesis, it is believed that initiators bind to DNA and that
tumor promoters such as TPA bind non-covalently to membrane-associated high
affinity receptors, most likely protein kinase C. Thus, TPA, the teleocidins, and
the Iyngbyatoxins as well as aplysiatoxin serve as diacylglycerol mimics,
15 binding to the diacylglycerol site of protein kinase C, thus activating the kinase.
Indeed, computer assisted molecular modeling studies of these
tumor promoters have revealed a commonality of their hydrophobic regions and
certain heteroatoms. On the basis of both solution NMR studies and molecular
mechanics calculations, it was additionally reported that the indolactam portion20 (indolactam V, 3) of the teleocidins and Iyngbyatoxins can exist in two
conformational states, the sofa or twist-like conformations. At equilibrium, theratio of twist/sofa was 2.8; the twist form of ILV represents the biologically
active conformation.
Compounds related to the teleocidins are disclosed in
25 Kozikowski, A. et al. Journal of the American Chemical Society. 1993, 1 15,
3957-3965; in PCT Application WO/95-09,160 (1995); in Endo Y. et al. Journal
of the American Chemical Society. 1996, 1 18, 1841-1855; and in Endo Y. et al.
Chem. Pharm. ~ull. 1997, 45, 424-426. However, a continuing need exists for
novel compounds which can selectively modulate PKC so as to effect the
30 selective killing of cancer cells.
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Sulllnl~r of the Invention
The present invention provides certain benzolactam PKC
modulators, which exhibit PKC isoform selectivity. The compounds are of
general formula (I):
Y
~ . OH
Z-N o ~ (I)
~ _--'R3
ll 71
~R1
R2
wherein
R, is H, (C,-C5)alkyl, ORa, SRa, N(Ra)(Rb), halo, NO2,
NHC(O)[(C,-C4)alkyl] or NHOH;
R2 is a (C5-C22) hydrocarbyl group, optionally conlplising 1-3
20 double bonds, 1-2 triple bonds or a mixture thereof, or (C6-C,2)aryl(C2-C,0)alkyl,
wherein the alkyl moiety optionally comprises 1-2 double bonds, 1-2 triple
bonds or a ~ e thereof; wherein said (C5-C22) hydrocarbyl group or said (C6-
C~2)aryl(C2-CI0)alkyl may optionally be substituted with 1 or 2 substituents
independently selected from the group con~i~ting of halo, hydroxy, cyano, nitro,25 (C,-C5)alkyl, (C~-C5)alkoxy, trifluoromethyl, trifluoromethoxy, -C(=O)O(C,-
C5)alkyl, and N(Re)(Rf);
R, and R2 together are -CH(RC)-CH2-C(O)-N(Rd)-, -C(RC)=CH-
C(O)N(Rd)-, -C(RC)=CH-N(Rd)- or-C(RC)=CH-O-;
R3 is H, OH or halo;
~ 30 Ra and Rb are independently H or (C,-C5)alkyl;
Rc is a (C5-C22) hydrocarbyl group;
~ ,
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Rd is H or (C,-C5)alkyl;
Re and Rf are independently hydrogen, (Cl-C5)alkyl, or
(C,-C5)alkanoyl, or together with the nitrogen to which they are attached are
pyrrolidino, piperidino or morpholino;
Z is H or (C,-C5)alkyl; and
Y is H or (C,-Cs)alkyl;
or a ph~ ceutically acceptable salt thereof.
Pharmaceutical compositions comprising an amount of one or
more compounds of formula (I) effective to treat m~mm~ n conditions
associated with pathological cellular proliferation, particularly human cancers,such as solid tumors and leukemias, are also an embodiment of the invention.
The present invention also provides a method to inhibit the pathological
proliferation of m~mm~ n cells, such as cancer cells, by ~-lmini.etering to a
m~mm~l afflicted with such a condition, an effective inhibitory amount of one ormore of the compounds of forrnula I, preferably formulated as said
pharmaceutical composition, i.e., in unit dosage form. Novel intermediates and
processes to prepare compounds of forrnula (I), as depicted in Figures 6-12 are
also embodiments of the invention.
The discovery of these new modulators of PKC that exhibit
isotype selectivity will permit elucidation of the functional importance of the
different PKC isoforms in the regulation of cell function, and can provide PKC
based therapeutics that may find use not only in the treatment of cancer, but
potentially autoimmune diseases, and infl~mm~tion.
Brief Description of the Fi~ures
FIG. I shows the structural organization of the PKC gene family.
- FIG. 2 shows the structures of telocidin B-4, A-l, A-2, and indolactam V.
FIG. 3 shows compounds of the invention.
FIG. 4 shows an electrophile useful for preparing compounds of the
invention.
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FIG. 5 shows an electrophile useful for ~ palhlg compounds of the
invention.
FIG. 6 shows a scheme for ple~alillg compounds of the invention.
FIG. 7 shows a scheme for ~,rc;p~;l g compounds of the invention.
FIG. 8 shows a scheme for ~l~p~illg compounds of the invention.
FIG. 9 shows a scheme for plcpalillg compounds of the invention.
FIG. 10 shows a scheme for plep~;llg compounds of the invention.
FIG. 1 I shows a scheme for l,le~alil1g compounds of the invention.
FIG. 12 shows a scheme for preparing compounds of the invention.
FIG. 13 shows the cytotoxicity of compound 17 and ILV in MCF-7 and
MDA-MB-23 1 breast carcinoma cells.
FIG. 14 shows the Western blot of PKC isoform levels 24 hours after
treatment with compound 17.
FIG. 15 shows the antitumor activity of compound 17 against the
MDA-MB-23 l xenograft in nude mice.
Detailed Description of the Invention
~ In the following description ofthe plerelled embodiments,
reference is made to the accompanying figures which form a part hereof7 and in
which is shown by way of illustration specific embodiments in which the
invention may be practiced. It is to be understood that other embodiments may
be utilized and structural changes may be made without departing from the scope
of the present invention.
The following definitions are used, unless otherwise described.
Halo is fluoro, chloro, bromo, or iodo. The term "alkyl" encompasses branched
or unbranched alkyl, cycloalkyl or (cycloalkyl)alkyl, but reference to an
individual radical such as "propyl" embraces only the straight chain radical, a
branched chain isomer such as "isopropyl" being specifically referred to. Aryl
comprises a phenyl radical, an ortho-fused bicyclic carbocyclic radical having
about nine to ten ring atoms in which at least one ring is aromatic, as well as
simple (C,-C4)n alkylaryl wherein n is 1-3.
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It will be appreciated by those skilled in the art that compounds of
the invention having a chiral center may exist in and be isolated in optically
active and racemic forms. Some compounds may exhibit polymorphism. It is to
be understood that the present invention encompasses any racemic, optically-
5 active, polymorphic, or stereoisomeric forrn, or mixtures thereof, of a compoundof the invention, which possess the useful properties described herein, it being
well known in the art how to prepare optically active forms (for example, by
resolution of the racemic form by recryst~lli7~fion techniques, by synthesis from
optically-active starting materials, by chiral synthesis, or by chromatographic
10 separation using a chiral stationary phase).
Specific values listed below for radicals, substituents, and ranges,
are for illustration only and they do not exclude other defined values or other
values within defined ranges for the radicals and substituents
Specifically, (C,-C5)alkyl is methyl, ethyl, propyl, isopropyl,
15 butyl, iso-butyl, sec-butyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, or
cyclopentyl; and aryl is phenyl, methylphenyl, ethylphenyl, propylphenyl,
dimethylphenyl, diethylphenyl, indenyl, methylindenyl, dimethylindenyl,
- naphthyl, methylnaphthyl, or dimethylnaphthyl.
