Note: Descriptions are shown in the official language in which they were submitted.
Wo 95/06031 2 ~ 7 ~` ~ 5 8 PCT/US94/09343
TIT~ E
"Inhibitors of TNF-alpha Secretion"
~ROSS-RF.F~.RF.NC~. TO RF.~,ATFD APPT ICATTONS
This application is a continuation-in-part of application serial no. 08/110,601, filed
August 23, 1993, pending.
F~FT,D OF THF. INVF.NT~ON
The invention pertains to compounds which are inhikitnrs of metalloproteases and, in
particular, to compounds which inhibit the TNF-a converting enzyme.
RACKGROUND OF THF. INVF.NT~ON
Tumor necrosis factor-a (TNF-a, also known as cachectin) is a m~mm~ n protein
capable of inducing a variety of effects on numerous cell types. TNF-a was initially
20 characterized by its ability to cause lysis of tumor cells and is produced by activated cells
such as mononucLear phagocytes, T-cells, B-cells, mast cells and NK cells. In mononuclear
phagocytes, TNF-a is initially synthesized as a membrane-bound protein of a~ro~ lalely
26 kD. A 17 kD fragment of the 26 kD membrane-bound TNF-a is "secreted" and
combines with two other secreted TNF-a molecules to form a circulating 51 kD homotrimer.
25 TNF- a is a principal mediator of the host response to gram-negative bacteria.
Lipopolysaccharide (LPS, also called endotoxin), derived from the cell wall of gram-
negative bacteria, is a potent stimulator of TNF-a synthesis. Because the deleterious effects
which can result from an over-production or an unregulated-production of TNF aree~lGmely serious, considerable efforts have been made to control or regulate the serum level
30 of TNF. An important part in the effort to effectively control serum TNF levels is the
und~ ndillg of the mechanism of TNF biosynthesis.
The mechanism by which TNF-a is secreted has only been recently elucidated.
Kriegler et al. Cell, 53, 45-53, (1988) conjectured that TNF-a "secretion" is due to the
35 cleaving of the 26 kD membrane-bound molecule by a proteolytic enzyme or protease.
Scuderi et. al., J. Immunology, 143, 168-173 (1989), suggested that the release of TNF-a
from human leukocyte cells is dependent on one or more serine proteases, e.g., a leukocyte
el~st~e or trypsin. A serine protease inhibitor, p-toluenesulfonyl-L-arginine methyl ester,
was found to suppress human leukocyte TNF release in a concentration-dependent manner.
40 Scuderi et. al. suggested that the arginine methyl ester competes for the arginine-binding site
WO 95/06031 2 ~ PCT/US94/09343
in the enzyme's reactive center and thereby blocks hydrolysis. The lysine and phenylalanine
analogs of the inhibitor reportedly failed to mimic the arginine methyl ester.
We have discovered that the protease which causes the cleavage of the TNF-a
S molecule into the 17 kD protein is, in fact, a metalloprotease which is believed to reside in
the plasma membrane of cells producing TNF-a. The physicochemical characteristics of the
enzyme have not been published.
Most, but not all, proteases recognize a specific amino acid sequence. Some
10 proteases primarily recognize residues located N-terminal of the cleaved bond, some
recognize residues located C-terminal of the cleaved bond, and some proteases recognize
residues on both sides of the cleaved bond. Metalloprotease enzymes utilize a bound metal
ion, generally Zn2+, to catalyze the hydrolysis of the peptide bond. Metalloproteases are
implicated in joint destruction (the matrix metalloproteases), blood pressure regulation
15 (angiotensin converting enzyme), and regulation of peptide-hormone levels (neutral
endopeptidase-24.1 1).
Numerous inhibitors have been developed against the previously described
metalloproteases. A general family of inhibitors against matrix-metalloproteases, and in
20 particular collagenase, is reported in WO 92/09563. This document shows compounds
having the general structure of a reverse hydroxamate - or a hydroxyurea - linked via an
amide to an amino acid derivative, such as tryptophan or 2-naphthyl alanine. Inhibitors of
collagenase are also reported in WO 88/06890; these compounds contain sulfhydryl moieties
as well as phenylalanine and tryptophan analogs. Collagenase inhibitors are reported in WO
25 92/09556 and U.S. Patent No. 5,114,953 and possess hydroxamate moities and fused or
conjugated bicycloaryl substituents. The myriad potential gelatinase inhibitors covered by
the generic formula in EPA 489,577 are amino acid derivatives optionally possessing a
hydroxamate group. Hydroxamate derivatives useful as angiotensin converting enzyme
(ACE) inhibitors are reported in EPO 498,665.
Inhibition of the TNF-a converting enzyme (hereinafter referred to as "TACE"), anovel metalloprotease, inhibits release of TNF-a into the serum and other extracellular
spaces. TACE inhibitors would therefore have clinical utility in treating conditions
characterized by over-production or unregulated production of TNF-a. A particularly useful
35 TACE inhibitor for certain pathological conditions would selectively inhibit TACE while not
affecting TNF-13 (also known as Iymphotoxin) serum levels. The over-production or
unregulated production of TNF-a has been implicated in certain conditions and ~ e~es, for
example:
wo 95/06031 2 17 015 g PCT/US94/09343
I. Systemic Tnfl~mm~tory Response Syndrome, which includes:
Sepsis syndrome
gram positive sepsis
gram negative sepsis
culture negative sepsis
fungal sepsis
n~uLI~penic fever
urosepsis
meningococcemia
Trauma/h~,l.c",hage
Burns
Ionizing radiation exposure
Acute ~lc,-,alilis
Adult respiratory distress syndrome.
II. Reperfusion Injury, which includes:
Post pump syndrome
Ischemia-reperfusion injury
III. Cardiovascular Disease, which includes:
Cardiac stun syndrome
Myocardial infarction
Congestive heart failure
IV. Infectious Disease, which includes:
HIV infection/ HIV neuropathy
Meningitis
Hepatitis
Septic arthritis
Peritonitis
Pneumonia
Epiglottitis
E. coli 0157:H7
Hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura
Malaria
Dengue hemorrhagic fever
T f~ hm~ni~cic
Leprosy
WO 95/06031~ 2 ~ 7 ~15 8 PCT/US94/09343
Toxic shock syndrome
Streptococcal myositis
Gas gangrene
Mycobacterium tuberculosis
Mycobacterium avium intr~cell~ re
Pneumocystis carinii pnt~llmoni~
Pelvic infl~ y disease
Orchitis/epidydimitis
Legionella
Lyme disease
Influenza A
Epstein-Barr Virus
Viral-associated hemaphagocytic syndrome
Viral encephalitis/aseptic meningitis
V. Obstetrics/Gynecology, including:
Gl I l~lt Ul G labor
Miscarriage
Infertility
VI. Tnfl~mm~toryDisease/Au~illlllllll~ity, which includes:
Rheumatoid arthritis/seronegative al ~l~opathies
Osteoarthritis
Tnfl~mm~tory bowel disease
Systemic lupus erythematosis
Iridocyclitis/uveitis/optic neuritis
Idiopathic pulmonary fibrosis
Systemic vasculitis/Wegener's granulomatosis
Sarcoidosis
Orchitis/vasectomy reversal procedures
VII. Allergic/Atopic Diseases, which includes:
Asthma
Allergic rhinitis
F~.7~.m~
Allergic contact dermatitis
Allergic conjunctivitis
Hypersensitivity pneumonitis
WO 95/06031 2 ~ 7 ~ i 5 8 PCT/US94/09343
VIII. ~lign~ncy, which includes:
A~
A~L
CML
~.
Hodgkin's disease, non-Hodgkin's lymphoma
MM[ '
Kaposi's s~;o,l,a
Colorectal ;~.;i,l~ ,lla
Nasopharyngeal carcinoma
Malignant histiocytosis
Paraneoplastic syndrome/hypercalcemia of malignancy
IX. Transplants, including:
Organ transplant rejection
Graft-versus-host disease
X. Cachexia
XI. Congenital, which includes:
Cystic fibrosis
Familial h~llla~c,phagocytic lymphohistiocytosis
Sickle cell anemia
XII. Dermatologic, which includes:
Psoriasis
Alopecia
XIII. Neurologic, which includes:
Multiple sclerosis
Migraine he~ he
" XIV. Renal,which includes:
Nephrotic syndrome
Hemodialysis
Uremia
WO95/06031 2 ~7 01~ 8 PCT/USg4/09343
XV. Toxicity, which includes:
OKT3 therapy
Anti-CD3 therapy
Cytokine therapy
Chemotherapy
Radiation therapy
Chronic salicylate intoxication
XVI. Metabolic/ldiopathic, which includes:
Wilson's disease
Hemachromatosis
Alpha-1-antitrypsin deficiency
Diabetes
H~chimoto's thyroiditis
Osteoporosis
Hypoth~l~mic-~iLui~ y-adrenal axis evaluation
Primary biliary cirrhosis
Inhibitors of TACE would prevent the cleavage of cell-bound TNF-a thereby
20 reducing the level of TNF-a in serum and tissues. Such inhibitors would be of significant
clirlical utility and could be potential therapeutics for treating the above TNF-a-related
disorders.
SUMMARY OF THF, INVF.NTION
The invention relates to compounds of formula I:
O O
11 11
X-[CH]m-CH-C-N-CH-C-[A]n-N-B-NH2
(I) I I H I H
R1 R2 R3
wherein:
X is hydroxamic acid, thiol, phosphoryl or carboxyl;
misO, 1 or2;
Rl, R2 and R3 each independent of the other is hydrogen, alkylene(cycloalkyl),
oR4, SR4, N(R4)(R5), halogen, substituted or unsubstituted Cl to C8 alkyl, Cl to
Wo 95/06031 Z ~ ~ O l S 8 PCT/US94/09343
C8 aL~-ylenearyl, aryl, a p,oLe~Led or unprotected side chain of a naturally occurring
a-amino acid; or the group -R6R7, wherein R6 is substituted or unsubstituted Cl to
C8 alkyl and R7 is oR4, SR4, N(R4)(R5) or halogen, wherein R4 and R5 are, each
independent of the other, hydrogen or substituted or unsubstituted C1 to C8 alkyl;
nisO, 1 or2;
provided that when n is 1, A is a ~ro~ecled or an unprotected a-amino acid radical;
when n is 2, A is the same or different protected or unprotected a-amino acid radical;
and
B is unsubstituted or substituted C2 to C8 alkylene;
and the phaTm~elltir~lly acceptable salts thereof.
The compounds of formula I are useful as metalloprotease inhibitors, and particularly
15 useful as inhibitors of the TNF-a converting enzyme (TACE).
The invention also relates to a method of treating a mammal having a disease
characterized by an overproduction or an unregulated production of TNF-a. The method
comprises the steps of ~lmini~tering to the m~mm~l a composition comprising an effective
amount of a biologically active compound of formula II:
O O
Il 11
X-[CH]m-CH-C-N-CH-C-[A]n-N-y (II)
l l H l H
R 1 R2 R3
wherein:
X is hydroxamic acid, thiol, phosphoryl or carboxyl;
misO, 1 or2;
R1, R2 and R3 each independent of the other is hydrogen, alkylene(cycloalkyl),
oR4, SR4, N(R4)(R5), halogen, substituted or unsubstituted Cl to C8 alkyl, C1 toC8 aLkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring
a-amino acid; or the group -R6R7, wherein R6 is Cl to C8 aLkyl and R7 is oR4,
SR4, N(R4)(R5) or halogen, wherein R4 and R5 are each, independent of the other,hydrogen or substituted or unsubstituted Cl to C8 alkyl;
n is 0, 1 or 2;
Y is hydrogen, unsubstituted or substituted Cl to C8 alkyl, alkylene(cycloalkyl), the
group -R8-COOR9 or the group -RlON(Rl l)(R12); wherein R8 is Cl to C8
WO 95/06031 2 17 0 1~ 8 PCTIUS94/09343
aL~ylene; R9 is hydrogen or Cl to C8 aL~cyl; R10 is unsubstituted or substituted Cl to
C8 alkylene; and Rl 1 and R12 are each, independent of the other, hydrogen or Cl to
C8 aL~cyl;
provided that when n is 1, A is a protected or an unprotected a-amino acid radical;
and
when n is 2, A is the same or different protected or unprotected a-arnino acid radical;
and the pharm~ellti~lly acceptable salts thereof;
wherein the colll~oul,d is capable of reducing serum TNF-a levels by at least 80%
when ~tlmini~tered at 25mg/kg in a murine model of LPS-induced sepsis syndrome;
and a ~hal ,. ,~eutir~lly acceptable calrier.
The discovery of useful inhibitors of the TACE metalloprotease has led to the
discovery of further embodiments of the invention, including pharmaceutical compositions
for treating the above-listed disorders comprising a compound according to formula II and
15 protein having TNF-binding activity.
T)FT~IJ,FD DFSCRIPTTON OF THF INVFNTI20
The invention is directed to a compound of formula I:
O O
Il 11
X-rCH]m-CH-C-N-CH-C-[A]n-N-B-NH2
(I) I I H I H
R 1 R2 R3
wherein:
X is hydroxamic acid, thiol, phosphoryl or carboxyl;
mis 0, 1 or 2;
Rl, R2 and R3 each independent of the other is hydrogen, alkylene(cycloalkyl),
oR4, SR4, N(R4)(R5), halogen, substituted or unsubstituted Cl to C8 aL~yl, Cl toC8 alkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring
oc-amino acid; or the group -R6R7, wherein R6 is substituted or unsubstituted Cl to
C8 aL~yl and R7 is oR4, SR4, N(R4)(R5) or halogen, wherein R4 and R5 are each,
independent of the other, hydrogen or substituted or unsubstituted C1 to C8 alkyl;
n is 0, 1 or 2;
provided that when n is 1, A is a pl.~lecled or an unprotected oc-amino acid radical;
WO 95/06031 . r - PCT/US94/09343
~170158
provided that when n is 1, A is a ,~lvle~;led or an unprotected oc-amino acid radical;
when n is 2, A is the same or different protected or unprotected a-amino acid radical;
and
B is unsubstituted or substituted C2 to C8 alkylene;
and the pharmaceutically acceptable salts thereof.
J The compounds of formula I are useful as inhibitors of TNF-oc secretion, and
pa~ticularly useful as inhibitors of the TNF-o~ converting enzyme (TAOE).
The invention also relates to a method for treating a m~mm~l having a condition or a
disease characterized by overproduction or unregulated production of TNF-oc, comprising
~-lmini~tering to the ",~.1"~1 a composition comprising an effective amount of a biologically
active compound of formula II:
O O
Il 11
X-[CH]m-CH-C-N-CH-C-[A]n-N-Y (II)
H I H
R 1 R2 R3
wherein:
X is hydroxarnic acid, thiol, phosphoryl or carboxyl;
misO, 1 or2;
Rl, R~2 and R3 each independent of the other is hydrogen, alkylene(cycloalkyl),
oR4, SR4, N(R4)(R5), halogen, substituted or unsubstituted Cl to C8 alkyl, Cl toC8 alkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring
a-amino acid; or the group -R6R7, wherein R6 is C1 to C8 alkyl and R7 is oR4,
SR4, N(R4)(R5) or halogen, wherein R4 and R5 are each, independent of the other,hydrogen or substituted or unsubstituted C1 to C8 alkyl;
n is 0, 1 or2;
Y is hydrogen, unsubstituted or substituted C1 to C8 alkyl, alkylene(cycloalkyl), the
group -R8-COOR9 or the group -R1ON(R1 1)(R12); wherein R8 is Cl to C8
alkylene; R9 is hydrogen or Cl to C8 alkyl; R10 is unsubstituted or substituted Cl to
C8 alkylene; and Rl 1 and R12 are each, independent of the other, hydrogen or Cl to
C8 alkyl;
provided that when n is 1, A is a protected or an unprotected oc-arnino acid radical;
and
W 095/06031 ~ ~ 7 ~ ~ ~ 8 PCTrUS94/09343
when n is 2, A is the same or different protected or unprotected a-amino acid radical;
and the pharmaceutically acceptable salts thereof;
wherein the co~ ulld is capable of reducing serum TNF levels by at least 80%
when administered at 25mg/kg in a murine model of LPS-induced sepsis syndrome;
and a ph~"laceutically acceptable carrier.
