Note: Descriptions are shown in the official language in which they were submitted.
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NEW CYSTEINE DERIVATIVES, PROCESSES FOR THEIR PRODUCTION,
AND PHARMACEUTICALS CONTAIrIING THEM
The invention comprises new matrix metalloproteinase inhibitors that are based
on the
use of the amino acid L- or v-cysteine which is derivatized at the amino and
carboxyl
function with non-peptidic type groups (formula I). The invention comprises
methods for
the production of the inhibitors and their use in the field of therapeutics.
The family of matrix metalloproteases (MIvv>Ps) has become a major target for
drug
design, since these enzymes are involved in tissue remodeling and connective
tissue
turnover, and thus in several diseases where (i) rapid extracellular matrix
degradation is
taking place, e.g. during congestive heart failure and extravasion of highly
metastatic
tumor cells, or (ii) slow extracellular matrix degradation is occurring, e.g.
artherosclerotic lesion formation and rupture. cartilage matrix loss in
osteoarthritis, bone
?0 matrix degradation in osteoporosis, gingival degradation in periodontal
disease. matrix
remodeling and deposition in Alzheimer plaque formation and rheumatoid
arthritis.
The MIVVIP family currently includes fourteen members, ten of which are
secreted from
the cells in a soluble form and four members are membrane-bound enzymes. The
MNiPs
?5 are zinc dependent and calcium requiring enzymes which are inhibited by one
of the
members of the tissue inhibitor of metalloproteinase (TINiP) family. Synthetic
inhibitors
of this class of enzymes have been developed as hydroxamates, N-carboxvalkyl
derivatives, phosphonamidates and phosphinates as well as by using thiol
groups as
ligands for the active-site zinc atom.
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an N-terminal propeptide of about 80 residues. The propeptide forms a separate
smaller
domain that contains three oc-helices and an extended peptide that occupies
the active
site. The catalytic domain in all these structures contains two Zn2+ ions, i.
e. a "structural"
zinc ion and a "catalytic" zinc ion. The "catalytic" zink ion is coordinated
by the side
chains of three histidyl residues of the consensus sequence HEX~GXXI~i. The
fourth ligand of the "catalytic" zink in the inhibited enzymes is a
coordinating group of
the inhibitors like the hydroxamate or carboxylate; in the pro-N» propeptide
it is the
thiol group of the cysteine residue( 1 ).
Correspondingly, the thiol-based collagenase inhibitors, proposed so far, are
generally of
peptidic structure containing cysteine or cysteine-like amino acids and their
design was
based on the binding mode of the substrate and more recently, of the the
cysteine-
containing propeptide.
The present invention relates to a new class of M1VVIP inhibitors which were
derived from
cysteine in a non-peptidic manner as shown in the general formula I, processes
for the
preparations, and pharmaceutical compositions containing these compounds and
their
therapeutical use in medicine,
H O
I
Ri,A~N NCR
I
H
SH tn
wherein
A denotes -CO-, -S02-, -NH-CO-, or -O-CO-
R, denotes hydrogen, a linear or branched saturated or unsaturated alkyl group
of 1 to
15 carbon atoms or a C,-C~s alkyl group substituted by halogen, mercapto,
hydroxy,
alkoxy, amino or nitro, or by carbocyclic aromatic or non aromatic ring
systems which
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3
are optionally substituted once or several times or aromatic or non aromatic
heterocycles, optionally substituted, their pharmacologically acceptable
salts, or optically
active forms thereof.
R denotes hydroxy, a linear or branched saturated or unsaturated alkyl group
of 1 to 15
carbon atoms or a C,-C,3 alkyl group substituted by carbocycGc aromatic or non
aromatic ring systems which are optionally substituted once or several times
or aromatic
or non aromatic heterocycles, optionally substituted, their pharmacologically
acceptable
salts, or optically active forms thereof.
With respect to formula I R or/and R, represent a branched saturated or
unsaturated
alkyl group of 1 to 15 carbon atoms selected from methyl, ethyl, propyl, n-
butyl, tert-
butyl, pentyl, hexyl, heptyl, octyi, nonyl, decyl, undecyl, dodecyi, etc.,
vinyl, etc. as well
as the corresponding alkinyl groups e. g. acetylene.
The carbocyclic aromatic or non aromatic substituents for said alkyl groups
are selected
from C3-Cs cycioalkyls such as cyclopropyl, cyclobutyl, cyclopentyl, or
cyclohexyl, or
1 S C6-C,4 carbocyclic aromatic substituents such as phenyl, naphtyl, or
anthranyl, or
heierocyclic non aromatic substituents such as pyrolidinyl, piperidinyl,
piperazinyl, or
morpholinyl, or heterocyclic aromatic substituents such as pyrrolyl,
pyridinyl, furyl,
thienyl, thiazolyl, imidazolyl, pyrimidinyi, purinyl, indolyl, quinolyl,
carbazolyl.