A specific value for R, is ORa, SRa, N(Ra~(Rb), halo, NO~,
20 NHC(O)[(CI-C4)alkyl] or NHOH; for R2 is l-decynyl or decyl; for R3 is H; for
Rc is (C5-C~5)alkyl; for Y is H; and for Z is methyl.
A more specific value for R, is ORa.
A specific group of compounds are compounds of formula I
wherein R, and R2 together are -CH(RC)-CH2-C(O)-N(Rd)-, -C(RC)=CH-
25 C(O)N(Rd)-, -C(RC)=CH-N(Rd)- or -C(R9=CH-O-H; or a pharmaceutically
acceptable salt thereof.
Another specific group of compounds are compounds of formula
I wherein Z is CH3; Y is H; R, is ORa, SRa, N(Ra)(Rb), halo, NO2, NHC(O)[(C,-
C4)alkyl] or NHOH; R2 is (C5-C,5)alkyl, optionally comprising 1-3 double bonds,
30 1-2 triple bonds or a mixture thereof; and R3 is H; or a pharrnaceutically
acceptable salt thereof.
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Another specif1c group of compounds are compounds of formula
I wherein: R, is ORn, SRa, N(Ra)(Rb), halo, NO2, NHC(O)[(Cl-C4)alkyl] or
NHOH; and R2 is a hydrophobic (C5-C22) hydrocarbyl group, optionally
comprising 1-3 double bonds, 1-2 triple bonds or a mixture thereof, or (C6-
S C,2)aryl(C2-C,0)alkyl, wherein the alkyl moiety optionally comprises 1-2 double
bonds, 1-2 triple bonds or a mixture thereof; or R, and R2 together are -CH(RC)-CH2-C(O)-N(Rd)-, -C(RC)=CH-C(O)N(Rd)-, -C(RC)=CH-N(Rd)- or -C(RC)=CH-O-
H; or a ph~ .eutically acceptable salt thereof.
Another specific group of compounds are compounds of formula
I wherein: R2 is (C5-C,5)alkyl, optionally comprising 1-3 double bonds, 1-2
triple bonds or a mixture thereof; or a pharmaceutically acceptable salt thereof.
Another specific group of compounds are compounds of formula
I wherein: R2 is (C6-C,2)aryl(C2-C,0)alkyl, wherein the alkyl moiety optionally
comprises 1-2 double bonds, 1-2 triple bonds or a mixture thereof; or a
pharmaceutically acceptable salt thereof.
A preferred group of compounds are compounds of formula I
wherein Z is CH3; Y is H; R2 is (C5-C,5)alkyl, optionally comprising 1-3 double
bonds, l-2 triple bonds or a mixture thereof; and R3 is H; or a pharmaceuticallyacceptable salt thereof.
Another preferred group of compounds are compounds of formula
I wherein R2 is l-decynyl; or a ph~ ceutically acceptable salt thereof.
Processes for preparing compounds of formula I are provided as
further embodiments of the invention and are illustrated by the following
procedures in which the meanings of the generic radicals are as given above
unless otherwise qualified.
Compounds of formula I wherein R, is 1-alkynyl can be prepared
from a corresponding compound wherein R, is iodide by coupling with the
requisite alkyne using a suitable catalyst, such as for example palladium.
Suitable conditions for such a coupling reaction are illustrated in Example 1.
Compounds of formula I wherein R, is l-alkyl can be prepared
from a co,le~yol1ding compound wherein R, is l-alkynyl by hydrogenation of
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the alkyne bond using a suitable catalyst, such as for example palladium on
carbon. Suitable conditions for such a hydrogenation are illustrated in
Example 2.
Compounds of formula I can generally be prepared by the
5 reaction of 2,6-disubstituted arylmetal compounds as nucleophiles with
enantiomerically pure three carbon electrophiles incorporating the necessary
amino and hydroxyl groups in protected form.
Electrophiles.
Two readily available electrophiles (Figures 4 and 5) are suitable
for preparing compounds of the invention, the protected aziridinemethanol 21,
and the protected serine aldehyde 23 ("Enantiospecific Synthesis of
D-a,~-Diaminoalkanoic Acids" Beaulieu, P. L.; Schiller, P. W. Tetrahedron
Lett. 1988, 29, 2019-2022). To obtain compound 21, the known
15 L-serine-derived methyl or benzyl esters 20 can be reduced to the
aziridinemethanol, e. g., with NaBH4 and the hydroxyl group silylated [(a)
"Construction of Optically Pure Tryptophans from Serine Derived
Aziridine-2-carboxylates" Sato, K.; Kozikowski, A. P. Tetrahedron Lett. 1989,
31, 4073-4076. (b) "One-Step Synthesis of Optically Active Benzyl
20 N-Trityl-L-Aziridine-2-Carboxylic Esters" Kyul-Yeheskiely, E.; et al.
Tetrahedron Lett. 1992, 33, 3013-3016] ("Derivatives of heterocyclic
a-iminocarboxylic acids. 4. Reduction of N-alkoxycarbonyl derivatives of
a-iminocarboxylic acids" Nurdinov, R.; et al. Khim. Geterotsikl. Soedin. 1993,
1567-1573; Chem. Abstr. 1995, 123, 83337x). Since cuprates exhibit enhanced
25 reactivity towards aziridines compared with organolithium reagents,
stoichiometric or catalytic amounts of copper(I) salts may be included in reaction
- mixtures involving 21.
Nucleophiles.
The most convenient nitrogen substituent on the aromatic ring
would be a free amino group (NH2). While, N-alkylarylamines have been
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successfully ortho-metalated by N-lithiation, reacted with CO2 to form the
lithium carbamate, and further treated with tert-butyllithium, this procedure
failed in the case of aniline ("Carbon Dioxide: A Reagent for the Simultaneous
Protection of Nucleophilic Centers and the Activation of Alternative Locations
to Electrophilic Attack. 17. Substitution of N-Methyl-l- and
N-Methyl-2-naphthylamine and Side-Chain Functionalization of o-Toluidine"
Katritzky, A. R.; et al. J. Org Chem. 1991, 56, 5045-5048).
N-(methoxycarbonyl)-O-(methoxymethyl)-m-aminophenol has been reported to
undergo directed metalation mainly in the 2-position, whereas the corresponding
N-Boc derivative reacted on the opposite side of the nitrogen in position 4 (
"Biosynthesis of Sarubicin A. Synthesis and Incorporation of
6-Hydroxy[l3COI5NH2]anthranilamide'' Gould, S. J.; Eisenberg, R. L. J. Org.
Chem. 1991, 56, 6666-6671). Since the nitrogen may need to be deprotected in
the presence of protecting groups such as N-Cbz and O-TBDMS, the
N-(allyloxycarbonyl) derivative 24 (Figure 6) is a convenient starting material
for pl~pa~ g compounds of the invention. While the allyloxycarbonyl group is
not bulky, it is readily removed by various nucleophiles or hydride donors in the
presence of a Pd catalyst.
An intermediate of formula 28 is particularly useful for ~rep~l ;llg
compounds of formula I. An intermediate of formula 28 can be ~ aled as
shown in Figure 6 by reaction of a nucleophile of formula 24 and an aziridine
(electrophile) of formula 21 followed by deprotection of the aniline nitrogen.