The invention includes pharmaceutical compositions containing a compound
according to formula I as the active component. In addition, pharmaceutical compositions
comprising a compound according to formula II and a protein which binds TNF are
10 described. An example of a protein which binds TNF is an anti-TNF antibody or a soluble
TNF receptor which is described in EPA 0418014, ~ignecl to the assignee of the instant
application. The disclosure of EPA 0418014iS incorporated herein by reference.
The following definitions are used herein. "Alkyl" means a straight or branched,15 univalent, saturated or unsaturated hydrocarbon group of 1 to 8 carbon atoms. Alkyl groups
include the straight-chain groups methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl,
vinyl, allyl, butenyl, pentenyl, hexenyl, heptenyl and octenyl as well as the branched
isomers thereof.
"Substituted alkyl" means an alkyl group substituted with one or more of hydroxy,
amino, halogen, or thiol.
"Alkylene" means a bivalent alkyl group as defined above.
"Substituted alkylene" means an alkylene group substituted with one or more of
hydroxy, amino, halogen or thiol groups.
"Aryl" means an aromatic or heteroaromatic group, including for example, phenyl,naphthyl, pyridyl, quinolyl, thienyl, furyl and the like, optionally substituted with one or
more of Cl to C8 alkyl, hydroxy, amino, halogen, thiol or alkyl groups.
"Alkylene(cycloalkyl)" refers to groups of the structure -R13-R14 wherein R13 is an
alkylene as defined above, and R14 is a univalent cyclic alkane radical, for example,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.
"Alkylenearyl" means the group -R15-R16, wherein R15 is a substituted or
-n.cllbstitllte~l alkylene group as defined above, and R16 is a substituted or unsubstituted aryl
group as defined above.
WO 95/06031 PCT/US94/09343
~ 21~01~
"a-Amino acid" refers to any of the 22 common amino acids, e.g., alanine, arginine,
asparagine, aspartic acid, cysteine, cystine, glutamine, glutamic acid, glycine, histidine,
hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,
S ~I~eulline, tryptophan, tyrosine and valine.
"~olecled amino acid" and "~lulecled side chain of an a-amino acid" means the side
chains of the amino acid are permanently or L~ ,ol~ily coupled to a chemical group which
protects or prevents the side chain from undesired branching, structural modification or
10 rearrangement which can occur during subsequent synthetic steps. Use of such protecting
groups for these purposes is well known in the art, as are the protecting groups themselves.
Examples of common protecting groups are N-tert-butyloxycarbonyl (Boc) and N-9-
fluorenylmethyloxycarbonyl (Fmoc).
"Biologically active" as used in defining certain compounds of formula II, designates
a compound capable of (a) inhibiting secretion of TNF-a; (b) preventing cleavage of
membrane-bound TNF-a by TACE; or (c) reducing serum TNF levels by at least 80% when
~rlministered at 25 mg/kg in a standard murine model of LPS-indl~e-l sepsis syndrome.
In the compounds of formulas I and II, pl~rt;llc;d radicals for X are hydroxamic acid,
thiol and phosphoryl. More preferred X radicals are hydroxamic acid and thiol, while the
most preferred radical is hydroxamic acid. The plere~ d value for m is 1.
Preferred R1 or R2 radicals are hydrogen, C1 to C8 alkyl and Cl to C8
alkylenearyl. Where Rl or R2 is alkyl, preferred is Cl to C6 alkyl and most ~l~rell~,d is Cl
to C4 alkyl. Where R1 or R2 is alkylenearyl, preferred alkylene groups are C1 to C6
alkylene, and more ~iefelled is Cl to C4 alkylene; and preferred aryl groups are phenyl and
substituted phenyl. The most preferred alkylenearyl group for R1 or R2 is C1 to C4
alkylenephenyl. The most preferred group for R1 is hydrogen and the most preferred
group for R2 is isobutyl.
Preferred R3 radicals are substituted and unsubstituted C1 to C8 alkyl and C1 to C8
alkylenearyl. Where R3 is alkyl, pl~fellt;d is Cl to C6 alkyl and more preferred is Cl to C4
alkyl, with t-butyl being most preferred. Where R3 is C1 to C8 alkylenearyl, preferred
alkylene groups are Cl to C6 alkylene, and more pl~;fell ;;d is Cl to C4 alkylene; and
preferred aryl groups are phenyl, naphthyl, and thienyl, each optionally substituted with
hydlu~y, amino, halogen, thiol or alkyl groups. F'lcfelled groups for R3 are therefore Cl to
C4 alkylenephenyl, Cl to C4 alkylenenaphthyl, and C1 to C4 alkylenethienyl. More
_
WO 95/06031 ~17 01~ 8 PCT/US94/09343 ~
plcrell~d is C1 to C4 alkylenenaphthyl, with methylenenaphthyl being most ~lefel,cd.
Where R3 is a protected or unprotected side chain of a naturally occurring a-amino acid, R3
preferably is an arginine, lysine, tryptophan or tyrosine side chain. However, the most
rellGd radicals for R3 are t-buyl, methylene(cyclohexyl) and methylene-(2'naphthyl).
S r
The radical A is preferably an unprotected naturally-occurring amino acid residue.
More preferred naturally-occurring residues are the alanyl radical or an wlplolec~ed seryl
radical. The most plc~ll~d radical for A is an alanyl residue. Further preferred compounds
are those where n is 0 or 1, while most preferably n is 1.
~er~ d radicals for B are C2 to C6 alkylene. More ~lc;~lled radicals are C2 to C4
aLkylene, with dimethylene being most ~lt;rt;lled.
For compounds according to formula II, Y is preferably hydrogen, unsubstituted or
substituted Cl to C8 alkyl or the group -R10N(Rl l)(R12). Most preferred is the group
-R10N(R11)(R12) with Rl0 preferably being unsubstituted or substituted C1 to C6
alkylene, Rl 1 and R12 preferably are each independently hydrogen or Cl to C6 alkyl. More
pl~rtill ;;d R10 radicals are unsubstituted or substituted Cl to C4 alkylene, with dimethylene
'oeing most preferred. More preferred radicals for R10 and Rl 1 are hydrogen or Cl to C4
alkyl, with hydrogen being most plef~llGd.
Compounds according to the invention can be prepared utilizing the procedures
outlined below, the appended reaction Schemes and the procedures detailed in the Examples
below.
General Synthesis
With reference to Scheme 1, the inhibitor compounds may be prepared by converting
the carboxylic acid or ester compound (Io), wherein R is H or C1 to C8 alkyl, and P is
CBZ, BOC, FMOC or other suitable protective group (Greene T., Wuts P., "Protective
Groups in Organic Synthesis", 2nd Ed.; Wiley: New York, 1991; Chapter 7), to thecorresponding hydroxamic acid or hydroxamic ester compound (Ip). In compound (Ip), R'
is H, TMS, t-Bu, Bzl or other group made by treating these compounds, or an activated
form of the carboxylic acid, (Bodanszky, M., Bodanszky, A., "The Practice of Peptide
Synthesis"; Springer-Verlag: Berlin, 1984; Chapter II) with a hydroxylamine reagent under
conditions which effect the conversion. This is followed by the subsequent removal of the
protective group P and R' to generate compound (Iq). The abbreviations used above
correspond to the following: Bzl=benzyl; BOC=t-butoxycarbonyl; tBu=t-butyl;
CB~ben~yloxyc~'~nyl; FMOC=9-fluorenylmethoxycarbonyl; TMS=trimethylsilyl.
~ WO 95/06031 - . PCT/US94/09343
2~0158
A hydl~xylamine reagent described above can be hydroxylamine or ~ltern;ltively, it
can be an O-protected hydroxylamine such as commercially available O-trimethylsilyl
hydroxylamine, O-tert-butylhydroxylamine, or O-benzylhydroxylamine.
The ~.re~)aldlion of precursor compound (Io) may be carried out by condensing the
dicarboxylate compound (Ie), with the amine (In), wherein R" is an activating group
(Bod~n~7~y, M.; et al., suprà.) such as an active ester, anhydride or other group that causes
con-1~n~tion with the amine terminus of compound (In) to occur with formation of a peptide
10 bond.
The preparation of compound (Ie) may be typically carried out as follows: the
sodium salt of the 2-oxocarboxylate compound (Ia), is esterified with benzyl bromide to
produce the benzyl ester (Ib). Several examples of compound (Ia) are commercially
15 available as various salts or carboxylic acids. Others can be made synthetically (see, for
example, Nimitz, J. et al., J. Org. Chem. 46:211, 1981; and Weinstock, L.et al., Synth.
Commun. 11:943, 1981). The benzyl ester compound (Ib) is treated with a Wittig reagent,
typically methyl or tert-butyl triphenylphosphoranylidene acetate, to form the alkene (Ic), as
a mi~Lule of E- and Z- isomers. Reduction of the alkene compound (Ic) is carried out with
20 hydrogen, in the presence of an appropriate catalyst (typically palladium on activated
charcoal), to both hydrogenate the double bond and to remove the benzyl ester, giving the
mono-ester compound (Id) as a enantiomeric mixture. Compound (Ie) is obtained bytreating the mono-ester compound (Id) using any of a variety of conventional carboxylate
activation procedures.
The preparation of the amine compound (In) is achieved by condensing the
com~ou,ld (Il~ with the amine compound (Ik), wherein P' is an amine protective group other
than P, and R" is an activating group such as an active ester, anhydride or other group that
causes condensation with the amine terminus of (Ik) to occur with formation of a peptide
30 bond, to give compound (Im). Removal of P' is accomplished under a~p~ iate conditions
(Bodanszky, M.; Bodanszky, A., "The Practice of Peptide Synthesis"; Springer-Verlag:
Berlin, 1984; Chapter III) to produce compound (In), either as corresponding amine or the
amine salt.
3~ Compound (Il) is prepared from the commercially available N-protected carboxylic
acid, or which can be synthesized by standard methods.
WO 9S/06031 ~ ~ 7 (1 15 8 PCTIUS94/09343
Preparation of (Ik) is carried out by condensing the compound (Ii) with mono-
protected diamine (Ih) wherein P is an amine protective group such as CBZ, BOC, FMOC or
other suitable protective group; and P' is an amine protective group other than P, and R" is
an activating group such as an active ester, anhydride or other group that causes
5 con~ens~tion with the unprotected amine terminus of compound (Ih) to occur with formation
of a amide bond to give compound (Ij). Removal of P' under a~ .-iate conditions is
accomplished to produce compound (Ik), either as the corresponding amine or the arnine
salt.
Precursor compound (Ih) is ~ paled in two steps from the amine-nitrile (If).
Several examples of compound (If) are available commercially and others can be easily
synthesized by classical methods. The amine-nitrile (If) is protected with an a~p-u~-iate
ploleclive group reagent to produce the protected amine-nitrile (Ig). In compound Ig, P is
typically CBZ, BOC or FMOC groups, but can be any other suitable group. The protected
15 amine-nitrile (Ig) undergoes reduction with a reagent such as borane-methyl sulfide complex
or sodium borohydride/cobalt (II) chloride, to give the mono-protected diamine (Ih) which
can be isolated as its amine salt.
Compound (Ii) is p-~l)al~d from the carboxyl form of the co--Gs~-onding P'-pl~ule~;led
20 dipeptide or P'-protected amino acid by conventional methods, or can be purchased
cc" . " "~ ;ially.
The compounds of formula II may be aclmini~tered orally, parenterally, via
inh~l~tion, transdermally, intra-nasally, intra-ocularlly, mucosally, rectally and topically.
25 Such a~lminictration may be in dosage unit formulations containing conventional adjuvants
and carrier materials. The term "pal~nte-~l" as used herein includes subcutaneous injections,
intravenous, intramuscular, intracisternal injection or infusion techniques.
The amount of active ingredient that may be combined with the carrier materials to
30 produce a single dosage form will vary depending upon the host treated and the particular
mode of administration. Such carrier materials are well known, and are described, for
example, in European Patent Application No. 0 519 748, incorporated herein by reference.
It will be understood, however, that the specific dose level for any particular patient will
depend upon a variety of factors including the activity of the specific compound employed,
35 the age, body weight, general health, sex, diet, time of administration, route of
arlmini~tration, rate of excretion, drug combination and the severity of the particular disease
undergoing therapy.
14
WO95/060~ Q 1~ g PCT/US94/09343
The following examples are illustrative of the invention. Thin layer chromotagraphy
was ~elrcl"led using silica gel 60 F2s4 plates. Reaction schemes for Examples 1 through 9
are appende~d and follow Example 14. As used heren, "Compound A" refers to the
compound N- { D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl } L-3-(2'naphthyl)-
5 alanyl-L-alanine amide described by Spatola et. al., Peptides: Chemistry and Biology,
Proceedings of the 12th American Peptide Symposium, eds. Smith, J.A., Rivier, J.E.,
ESCOM, Leiden, Netherlands. Compound A was ~ ,ar~d using the following procedure,
and a reaction scheme therefor is appended as reaction scheme A.
10 ~ ion of Compound A
Referring to reaction scheme A and scheme 2, a mixture of 2.0g (6.3 mmol) of N-
BOC-L-3-(2'-naphthyl)alanine and 0.80g (6.9 mmol) of N-hydroxysuccinimi(le7 and 1.8g
(9.5 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in anhydrous
N,N-dimethylfo,,.~ -ifle (10 ml) was stirred for 90 minutes at room lelllp~ldture. To this
was added 1.2g (9.5 mmol) of L-alanine amide hydrochloride, followed by 1.4 ml (9.5
mmol) of triethylamine dissolved in S ml of anhydrous N,N-dimethylful".~...i(le. After
stirring at room temperature for 14 hours, the solvent was removed in vacuo. The residue
was dissolved in ethyl acetate (200 ml) and washed with lM HCl (3x50 ml), water (2x50
ml), saturated sodium bicarbonate (2x50 ml) and finally brine (50 ml). After drying over
anhydrous magnesium sulfate, the solution was filtered and concentrated in vacuo to give
2.1g (86%) yield) of N-BOC-L-3-(2'-naphthyl)alanyl-L-alanine amide (Al) as a white
solid. TLC: Rf 0.16 (chloroform-isopropanol 19:1); NMR (d6-DMSO) o 1.15 (m,3H),
1.24(s,9H), 3.05(m,2H), 4.23(m,2H), 7.02(s,1H), 7.07(s,2H), 7.35(s,1H), 7.47(m,2H),
7.71(s,1H), 7.82(m,3H), 7.98(d,1H).