The carbocyclic aromatic or non aromatic ring systems respectively
heterocycles can
optionally be substituted once or several times for example by halogen-, vitro-
, hydroxy-,
C,-C6 alkyl-, C,-C6 alkoxy-, amino-, mercapto-, carboxyl-, cyano-, or sulfonyl
groups.
If A denotes -CO-, the residue R,CO- is preferably selected from the residues
of the
following carboxyclic acids:
Formic Acid, Acetic acid, Propionic acid, Hexanoic acid, Lauric acid, Myrisic
acid,
Paimitic acid, Stearic acid, Arachidonic acid, Behenic acid, Octadecenoic
acid, Linoleic
acid, Linolenic acid, 3-Mercaptopropionic acid, Glyoxylic acid, Malonic acid,
Succinic
acid, 4-Aminobutanoic acid, 6-Aminocaproic acid, 3-Nitropropionic acid,
Naphthylacetic
acid, 4-Aminophenylacetic acid, Acrylic acid, Cinnamic acid, 4-Amino-cinnamic
acid,
Aminocrotonic acid, Fumaric acid, Maleinic acid, Phthalic acid, Benzoic acid,
Nitrobenzoic acid, 3-Aminobenzoic acid, 4-Aminobenzoic acid, Anthranilic acid,
Salicilic
acid, 3-Amino-salicilic acid, 4-Amino-salicilic acid, 5-Amino-salicilic acid,
Naphthoic
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acid, p-Phenylbenzoic acid, Phenanthroic acid, Nicotinic acid, 3-Aminopyrazin-
2-
carbonic acid, Pyridine carboxylic acid, Piperazine carboxylic acid,
Piperidine carboxylic
acid.
If A denotes -SOZ, the residue R,SOz- is preferably selected from the residues
of the
following sulforlic acids:
Methanesuifonic acid, Ethanesulfonic acid, Propane sulfonic acid, 3-
Hydroxypropane
sulfonic acid, Ortharulic acid (aniline- 2- sulfonic acid),
Naphthalenesulfonic acid,
Naphthylamine sulfonic acid, Aminomethane sulfonic acid, 2-Mercaptoethane
sulfonic
acid, 2-Chloroethane sulfonic acid, N,N'-Dimethylsulfamic acid, Piperidine
sulfonic acid,
5- (2- Aminoethylamino)- 1- naphthalene- sulfonic acid ,
Iodoxyquinolinesulfonic acid,
Pyridine- 3- sulfonic acid, p- Toluenesulfonic acid, 2- (p-
Toluidino)naphthalene- 6-
sulfonic acid, Decyl methanesulfonic acid, 2- [(2- Amino- 2-
oxoethyl)amino]ethanesulfonic acid, 2- (N- Cyclohexylamino)ethanesulfonic
acid, 2-
[bis(2- Hydroxyethyl)amino]ethanesulfonic acid, N- 2- Hydroxyethylpiperazine-
N- 2-
ethanesulfonic acid, N- tris(Hydroxymethyl)methyl- 2- amino- ethanesulfonic
acid, 2- (N-
Morpholino)ethanesulfonic acid, Piperazine- N,N- bis(2- ethanesulfonic acid),
3- (2-
Pyridyl)- 5,6- bis(4- phenylsulfonic acid)- 1,2,4- triazine.
If A denotes -NHCO-, the residue R,-NH-CO- is preferably selected from the
residues of
the following urea derivatives:
n- Butyl-, R- (+)- alpha- Phenylethyl-, R-(-)-1-(1-Naphthyl)-ethyl-, Ethyl-,
Propyl-,
Hexyl-, Octyl-, Benzyl-, Chlorobenry!-, Methylbenzyl-, 3-Picolyl-, 2-
(Aminomethyl)-
pyridyl urea.
If A denotes -O-CO-, the residue R,-O-CO- is preferably selected from the
residues of
the following Carbamates:
Methyl carbamate, Ethyl carbamate, 9-Fluorenylmethyl carbamate, 9-(2-
Sulfo)fluorenylmethyl carbamate, 9-(2,7-Dibromo)fluorenylmethyl carbamate, 4-
Methoxyphenacyl carbamate, 2,2,2-Trichloroethyl carbamate, 2-Phenylethyl
carbamate,
1-(1-Adamantyl)-1-methylethyl carbamate, I,1-Dimethyl-2-haloethyi carbamate, 1-
Methyl-1-(4-biphenyl)-1-methylethyl carbamate, 2-(2'-Pyridyl)ethyl carbamate,
1-
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Adamantyl carbamate, Vinyl carbamate, Allyi carbamate, 1-Isopropylallyl
carbamate, 4-
Nitrocinnamyl carbamate, 8-Quinolyl carbamate, Cyclohexyl carbamate, Benzyl
carbamate, p-Methoxybenzyi carbamate, p-Nitrobenzyl carbamate, p-Bromobenzyl
carbamate, 9-Anthrylmethyi carbamate, diphenylmethyl carbamate, 2-
Methylsulfonylethyl
5 carbamate, 2-(p-Toluenesulfonyl)ethyl carbamate, 4-Methylthiophenyl
carbamate.