The resulting aniline 25 can be alkylated to give a compound of formula 26.
Hydrogenation of 26 followed by lactam formation yields an intermediate of
formula 28.
The methoxymethoxy substituent in intermediate 28 provides
access not only to 7-hydroxy- and 7-alkoxybenzolactams but, as discussed
below, to a variety of other compounds of the invention via aryl triflate
chemistry.
Benzolactams Cont~ining 7-halo or 7-CF3 substituents can
conveniently be synthesized from the requsite N-(allyloxycarbonyl)-m-
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11
substituted ~nilin~s using a procedure similar to the one described above. A
variety of potentially suitable substrates have been reported which differ in their
thermal stability as well as in the ease or difficulty with which the second
substituent can be transformed into NH2. 3-Chlorobenzonitrile and
N-tert-butyl-3-chlorobenzamide undergo directed lithiation in position 2 at
-70~C, and the resulting organolithiums can be trapped with an electrophile
("Heteroatom-Facilitated Lithiations" Gschwend, H. W.; Rodriguez, H. R. Org
React. 1979, 26, 1-360 (unpublished results by Rodriguez, H. R.)). 3-Fluoro-
and 3-chlorophenyloxazolines have been metalated in position 2 ("A New Route
to 3-Hydroxyphthalides: Application to the Synthesis of Racemic [5-'3C]
Daunomycinone" Becker, A. M.; et al. Tetrahedron Lett. 1986, 27~ 3431-3434.)
and ("The Oxazoline-Benzyne Route to 1,2,3-Trisubstituted Benzenes. Tandem
Addition of Organolithiums and a-Lithionitriles to Benzynes" Pansegrau, P. D.;
et al. J. Am. Chem. Soc. 1988, 110, 7178-7184). Additionally,
3-Fluorobenzaldehyde dimethyl acetal undergoes metalation and subsequent
carboxylation in high yield ("Synthesis of Functionalized Hydroxyphthalides and
Their Conversion to 3-Cyano- I (3H)-isobenzofuranones. The Diels-Alder
Reaction of Methyl 4,4-Diethoxybutynoate and Cyclohexadienes" Freskos, J. N.;
Morrow, G. W.; Swenton, J. S. J: Org Chem. 198S, 50, 805-810).
The 7-halo-6-hydroxybenzolactams of the invention can
conveniently be prepared as illustrated in Figure 7. Lithiated 3-fluoro- and
3-chlorobenzaldehyde dimethyl acetal 35a,b can be reacted with aldehyde 23.
Separation of the resulting stereoisomers followed by acid hydrolysis of the
aryllithium addition product 36, yields an aldehyde, which forms a hemiacetal
with the benzylic hydroxyl group (37). Benzylic alcohol 37 can be selectively
oxidized to a lactone using, for example, MnO7. Acetonide protection can then
~ be restored. The lactam can be ammonolyzed to yield 39, and the liberated
benzylic hydroxyl group protected by silylation. Hofmann degradation gives an
aniline of formula 40, which can be converted to the bis-tert-butyldimethylsilylether 41. Using a sequence similar to that illustrated in Figure 6, a compound of
formula 41 can be converted to a 7-halo-6-hydroxybenzolactam of formula 42.
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12
Compounds of the invention wherein R3 is fluoro (such as for
example a compound of formula 43 can be prepared by treatment of a
corresponding compound wherein R3 is hydroxy (such as for example a
compound of formula 42) with (diethylaminosulfur)trifluoride (DAST) as sho~vn
in Figure 7.
A compound of formula I wherein R3 is hydrogen can be prepared
from a corresponding compound of formula I wherein R3 is hydroxy, such as for
example a compound of formula 42, by formation of a cyclic thionocarbonate of
formula 44 followed by Barton deoxygenation ("Synthesis of Deoxysugars and
Deoxynucleosides from Diol Thiocarbonates" Barton, D. H. R.; Subr~m~niAn, R.
J. Chem. Soc., Chem. Commun. 1976, 867-868).
As illustrated in Figure 8, the interrnediate aryl triflate 46, can be
used to prepare compounds of formula I having a variety of 7-substituents.
Methoxycarbonylation of 46 ( "Palladium Catalysed Alkoxycarbonylation of
Phenols to Benzoate Esters" Dolle, R. E.; et al. J. Chem. Soc., Chem. Commun.
1987, 904-905) gives the ester 47 which can be transformed into the derivatives
48 and subsequently compounds of forrnula 49, by selective reduction of the
ester moiety, followed by cuprate alkylation of the derived triflate.
The ester function of a compound of formula 47 can also be used
to introduce a nitrogen atom into position 7 by means of a Curtius degradation.
The product 50 can be alkylated in position 8 as previously illustrated in Figure
6 to obtain compound 51. This intermediate can be used to prepare 7-nitro- and
7-(hydroxyamino)benzolactams 53, 54 by peracid oxidation and reduction with
zinc.
Diazonium chemistry can be applied to 51 to prepare the 7-iodo-
and 7-mercapto derivatives 55, 56. The corresponding chlorides and bromides
may be obtained from 51 by action of tert-butyl nitrite and the anhydrous
copper(II) halides ("Alkyl Nitrite-Metal Halide De~min~tion Reactions. 2.
Substitutive De~min~tion of Arylamines by Alkyl Nitrites and Copper(II)
Halides A Direct and Remarkably Efficient Conversion of Arylamines to Aryl
Halides" Doyle, M. P.; et al. J. Org Chem. 1977, 42, 2426-2431). The
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13
corresponding fluoride may be obtained from 51 using a procedure similar to
that described in "A Mild and Efficient Method of Aromatic Fluorination"
Rosenfeld, M. N.; Widdowson, D. A. ~ Chem. Soc., Chem. Commun. 1979,
914-916.
Compounds of formula I wherein R, and R2 together are -
CH(RC)-CH2-C(O)-N(Rd)-, -C(RC)=CH-C(O)N(Rd)-, -C(RC)=CH-N(Rd)- or-
C(RC)=CH-O- can be prepared using procedures similar to those illustrated in in
Figure 9. Iodophenol 58 and the iodoaniline 59 can be alkylated with the allylichalide 60, and the resulting intermediates cyclized under Pd catalysis to obtainthe benzofuran 63 ("Synthesis of Benzofurans, Tetrahydrobenzopyrans, and
Related Cyclic Ethers via Cyclic Carbop~ tionll Negishi, E.; et al.
Heterocycles 1989, 2~, 55-58) and the indole 64 ("Conversion of
2-Halo-N-allyl~nilin~s to Indoles via Palladium(0) Oxidative Addition-Insertion
Reactions" Odle, R.; et al. J. Org. Chem. 1980, 45, 2709-2710). An
intermolecular Heck reaction between 59 and dimethyl maleate gives
quinolinone 65 ( "Palladium-Catalyzed Synthesis of 2-Quinolone Derivatives
from 2-Iodo~nilines" Cortese, N. A.; et al. J. Org. Chem. 1978, 43, 2952-2958).
Elaboration of the methoxycarbonyl side chain in quinolinone 65 to an alkyl
group, using standard conditions, yields the alkylated compound 66.
Hydrogenation of the unsaturated heterocyclic ring of compound 66 gives the
lactam stereoisomers 67.
An intermediate of formula 28 can alternatively be prepared as
illustrated in Figure 10. 1,3-Cyclohexanedione 69 can be alkylated with
aziridine 68 obtained by coupling the serine and valine building blocks.