A suspension of 1.8g (4.7 mmol) of (Al) in dichloromethane (15 ml) was cooled
with an ice bath. Trifluoroacetic acid (15 ml) was added and the homogeneous solution was
stirred at ca.5 C for 5 minutes, then allowed to warm to room temperature. After 1 hour the
dichloromethane and the trifluoroacetic acid were removed in vacuo. The residue was
dissolved in anhydrous N,N-dimethylform~micle (18 ml) containing 5.6 ml (33 mmol) of
triethylamine. To this was added 1.2g (4.2 mmol) of (ld) in one portion. After stirring for
14 hours, the N,N-dimethylformamide was removed in vacuo to give a residue. The
residue was dissolved in ethyl acetate (250 ml) and washed with lM HCl (2x75 ml), water
(75 ml), saturated sodium bicarbonate solution (2x75 ml) and finally brine (75 ml). After
drying over anhydrous magnesium sulfate, the solution was filtered and concentrated to
produce l.5g (89% yield) of N-{D,L-2-(methoxycarbonyl)methyl-4-methylpentanoyl}-L-3-
(2'-naphthyl)alanyl-L-alanine amide (A23 as a white solid. TLC: Rf 0.57 (chloroform-
iS~~ ol 9:1);
MS: mle 455 (M+)
WO 95/06031 PCT/US94/09343
~170~ 58
Under an atmosphere of argon, a mixture of 0.62g (11 mmol) of KOH in 2.8ml of
hot methanol was combined with a mixture of 0.61g (8.8 mmol) of hydroxylamine
hydrochloride in 2.8 ml of hot methanol. After cooling in an ice bath, the reaction was
filtered into a flask containing 1.0g (2.2 mmol) of (A2) and 1 ml of anhydrous N,N-
dimethylf~ .icle After stirring for 18 hours, the solvent was removed in vac~o . The
solid was dissolved in hot ethyl acetate (250 ml) and washed with 16 ml of 10% potassium
bisulfate solution. The organic phase was heated to its boiling point before drying over
anhydrous sodium sulfate. Filtration and subsequent concentration of the filtrate in vacuo
10 produced a solid, which was ~ uldled with ether (50 ml) and collected by filtration to give
0.77g (77% yield) of N-{D,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl}-~3-
(2'-naphthyl)alanyl-L-alanine amide (A) as a white solid The diastereomers of (A) were
separated and purified by reverse phase HPLC using a C1g column, eluting with water
col~ i,-g 0 1% trifluoracetic acid with a gradient of acetonitrile (0-60% in 30 minutes) and
15 also containing 0 1% ~ifluoroacetic acid, ("Method A"), to give a purified early eluting
dia~Lcreolller and a purified late eluting diastereomer, which had retention times of 21 and 23
esrespectively. TLC: Rf0.13(chloroform-methanol9:1)
lH NMR(d6-DMSO) o 0.63~d,3H), 0.72(d,3H), 0.90(m,1H), 1.21(d,3H), 1.26(m,2H),
1.86(m,2H), 2.63(m,1H), 2.99(m,1H), 3.24(m,1H), 4.18(q,1H), 4.55 (m,lH),
20 7.05(s,1H), 7.28(s,1H), 7.48(m,3H), 7.72(s,1H), 7.83(m,3H), 7.91(d,1H), 8.27(d,1H);
13C NMR (D2O/CD3CN) o 17.7, 21.8, 23.1, 26.0, 36.3, 37.4, 41.5, 42.2, 50.1, 55.5,
126.7, 127.1, 128.2, 128.5, 128.8, 129.0, 133.2, 134.2, 135.6, 170.4, 173.0, 177.4,
177.5.
MS: mle 456 (M+)
FXA~PJ,F 1
Synthesis of N- ~ D.L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl ~ -L-3-(2'-
naphthyl)alanyl-L-alanine.2-aminoethyl amide (Compound 1)
HO-H N ~N V--N ~ N _ N H2 HOA~
0~< 0
~
16
WO 95/06031 2 ~ 7 0 i 5 ~ PCT/US94/09343
With reference to reaction Scheme 2, a slurry of 25g (0.164 mol) of the sodium salt
of 4-methyl-2-oxopentanoic acid, sodium salt in anhydrous N,N-dillleLhylfc" ."~.-,;de (50 ml)
cont~ining 19.6 ml (0.164 mol) of benzyl bromide was ~git~te,(l at room temperature for 4
days. The solvent was removed in vacuo. The residue was dissolved in 250 ml of hexane
S and washed with water (3x50 ml) and brine (50 ml) . After drying over anhydrous
m~nçsillm sulfate, the solution was filtered and concentrated in vacuo to give 33.2g (92%
yield) of benzyl 4-methyl-2-oxopentanoate (la) as a viscous, colorless oil. TLC: Rf 0.70
(ethyl acetate-hexane 1:4); 1H NMR(CDCl3) o 0.94(d, 6H), 2.18(m,1H), 2.71(d, 2H),
5.26(s, 2H), 7.37(m, 5H); 13C NMR (CDCl3) o 22.5, 24.2, 48.1, 67.9, 128.7, 128.8,
128.9, 134.7, 161.3, 194Ø
A solution of 26.4g (0.120 mol) of benzyl ester (la) and 40.1g (0.120 mol) of
methyl (triphenylphosphoranylidene)acetate in dichl~ l~",lethane (410 rnl) was sti~red at room
t~ p~ UlG for 18 hours. Removal of the dichloromethane in vacuo produced a solid which
lS was triturated with several volumes of hexane (4xlO0 ml). The hexane volumes were
collected by filtration, combined and concentrated in vacuo to produce an oil which was
fli~tille~l at reduced pressure (bp.138-157 C/0.8mm Hg) to obtain 27.8g (84% yield) of
purified benzyl E,Z-2-isobutyl-3-(methoxycarbonyl)propenoate (lb) as a yellow oil. TLC:
Rf 0.53 and 0~67; E and Z isomers (ethyl acetate-hexane 1:4); NMR(CDC13) o O.91(m, 6H,
CH(CH3)2), 1.85(m,1H, CH(CH3)2), 2.23(Z) and 2.79(E) (d, 2H, C=CCH2), 3.62(Z)
and 3.74(O (s, 3H, C02CH3), 5.23(E) and 5.27(Z) (s, 2H, CO2CH2C6Hs), 5.82(Z) and6.82(E) (s, 1~, CH=C), 7.35(m, SH, C6Hs).
A suspension of 4.0g of 10% palladium on activated carbon in a solution of 27.2g(0.098 mol) of (lb) dissolved in 75 ml of methanol was agit~te~l under 4 atmospheres of
hydrogen for 24 hours. Removal of the catalyst by filtration and conce~ tion of the filtrate
invacuo gave an oil which was distilled at reduced pressure (bp.115-121C/O.Smm Hg) to
obtain 12.7g (68%) of D,L-2-isobutyl-3-(methoxycarbonyi)propionic acid (lc) as acolorless oil. lH NMR(CDC13) â 0.94 (m, 6H), 1.36(m,1H), 1.63(m, 2H), 2.58(m, 2H),
2.95(m, lH), 3.70(s, 3H), 10.8(bs,1H); 13C NMR (CDCl3) o 22.1, 22.3, 25.6, 35.8,39.2, 40.8, 51.7, 172.2, 181.3.
A solution of 12.3g (0.065 mol) of (lc) and 7.5g (0.065 mol) of N-
hydroxysuccinimitle dissolved in anhydrous tetrahydrofuran (100 ml) was cooled to ca. 5 C
with an ice bath. A solution of 13.5g (0.065 mol) of 1,3-dicyclohexylcarbodiimide
dissolved in anhydrous tetrahydlofuldn (50 ml) was added. The n~ ul~; was stirred at ca. 5
C for 1 hour, then allowed to stand overnight under refrigeration. After removal of the
dicyclohexylurea by-product by filtration, the filtrate was concentrated invacuo to produce a
WO 95/06031 ~ 1 7 0 ~ PCT/US94/09343
solid, which was recryst:~lli7çd from ethyl acetate-hexane to give 14.5g (78% yield) of D,L-
2-isobutyl-3-(methoxycarbonyl)propionic acid, N-hydroxysuccinimidyl ester (ld) as a
white solid. TLC: Rf 0.46 (chloroforrn-isopropanol 19:1); lH NMR(CDC13) ~ 0.97(m,
6H), 1.61(m,2H), 1.80(m, lH), 2.72(m, 2H), 2.84(s, 4H), 3.74(s, 3H); 13C NMR
(CDC13) ~ 21.9, 22.5, 25.5, 36.2, 37.2, 41.0, 52.0, 168.8, 170.6, 171Ø
To a solution of 24.9g (0.10 mol) of benzyl succinimidylcarbonate and 10.2g
(0.llmol) of aminoacetonitrile hydrochloride dissolved in anhydrous N,N-
dimethylrc,~ ,.-ide (100 ml) was added 15.4 ml (0.1 lmol) of triethylamine over a period of
10 30 l~ u~es at room temperature. The Il~ ul~ was stirred at room temperature for 12 hours.
Removal of the N,N-dimethylformamide in vacuo produced a residue which was dissolved
in 350 ml of ethyl acetate. The solution was washed with water (350 ml), 2M HCl (3x50
ml) and brine (50 ml). After drying over anhydrous magnesium sulfate, the solution was
~lltered and concentrated in vacuo to give 17.3g (91% yield) of N-CBZ-aminoacetonitrile
15 (le) as an amber solid. TLC: Rf 0.65 (ethyl acetate-hexane 1:1); lH NMR(CDC13)
4.05(d, 2H), 5.13(s, 2H), 5.46(bt, lH), 7.35(bs, 5H); 13C NMR (CDCl3) ~ 29.5, 67.9,
116.2, 128.3, 128.5, 128.7, 135.5, 155.7.
Under an atmosphere of dry argon, 24.3g (0.128 mol) of N-CBZ-aminoacetonitrile
20 (le) was dissolved in anhydrous tetrahydrofuran (32 ml). The solution was stirred and 64
ml of borane-methylsulfide complex (2M in tetrahydrofuran) was added via syringe. The
Lulc was heated to reflux and stirred overnight. The llli~UlG was cooled with an ice bath
as 5 ml of water was added slowly, with vigorous stirring. The stirring was continued for
ca. 5 minutes, then 75 ml of 6M HCl was slowly added. The mixture was stirred for 1
25 hour, then the excess tetrahydroruldn and dimethyl sulfide was removed ~n vacuo. The
aqueous residue was extracted with ether (2x50 ml). The ether extracts were then discarded.
The pH of the aqueous residue was raised to 11 by adding concentrated NH40H. Theresulting aqueous solution was extracted with ethyl acetate (3x100 ml) and the ethyl acetate
extracts were combined and washed with brine (50 ml). After drying over anhydrous
30 magnesium sulfate, the solution was filtered and concentrated in vacuo. The resulting oil
was dissolved in 30 ml of anhydrous methanol, treated with cold methanolic HCl and
concentrated invaCuo to produce a solid. The solid was triturated with ether and collected
by filtration to give 15.1g (51% yield) of N-CBZ-ethylenefli~mine hydrochloride tlf) as a
white powder. lH NMR(D20) â 3.15(m, 2H), 3.46(m, 2H), 5.14(s, 2H), 7.46(bs, 5H);35 13C NMR (D2O) ~ 41.1, 42.6, 70.4, 131.0, 131.3, 131.7, 132.0, 139.4, 161.7.
A solution of 10.0g (0.043 mol) of (lf) and 10.3g (0.036 mol) of N-BOC-L-
~l~nine7 N-hydroxysuccinimide ester in anhydrous N,N-dimethylformamide (50 ml) was
18
WO 9S/06031 ~ ~ 7 ~ ~ 5 8 PCTIUS94/09343
cooled with an ice bath. To this was added 7.6 ml (0.054 mol) of triethylamine in
anhydrous N,N-dimethylformamide (20 ml) over a period of 30 minutes. The reaction was
stirred at ca. 5 C for 1 hour, then at room temperature for 1 hour. The N,N-
dimethylrc,~ ,-lide was removed in vacuo and the resulting residue was dissolved in 300
ml of ethyl acetate. The solution was washed with lM HCl (3x100 ml), water (100 ml),
saturated sodium bicarbonate solution (3x100 ml) and finally, with brine (100 ml). After
drying over anhydrous magnesium sulfate, the solution was ffltered and concentrated in
vacuo to give 12.4g (94% yield) of N-BOC-L-alanine, 2-(benzyloxycarbonylamino)ethyl
amide (lg) as a white solid. TLC: Rf 0.67 (chloroform-iso~r~allol 9:1); lH NMR(CDC13)
~ 1.27(d, 3H), 1.40(s, 9H), 3.32(m, 4H), 4.15(m, lH), 5.06(s, 2H), 5.51(d, lH),
5.90(m,1H), 7.19(m, lH), 7.31(bs, 5H); 13C NMR (CDC13) ~ 18.5, 28.2, 39.6, 40.5,50.1, 66.5, 79.8, 127.9, 128.3, 136.3, 155.4, 156.9, 173.7.
A solution of 12.0g (0.033 mol) of (lg) in 25 ml of dichloromethane was cooled
with an ice bath and 25 ml of trifluoroacetic acid was added. The solution was stirred at ca 5
C for 20 minutes, then allowed to stir to room telll~cl~ture. After 90 minutes, the
dichlc,~ Ll.~l-e and trifluoroacetic acid were removed in vacuo . The resl-lting residue was
dissolved in 200 ml of ethyl acetate and washed with 2M sodium hydroxide (200 ml) and
brine (100 ml). After drying over anhydrous magnesium sulfate, the solution was filtered
and concentrated in vacuo to produce 7.86g (90% yield) of L-alanine, 2-
(benzyloxycarbonylamino)ethyl amide (lh) as a white solid. 1H NMR(CDCl3) o 1.28(d,
3H), 2.09(m, 2H), 3.33(m, 4H), 3.47(q, lH), 5.07(s, 2H), 5.59(bt, lH), 7.33(bs, 5H),
7.69(bt, lH);13C NMR (CDC13) ~ 21.3, 39.5, 40.9, 50.4, 66.6, 128.0, 128.1, 128.4,
136.4, 156.9, 176.7.
Under an atmosphere of dry argon, a solution of 8.9g (0.028 mol) of N-BOC-L-3-
(2'-naphthyl)alanine and 3.2 ml (0.028 mol) of 4-methylmorpholine in anhydrous N,N-
dimethylform~mide (20 ml) was cooled to -15 C and treated with 3.67 ml (0.028 mol) of
isobutyl chlcloru~ a~e. The Illi~Lul~; was stirred at -15 C for 30 minutes, then a solution of
7.5g (0.028 mol) of (lh) and 3.2 ml (0.028 mol) of 4-methylmorpholine in anhydrous
N,N-dimethylfollllalllide (20 ml) was added slowly, over 10 minutes. The reaction was
stirred at -15 C for 2 hours, then at room temperature for 18 hours. The N,N-
dimethylform~mi-1e was removed in vaCuo and the resulting solid was dissolved in 1 liter of
hot ethyl acetate. The hot solution was washed with lM HCl (3x150 ml), water (150 ml),
saLul~ted sodium bicarbonate (3x150 ml) and finally with brine (150 ml). After drying over
anhydrous magnesium sulfate, the hot solution was concentrated in vacuo. The resulting
yellow solid was triturated with 400 ml of cold 1:3 ethyl acetate-hexane and collected by
filtration to give 14.5g (91% yield) of N-BOC-L-3-(2'-naphthyl)alanyl-L-alanine, 2-
19
WO 95/06031 2 ~ 7 ~ 1 ~ 8 PCT/US94/09343 l
(benzyloxycarbonylamino)ethyl amide (li) as a white solid. TLC: Rf 0.59 (chlol~ fol.n-
isopropanol 9:1); lH NMR(CDCl3) ~ 1.26(d, 3H), 1.35(s, 9H), 3.16(m, 6H), 4.42(m,lH), 4.50(m, lH), 5.07(s, 2H), 5.25(d, lH), 5.69(m, lH), 6.82(m, lH), 6.90(d, lH),
7.29(s, lH), 7.31(bs, SH), 7.45(m, 2H), 7.61(s, lH), 7.76(m,3H);13C NMR (CDCl3)
S 18.0, 28.2, 38.2, 39.7, 40.6, 49.0, 55.9, 66.6, 80.6, 125.8, 126.2, 127.2, 127.5, 127.6,
127.9, 128.0, 128.4, 132.4, 133.3, 133.8, 134.2, 155.4, 156.7, 171.4, 172.4.