Removal of the N-protective group yields a compound which can close to the
eight-membered lactam ring under conditions of enamine formation (high
dilution), to give enaminone 73. Arom~ti7~tion of enaminone 73, using standard
conditions, gives compound 75, which can be protected to give an intermediate
of formula 28.
Compounds of formula I can also be prepared using a sequence
similar to that described in the previous paragraph. As shown in Figure 10,
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14
alkylation of the dianion of 69 gives the substituted cyclohexanedione 70.
Subsequent alkylation with the aziridine 68, followed by N-deprotection gives
the cyclic enaminone 74, which can be elaborated to compounds of formula 76
and 33.
Compounds of formula I wherein R, is H can generally be
prepared using procedures similar to those described in Examples l and 2, as
illustrated in Figure 11.
Compounds of formula I wherein Rl is ORa can generally be
prepared using procedures similar to that described in Example 3, as illustratedin Figure 12.
It is noted that many of the starting materials employed in the
synthetic methods described above are commercially available or are reported in
the scientific literature.
In cases where compounds are sufficiently basic or acidic to form
stable nontoxic acid or base salts, a~lmini~tration ofthe compounds as salts maybe appropl;ate. Examples of pharmaceutically acceptable salts are organic acid
addition salts formed with acids which form a physiological acceptable anion,
for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,
succinate, ben~o~t~, ascorbate, a-ketoglutarate, and oc-glycerophosphate.
Suitable inorganic salts may also be formed, including hydrochloride, sulfate,
nitrate, bicarbonate, and carbonate salts.
Ph~rm~reutically acceptable salts may be obtained using standard
procedures well known in the art, for example by reacting a sufficiently basic
compound such as an amine with a suitable acid affording a physiologically
acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or
alkaline earth metal (for example calcium) salts of carboxylic acids can also bemade.
The compounds of formula I can be formulated as pharmaceutical
compositions and aflmini~tered to a m~mm~ host, such as a human patient in
a variety of forms adapted to the chosen route of a-lmini~tration, i.e., orally or
parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
.. . . .... . .
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Thus, the present compounds may be systemically ~lministered,
e.g., orally, in combination with a ph~rm~eutically acceptable vehicle such as
an inert diluent or an ~imil~ble edible carrier. They may be enclosed in hard orsoft shell gelatin capsules, may be co~ ,ssed into tablets, or may be
5 incorporated directly with the food of the patient's diet. For oral therapeutic
iq~lmini~tration, the active compound may be combined with one or more
excipients and used in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions
and preparalions should contain at least 0.1% of active compound. The
10 percentage of the compositions and plel)a,alions may, of course, be varied and
may conveniently be between about 2 to about 60% of the weight of a given unit
dosage form. The amount of active compound in such therapeutically useful
compositions is such that an effective dosage level will be obtained.
The tablets, troches, pills, capsules, and the like may also contain
15 the following: binders such as gum tr~g~c~nth, acacia, corn starch or gelatin;
excipients such as dicalcium phosphate; a disintegrating agent such as corn
starch, potato starch, alginic acid and the like; a lubricant such as magnesium
stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartameor a flavoring agent such as pepperrnint, oil of wintergreen, or cherry flavoring
20 may be added. When the unit dosage form is a capsule, it may contain, in
addition to materials of the above type, a liquid carrier, such as a vegetable oil or
a polyethylene glycol. Various other materials may be present as coatings or to
otherwise modify the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and
25 the like. A syrup or elixir may contain the active compound, sucrose or fructose
as a sweetening agent, methyl and propylparabens as preservatives, a dye and
- flavoring such as cherry or orange flavor. Of course, any material used in
p~ )a~ g any unit dosage form should be ph~rm:lçeutically acceptable and
subst~nti~lly non-toxic in the amounts employed. In addition, the active
30 compound may be incorporated into sustained-release prepaldLions and devices.
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16
The active compound may also be ~(lmini~tered intravenously or
intraperitoneally by infusion or injection. Solutions of the active compound or
its salts can be prepared in water, optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethylene glycols,
5 triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage
and use, these preparations contain a preservative to prevent the growth of
microorg~ni ~m ~
The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or sterile powders10 comprising the active ingredient which are adapted for the extemporaneous
plepal~lion of sterile injectable or infusible solutions or dispersions, optionally
encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile,
fluid and stable under the conditions of manufacture and storage. The liquid
carrier or vehicle can be a solvent or liquid dispersion medium comprising, for
15 example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid
polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and
suitable mixtures thereof. The proper fluidity can be m~int~ined, for example,
by the formation of liposomes, by the maintenance of the required particle size in
the case of dispersions or by the use of surfactants. The prevention of the action
20 of microorg~ni~m~ can be brought about by various antibacterial and antifungal
agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include isotonic agents, for
example, sugars, buffers or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the compositions of
25 agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the
active compound in the required amount in the appropriate solvent with various
of the other ingredients enumerated above, as required, followed by filter
sterilization. In the case of sterile powders for the preparation of sterile
30 injectable solutions, the preferred methods of preparation are vacuum drying and
the freeze drying techniques, which yield a powder of the active ingredient plus
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17
any additional desired ingredient present in the previously sterile-filtered
solutions.
For topical ~mini~tration, the present compounds may be applied
in pure form, i.e., when they are liquids. However, it will generally be desirable
5 to ~rlmini~ter them to the skin as compositions or formu}ations, in combination
with a dermatologically acceptable carrier, which may be a solid or a liquid.
Useful dosages of the compounds of formula I can be determined
by comparing their in vitro activity, and in vivo activity in animal models.
Methods for the extrapolation of effective dosages in mice, and other ~nim~l~, to
10 hum~n~ are known to the art; for example, see U.S. Pat. No. 4,938,949.
Generally, the concentration of the compound(s) of formula I in a
liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferablyfrom about 0.5-10 wt-%. The concentration in a semi-solid or solid composition
such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-
15 %. Single dosages for injection, infusion or ingestion will generally varybetween 50-1500 mg, and may be ~mini~tered, i.e., 1-3 times daily, to yield
levels of about 0.5 - 50 mg/kg, for adults.
Accordingly, the invention includes a pharmaceutical
composition comprising a compound of forrnula I as described hereinabove; or a
20 ph~ reutically acceptable salt thereof; and a pharmaceutically acceptable
diluent or carrier.
The ability of compounds of the invention to modulate PKC can
be demonstrated using standard models which are well known in the art, or can
be demonstrated using the tests described hereinbelow.
Isozyme Studies.
- The isozyme selectivity of representative compounds of theinvention and ILV (3) was determined by investigating their ability to displace
[3HlPDBU binding to recombinant PKC isozymes expressed in the baculovirus
30 system, as described by Kazanietz, M. G.; et al. Characterization of ligand and
substrate specificity for the calcium-dependent and calcium-independent PKC
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18
isozymes. Mol. Pharmacol. 1993, 44, 298-307. As shown in Table 1, the
compound of Example 1 (17) showed a higher affinity for the a and ,B isozymes,
in comparison to y, ~, and ~, with approximately a ten-fold difference in affinity
between PKC oc and ~. It is appd,~ll that the ectronic effect of the acetylenic
5 group influences isozyme selectivity, since the saturated alkyl compound of
Example 2 (18) shows only a four-fold difference in the Kj for a versus ~.
Tablc 1. K; values ~ SEM for the inhibition of [3H]PBDU binding b~ the
compounds tested.
Compound
Acetylene- 14.7 ~t 1.3 17.4 ~ 2.2 40.7 ~ 8.9 122 ~ 22 142 ~ 3
Benzolactam
15 17
Saturated- 46.6 ~ 8.0 58.2 ~ 12.6 145 ~ 25 185 ~ 30 187 ~ 22
Benzolactam
18
ILV* 11.0 6.1 19.4 8.2 21.9
*Data taken from Mol. Pharmacol. 1993, 44, 298-307.
Cell proliferation assay and PKC Downre~ulation.
Representative compounds of the invention were also tested for
25 antiproliferative activity against breast carcinoma cell lines MCF-7 and MDA-MB-231 (Yu, G.; et al. Transfection with protein kinase Coc confers increased
multidrug resistance to MCF-7 cells expressing P-glycoprotein. Cancer
Commun. 1991, 3, 181-189). Exposure of MCF-7 and MDA-MB-231 cells to
the compound of Example I (17) for four days resulted in IC50 values of 20 and
30 30 !lM, respectively~ whereas ILV (3) was inactive (Figure 13).
PKC isozyme levels were determined in MCF-7 and MDA-MB-
231 cells exposed to the compound of Example I (17) for 24 hours (Figure 14).
In MCF-7 cells, PKC~ and PKC~ were virtually elimin~te~l, PKC~ was reduced
to a lesser extent, and PKC~, was unchanged. MDA-MB-231 cells exhibited a
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19
similar reduction in PKC~, whereas PKC~ and ~ were slightly reduced, while
PKCa and PKC~ remained unchanged. These results indicate that the compound
of Example 1 (17), while not completely selective, preferentially downregulates
PKC~ in both cell lines. The varying degree of selectivity of the compound of
S Example 1 for other PKC isozymes was to some degree cell type specific. This
result was not completely unexpected, since different tumors would be expected
to exhibit different PKC isozyme patterns as well as different pathways
governing their stability and turnover.
Using a procedure similar to that described in: Price, J.E., et al.
10 Cancer Res. 50:717-721, 1990, the antitumor activity of compound 17 was
evaluated in vivo in MDA-MB-23 I human breast carcinoma xenographs at the
maximum dose evaluated thus far for toxicity, but not necessarily the maximum
tolerated dose (Figure 15). Daily i.p. ~lmini~tration of the compound for three
consecutive weeks resulted in 65% inhibition of tumor growth three weeks after
15 treatment was initiated. No overt general cytotoxic effects were observed.
Compounds of the invention have been shown to be
downregulators of PKC, and are therefore useful to treat conditions ameliorated
by reduction of PKC activity. Such conditions include but are not limited to
cancer, autoimmune ~i~e~e~, and infl~mm~tion. Accordingly, the invention
20 includes a method for modl]l~ting PKC in a m~mm;~l comprising ~mini~tering
to said m~mm~l a pharmaceutically effective dose of a compound of formula I;
or a ph~rm~ceutically acceptable salt thereof. The invention also includes a
method for the treatment of cancer in a m~mm~l comprising a~mini~tering to
said m~mm~3l a pharmaceutically effective dose of a compound of formula I, or a
25 pharmaceutically acceptable salt thereof.
Compounds of formula (I) are generally effective to treat
m~mm~ n conditions associated with pathological cellular proliferation. In
addition to the utilities described above, they may also be useful to treat
conditions which include restenosis, atherosclerosis, coronary heart disease,
30 thrombosis, myocardial infarction, stroke, uterine fibroid or fibroma, and
obliterative disease of vascular grafts and transplanted organs.
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The invention will now be illustrated by way of the following
non-limiting Examples.
Examples
Example 1. (25,5S)-8-(1-Decynyl)benzolactam (Figure 11, compound 17).
To a mixture of 16 (Figure 11 ) ( 1 16 mg, 0.27 mmol), Et2NH (2
mL), and PdCI2(PPh3)2 (26 mg, 0.018 mmol) were added 1-decyne (0.13 mL,
0.74 mmol) and CuI (4 mg, 0.01 mmol). The resulting solution was stirred at
room temperature for 24 hours. The solvent was evaporated, the residue was
10 dissolved in 1 mL of 20% aqueous NaOH and 4 mL of MeOH, and the solution
was stirred at room temperature for 0.5 hours. The mixture was partitioned
between 10 mL of water and 100 mL of EtOAc. The organic layer was separated,
washed with brine, and dried over Na2SO4. Evaporation and chromatography on
silica gel (2/1 ethyl acetate/petroleum ether as eluent) afforded 87 mg (81%) of15 the title compound (17): [a]20D -303.3~ (c 0.5, ethanol); 'H NMR (300 MHz,
CDCI3) ~ 7.18 (d, J= 7.8 Hz, IH), 7.09 (s, lH), 6.85 (d, J= 7.8 Hz, lH), 6.55 (s,
lH), 3.82 ~m, lH), 3.58 (m, lH), 3.48 (m, 2H), 3.12 (dd, J= 16.3, 8.1 Hz, lH),
2.80 (s, 3H), 2.73 (d, J= 16.2 Hz, lH), 2.33 (m, IH), 2.30 (t, J= 7.8 Hz. 2H),
1.68-1.15 (m, 12H), 1.05 (d, J= 7.2 Hz, 3H), 0.88 (t, J= 7.4 Hz, 3H), 0.85 (d, J20 = 7.2 Hz, 3H); MS m/z 398 (M+), 312, 91; HRMS calc. for C25H38N2O, 398.293,
found 398.294. Anal. calcd. for C2sH38N2O2: C, 75.34; H, 9.61; N, 7.03. Found: C,
74.98, H, 9.86, N, 6.71 .
The intermediate 16 was prepared as follows (Figure 11).
25 a. (S)-O-Acetyl-2-[(ethoxycarbonyl)amino]-3-phenyl-1-propanol (Figure
11, compound 78). To a suspension of (S)-2-amino-3-phenyl-1-propanol 77
(25.0 g, 0.165 mol) and Na2CO3 (20.1 g, 0.19 mol) in 80 mL of water was added
EtOCOCI (26.0 mL, 0.27 mol) dropwise at room temperature. The solution was
stirred at room temperature for 4 hours and extracted with CH2CI2 (4 x 250 mL).
30 The organic layer was dried over Na2SO4 and evaporated to give an oil which
was dissolved in Et3N (100 mL, 0.72 mol) and Ac2O (60 mL, 0.5~ mol). The
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21
mixture was stirred overnight at room temperature and partitioned between 400
mL of EtOAc and 150 mL of water. The organic layer was washed with 50 mL
of brine and dried over Na2SO4. Evaporation and chromatography (I /2 ethyl
acetate-petroleum ether as eluent) afforded 40.2 g (91%) ofthe di-acetate: [aJ20D -
5 17.9~ (c 0.9, CHCI3); IR (KBr) 2980, 1700, 1500, 1200 cm-~; 'H NMR (300
MHz, CDC13) o 7.31-7.15 (m, 4H), 4.72 (br s, IH), 4.09 (q, J = 7.3 Hz, 2H),
4.07-4.02 (m, 3H), 2.80 (m, 2H), 2.04 (s, 3H), 1.18 (t, J = 7.3 Hz, 3H); MS m/z
265 (M+), 165, 91. Anal. Calcd for C~4H~gNO4 C, 63.38; H, 7.22; N, 5.28. Found:
C, 63.36; H, 7.30; N, 5.06.