A suspension of 2.5g (0.0044 mol) of (li) in dichlol~"llethane (10 ml) was cooled
with an ice bath and 10 ml of trifluoroacetic acid was added. The homogeneous solution
was stirred at ca. 5 C for 20 minutes, then allowed to warm to room temperature. After 90
minlltes the dichlolu..~ nt- and trifluoroacetic acid were removed in vacuo . The resulting
residue was dissolved in 100 ml of ethyl acetate and washed with 2M NaOH (3x50 ml),
water (50 ml) and brine (50 ml). The non-homogeneous solution was transferred to a flask
containing 100 ml of absolute ethanol, and heated until it became homogeneous. The hot
solution was dried over a small amount of anhydrous sodium sulfate, filtered, and
conce~ ted in vacuo to obtain a solid. The solid was triturated with cold 1:3 ethyl acetate-
hexane and collected by filtration to give 1.46g (71% yield) of L-3-(2'-naphthyl)alanyl-L-
~l~nine, 2-(benzyloxy-carbonyl-amino)-ethyl amide (lj) as a white solid. lH NMR(CDC13)
~ 1.33(d, 3H), 1.60(bs, 2H), 2.83(m, lH), 3.34(m, 5H), 3.82(m, lH), 4.44(m, lH),5.07(s, 2H), 5.33(t, lH), 6.92(t, lH), 7.31(bs, 5H), 7.36(s, lH), 7.48(m, 2H), 7.65(s,
lH), 7.72(d, lH), 7.81(m, 3H);13C NMR (CDC13) ~ 17.6, 40.6, 40.7, 40.9, 48.6, 56.1,
66.9, 125.4, 125.8, 127.2, 127.4, 127.5, 127.8, 127.9, 128.4, 132.4, 133.4, 135.1,
136.5, 156.1, 172.7, 174.7.
To a solution of 1.4g (0.003 mol) of (lj) and 0.42 ml (0.003 mol) of triethylamine
dissolved in anhydrous N,N-dimethylform~mide (2 ml) was added 0.87g (0.003 mol) of
(ld). The mixture was stirred at room temperature for 18 hours. The N,N-
dimethylform~mide was removed in vacuo. The resulting residue was dissolved in 200 ml
of hot ethyl acetate and washed with lM HCI (3x50 ml), water (50 ml), saturated sodium
bicarbonate solution (3x50 ml) and finally brine (50 ml). After drying over anhydrous
m~.lesil.." sulfate, the hot ethyl acetate solution was filtered and concentrated in vacuo to
give 1.7g (89% yield) of D,L-2-(methoxycarbonyl)methyl-4-methylpentanoyl-L-3-(2'-
naphthyl)-alanyl-L-alanine, 2-(benzyloxycarbonylamino)ethyl amide (lk) as an off-white
solid. TLC: Rf 0.32 (chlororol,ll-isopropanol 19
Under an atmosphere of argon, a I~ ul~; of 2.66g (0.047 mol) of KOH in 12 ml ofhot methanol was combined with a mixture of 2.63g (0.037 mol) of hydroxylamine
hydrochloride in 12 ml of hot methanol. After cooling in an ice bath, the reaction was
WO 95/06031 ,~ ~ 7 ~ 1 58 Pcrtuss4/o9343
filtered into a flask containing 6.0g (0.0095 mol) of (lk) and 12 ml of anhydrous N,N-
dimethylro-..l~.-.i~le. After stirring under argon for 18 hours, the solvent was removed in
vacuo. The resulting solid was triturated with 100 ml of ethyl acetate and collected by
filtration to give 5.2g (86% yield) of D,L-2-(hydroxyaminocarbonyl)methyl-4-
methylpentanoyl-L-3-(2'-naphthyl)alanyl-L-alan-ine, 2-(benzyloxycarbonylamino)ethyl
amide (lm) as an off white solid. TLC: Rf 0.23 and 0.36 (chlolofol,.,-isopropanol 9:1);
13C NMR(d6-DMSO) o 18.0, 21.7, 23.2, 25.1, 35.7, 36.6, 37.3, 38.7, 40.7, 40.8, 48.5,
54.0, 65.3, 125.3, 125.9, 127.3, 127.4, 127.7, 127.9, 128.3, 131.8, 132.9, 135.7,
136.0, 137.1, 156.1, 167.1, 170.7, 172.7, 174.7.
MS: )nle 634 (M+).
A suspension of l.Og of 10% palladium on activated carbon in a solution of 2.0g
(0.0031 mol) of (lm) dissolved in glacial acetic acid (75 ml) was agitated under 4
atmospheres of hydrogen for 24 hours. Removal of the catalyst by filtration, andconcentration of the filtrate in vacuo produced a residue which was triturated with 50 ml of
ether and dried in vacuo to give 2.0g of crude D,L-2-(hydroxyaminocarbonyl)methyl-4-
methylpentanoyl-L-3-(2'-naphthyl)alanyl-L-~l~ninç, 2-(amino)ethyl amide (1).
The diastereomers of (1) were separated by reverse phase HPLC using a Clg
column and eluting with water containing 0.1% trifluoroacetic acid with a gradient of
acetonitrile (0-60% in 30 minutes) also containing 0.1% trifluoroacetic acid (hereinafter
"Method A"). The purified diastereomers (ln) and (lo) had retention times of 20 and 22
minutes, respectively. Diastereomer (ln) showed the following NMR data. 13C
NMR(D2O) o 24.6, 28.9, 29.1, 30.3, 33.2, 43.4, 44.8, 47.0, 48.6, 49.1, 57.6, 62.8,
134.2, 134.6, 135.3, 135.6, 135.8, 135.9, 136.4, 140.2, 141.2, 142.1, 178.3, 180.8,
183.1, 185.4.
MS: mle 500 (M+).
The following is an alternative method, which is a plerell~id method, for preparing
compound ~(c) such that a greater ratio of the desired stereoisomer (R) is produced as
coll~paled to the undesired stereoisomer (S). The reaction steps and reference numerals for
the respective compounds are shown in Reaction Scheme 10.
- By following the procedure of Newman, M. S.; Kutner, A. J. Am. Chem. Soc.
1951, 73, 4199, a solution of sodium methoxide was prepared by dissolving 1.29g (0.056
mol) of sodium in l5ml of anhydrous methanol, which was added to a slurry of 25g (0.242
mol) of L-valinol in 500 ml of diethyl carbonate. The reaction mixture was then heated for 2
hours, with 200ml of distillate collected in the temperature range of 75-123 C. The distillate
WO 95/06031 2 ~ 5 ~ PCT/US94/09343
was discarded and the reaction n~ ulc was allowed to cool to room l~l"~e-~ture and stand
overnight. The excess diethyl carbonate was removed from the reaction ",i~ in vacuo by
rotary evaporation to give a residue. The residue was dissolved in 500ml of ethyl acetate and
washed with water (3 x 200ml) and brine (200ml). After drying over anhydrous magnesium
S sulfate, the solution was filtered and concentrated in vacuo to give a white solid. The solid
was recryst~lli7e-1 from ethyl acetate-hexane to produce 23.2g (74% yield) of (S)-4-
isopropyl-2-oxazolidinone 12(a) as white needles. TLC of 12(a): Rf 0.50 (ethyl acetate-
hexane 3:1); lH NMR (CDC13) d 0.90(d, J = 6.7Hz, 3H), 0.97(d, J = 6.7Hz, 3H),
1.72(m, lH), 3.63(m, lH), 4.10(dd, J = 8.7, 6.4Hz, lH), 4.45(m, lH), 7.32(bs, lH);
10 13C NMR (CDC13) d 17.5, 17.8, 32.6, 58.3, 68.5, 160.7.
Following the procedure of Vogel, A. In Vogel's Practical Organic Chemistry, 4thEd.; Wiley & Sons: New York, 1978; p 498 and 1208, 4-methylpentanoyl chloride 12(b)
was ~lc~aled by adding dropwise with stirring, 38ml (0.52 mol) of thionyl chloride to 50g
15 (0.43 mol) of 4-methylvaleric acid over 30 minutes. The mixture was heated during the
addition, leading to vigorous HCl gas evolution. When the thionyl chloride addition was
completed, the reaction IlliX.~UlG was refluxed for 1 hour. The reaction ll~ G was ~ tilled,
with collection of the ~ till~te between 135 and 148 C. The material was re-distilled and
47.3g (81% yield) of 4-methylvaleroyl chloride 12(b) was collected between 143 and 148
20 C as a colorless liquid. lH NMR (CDC13) d 0.92(d, J = 6.2 Hz, 6H), 1.62(m, 3H), 2.90(t,
J = 7.4 Hz, 2H); 13C NMR (CDC13) d 22.0, 27.2, 33.6, 45.3, 173.9.
Following the procdure of Evans, D. A.; Bartroli, J.; Shih, T. L. J. Am. Chem.
Soc. 1981,103, 2127, a solution of 32.3g (0.25 mol) of 12(a) in 500ml of anhydrous
25 tetrahydrofuran was cooled to -78 C and lOOml of 2.5M (0.25 mol) n-butyllithium in
hexanes was added. When the addition was complete, the mixture was stirred at -78 C for
10 minutes, then warmed to 0 C and stirred for 20 minutes. The reaction mixture was
cooled to -78 C and 34.6ml (0.25 mol) of 12(b) was added over 10 minutes. Stirring was
continued at -78 C for one hour, then the reaction mixture was allowed to stir at room
30 t~ ture overnight. The tetrahydrofuran was removed in vacuo by rotary evaporation to
produce an orange residue.
The residue was dissolved in 750ml of ethyl acetate and washed with water (2 x
250ml) and brine (3 x lOOml). After drying over anhydrous magnesium sulfate, the solution
35 was filtered and concenlldled in ~acuo to give 60g of orange oil.
o 95/06031 ~ ~ 7 01 ~ B . PCT/US94/09343
The oil was purified in two batches by flash cl~ atography on silica gel 60 (500 g).
The product was eluted with 1 :4 ethyl acetate:hexane to produce 48.6g (86%) of 12(c) as a
pale yellow oil. TLC: Rf 0.42 (1 :4 ethyl acetate-hexane)
lH NMR (CDCL3) d 0.88(d, J = 6.9Hz, 3H), 0.92(m, 9H), 1.57(m, 3H), 2.37(m,
lH), 2.93(m, 2H), 4.25(m, 2H), 4.44(m, lH); 13C NMR (CDCl3) d 14.5, 17.9, 22.2,
27.6, 28.3, 33.2, 33.5, 58.3, 63.2, 153.9, 173.5.
Following the procedure of Evans, D.A.; Ennis, M.D.; Mathre, D.J. J. Am. C~em.
Soc. 1982, 104, 1737, a mixture of 16.3ml (0.116 mol) of diisopropylamine and 200ml
of anhydrous tetrahydrofuran was cooled to -S C under an atmosphere of dry argon, and
46.5ml (0.116 mol) of n-butyllithium (2.5 M in hexanes) was added. The mixture was
stirred at -S C for 25 minutes, then cooled to -78 C. A solution of 24.0g (0.106 mol) of
12(c) in 67ml of anhydrous tetrahydrofuran was added, and the reaction mixture was
stirred at -78 C for 30 minutes. The reaction was allowed to warm to -5 C and 27.4 ml
(0.317 mol) of allyl bromide was added. The Illi~LLllG was stirred at -S C for4 hours then 10
ml of water was added, followed by removal of the tetrahydlc~rulall by rotary evaporation to
give an oil. The oil was dissolved in ethyl acetate (SOOml) and washed with water (125 ml)
and brine ~3 x 125 ml). After drying over anhydrous magnesium sulfate, the solution was
filtered and concentrated in vacuo by rotary evaporation to produce an oil. The oil was
puri~led by filtering it through lOOg of silica gel 60 with 1.25 liters of 1:4 ethyl acetate-
hexane. Five fractions of 250 ml each were collected. Each fraction was checked by TLC.
The fractions containing purified product were combined and the solvent was removed by
rotary evaporation to give 26.8g (95% yield) of 12~d3 as a colorless oil. TLC: Rf 0.52
(1:4 ethyl acetate-hexane). IH NMR (CDCl3) d 0.89(m, 12H), 1.28(m,1H), 1.53(m, lH),
1.65(m, lH), 2.33(m, 3H), 4.06(m, lH), 4.23(m, 2H), 4.46(m, lH), 5.04(m, 2H),
5.80(m, lH); 13C NMR (CDC13) d 14.5, 18.0, 22.5, 22.8, 26.0, 28.3, 37.5, 40.2, 40.3,
58.5, 62.9, 117.0, 135.1, 153.6, 176.1.
Generally following the methods of Evans, D.A.; Ennis, M.D.; Mathre, D.J. J. Am.Chem. Soc. 1982,104, 1737, a solution of 20.2g (0.187 mol) of anhydrous benzyl
alcohol dissolived in 63ml of anhydrous tetrahydrofuran was cooled to -5 C under a dry
argon atmosphere and 56.1ml (0.140 mol) of n-butyllithium (2.5 M in hexanes) was added
over 10 minlltes~ The reaction mixture was stirred at -5 C for 20 minutes, then a solution of
25.0g (0.0934 mol) of 12(d) dissolved in 380 ml of anhydrous tetrahydror.lldn (pre-cooled
to -5C) was added. The reaction was stirred at -5 C for 2 hours, then water (SOml) was
added. The reaction was allowed to warm to room temperature. The tetrahydrofuran was
removed by rotary evaporation to produce a residue. The residue was dissolved in ethyl
acetate (250nnl) and washed with water (125ml) and brine (125ml). After drying over
WO 95/06031 ~ ~ 7 ~1 5 8 PCT/US94/09343
anhydrous magnesium sulfate, the solution was filtered and concentrated by rotary
evaporation to produce an oil. The oil was purified by flash chromatography on silica gel
(240g). The product was eluted with 97:3 hexane-ethyl acetate to give 38.9g (85%) of
12(e) as a pale yellow oil. The chiral auxiliary 12(a) was eluted with ethyl acetate for re-
use (40% recovery). TLC of 12(e): Rf 0.80 (1:4 ethyl acetate-hexane). lH NMR
(CDC13) d 0.86(d, J = 6.8 Hz, 3H), 0.88(d, J = 6.8 Hz, 3H), 1.27(m, lH), 1.57(m, 2H),
2.23(m, lH), 2.33(m, lH), 2.58(m, lH), S.Ol(m, 2H), 5.10(s, 2H), 5.71(m, lH),
7.33(m, SH); 13C NMR (CDCl3) d 21.9, 22.9, 26.0, 37.0, 41.0, 43.4, 65.9, 116.7,
128.0, 128.1, 128.4, 135.3, 136.0, 175.5.