b. (S)-O-Acetyl-3-(2-aminophenyl)-2-[(ethoxycarbonyl)amino]-1-propanol
(Figure 11, compound 9). To a solution of (S)-O-Acetyl-2-
[(ethoxycarbonyl)amino]-3-phenyl-1-propanol (25.0 g, 0.094 mol) in Ac.O (25
mL) was added nitric acid (10 mL, 0.24 mol) dropwise at 0 ~C. The mixture was
15 stirred at room temperature for 24 hours, poured into 200 mL of ice-water, aIId
extracted with EtOAc (3 x 250 mL). The organic layers were washed with brine,
dried over Na2SO4, and evaporated. The residue was dissolved in 200 mL of
EtOAc, and 500 mg of Pd/C was added. The mixture was stirred under 1 atm of
H2 at room temperature for 24 hours. Filtration from the catalyst, evaporation,
20 and chromatography (2/3 ethyl acetate/petroleum ether as eluent) provided 9
(6.02 g, 23%) and its isomer 10 (13.2 g, 50%). Compound 9: [~]20D +4.6~ (c 0.5,
CHC13); IR (KBr) 3360, 1720, 1700, 1500, 1200, 730 cm-'; 'E~ NMR (300 MHz,
CDC13) ~ 7.06 (t, J= 7.5 Hz, lH), 6.93 (d, J= 7.6 Hz, lH), 6.67 (m, 2H), 5.26
(br s, lH), 4.14 (q, J= 7.3 Hz, 2H), 4.10-4.05 (m, 3H), 4.01 (br s, 2H), 2.94 and
25 2.61 (ABq, 2 H, J= 13.6 Hz), 2.10 (s, 3H), 1.25 (t, J= 7.3 Hz, 3H); MS m/z 280
(M+), 191, 132, 106. Anal. Calcd for C,4H20N2O4: C, 59.98; H, 7.19; N, 9.99.
Found: C, 59.65; H, 7.19; N, 9.89.
c. (2S,2'S)-N-[3'-Acetoxy-2'-((ethoxycarbonyl)amino)phenyl]valine benzyl
30 ester (Figure 11, compound 12). A mixture of (R)-benzyl a-[[(trifluoromethyl)-
sulfonyl~oxy]isovalerate (11, 5.03 g, 18.1 mmol), 9 (5.02 g, 18.1 mmol), and
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22
2,6-lutidine (2.4 mL, 20 mmol) in 40 mL of 1,2-dichloroethane was stirred at 70
~C for 10 hours. Evaporation and chromatography on silica gel (1/5 ethyl
acetate/petroleum as eluent) gave compound 12 (6.05g, 70%) as a colorless oil:
[a]20D -20.6~ (c 0.07, CHCI3); IR (KBr) 3300, 1725, 1710, 1520 cm-'; 'H NMR
5 (300 MHz, CDCI3) ~ 7.29 (m, SH), 7.05 (t, J= 7.5 Hz, lH), 6.95 (d, J= 7.6 Hz,
lH),6.67(t,J=7.5Hz, lH),6.68(d,J=7.6Hz, lH),5.14(m,3H),4.12(m,
5H), 3.91 (d, J= 6.2 Hz, lH), 2.98 (m, lH), 2.67 (m, lH), 2.23 (m, lH), 2.02 (s,3H), 1.16(t,J=7.2Hz,3H), 1.03(d,J=7.0Hz,3H),0.84(d,J=7.0Hz,3H);
MS m/z 471 (M + Ht), 335, 275, 186, 91; HRMS calcd for C26H34N2O6 470.241;
10 found 470.241.
d. (2S,5S)-Benzolactam (Figure 11, compound 15). A mixture of 12 (6.0 g,
12.8 mmol) and KOH (5.0 g, 89 mmol) in 40 mL of MeOH/H2O (1:1) was
stirred at 30 ~C for 3 days. After neutralization to pH approx. 7 with conc. HCl,
15 di-tert-butyl dicarbonate (2.7 g, 12.5 mmol) and NaHCO3 (1.5 g, 18 mmol) wereadded. The I~ Ule was stirred at room temperature for 24 hours, washed with
petroleum ether, and adjusted to approximately pH 2. Extraction with EtOAc (4
x 150 mL), drying over Na2SO4, and evaporation gave 4.1 g of crude 13. This
product (1.61g, 4.37 mmol) and N-hydroxysuccinimide (0.52 g, 4.41 mmol)
20 were dissolved in 20 mL of CH3CN. Dicyclohexylcarbodiimide (DCC) (1.21 g,
5.81 mmol) in 20 mL of CH3CN was added dropwise at 0 ~C, and the mixture
was stirred at room temperature overnight. Filtration from the pleci~ e,
evaporation, and chromatography on silica gel (1/1 CH2Cl2/EtOAc as eluent
provided 1.79 g of 14. Without further purification, this product was directly
25 dissolved in 10 mL of dried CH2Cl2 and the solution was cooled to 0 ~C before10 mL of CF3COOH was added. The mixture was stirred at 0 ~C for 2 hours, and
the volatiles were removed in vacuo below 30 ~C. The residue was dissolved in
100 mL of EtOAc, and 5 mL of saturated aqueous NaHCO3 solution was added.
The mixture was heated at 80 ~C for 24 hours with vigorous stirring. After
30 cooling to room temperature, 40 mL of water was added. The organic layer was
separated, and the aqueous layer was extracted with EtOAc (4 x 20 mL). Drying
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over Na2SO4 and evaporation produced a yellow oil, which was dissolved in 10
mL of CH3CN. This solution was cooled to 0 ~C, and 4.0 mL (40 mmol) of
formalin, 1.0 g (16 mmol) of NaBH3CN, and 0.27 mL of AcOH were added. The
resulting mixture was stirred at 0 ~C for 2 hours before quenching with
phosphate buffer (pH = 2). The solvent was evaporated, and the residue was
dissolved in EtOAc, washed with water, saturated NaHCO3, and brine, and dried
over Na2SO,. Evaporation and chromatography on silica gel (1/10 MeOH/CH2CI2
as eluent) gave 15 (480 mg, 44% overall from 12): [a]2CD -271~ (c 0.08, CHCl3);
IH NMR (300 MHz, CDCl3) ~ 6.80-7.20 (m, 4H), 6.72 (br s, lH), 4.10 (m, lH),
3.73 (m, lH), 3.61 (m, lH), 3.46 (d,J= 8.1 Hz, lH), 3.11 (dd,J= 15.8, 8.1 Hz,
lH), 2.85 (dd, J= 15.8, 3.2 Hz, lH), 2.84 (s, 3H), 2.48 (m, lH), 1.15 (d, J= 6.4Hz, 3H), 0.94 (d, J= 6.6 Hz, 3H); MS m/z 262 (M+), 245, 117, 91.
e. (2S,SS)-8-Iodobenzolactam (Figure 11, compound 16). Amixture of 15
(850 mg, 3.2 mmol), Et3N (15 mL), and Ac2O (5 mL) was stirred at room
t~ dlure for 24 hours. The mixture was poured into 100 mL of water,
extracted with EtOAc (4 x 100 mL), washed with brine, and dried over Na2SO4.