By generally following the procedures of Carlsen, P.H.J.; Katsuki, T.; Martin,
V.S.; Sharpless, K.B. J. Org. Chem. 1981, 46, 3936, a suspension of 38.0g (0.154 mol)
of 12(e~ and 145g (0.679 mol) of sodium periodate in 330 ml of acetonitrile, 330 ml of
carbon tetrachloride and 497 ml water was stirred at O C, while 0.83g (2.4 mol%) of
15 r~lthenillm trichloride hydrate was added. The mixture was stirred at O C for lS minutes,
then allowed to stir to room temperature for 4 hours. The reaction was filtered to remove the
solid, using SOOml of dichloromethane and 250ml of water to rinse the solid collected. The
filtrate was Lldn~ll~d to a separatory funnel and the layers were separated. After drying over
anhydrous magnesium sulfate, the lower(dichloromethane) layer was filtered and
20 cf~.~ce~ Led in vacuo by rotary evaporation to produce a dark oil. The oil was purified with
two successive flash chromatography columns [each column: 500 grams of silica gel 60,
eluted with l900ml of 1:4 ethyl acetate:hexane, and 1000 ml of ethyl acetate] to produce 26.6
(65% yield) of 12(f~ as a viscous oil.
TLC of 12(f): Rf 0.10 (1:4 ethyl acetate-hexane). lH NMR (CDC13) d 0.88(d, J
25 = 6.2 Hz, 3H), 0.92(d, J = 6.4 Hz, 3H), 1.33(m, lH), 1.60(m, 2H), 2.49(dd, J = 17.0,
4.8 Hz, lH), 2.77(dd, J = 17.0, 9.S Hz, lH), 2.94(m, lH), 5.15(s, 2H), 7.35(m, SH),
ll.l(bs, lH); 13C NMR (CDC13) d 22.2, 22.4, 25.7, 36.1, 39.2, 41.0, 66.4, 128.0,128.1, 128.4, 135.8, 174.9, 178.2.
Ethereal diazo~ llane (Aldrich Chemical Co. Technical Information Bulletin No.
AL-180) was slowly added to a solution of 22g (0.083 mol) of 12(f) in 50 ml of diethyl
ether until the reaction mixture remained yellow with swirling. The reaction mixture was
back titrated to colorlessness with 1 :9 acetic acid-diethyl ether. After drying over anhydrous
magnesium sulfate the colorless solution was filtered and concentrated in vacuo by rotary
35 evaporation to produce a viscous oil. The oil was dissolved inlOOml of methanol and
transferred to a Parr bottle containing l.Og of 10% p~ r~ m on charcoal catalyst and shaken
under 4 atm. of hydrogen for 6 hours at room temperature. The IlliXlul~ was filtered through
celite and the filtrate was concentrated in vacuo by rotary evaporation to produce an oil. The
24
~ W095/06031 2~ 7~15~ ~ PCr/US94109343
oil was vacuu7m distilled to give 13.9 g (89% yield) of 12(f) as a colorless oil; b.p. 110-
123 C / 0.2mmHg.
TLCofl2(f~: Rf 0.15 (3:7 ethylacetate-hexane)
TLC of methyl ester intenneAi~te: Rf 0.73 (3:7 ethylacetate-hexane) TLC of l(c):Rf 0.23 (3:7 ethyl acetate-hexane). IH NMR of l(c) (CDC13) dO.91(d, J = 6.3 Hz, 3H),
0.95(67.4 Hz, 3H), 1.33(m, lH), 1.64(m, 2H), 2.45(dd, J = 16.7, 11.43(bs, lH);
3C NMR of ~I(c) (CDC13) d 22.2, 22.4, 25.7, 35.8, 39.3, 40.9, 51.8, 172.3, 181.6.
F.XAMPl,F 2
Synthesis of N-~D.L-2-(hydroxyaminocarbonvl)methvl-3-methylbutanoyl~-L-3-(2'-
naphthyl)-L-alanine amide (Compounds 2 and 3)
Y H
HO-HN ~ .~ NH2
0~<
Referring to Scheme 3, Compound (2d) was synthesized from the sodium salt of the3-methyl-2-oxobutanoic acid by the sequence of reactions used to prepare compound (ld)
from 4-methyl-2-oxol~e,~ oic acid, sodium salt.
Compound (2a): 73% yield; bp. 100-121 C/0.3mmHg;
lH NMR(CDC13) o 1.13(d,6H), 3.24(m,1H), 5.27(s,2H), 7.37(m,5H); 13C
NMR(CDC13) o 17.0, 37.0, 67.6, 128.4, 128.5, 128.6, 134.5, 161.5, 197.7.
Compound (2b): 58% yield; bp. 125-147 C/0.6mmHg;
TLC: Rf 0.54(ethyl acetate-hexane 1:4), lH NMR(CDC13) o l.ll(d,6H), 2.66(m,1H),
3.62(s, 3H), 5.27(s,2H), 5.79(s,1H), 7.35(m,5H); 13C NMR (CDC13) ~ 20.4, 32.7,
25 51.5, 67.0, 117.0, 128.2, 128.3, 128.5, 135.3, 156.2, 165.4, 168.4.
Compound (2c): 76% yield; bp. 115-119 C/0.7mmHg; TLC: Rf 0.09 (ethyl acetate-
hexane 1:4); lH NMR(CDC13) ~ 0.96(d,3H), O.99(d,3H), 2.09(m,1H), 2.43(m,1H),
2.76(m,3H), 3.69(s,3H); 13C NMR(CDC13) ~ 19.1, 19.8, 29.7, 32.1, 47.0, 51.7, 172.8,. 30 180.4.
WO 9S/06031 PCT/US94/09343 ~
Compound (2d3: 55% yield; TLC: Rf0.60(chloroform-isopropanol 19:1); 1H
NMR(CDC13) ~ 1.06(d,3H), 1.08(d,3H), 2.12(m,1H), 2.58(m,1H), 2.84(m,5H), 3.07(m,lH), 3.72(s,3H); 13C NMR(CDC13) o 19.4, 19.6, 25.6, 30.3, 33.1, 45.2, 52.1, 168.9,
169.6, 171.5.
The diastereomers (2) and ~3) can be made from L-3-(2'-naphthyl)zll~nine amide
hydrochloride (8b) and compound (2d), using the sequence of reactions used to prepare
Compound (1) from Compounds (lj) and (ld). Compounds (2) and (3) were separated
by reverse phase HPLC as described above.
Compound (2): HPLC retention time (Method A) 21 ,~inl-~es
lH NMR(CD3CN/D2O) ~ 0.19(d,3H), 0.50(d,3H), 1.38(m,1H), 2.24(m,3H),
2.95(m,1H), 3.50(m,1H), 4.68(m,1H), 7.48(m,3H), 7.76(s,1H), 7.83(m,3H); 13C NMR
(CD3CN/D2O) o 20.2, 20.3, 31.1, 33.4, 38.0, 50.2, 55.5, 126.7, 127.2, 128.4, 128.6,
129.1, 129.2, 133.8, 134.4, 136.6, 171.5, 176.3, 176.4.
MS: mle 371 (M+).
Compound (3): HPLC retention time (Method A) 23.1 minutes.
MS: mle 371 (MH+).
FXAMPI,F 3
Synthesis of N-r3-(hyd,v~cyaminocarbonyl)propanoyll-L-3-(2'-naphthyl~alanyl-L-alanine
amide (Compound 4)
HO-H N -~L~~r N _~--N I N H2
~<
~_d
Referring to Scheme 4, to a solution of 1.74g (10 mmol) of tert-butyl hydrogen
succinate (Buchi, G.; Roberts, C. J. Org Chem., 33:460, 1968) and 1.15g (10 mmol) of
N-hydroxy-succinimide in anhydrous tetrahydrofuran (20 ml) was added 2.06g (10 mmol)
30 of 1,3-dicyclohexylcarbodiimide After stirring at room temperature overnight, the reaction
was filtered to remove the dicyclohexylurea by-product. The filtrate was concentrated in
vacuo to give a residue. Chromatography on silica gel using ethyl acetate-hexane (1:1),
26
1~ Wo 95/06031 ~ ~ 7 0 1 ~ 8 PCT/US94/09343
provided 2.3g (84% yield) of tert-butyl succinimidyl succinate (4a) as a white solid. TLC:
Rf 0.50 (ethyl acetate-hexane 1: 1); NMR(d6-DMSO) ~ 1.39(s,9H), 2.56(m,2H),
2.80(bs,4H), 2.86 (m,2H).
A solution of 0.70g (1.8 mmol) of (A13 dissolved in 5.0 ml of trifluoroacetic acid
was stirred at room temperature for 90 minutes. The trifluoroacetic acid was removed in
vacuo to give a residue which was triturated with ether (20 ml) and dried in vacuo to give
0.72g of a pink solid. A portion (0.35g) of the solid was dissolved in 2.0 ml of anhydrous
N,N-dilllt;Ll,ylr~ --H.-.i~le. To this was added 0.24g (0.87 mmol) of (4a) and 0.18 ml (1.3
mmol) of triethylamine. After stirring at room temperature for 2 hours, the solvent was
removed in vacuo to produce a residue. Chromatography on silica gel using chloroform-
isopr~anol 9:1 provided 0.32g (84% yield) of N-[3-(tert-butoxycarbonyl)propanoyl]-L-3-
(2'-naphthyl)alanyl-L-alanine amide (4b) as white solid. TLC: Rf 0.33 (chloroform-
isopropanol 9:1); 1H NMR(d6-DMSO) ~ 1.23(d,3H), 1.30(s,9H), 2.27(m,4H),
2.93(m,1H), 3.20(m,1H), 4.22(m,1H), 4.61(m,1H), 7.03(s,1H), 7.22(s,1H),
7.46(m,3H), 7.75(s,1H), 7.83(m,3H), 8.07(d,1H), 8.19(d,1H); 13C NMR(d6-DMSO) o
18.3, 27.8, 30.1, 30.3, 37.6, 48.1, 54.1, 79.6, 125.4, 126.0, 127.4, 127.5, 127.9, 131.9,
133.0, 135.8, 170.8, 171.1, 171.6, 174.1.
A solution of 0.29g (0.64 mmol) of (4b) dissolved in lOml of trifluoroacetic acid
was stirred at room temperature for 30 minutes. The trifluoroacetic acid was removed in
vacuo to give a residue which was triturated with ether (20ml) and dried in vacuo to give
0.24g (95% yield) of N-[3-carboxypropanoyl]-L-3-(2'-naphthyl)alanyl-L-alanine amide
(4c) as a white solid. TLC: Rf 0.04 (chloroform-isopropanol 9:1); 1H NMR(d6-DMSO)
1.23(d,3H), 2.29(m,4H), 2.92(m,1H), 3.21(m,1H), 4.21(m,1H), 4.58(m,1H),
7.04(s,1H), 7.23(s,1H), 7.46(m,3H), 7.75(s,1H), 7.83(m,3H), 8.06(d,1H), 8.21(d,1H);
3C NMR (d6-DMSO) o 18.3, 29.1, 30.0, 37.6, 48.2, 54.1, 125.4, 126.0, 127.4, 127.5,
128.0, 131.9, 133.0, 135.8, 170.8, 171.3, 173.9, 174.1.
Under an atmosphere of dry argon, a solution of 0.22g (0.56 mmol) of (4c) and
0.062 ml (0.56 mmol) of 4-methylmorpholine anhydrous N,N-dimethylrc"",al,lide (2 ml)
was cooled to -15 C and treated with 0.073 ml (0.56 mmol) of isobutyl chloroformate. The
ulc was stirred at -15 C for 15 minutes, then a solution of O.lOg (0.81 mmol) of (O-
benzyl)hydroxylamine in anhydrous N,N-dimethylformamide (0.5 ml) was added. The
llli~Lul~ was stirred at -15 C for 1 hour, then at room temperature for 1 hour. The solvent
was removed in vacuo. The resulting solid was triturated with ethyl acetate and collected by
filtration to obtain 0.20g (73% yield) of N-[3-(benzyloxyaminocarbonyl)propanoyl]-L-3-(2'-
naphthyl)alanyl-L-alanine amide (4d) as a white solid. TLC: Rf 0.46 (chloroform-
WO 95/06031 ~ 17 ~ ~ ~ 8 PCT/US94/09343
iso~r~anol 8:2); lH NMR (d6-DMSO) ~1.26(d,3H), 2.25(m,4H), 2.95(m,1H), 3.22
(m,lH), 4.23(m,1H), 4.57(m,1H), 4.74(s,2H), 7.03(s,1H), 7.16(s,1H), 7.36(bs,5H),7.46(m,3H), 7.77(s,1H), 7.83(m,3H), 8.12(d,1H), 8.32(d,1H), 11.03(s,1H); 13C
NMR(d6-DMSO) ~18.3, 27.9, 30.4, 37.6, 48.4, 54.5, 77.0, 125.6, 126.1, 127.6, 128.1,
128.4, 128.5, 129.0, 132.0, 133.2, 136.0, 136.2, 169.0, 171.0, 171.7, 174.3.
A suspension of 0.20g of 5% palladium on activated carbon in a solution of O.lOg(0.20 mmol) of (4d) in 4 ml of glacial acetic acid was agitated under 4 atmospheres of
hydrogen for 18 hours. Removal of the catalyst by filtration, and col~ce.,lldlion of the filtrate
in vacuo produced a residue which was Llilulated with 10 ml of ether and dried in vacuo to
give a solid. Ch,u",alography on Baker octadecyl reverse phase gel, eluting with water-
acetonitrile-acetic acid(57:40:3), provided 0.065g (79% yield) of N-[3-
(hydlo~ya~l~inocarbonyl)-propan-oyl]-L-3-(2'-naphthyl)alanyl-L-alanine amide (4), as a
white solid. TLC: Rf 0.05 (chloroform-isopropanol 8:2); lH NMR(d6-DMSO) ~
1.24(d,3H), 2.08(m,2H), 2.28(m,2H), 2.92(m,1H), 3.22(m,1H), 4.20(q,1H),
4.54(m,1H), 7.02(s,1H), 7.20(s,1H), 7.46(m,3H), 7.76(s,1H), 7.84(m,3H), 8.12(d,1H),
8.27(m,1H), 10.39(s,1H); 13c NMR(d6-DMSO) ~18.0, 27.6, 30.4, 37.3, 47.9, 54.0,
125.3, 125.8, 127.2, 127.3, 127.7, 131.7, 132.8, 135.7, 168.3, 170.5, 171.3, 174Ø
F~AMPT F 4
Synthesis of N -~DL-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl~-L-ar~inyl-T -
alanine, 2-aminoethyl amide (Co~ ,ound 5)
HO-HN~--~N--LN~N--~NH2
~NH
HN~ NH2
With reference to Scheme 5, Compound (5a) was synthesized from Compound
(lh) and Na-Boc-Ng-(di-cBz)-L-arginine in 79% yield, by following the method used to
prepare Compound (li). TLC: Rf 0.59 (chloroform-isopropanol 9:1); lH NMR (CDC13)~ 1.18(d,3H), 1.40(s,9H), 1.62(m,4H), 3.27(m,4H), 3.89(m,2H), 4.09(m,1H),
4.21(m,1H), 5.06(s,2H), 5.13(m,2H), 5.22(s,2H), 5.58(m,1H), 5.67(m,1H),
6.70(d,1H), 6.80(m,1H), 7.33(bm,15H), 9.30(m,1H), 9.42(m,1H); 13C NMR (CDC13)
17.3, 25.0, 27.9, 28.3, 39.8, 40.7, 44.0, 49.3, 54.7, 66.6, 67.1, 69.0, 80.4, 127.9,
28
WO 95/06031 ~ 1 7 0 :~ 5 ~ PCT/US94/09343
128.0, 128.3, 128.4, 128.5, 128.8, 128.9, 134.5, 136.6, 155.7, 156.9, 160.7, 163.5,
172.2, 172.4.
Compound (5b) was ~ Gd from Compound (5a) in 87% yield, by the method
5 used to ~JlG~)al~;; Compound (lj). TLC: Rf 0.11 (chlol~rc~ isopropanol 9:1); lH NMR
(CDC13) ~ 8(d,3H), 1.43(m,1H), 1.70(m,4H), 3.30(m,6H), 3.91(m,2H) 4.34(m,1H),
5.03(s,2H), 5.1 l(s,2H), 5.22(s,2H), 5.50(m,1H), 7.01(m,1H), 7.33(bm,15H),
7.76(d,1H), 9.25(m,1H), 9.41(m,1H); 13C N~R (CDC13) ~ 17.7, 24.5, 31.1, 40.3, 40.6,
44.1, 48.6, 54.1, 66.7, 66.9, 68.9, 127.9, 128.0, 128.1, 128.2, 128.3, 128.4, 128.5,
10 128.8, 134.6, 136.3, 136.8, 155.7, 157.1, 160.4, 163.7, 172.8, 175.4.
Compound (5c) was ~ alGd from Compounds (5b) and (Id) in 88% yield, as a
ule of diastereomers, with the method used to prepare Compound (lk).
1HNMR (d6-DMSO; mixture of diastereomers) o 0.79(bm,6H), 1.06(m,1H), 1.13 &
1.20(d, 3H), 1.52(bm,6H), 2.40(m,1H), 2.71(m,1H), 3.03(bm,5H), 3.47 & 3.54(s,3H),
3.88(m, 2H), 4.18(m,2H), 5.00(s,2H), 5.04(s,2H), 5.24(s,2H), 7.35(bm,18H), 7.59 &
7.71(d,1H), 7.66 & 7.94(t,1H), 8.13 & 8.45(d,1H); 13C NMR(d6-DMSO); mixture of
diastereomers) ~ 17.8 & 18.3, 21.8 & 22.2, 22.9 & 23.0, 25.0 & 25.2, 25.4, 28.4 &
28.7, 36.4 & 36.5, 39.6, 40.0, 41.2 & 41.3, 44.3 & 44.4, 48.1 & 48.2, 51.1 & 51.4, 52.4
& 53.1, 65.3, 66.1, 68.2, 127.5, 127.6, 128.3, 128.6, 135.2, 135.3, 137.0, 155.0,
156.1, 156.2, 159.5, 162.8, 162.9, 170.9, 171.0, 171.9, 172.0, 172.8, 174.0, 174.8.
Hydroxamate (5d) was prepared from Compound (~c) in 78% yield as a Illi~Ul~ of
diastereomers.
Hydroxamate ~5d) was de~rotec~ed by hydrogenolysis to give Compound (5) in
59% yield as a IllixLulG of diastereomers. HPLC retention times (method A) 10.1 and 10.3
minutes; lH NMR(D2O; mixture of diastereomers) o 0.89(m,6H), 1.25(m,1H),
1.39(m,3H), 1.69(bm,6H), 2.38(m,2H), 2.85(m,1H), 3.15(dd,2H), 3.22(dd,2H),
3.53(m,2H), 4.32(m, 2H); 13C NMR (D2O; mixture of diastereomers) o 24.3 & 24.5,
28.9 & 29.1, 30.4 & 30.5, 32.4 & 32.6, 33.4 & 33.5, 35.7 & 35.8, 43.4 & 43.6, 44.9,
47.0 & 47.1, 48.4 & 48.5, 49.0 & 49.1, 49.2, 57.8 & 58.0, 61.1 & 61.4, 164.8, 178.4 &
178.5, 181.4 & 181.8, 183.5 & 183.8, 185.6 & 186.4.
MS: mle 459 (M+).
29
WO 9S/06031 ~ i 8 PCTIUS94/09343
F,XAMPT.F, 5
Synthesis of N - ~D.L-2-(hydroxyaminocall~nyl)methyl-4-methylpentanoyl ~L-Iysinyl-L-
alanine amide (Compound 6)
O ~ H O CH3
HO-HN ~L~N----~N _~NH2
S NH2
Referring to Scheme 6, a solution of 5.0g (0.010 mol) of Na-BOC-N-CBZ-L-
lysine p-nitrophenyl ester and 1.5g (0.012 mol) of L-alanine amide hydrochloride and 1.67
ml (0.012 mol) of triethylamine in anhydrous N,N-dhnt;Lhylro~ ,,ide. (50 ml) was stirred at
10 room temperature for 16 hours before the solvent was removed in vacuo. The resulting
residue was dissolved in ethyl acetate (200 ml) and washed with 3M NaOH (3x100 ml),
water (3x100 ml), lM HCl (2x100 ml) and finally with brine (100 ml). After drying over
anhydrous sodium sulfate, the solution was filtered and concentrated in vacuo to give 4.3g
(96% yield) of Noc-BOC-N-CBZ-L-lysyl-L-alanine amide (6a) as a white solid.
15 TLC: Rf 0.32 (chlolorc,~,n-isopropanol 9:1); lH NMR (d6-DMSO) o 1.20(d,3H),
1.35(bm, 6H), 1.37(s,9H), 2.97(m,2H), 3.86(m,1H), 4.21(m,1H), 5.00(s,2H),
6.95(d,1H), 7.06(s, lH), 7.24(t,1H), 7.34(m,6H), 7.78(d,1H); 13C NMR (d6-DMSO)
18.6, 22.8, 28.2, 29.2, 31.4, 40.1, 47.8, 54.5, 65.2, 78.2, 127.8, 128.4, 137.3, 155.5,
156.1, 171.7, 174.2.
Compound (6b) was prepared from Compounds (6a) and (ld) in 69% yield using
the method previously described to prepare Compound (A2). TLC: Rf 0.21 and 0.29
(chloroform-isopropanol 9:1); lH NMR (d6-DMSO; mixture of diastereomers) ~
0.81(m,3H), 0.88(m,3H), 1.17 & 1.23(d,3H), 1.40(bm,8H), 2.46(m,3H), 2.78(m,1H),
25 2.98(m,2H), 3.54 & 3.56(s, 3H), 4.08(m,1H), 4.16(m,1H), 5.00(s,2H), 7.04(m,1H),
7.23(t,1H), 7.34(m,6H), 7.58 & 7.68(d,1H), 8.10 & 8.42(d,1H).
Compound (6c) was prepared from Compound (6b3 in 48% yield, using the
method previously described to prepare (A3). TLC: Rf 0.16 (chloroform-isopropanol 8:2).
30 MS: 7nle 522 (M+).
WO 95/06031 ~ 1 7 ~ Pcrluss4lo9343
The diastereomers (6A) and (6B) were l~el,ared from Compound (6c) by the
method used to prepare Co~ ound (1) from Compound (lm). HPLC purification (method
A) produced an early-eluting isomer (6A) and a late-eluting isomer (6~).
- 5 Compound (6A): HPLC retention time (method A): 9.2 minutes;
H NMR ~d6-DMSO) ~ 0.81(d,3H), 0.88(d,3H), 1.06(m,1H), 1.28(d,3H),
1.40(bm,7H), 1.75(m,1H), 2.03(m,1H), 2.22(m,1H), 2.73(m,3H), 4.01(m,1H),
4.13(m,1H), 7.04(s,1H), 7.11(s,1H), 7.78(bs,3H), 8.06(d,1H), 8.48(d,1H),
10.61(s,1H); 13C NMR(d6-DMSO) o 17.6, 21.8, 22.4, 23.5, 25.5, 26.4, 30.1, 35.7,
39.2, 40.0, 41.3, 48.4, 53.1, 168.1, 171.4, 174.8, 175.5;
MS: mle 387 (M+).
Cc,lll~x)ulld (6B): HPLC retention time (method A): 9.9 minutes;
1H NMR(d~-DMSO) o 0.81 (d,3H), 0.87(d,3H), 1.08(m, lH), 1.18(d,3H), 1.46(bm,7H),1.68(m,1H), 2.05(m,1H), 2.17(m,1H), 2.76(m,3H), 4.16(m,2H), 7.04(s,1H),
7.35(s,1H), 7.67(d,1H), 7.73(bs,3H), 8.08(d,1H), 10.58(s,1H); 13C NMR(d6-DMSO)
18.5, 22.1, 22.2, 23.2, 25.1, 26.3, 30.5, 35.5, 39.2, 40.1, 41.3, 47.8, 52.0, 167.9,
171.1, 174.0, 174.3;
MS: mle 387 (MH+).
FXAMPI,F 6
Synthesis of N- f D.L-2-(hydroxyaminocarbonyl)methyl-4-methylpen~ oyl ~L-tyrosyl-L-
alanine amide (Compound 7)
HO-HN)~_~N~ N~NH2
OH
With reference to Scheme 7, Compound (7a) was prepared from N-BOC-(O-
benzyl)-L-tyrosine p-nitrophenyl ester and L-alanine amide hydrochloride in 99% yield, with
- the method used to pl~pa-e Compound (6a). TLC: Rf 0.51 (chl~r~ fo"l~-is~,~allol 9:1);
1H NMR (d6-DMSO) o 1.22(d,3H), 1.30(s,9H), 2.67(m,1H), 2.91(m,1H), 4.09(m,1H),
4.22(m,1H), 5.05(s,2H), 6.90(m,3H), 7.06(s,1H), 7.18(m,2H), 7.28(s,1H),
WO 95/06031 ~ ~ 7 ~1~ g PCT/IJS94/09343
2.38(bm,5H), 7.88(d,1H); 13C NMR (d6-DMSO) ~ 18.5, 28.1, 36.4, 47.8, 56.0, 69.1,78.1, 114.3, 127.5, 127.7, 128.3, 130.1, 130.2, 137.2, 155.2, 156.8, 171.2, 174Ø
Compound ~7b) was plepal~,d from Compound ~7a3 as a nli~lule of dia~
in 64% yield with the method used to synthesize Compound (6b). TLC: Rf 0.53 and 0.57
(chlorof{,.lll-is~",lol)anol 9:1); lH NMR (d6-DMSO; nlixLu~e of dia~l~re~ 0.60 &0.68(d,3H), 0.76 & 0.82(d,3H), 1.04(m,1H), 1.19 & 1.26(d,3H), 1.40(m,2H), 2.31(bm,
2H), 2.68(m,2H), 3.05(m,1H), 3.48 & 3.55(s,3H), 4.20(m,1H),4.44(m,1H), 5.03 &
5.04(s,2H), 6.87(m,2H), 7.06(bs,1H), 7.15(m,3H), 7.38(bm,5H), 7.69 & 7.78(d,1H),8.15 & 8.39 (d,lH); 13C NMR (d6-DMSO; IlliX.IUlG of diasl~l~olll~ 18.0 & 18.4,
21.9 & 22.1, 22.9 & 23.1, 24.6 & 25.1, 35.8 & 36.0, 36.4 & 36.6, 39.4 & 39.7, 41.1 &
41.2, 47.9 & 48.0, 51.2 & 51.4, 53.9 & 54.6, 69.1 & 69.2, 114.2 & 114.3, 127.5, 127.7,
128.4, 130.1, 130.2, 137.2, 156.8 & 156.9, 170.6 & 170.8, 171.9 & 172.7, 173.8 &173.9, 174.0 & 174 4
Compound (7c3 was prepared from Compound (7b) in 48% yield with the method
used to prepare Colllpoul~d (6c3. A single diastereomer of Compound (7c3 was isolated by
HPLC (method A). 1H NMR (CD30D). ~ 0.46(m,6H), 0.61(m,1H), 0.76(m,1H),
1.13(m,1H), 1.28(d,3H), 1.89(m,1H), 2.17(m,1H), 2.45(m,2H), 3.10(m,1H),
4.18(m,1H), 4.39(m,1H), 4.83(s,2H), 6.70(m,2H), 6.97(m,2H), 7.17(m,5H);
3C NMR(CD3OD) ~ 17.8, 22.2, 23.9, 26.3, 36.8, 37.2, 42.2, 43.0, 50.8, 56.7, 71.0,
115.9, 128.5, 128.9, 129.5, 131.1, 138.8, 159.1, 170.9, 173.8, 178.2, 178.6.
The diastereomer ~7c) was deprotected under 4 atmospheres of hydrogen in the
presence of 10% p~ m on carbon in methanol to produce C~,lllpound (73 in 92% yield.
FXAMP~,FSynthesis of N-rD~L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl~-L-3-(2'-
naphthyl)alanine amide (Compounds 8 and 9)
HO-HN ~N_~NH2
F<
WO 9S/06031 . PCT/US94/09343
2~iO~'S'8'
With reference to Scheme 3, a solution of 3.2g (0.010 mol) of N-BOC-L-3-(2'-
naphthyl)alanine and 1.3g (0.011 mol) of N-hydroxysuccinimide dissolved in 10 ml of
anhydrous tetrahy.lloruld,l was cooled to ca. 5 C. A solution of 2.3g (0.011 mol) of 1,3-
dicyclohexylcarbotliimide dissolved in 5 ml of anhydrous tetrahy(llofu~dn was added, and
the mi~lule was stirred at ca. 5 C for 30 minutes, then at room lelll~ldture for 30 minutes.
The dicyclohexylurea by-product was removed by filtration, and the filtrate was transferred
to a flask containing 1.5 ml (0.022 mol) of concentrated NH40H. After the ~ ul~; had
stirred at room temperaturè for 1 hour, the solvent was removed in vacuo to give a residue.
The residue was dissolved in ethyl acetate (350 ml) and washed with water (100 ml), lM
10 HCL (100 ml), water (100 ml), saturated sodium bicarbonate solution (100 ml) and finally
with brine (100 ml). After drying over anhydrous magnesium sulfate, the solution was
filtered and concentrated in vaCuo to produce a solid. The solid was recry~t~lli7ed from
ethyl acetate to give 2.2g (70% yield) of N-BOC-L-3-(2'-naphthyl)alanine amide (8a) as a
white solid. TLC: Rf 0.50 (chloroforrn-isopropanol 9: 1);
15 lH NMR(d6-DMSO) ~ 1.27(s,9H), 2.92(m,1H), 3.12(m,1H), 4.22(m,1H), 6.91(d,1H),7.07(s,1H), 7.44(s,1H), 7.50(m,3H), 7.75(s,1H), 7.85(m,3H); 13C NMR (d6-DMSO)
28.3, 37.9, 55.7, 78.1, 125.5, 126.1, 127.5, 127.6, 128.0, 132.0, 133.1, 136.2, 155.4,
173.7.
A stream of hydrogen chloride gas was bubbled into a solution of 1.95g (0.0062
mol) of N-BOC-L-3-(2'-naphthyl)alanine dissolved in 60 ml of anhydrous 1,4-dioxane, for
15 minutes. Ether (400 ml) was added, causing a solid to precipitate. The solid was
collected by filtration and dried in vacuo to give 1.36g (88% yield) of L-3-(2'-naphthyl)alanine amide hydrochloride (8b). 1H NMR(d6-DMSO) ~ 3.27(m,2H),
25 4.10(m,1H), 7.48(m,3H), 7.55(s,1H), 7.79(s,1H), 7.86(m,3H), 8.14(s,1H),
8.40(bm,3H); 13C NMR(d6-DMSO) ~ 37.0, 53.6, 125.9, 126.3, 127.7, 127.9, 128.1,
128.4, 132.4, 133.0, 133.1, 169.8.