After evaporation, the residue was dissolved in 10 mL of 1,4-dioxane and 2 mL
of pyridine. To this solution 1.70 g (6.7 mmol) of I2 was added, and the deep
brown solution was stirred at room temperature for 3 days. The mixture was
partitioned between EtOAc and water, and the organic layer was washed with 10
mL of 10% aqueous NaHSO3 and dried over Na2SO4. Chromatography on silica
gel provided 598 mg of 16 together with 338 mg of unreacted starting material
(83% of 16 based on conversion): [a]20D -279" (c 0.21, CHCIl); IR (KBr) 3200,
1740, 1680, 1260, 1240, 1065 cm-'; 'H NMR (300 MHz, CDCl3) ~ 7.18-7.00 (m,
3H), 6.05 (s, lH), 4.54 (m, lH), 4.13 and 3.98 (ABq, 2 H, J= 11.1 Hz, both partsd with J= 8.2 Hz), 3.45 (d, J= 8.2 Hz, lH), 3.01-2.87 (m, 2H), 2.76 (s, 3H),
2.46 (m, lH), 2.10 (s, 3H), 1.08 (d, J= 7.2 Hz, 3H), 0.92 (d, J= 7.2 Hz, 3H); MSm/z 430 (M+), 304, 261, 233, 158, 132; HRMS calcd for C,7H23N2O3I: 430.076,
found: 430.075.
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24
Example 2. (2S,5S)-8-Decylbenzolactam (Figure 11, compound 18).
A mixture of the compound of Example 1 (17) (20 mg, 0.05
mmol), Pd/C (10 mg), and EtOAc (5 mL) was hydrogenated under 10 atm of H~
at room telllp~ldlu~e for 2 hours. Filtration from the catalyst, evaporation, and
chromatography gave 18 mg (90%) of the title compound 18. [O~]2OD -247.5 ~ (c
1.0, ethanol); 'H NMR (300 MHz, CDCI3) ~ 6.93 (m, 2H), 6.87 (s, lH), 6.45 (s,
lH), 4.14 (m, lH), 3.78 and 3.51 (ABq, 2~I, J = 15.8 Hz, both parts d, J = 12.4
and 8.6 Hz, resp.), 3.39 (d, J= 8.3 Hz, lH), 2.96 (dd, J= 16.2, 10.6 Hz, lH),
2.83 (d, J= 16.2 Hz, IH), 2.70 (s, 3H), 2.36 (t, J= 7.6 Hz, 2H), 2.30 (m, IH),
1.60-1.06 (m, 16H), 1.01 (d, J= 7.6 Hz, 3H), 0.82 (d, J= 7.6 Hz, 3H), 0.79 (t, J= 7.4 Hz, 3H); MS m/z 402 (M+), 359, 331, 316; HRMS calc. for C25H4~N2O2
402.324, found 402.325. Anal. calcd. for C25H42N2O2: C, 74.58; H, 10.51; N, 6.96.
Found: C, 74.26, H, 10.82; N, 6.71.
Example 3. (2S,5S)-7-Methoxy-8-(1-decynyl) benzolactam (Figure 12,
compound 8).
To a suspension solution of 7(Figure 12) (25 mg, 0.085 mmol)
and HgC12 (23 mg, 0.085 mmol) in 2 mL of methylene chloride was added I~ (22
mg, 0.085 mmol). The mixture was stirred at room temperature overnight and
filtered. The filtrate was washed with aqueous 0.1 M sodium thiosulfate, and a
saturated aqueous solution of potassium iodide. The organic layer was dried and
concentrated by rotary evaporation.
To a mixture of the above iodide, 2 mL of diethylamine and
PdC12(Ph3P)2 (13 mg. 0.009 mmol) was added l-decyne (0.065 mL, 0.37 mmol)
and CuI (4 mg, 0.01 mmol). The resulting solution was stirred at room
temperature for 24 hours. The solvent was removed under reduced pressure, and
the residual oil was purified by chromatography (silica gel, 2/1 ethyl
acetate/petroleum ether as eluent) to afford 27 mg (74% yield) of the title
compound 8. [a]20D = -302 ~ (c 0.63, CHCI3); IH NMR (300 MHz, CDCI3) o 7.18
CA 022~3463 1998-11-03
WO 97/43268 PCT/US97/0814
(d, J = 8.3 Hz, lH), 6.70 (br s, IH), 6.62 (d, J = 8.3 Hz, lH), 3.89 (s, 3H), 3.72-
3.53(m,3H),3.46(d,J=9.2Hz,lH),3.26and2.78(ABq,d,J=17.3Hz,2H),
2.79 (s, 3H), 2.48 (t, J = 7.5 Hz, 2H), 2.46 (m, lH), 1.53 (m, 2H), 1.46(m, 2H),1.37(m,8H), 1.02(d,J=7.4Hz,3H),0.87(t,J=7.2Hz,3H),0.78(d,J=7.4
S Hz, 3H): MS m/z 428 (M~), 329, 291, 249, 221, 104, 77. HRMS calcd for
C26H40N2O3: 428.304, found: 428.302.
a) 3-Hydroxy-2-(hydroxymethyl)nitrobenzene (Figure 12, compound 80).
A mixture of 15.0 g of 5-nitro-1,3-benzodioxane79 (Ando, M.; Emoto, S., Bull.
10 Chem. Soc. Jpn. 46, 2093, 1973) and 800 mL of I N HCI was allowed to reflux
for 24 hours. The cooled suspension was extracted with ethyl acetate (400 mL x
3). The combined organic layers were washed with water and brine, and dried
over MgSO4. After removal of solvent, the residual oil was purified by
chromatography to afford 9.7 g of the diol. 'H NMR (300 MHz CDCl3) o 7.41
15 (d,J=7.2Hz, IH),7.28(dd,J=7.2Hz, lH),7.13d,J=7.2Hz),S.O9(s,2H);
MS Mtz 169 (M+), 151, 133, 121, 105, 93, 77.
b) 2-(Hydroymethyl)-3-(methoxy)nitrobenzene (Figure 12, compound 81).
To a solution of 9.7 g of 80 (57 mmol) in 300 mL of dry acetone was added 81 g
20 of idomethane (57 mmol) and 11.8 g of potassium carbonate (85 mmol). The
mixture was allowed to reflux overnight, and the cooled solution was partitionedbetween 300 mL of ethyl acetate and 100 mL of water. The organic layer was
washed with water and brine, and dried over Na2SO4. After removal of solvent,
the residual oil was passed a short column (silica gel, 1/1 ethyl acetate/petroleum
25 ether as eluent) to afford 10.3 g (98%) of the methoxy alcohol; 'H NMR (300
MHz, CDC13) o 7.38 (d, J = 7.2 Hz, IH), 7.19 (dd, J = 7.2 Hz, IH), 7.09 (2, J =
7.2 Hz), 5.10 (s, 2H), 3.81 (s, 3H); MS m/z 183 (M+), 166, 150, 108, 92, 77;
HRMS calcd for C8H9NO4: 183.053, found: 183.052.
30 c) 2-[2-(tert-Butoxycarbonylamino)-2-(ethoxycarbonyl)ethyl]-3-(methoxy)
nitrobenzene (Figure 12, compound 4). To a solution of 81 (4.0 g, 22 mmol) and
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triethylamine (3.3 g, 33 mmol) in 160 mL of THF was added mesyl chloride (3.8
g, 33 mmol), dropwise with cooling at -20 ~C. The solution was warrned to
room temperature slowly, and the THF was removed by rotary evaporation. The
residue was partitioned between 200 mL of ethyl acetate and 70 mL of water.