The diastereomers (8) and (9) can be made from L-3-(2'-naphthyl)alanine amide
30 hydrochloride (8b) and (ld), using the sequence of reactions used to prepare Compound
(1) from Compounds (lj) and (ld).
Compound (8): HPLC retention time (method A) 22.6 minutes. 1H NMR
(CD3CN/D2O) ~ 0.71(m,6H), 1.09(m,2H), 1.28(m,1H), 2.12(m,2H), 2.59(m,1H),
35 2.84(m,1H), 3.11(m,1H), 4.45(m,1H), 6.94(m,7H).
MS: mle 385 (M+).
Compound (9): HPLC retention time (method A) 24.3 minutes, MS: mle 385
(M+)-
W O 95/06031 ~17 0 t ~ 8 ; PCTrUS94/09343
~". 1 ' ''' '
~XAlV~PT,F, X
Synthesis of N- rD~L-2-fhydroxyaminocarbonyl)methyl-4-methylpentanovl ~ -L-3-(2'-
naphthyl~-alanyl-L-serine amide (C~ )ound 10)
HO-HN~N _N~NH2
0~9< 0
With reference to Scheme 8, N-BOC-L-3-(2'-naphthyl)alanyl-L-(O-benzyl)serine
amide (lOa) was prepared from N-BOC-L-3-(2'-naphthyl)alanine and L-(O-benzyl)serine
10 amide in 80% yield with the method used to prepare (7a). TLC: Rf 0.51 (chloroform-
isopropanol 9:1); lH NMR (d6-DMSO) o 1.24(s,9H), 2.93(m,1H), 3.19(m,1H),
3.65(m,2H), 4.34(m,1H), 4.48(m,1H), 4.51(s,2H), 7.16(d,1H), 7.27(s,1H),
7.34(m,5H), 7.46(m,4H), 7.78(s,1H), 7.82(m,3H), 8.04(d,1H); 13C NMR (d6-DMSO)
~ 28.0, 37.4, 52.5, 55.9, 70.0, 72.1, 78.2, 125.4, 125.9, 127.3, 127.4, 127.5, 127.8,
15 128.2, 131.8, 132.9, 135.9, 138.2, 155.4, 171.3, 171.5.
L-3-(2'-naphthyl)alanyl-L-(O-benzyl)serine amide (lOb) was prepared from Com-
pound (lOa) in 95% yield with the method used to prepare Compound (lj). TLC: Rf
0.08 (chloroform-isopropanol 9:1); IH NMR d6-DMSO) o2.81(m,1H), 3.15(m,1H),
20 3.42(m,3H), 3.63(m,2H), 4.37(s,2H), 4.43(m,1H), 7.32(m,6H), 7.46(m,4H),
7.72(s,1H), 7.82(m,3H), 8.14(d,1H); 13C NMR (d6-DMSO) ~ 40.6, 52.0, 55.8, 70.0,
72.0, 125.3, 125.9, 127.4, 127.5, 127.7, 128.0, 128.2, 131.8, 133.0, 136.2, 138.1,
171.5, 174Ø
Compound (lOc) was prepared from Compounds (lOb) and (ld) as a mixture of
diastereomers in 97% yield following the method used to prepare Compound (lk). TLC:
Rf 0.69 and 0.73 (chloroform-isopropanol 9:1); 1H NMR (d6-DMSO; mixture of
diastereomers) oO.25 & 0.40(d,3H), 0.68 & 0.79(d,3H), l.OO(m,lH), 1.32(m,2H),
2.31(bm,3H), 2.~S4(m,1H), 2.98(m,1H), 3.37 & 3.50(s,3H), 3.68(m,2H), 4.48(m,1H),30 4.49 & 4.53(s,2H), 4.72(m,1H), 7.35(bm,6H), 7.44(m,4H), 7.78(m,4H), 7.93 &
7.99(d,1H), 8.30 & 8.49(d,1H); 13C NMR (d6-DMSO; mixture of diastereomers) ~ 21.4 &
34
~ WO 95/06031 2 ~ 7 0 1 ~ 8 Pcr/uss4l09343
22.1, 22.8, 24.5 & 25.1, 36.3 & 36.6, 37.1, 39.6, 41.0 & 41.1, 51.1 & 51.4, 52.6 &
52.7, 53.7 & 54.2, 69.8 & 69.9, 72.1, 125.3, 125.8, 127.4, 127.5, 127.6, 127.8, 128.2,
131.8 & 131.9, 132.9 & 133.0, 135.7 & 135.8, 138.1, 170.0, 171.2, 171.3, 171.8,
172.5, 174.0, 174.2.
5
Compound (lOd~ was prepared from Compound (lOc) in 74% yield with the
method used to ,~lG~al`e Co-llpoul~d (lm). TLC: Rf 0.12 (chlclo~l"l-isopl~opallol 9:1).
Compound (10) was l~,Gpa,~;d from Compound (lOd) in 84% yield with the
metho~l used to pl~l.ale Compound (ln). HPLC retention times: 25.2 and 27.1 minutes
(method A).
MS: mle 472 (M+).
FXAMPI F 9
Synthesis of N- rD,L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanovl ~ -L-3-(2'-
naphthyl)-alanyl-L-alanine methylamide (Compound 11)
HO-HN~N~LN~NH-CH3
1~<
~_q
Referring to Scheme 9, Compound (lla) was prepared from N-BOC-L-3-(2'-
naphthyl)alanine and L-alanine methylamide hydrochloride, in 89% yield using the method
previously described to prepare Compound (li).
TLC: Rf 0.58 (chloroform-isopropanol 9:1); 1H NMR (d6-DMSO) ~ 1.21(d,3H),
1.25(s,9H), 2.54(d,3H), 2.91(m,1H), 3.18(m,1H), 4.28(m,2H), 7.04(d,1H),
7.46(m,3H~, 7.75(s,1H), 7.83(m,4H), 8.07(d,1H); 13C NMR (d6-DMSO) o 18.5, 25.5,
28.0, 37.5, 48.1, 55.7, 78.1, 125.4, 125.9, 127.3, 127.4, 127.5, 127.9, 131.8, 132.9,
135.9, 155.3, 171.1, 172.3.
Cornpound (11~) was prepared from Compounds (lla) and (ld), in 86% yield
using the method previously described to prepare Compound (A2).
TLC: Rf 0.57 and 0.62 (chloroform-isol~lol~anol 9:1);
WO 95106031 ~ 1 7 ~1 5 8 PCT/US94/09343
1H NMR (d6-DMSO; mixture of diastèreomers) ~ 0.23 & 0.40(d,3H), 0.70 & 0.79(d,3H),
1.01(m,2H), 1.18 & 1.26(d,3H), 1.32(m,2H), 2.22(m,2H), 2.53(d,3H), 2.92(m,1H),
3.22(m,1H), 3.38 & 3.39(s,3H), 4.22(m,1H), 4.63(m,1H), 7.44(m,4H), 7.73(s,1H),
7.81(m,4H), 8.22 & 8.46(d,1H).
Compound (11~ was prepared from Compound (llb) in 23% yield using the
method previously described to prepare Compound (A3). TLC: Rf 0.18 (chlorofo
iso~r~,~anol 9: 1).
~XAMP~,F 10
Synthesis of N- ~D.L-2-(hydroxyaminocarbonyl)methyl-4-methylpentanoyl ~ -L-3-amino-2-
dimethylbutanoyl-L-alanine~ 2-aminoethyl amide (Compound 13)
l ~H CH3 H
HO-HN- ~N~N~N- - ~JI ij~
o - H o
7~
Following Reaction Scheme 10, N-Boc-L-tert-leucine 13(b) was prepared by
¢eating L-tert-leucine (Aldrich Chemical) with di-tert-butyl dicarbonate and diisopropylethyl
an~ine in dimethylfluoride (DMF). Then (13b) was treated with NHS and
dicyclohexylcarbodiimide (DCC) in anhydrous tertrahyd-oru,~n to produce N-Boc-L-tert-
20 leucine N-hydroxysuccinimidyl ester, which then is coupled with (lh) from Reaction
Scheme 2 and Example 1 to produce (13c). Compound (13) was prepared from (13c) by
following procedures similar to those described in Example 1 and shown in Reaction
Scheme 2 for the synthesis of compound (1). 1H NMR (d6-DMSO) o 0.76(d, J = 5.6 Hz,
3H), 0.82(d, J = 6.1 Hz, 3H), 0.90(s,9H), 1.06(m, lH), 1.17(d, J = 6.6 Hz, 3H),
1.39(m, 2H), 2.08(m, 2H), 2.69(m, 2H), 2.86(m, lH), 3.18(m, 2H), 4.19(m, 2H),
8.30(m, lH), 8.03(d, J = 7.0 Hz, lH), 7.86(d, J = 8.9 Hz, lH), 13C NMR (d6-DMSO)18.4, 22.6, 23.5, 25.7, 27.1, 34.5, 36.2, 39.2, 40.0, 41.1, 48.8, 60.3, 167.8, 170.1,
172.6, 174.5.
F.XAI~P~,li`. 11
Inhibition of TNF-a Release by T-cells
The following example demonstrates the selective in vitro inhibition of T-cell TNF-a
secretion, as co~ Jal~d to TNF-~ and IFN-~ secretion, by Compound 1.
36
WOg5/06031 21 ~ ~15 8 PCT/US94/09343
Human peripheral blood T-cells were purified from peripheral blood mononuclear
cells by rosetting with 2-aminoethylisothiouronium bromide hydrobromide-treated sheep
erythrocytes. After hypotonic lysis of sheep erythrocytes, monocytes were depleted by
5 plastic adherence for one hour at 37 C. The peripheral blood T-cells were stim~ t~(l with
anti-CD3 antibody (OKT3) which was i""llobilized on the culture wells at 10 ~Lg/ml in PBS
plus 10 ng/ml of the phorbol ester, PMA. Culture medium comprised RPMI 1640 medium
containing 10% fetal bovine serum, 50 U/ml penicillin, and 50 ,ug/ml ~L,eptc""ycin. The
stim~ tion was performed in the presence or absence of the inhibitor Compound 1 t200
10 ~lM), and TN~-a in the metlil-m was assayed by ELISA. Results are shown in Table I.
TABLE I
Effect of Compound l on Cytokine Production by Peripheral Blood T Cells
TNF-a(pg/ml) 3 Hrs. 24 Hrs.48 Hrs.
with Compound 1 ~ 100 300
without Compound ll00 325 800
TNF-~ (pg/ml)
with Co",~ou,ld 1 ~ 160 1050
without Compoulld 1 ~ 160 830
T~N- y (ELTSA OD)
with Compound 1 0.2 0.9 1.08
without Compound l0.3 0.65 1.15
~ lln~et~ct~ble
After 3 hours, there was 100 pg/ml of TNF-a in the m~ lm of cells without
Compound 1 and no detectable TNF-a in the medium of cells with 200 ,uM of Compound 1.
At 24 and 48 hours, Compound 1 inhibited TNF-a release by 72% and 63%, respectively,
while there was no inhibitory effect on the release of TNF-13 or interferon-~. Compound 1
clearly demon~l,dles selective inhibition of TNF-a secretion and has no effect on either
TNF-~ or interferon-~y secretion.
FX~MP~,F 12
Compound l Induced Increase in Cell Surface TNF-a on PMA+Ionomycin-Stimulated
Human T-cells
This example describes the effects of Compound 1 on cell surface TNF-a for humanT-cells which have been stim~ tecl by PMA and ionomycin.
WO 95/06031 ' . . ~ PCT/US94/09343
The alloreactive human T-cell clone, PL-l, does not express cell surface TNF-a in
the absence of stimulation. However, after stim~ tion with PMA plus ionomycin, cell
surface TNF-a, as well as the ligands for CD40 and 41BB, are rapidly induced on the cell
S s--rfa~e Detection of cell surface TNF-a was performed by staining with an Fc fusion
protein consisting of an Fc portion of a human IgG1 molecule (IgGFc) coupled with an
extr~cell~ r domain of TNF receptor (p80). Detection of cell surface ligands for 41BB and
CD40 was pe rc ",led by st~ining with analogous Fc fusion proteins consisting of IgGFc and
extracellular domains of 41BB and CD40, respectively. A fusion molecule consisting of
10 IgGFc and the extr~cell~ r portion of the IL-4 receptor (IL-4R:Fc) was utilized as a negative
control for staining, since PL-l cells do not express cell-surface IL-4 in response to PMA
stim--l~tion. TNFR:Fc and IL-4R:Fc fusion proteins are described in EP 0 464 533,
incorporated herein by reference. The same general procedures used to construct the
~NFR:Fc and IL-4R:Fc fusion molecules were utilized in the construction of the 41BB:Fc
15 and CD40:Fc molecules. Fc fusion proteins bound to their respective cell-surface ligands
were then detected with a biotinylated anti-human IgG1 followed by streptavidin-phycoerythrin. The intensity of staining was measured by a FACS (fluorescence activated
cell sorting) scan flow cyLoll-etel. The results are shown in Table II.
TABLE II
Effects of Compound 1 on Expression of Cell Surface TNF-a, IL-4, 41BBL and CD40L on
PMA and lonomvcin-Stimulated Human T-Cells (MF~. arbitrary units)
TNF-a 41BBL C~40L IL-4
No stimulation 10 10 10 10
4h after stimulation
+ Compound 13040 344 107 10
- Compound 1 83 428 107 10
18h after stimulation
+ Compound 1 616 9 46 10
- Compound 1 7 5 19 10
The specificity of Compound 1 for increasing cell surface TNF-a is apparent. Cells
3~ stim--l~te~l with PMA and ionomycin for four hours in the presence of Compound 1,
followed by staining with TNFR:Fc as described above, displayed a MFI of 3040 ascolllp~d to 83 in the absence of Compound 1. The effect of Compound 1 was specific for
TNFR:Fc binding as no increase on 41BB:Fc or CD40:Fc binding was detected. A
substantial increase in cell-surface TNF-a resulted in a 100-fold increase in TNFR:Fc
38
WO 95/06031 ~ :L 7 0 1 5 ~ PCTIIJS94/09343
binding in the presence of Compound 1 (MFI was 616) as compared to an MFI of 7 in
absence of Compound 1, after 18 hours of stim~ tion. Under the same conditions,
41BB:Fc and CD40:Fc binding were enhanced only approximately 2-fold.
F.XAMPT.F 13
In vivo Inhibition of TACE
~g Compound A versus Compound 1 versus control
Female Balb/c mice (18-20g) were injected i.v. with 400 ~lg of LPS.
Simnlt~neously, the mice were injected subcutaneously with 500 ,ug of Compound A or
Compound 1 in 0.5 ml of saline containing 0.02% DMSO. Control mice received LPS
intravenously and saline/DMSO subcutaneously. Two hours following the LPS injection,
serum was obtained and pooled from two mice in each treatment group. TNF-a levels were
~let~,rmined by ELISA and are shown in the following Table m.
TABLE III
Comparison of 500 ~g Each of Compound 1 versus Compound A on LPS-Induced Serum
TNF Levels in Balb/c Mice (pglml)
Compound 1 CompoundA Saline/DMSO
Serum TNF-a level lln~ tect~hle 65 157
Compound 1 inhibits the secretion of TNF-a at least by 80%, and essentially by
100%, as the TNF-a levels were undetectable. Comparatively, Compound A reduced serum
25 TNF-a levels by ~p-~ ~i,ately 60% as cc,~ ,d to the saline/DMSO control.
In a similar manner to the procedure described above, mice were injected i.v. with
400 ~g LPS. Simultaneously, the mice were injected subcutaneously with 500 ~Lg
Compound 1 in 0.5 ml saline containing 0.02% DMSO. Two hours later, serum was
30 obtained and pooled. TNF-a levels were determined by ELISA. Results are shown in
Table IV in pg/ml.
TABLE IV
Effect of 500 ~g Compound 1 on LPS-Induced Serum TNF Levels in Balb/c Mice (pg/ml)
ExperimentNo. LPS + Cpmd 1 LPS only LPS + Saline
301 1696 1268
2 269 2527 1768
3 281 1833 1732
39
WO 95/06031 ~ 'I 7 ~ 1 ~ 8 - PCT/US94/09343 ~
In e~y~,lhllent 1, Compound 1 reduced serum TNF-a levels by 82% as compared to
TNF-a levels in mice that received LPS only. As compal~d to mice that received LPS +
saline, Compound 1 reduced serum TNF-a levels by 76%. In experiment 2, Compound 1
S reduced serum TNF-a levels by 89% as compared to TNF-a levels in mice that received
LPS only. As compared to mice that received LPS + saline, Compound 1 reduced serum
TNF-a levels by 85%. In experiment 3, Compound 1 reduced serum TNF-a levels by 85%
as cc~llp~,d to TNF-a levels in mice that received LPS only. As coll~ ed to mice that
received LPS + saline, Compound 1 reduced serum TNF-a levels by 84%. Overall,
10Cc,l-lpou~ld 1 reduced serum TNF-a levels by 85.4 i 2.98% as compalGd to TNF-a levels
in mice that received LPS only. From Tables III and IV, Compound 1 effectively reduces
serum TNF-a levels by at least 80% when ~clmini~tered at 25 mg/kg in a murine model of
LPS-induced sepsis syndrome.
15 j~ Compound A versus Compound 1 versus control
Female Balb/c mice (18-20g) were injected i.v. with 450 ~Lg of LPS.
Simnlt~neously, the mice were injected subcutaneously with 250 ~g of Compound A or
Compound 1 in 0.25 ml of saline containing 0.02% DMSO. Control mice received LPSintravenously and saline/DMSO subcutaneously. Two hours following the LPS injection,
20 serum was obtained from three mice in each tre:~tment group. TNF-a levels were deterrnined
by ELISA. The results are expressed as the mean optical density (OD) obtained in the
ELISA from each treatment group, and are shown in Table V. The background OD of the
control sample was 0.162 + 0.003.
25TABLE V
Comparison of 250 ~g Each of Compound l versus Compound A on LPS-Induced Serum
TNF Levels in Balb/c Mice
T PS+Saline LPS+Saline+DMSO Cmpd 1+DMSO Cmpd A+DMSO
300.271 + 0.022 0.268 + 0.040 0.147 + 0.004 0.299 + 0.023
Table V illustrates the effect of Compound 1 and Compound A on inhibiting serum
TNF-a release in Lps-s~im~ te~l mice. Compound 1 reduced serum TNF-a levels to those
35 of the control, thereby indicating a complete inhibition of TNF-oc secretion at 250 ~lg/ml.
Compound A had no effect in reducing serum TNF-a levels as shown by the similarlity in
OD readings between LPS+Saline, LPS+Saline+DMSO, and Compound A.
WO9S/06031 1 7~ S8~ ~ PCT/~S94/09343
FXAMP~,F 14
Serum stability of Compound A and Compound 1
Each of Compound 1 and Compound A was diluted to 50 ~LM in normal mouse
S serum and incubated at 37 C. At various times, aliquots were withdrawn, diluted 100-fold
into ice-cold PBS, and tested for inhibitory efficacy against purified TACE. After 40
minutes, Cvlllpou-ld A showed a decrease in inhibitory effect corresponding to a 3-4 fold
loss in concentration of the col-,poulld, and Compound 1 showed no decrease in inhibitory
effect.
41
WO 95/06031 ! PCTIUS94/09343
217~158
SCHEME 1
o o
R2--C--C--ONa (la)
O O
R2_e--C--OCH2Ph (Ib)
O O
RO--e--CH=C--C--OCH2Ph (Ic)
R2
O O
RO--C [CH~m CH - C--OH (Id)
R1 R2
( R1 = H, m = 1 )
O O
RO--C [CH]m CH-C--OR" (Ie)
Rl R2
42
WO95106031 2 17 01~ 8PCT/US94/09343
SCHEME 1 -Continued
N _ C~(CH2)n~N H2 (If)
N_C-(CH2)n-NH-P(Ig)
H2N--B--N--P(Ih)
(Ii) P'--N--[A]n-C--OR"
P'--N--[A]n-C--N--B--N--P (Ij)
H2N--[A]n--C--N--B--N--P (Ik)
H H
43
WO 95/06031 ~ ~L 7 ~ l S 8 PCT/US94/09343
SCHEME 1-Continued
H2N--[A]n-C--N--B--N--P (Ik)
(Il) P'--N--CH-e--OR"
H
R3
O O
P--H--Cl H-C--NH--[A]n--C--H--B--H--P (Im)
R3
O O
H2N--ICH-e--H~[A]n~e--HN--B--HN--P (In~
R3
O O
Il 11
(Ie) RO--C [cH]m cH-C--OR''
R1 R2
O O O O
Il 11 11 11
RO--C [C IH]m~ ICH -C--HN ICH-C--H~[A]n~C--N--B--N--P ( lo )
R1 R2 R3
44
WO 95/06031 t~ S 8 PCTIUS94/09343
SCHEME 1-Continued
o o o o
11 11 11 11
RO--C [C I ]m~ ICH-C--H ICH-C--H~[A]n~C--H--B--H--P ( Io )
R1 R2 R3
O O O O
RO-HN--e [c IH]m~ jCH-C--H ICH-e--H~[A]n~C--N--B--H--P ( Ip )
Rl R2 R3
O O O O
Il ll 11 11
HO-HN--C [CH]m CH-C--N--ICH-C--N - [A]n - c--H--B--NH2 ( Iq )
R1 R2 R3
-
WO9~;106031 ;~ oi~i~ PCI/US94/09343
SCt~lEME A
O CH3
BOC-HN ~L t . NHS, EDC, DMF , BOC-HN _~L N ~ NH2
2. ~aanine amide ~ HCl, EbN, DMF ~ ( A,)
1. TFA, CH2C12
2. ~ld), Et3N, DMF
CH3O ~--H ~-
~ (A2)
~_Y
H2N-OH, CH30H,
KOH
Ho-HN)~$H ~_
~ ( A )
46
WO 95/06031 ;~ 17 015 8 PCT/US94/09343
SCHEME 2
O Bzl-Br, DMF I
- n-- Na+ ~ _ ~0_~
O (~) O
Ph3P=CH-CO2CH3 CH 0~~~
( 1b )
CH30~0_~ H2, Pd/C. CH30H CH o ~_~OH
H O O
( 1b) ( 1c)
O ~ O ~
CH30~0H NHS, DCC, THF ~ CH30~0Succ.
O O
( 1 C) ( ~)
WO 95/06031 PCT/US94/09343 ~I
~17~
SCHEME-2-Continued
--0 OSucc+HCI H2N--CN DMF, Et3N CB
(1e)
CBZ HN--CN 1- BH3 ~(CH3)2 . THFC NH2 HCI
( 1 e ) ( lf )
~, (1e). DMF- Et3N ~ BOc HN~,N ) NH-CBZ
BOC-HN~N_--NH-CBZ 1. CF3CO2H. CH2C12~ H NQ~N----NH-CBZ
( 1C ) 2. NaOHaq ( 1h )
O 1. iBuOCOCI 4-methyl,,,u,~l, 1 ,e DMF o CH3 H
BOC-HN _lLoH 2. ( lh ) 4-methyl"~ h ' ,e, DMF BOC HN--IL H ~N _--NH-CBZ
( 1 i)
O CH3 H o CH3 H
BOC-HN_ILN~N_--NH-CBZ H2N~LH~N----NH-cBz
1. CF3CO2H. CH2CI2 /~
( 1 i ) 2. NaOHaq. ~ ( 1j )
48
WO95/06031 ~ 7 ~15 ~ PCT/US94/09343
SCHEME-2-Continued
~ CH30~N--~Ln~N----NH-CnZ
CH30 OSucc. ~ H o
( 1d ) (~), DMF, Et3N ~ ( 1k )
/H2N-OH, CH30H,
~/ KOH
~H O CH3 H
HO-HN~o N H~N_~NH-CBZ
( 1m )
H2, Pd/C,
HOAc
O ~H O CH3 H
HO-HN~N .~LN~N~NH2- HOAc
~l .
(1)
49
217 ~158
WO 95/06031 PCTIUS94/09343
SCHEME 3
- ~ a-- Na+ Bzl-Br, DMF ~O_~
(~)
a Ph3p=cH-co2cH3 CH 0~~~
(~) (~)
O ~ _~ H2, Pd/C, CH3HCH o ~OH
H O ( ~ ) O
NHS, DCC, THF CH o~OSucc.
WO 9S/06031 2 :~ 7 ~15 8 PCT/US94109343
SCHEME-3-Continued
O ' O
BOC-HN_ILOH 1. NHS, DCC, THF BOC-HN~NH2
2. NH40H
HCI / dioxane
HO-HN~N--LNH2 o
0,~ 1. (ld), Et3N, DMF HCI H2N - LNH2
(~) & (~ 2. H2N-OH, KOH, ~ (8b)
1. (2d), Et3N, DMF
2. H2N-OH, KOH,
CH30H
Y H
HO-HN ~L~ NH2
(2) & (3) ~
Wo 95/06031 2 1 7 ~1 5 8 PCT/US94/09343
SCHEME 4
>Lo ll n-oH NHS, DCC, THF >L ~_ ~,O-N~
(.~La) O
BOC-HN~LNQ~NH2 ~ >LO~ N~LN~NH2
H o 2. (4a), Et3N, DMF O ' H o
( ~ ( 4b )
TFA
H CH3 HO~N__ILN~NH2
HO-H N J~ r N ~L N ~ N H2
o ~ H o ~
~(4) ~ (4C)
H2, Pd on C, HOAc 1. iBuOCOCI, DMF,
4-methylmorpholine
2. Bzl-O-NH2
~3-- H ~ N ~L N Q~ NH2
~ ( 4d )
~2
WO95/06031 ~ 1 7 0 15 ~ PCT/US94109343
SCHEME 5
BOC-HN--_ILOH BOC-HN_ILNQ~N_ NH-CBZ
1. iBuOCOCI ~ 4-m~tl ~ ,,u,~h " ,e DMF ~
~NH 2. (1h) 4-,nell,yl",o,~.h 1 ,e DMF NH (~a)
CBZ-N ~ NH-CBZ CBZ-N ~ NH-CBZ
1.CF3CO2H.CH2Cl2
2.NaOH~
O CH3 H
H2N ~ H ~ NH-CBZ
( ) ~ NH
CBZ-N NH-CBZ
(ld), Et3N DMF
~ H CH3 H
CH30~N~LN~N----NH-cBz
(5C) ~NH
CBZ-N~ NH-CBz
~3
WO 95/06031 ~ PCTIUS94/09343
SCHEME-5-Continued
CH20~N _~L N ~ N ~ NH-CBZ
( 5~) NH
CBZ-N~ NH-CBz
H2N-OH, CH30H,
KOH
HO-HN Q~NV - N~N - - NH-CBZ
~1) NH
CBz-N~ NH-CBz
H2, Pd/C,
CH30H. HCIaq.
HO-H N ~ N _~L N ~ N ~ NH2
2HCI
(5) NH
HN~ NH2
wo gS/06031 2 ~ 7 ~15 8 PCT/US94/09343
SCHEME 6
O CH3
BOC-HN__lLo_~No2 BOC-HNV_H o
L-alanine amide- HCI, ~
NH-CBZ Et3N, DMF NH-CBZ
1. TFA, CH2C12
2. (:~!) . Et3N, DMF
CH30~N _~L N ~f NH2
- H
(6b)
,~ NH-CBZ
HO-HN~H O ~_
( ~iA ) & ( 6B )
H2N-OH, CH30H,
NH2 KOH
~ H2, Pd on C, CH30H
HO-HN~H
O ~ O
(~) '
N H -CBz
WO9S/06031 ~ 70~5g PCT/US94/09343 ~b
SCHEME 7
O CH3
BOC-HN_Lo_~NO2 BOC-HN_~LN~NH2
L-alanineamide-HCI, ~ (~ )
O-Bzl Et3N, DMF O-Bzl
1. TFA. CH2C12
2. (~), Et3N, DMF
CH30 ~ H
O ~ O
(~) ~
J--` O O-Bzl
HO-HN~N~_LN~NH2
O ~ O
k~ ( 7 ) H2N-OH, CH30H,
KOH
OH ~\H2. Pd on C, CH30H
HO-HN--~ , H~
O ~ O
(7Ç)
O-Bzl
56
WO 95/06031 ~ 17 û 15 8 PCT/US94/09343
SCHEME 8
O O-Bzl
BOc-HN_JLOH 1. iBU0C0CI.4~ ul,ull ' ,e, DMF BOC-HN_~LH O
2. L-(O-benzyl)-serine amide, ~
&~ 4~ tl~ ul~JI ' ,e, DMF ~ ( 1 Oa )
1. CF3CO2H. CH2C12
2. NaoHaq.
CH30~ N_JLN~NH2 ~ (ld) - DMF, EbN H2N--~N~NH2
( 10c) ~ ( 10b )
H2N-OH, CH30H,
KOH
HO-HN~ - H~o HO-HN~N_~N~NH2
H2, Pd on C, ~
( 10d ) HOAC ~ ( 10 )
WO 95/06031 ~17 Q 1~ 8 PCTIUS94/09343
: S(~HEME 9
BOC-HN__ILOH
1. iBuOCOCI, 4-methylmorpholine, DMF ~
2. L-alanine, methylamide- HCI, ~ (~)
4-methylmorpholine, DMF ~
/ 1. TFA
2. ~), Et3N, DMF
CH3O~N_LNS~NH-CH3
0~0< 0
(11b)
H2N-OH, CH30H,
KOH
HO-HN ~ , N
o - H o
l( ( 11 )
~58
1~WO 95/06031 PCT/US94/09343
2i701~
SCHEME 10
HO NH EtO2CO, NaOCH3,~;74% OJ~NH
L - Valinol (1 2a)
SOCI2, ~; 81%
COOH ~ ~--COCI
4-Methylvaleric acid
(1 2b)
O O
O~NH a) nBuLi,THF.0C b)12b ,-78;C O~N~<
H` ~ 86/~
(1 2a) (1 2c)
O N~< a) LDA, OC O~_~N
\~ b) allyl bromide ~ 100% H
H ~~ (1 2c) /~ (1 2d)
0~ ~0~
(1 2d) /=\ ~
~~0- Li + NH
~HF, 0 C; 8~% \~ ~40% recovery
1--
59
WO 95/06031 ~ PCTIUS94/09343 ~It
~ 7~
SCHEME-1 O-Continued
~~'
H \~
( 1 2e)
RuCl3, NalO4,
CH3CN, CC4, H20; 65%
~~
CO2H
(1 2f)
a) CH2N2, Et20
b) H2, Pd / C, CH30H; 89%
H
--OH
CO2CH3
(1c)