5 The organic layer was washed with water and brine, and dried over Na2SO4.
After removal of solvent, the residue was dried in vacuo and then this residue
was dissolved in 50 mL of methylene chloride. To the resulting solution was
added Bu4NBr (6.16 g, 19.2 mmol), (5.0 g, 19.2 rnmol) and 50 mL of 10%
NaOH. The mixture was stirred for 36 hours at room temperature. The organic
10 layer was separated and aqueous layer was extracted with ether. The combined
organic layers were washed with brine, dried over Na2SO4, and concentrated by
rotary evaporation. The residue was dissolved in 50 mL of THF, and 50 mL of
5% HCI was added. The resulting solution was stirred overnight and then
saturated a~ueous NaHCO3 was added to adjust pH to l l . After removal of the
15 THF by rotary evaporation, the residue was extracted with ethyl acetate (3 x 100
mL). The combined organic layers were washed with brine, dried over Na2SO4
and concentrated. The residual oil was dissolved in 60 mL of acetonitrile, and
then 3.75 g of di-tert-butyl-dicarbonate (17.1 mmol) was added. After stirring
overnight, the solution was concentrated and the residue was chromatographed to
20 afford 3.80 g (49% yield from 3) of the ester. 'H NMR (300 MHz, CDC13) o
7.41 (d, J = 7.2 Hz, lH), 7.33 (dd, J = 7.2 Hz), 5.22 (br s, IH), 4.36-4.10 (m,
3H)7 3.92 (s, 3H), 3.04 (M, 2H), 1.01 (s, 9H), 0.99 (t, J + 7.2 Hz, 3H); MS m/z
368 (M+), 271, 150, 104, 77: HRMS calcd for Cl7H24N207; 368.159, found:
358.156.
d) 2-~2-(tert-Butoxycarbonylamino)-3-hydroxypropyl]-3-(methoxy)aniline
(Figure 12, compound 5). A mixture of 4 (5.0 g, 14.2 mmol), NaBH4 (1.62g,
42.6 mmol) in 350 mL of dry ethanol was heated at reflux for 2 hours. After
cooling to room temperature, the solvent was removed under reduced pressure
30 and the residue was partitioned between 400 mL of ethyl acetate and 100 mL ofwater. The organic layer was separated, washed with water and brine, and dried
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over Na2SO4. After removal of the solvent, the residual oil was
chromatographed to afford the nitro-alcohol.
A suspension of the nitro-alcohol and 100 mg of 10% Pd/C in 100
mL of methanol was exposed to hydrogen under atmospheric pressure with
5 vigorous stirring. After no more hydrogen was taken up, the Pd/C was filtered
off, and the filtrate was concentrated. Chromatography of the residual oil
afforded 3.65 g (92% yield) of 5. 'H NMR (300 MHz, CDCI3) ~6.98 (dd, J = 7.2
Hz, lH), 6.25 (d, J = 7.3 Hz, lH), 6.20 (d, J + 7.2 Hz, lH), 5.03 (br s, lH), 4.88
(br,s, lH),4.18(m, lH),3.79(s,3H),3.29and3.18(ABq,d,J=7.8Hz,2H),
10 2.85 and 2.74 (AB q, d, J = 15.4 Hz, 2H), 1.44 (s, 9H); MS m/z 296 (M+), 104, 77: HRMS calcd for C,5H24N2)(4: 296.174 found: 296.171.
e) N-[(S)-l-Ethoxycarbonyl-2-methylbutyl]-2-[2-(tert-
butoxycarbonylamino)-3-hydroxypropyl]-3-(methoxy)aniline (Figure 12,
15 compound 6). To a solution of (R)-ethyl-a-[[(trifluoromethyl)sulfonyl]oxy]
isovalerate (2.9 g, 10.5 mmol), 5 (2.8 g, 10 mmol in 35 mL of 1,2-dichloroethanewas added 2,6-lutidine (1.87 g, 11 mmol). The resulting solution was heated at
70 ~C for 40 hours, and the cooled solution was directly chromatographed to
afford 2.46 g (67%) of 6. 'H NMR (300 MHz, CDCI3) o 7.03 (dd, J = 7.2 Hz,
20 lH), 6.30 (d, J = 7.2 Hz, lH), 6.23 (d, J = 7.2 Hz) 5.29 ()br, s, IH), 4.11 (m, 3H),
3.84-3.44 (m, 2H), 3.79 (s, 3H), 2.98-2.72 (m, 2H), 1.86 (m, lH), 1.43 (s, 9H),
1.34-0.88 (m, 9H).
f) (2S,SS)-7-Methoxybenzolactam (Figure 12, compound 7). A mixture of
25 6 (2.1 g, 5.15 mmol), 20 mL of 0.5 N aqueous NaOH, and 20 mL of ethanol was
stirred for 10 hours at room temperature. The resulting solution was neutralizedto a pH of ~5 with concentrated hydrochloride acid and extracted with ether to
afford 1.52 g of crude acid.
This acid (1.5 g, 4.0 mmol) and N-hydroxysuccinimide (1.2 g,
30 10.0 mmol) were dissolved in 20 mL of acetonitrile. To this solution DCC (1.5g, 7.5 mmol) in 20 mL of acetonikile was added dropwise at 0 DC. The mixture
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was stirred at room temperature overnight. After filtering off the resulting solid,
the solution was evaporated under reduced pressure, and the residue was
chromatographed on silica gel (1/1 methylene chloride/ethyl acetate as eluent) to
provide 1.34 g of the activated ester. Without further purification, this product
5 was directly dissolved in 10 mL of dried methylene chloride, and the resultingsolution was cooled at 0 ~C prior to the addition of 10 mL of trifluoroacetic acid.
The reaction mixture was stirred at 0 ~C for 2 hours, and the trifluoroactic acid
was removed in vacuo at below 30 ~C. The residue was dissolved in 100 mL of
ethyl acetate and 10 mL of a saturated aqueous NaHCO3 solution. The mixture
10 was heated at 80 ~C for 24 hours with vigorous stirring. After cooling to room
temperature, 40 mL of water was added to the mixture, and the mixture was
extracted with ethyl acetate (4 x 100 mL) and dried over Na2SO4. The solvent
was removed under reduced pressure. The residue was dissolved in 10 mL of
acetonitrile, and 4 mL (40 mmol) of forrnalin, 1.0 g (16 mmol) of sodium
15 cyanoborohydride, and 0.27 mL of acetic acid were added sequentially at 0 ~C.The resulting solution was stirred at 0 ~C for 2 hours. After quenching with
phosphate buffer (pH = 2), the solvent was removed by evaporation. The residue
was dissolved in ethyl acetate, and the solution was washed with water, saturated
sodium bicarbonate, and brine, and dried over Na,SO4. After concentration, the
20 residue was purified by chromatography on silica gel (9/1 ethyl
acetate/methylene chloride as eluent) to give 7 (280 mg, 20% overall yield from
6). ~a]20D = -252~ (c 1.3, CHCI3); H NMR (300 MHz, CDCI3) ~ 7.15 (dd, J = 8.1
Hz, lH), 6.83 (br s, IH), 6..67 (d, J = 8.1 Hz, lH), 6.51 (d, J = 8.1 Hz, lH), 3.92
(m, lH), 3.82 (s, 3H). 3.75-3.18 (m, 4H), 2.78 (s, 3H), 2.70 (m, lH), 2.38 (m,
25 lH), 1.08(d,J=7.2Hz,3H),(d,J=7.2Hz,3H);MSm/z292(M~),261,249,
221, 174, 162, 137, 114; IIRMS calcd for Cl6H24N2O3: 292.179, found: 292.173.
All patents, patent applications, and publications cited herein, and
specifically, U.S. Provisional Patent Application Number 60/017,532, are
incorporated by reference herein as though fully set forth.