R is preferably selected from the following residues:
Ethyl-, Propyl-, Hexyl-, Octyl-, Benzyi-, 4-Chlorobenzyl-, 4-Methylbenzyl-, 3-
Picolyl-,
2-(methyl)-pyridyl-, 4-(methyl)pyridyl-, 3-Phenylpropyl-, 4-Phenylbutyl-, 2-(p-
Tolyl)ethyl- , 3-Nitrobenzyl-, Benzylethyl-, 2-Phenylethyl-, Adamantyl-,
Pyridyl-, Phenyl,
Cholestenyi-, Naphthyl-, 4-Phenoxy-phenyl or indolylethyl.
Preferred compounds according to formula I are compounds of example 5-22, and
of the
following table:
R,-CO R
Form 1- Be 1-
Phen 1- Na hth 1-
Na hth facet 1- 3-Picol 1-
4-Bi hen 1- Z-Phen leth 1-
R,-SO= R
'dine-3-sulfon i- Phen i ro 1-
-Toluenesulfon 1- -Chlorobe 1-
R~-NH-CO R
Be 1- P 'd 1-
R,-O-CO R
2-Phen leth 1- 4-Phen o hen
1-
Be 1- 2-Phen leth l-
2-(p-Toluenesulfonyi)ethyl--Benzyl-
The compounds according to the formula I consist of three parts which have
different
structures and different properties. The chelating group SH for the active-
site Zn2+ ion,
the hydrophobic groups R, or R to interact with the hydrophobic S~, protein
pocket as
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well as to contribute to additional hydrophobic interactions along the P'
substrate
binding cleft.
Inhibitors of the general formula I allow for developing selective inhibitors
of the
different MMPs as required for their differentiated pathological implications
e.g.
osteoporosis rheumatoid arthritis, periodontal disease, artherosclerosis,
congestive heart
failure, tumor invasion and metastasis and angiogenesis.
It is known that there are similar compounds described in the literature.
However, these
are peptidic compounds and they display a worse half life time in human
plasma.
The chemical structure of the cysteine derivatives was choosen in view of
increased
metabolic stability. Correspondingly, the amino group was acylated with
carboxylic acids
to produce amides, but preferentially urethanyl derivatives known to be more
stable to
enzymatic metabolism. Similarly, the C-terminal carboxyl function was
derivatized as
amide instead of esterification to avoid fast clearance rates due to
hydrolysis of the esters
by lipases. Moreover, N-alkyl and N-aryl amides were selected which are known
to be
more stable towards peptidases.
For the synthesis of the inhibitors classical procedures of organic synthesis
were applied
(2). Related L- and D-cystine derivatives of formula II were prepared
according to
standard procedures of peptide chemistry and then amidated with EDCIlHOBt
according
to Scheme 1. Subsequent reduction of the cystine compounds of formula III to
the
cysteine derivatives of Formula I was performed with reducing agents like
mercaptanes
or preferentially with phosphines like tributylphosphine, as shown in scheme
1.
______ _. __.__ _. _ ~_.~_ _ ~_ ~_ _._. T
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0 0
R1~A/~ OH RmA/~ NHR
O
H EDCl'HCl
S ~ HOBt S Bu P Rm /~ _ .R
+ 2 ~N-R --~ S Triflou hanol 95% 2 A
H
HS
/A~ OH /A~ NHR
R1 NH R1 NH
O O
II 111 I
Scheme 1. General route for the synthesis of the MMP inhibitors of the present
invention
The compounds of the present invention, which specifically inhibit MMPs, are
pharmacologically useful in the treatment of rheumatoid arthritis and related
diseases in
which collagenolytic activity is a contributing factor, such as, for example,
corneal
ulceration, osteoporosis, periodontitis, Paget's disease, gingivitis, tumor
invasion,
dystrophic epidermolysis, bullosa, systemic ulceration, epidermal ulceration,
gastric
ulceration, and the like. These compounds are particularly useful in the
treatment of
rheumatoid arthritis (primary chronic polyarthritis, PCP), systemic lupus
erythematosus
(SLE), juvenile rheumatoid arthritis, Sjogren's syndrome (RA + sicca
syndrome),
polyarteritis nodosa and related vasculities, e. g. Wegener's granulomatosis,
giant-cell
arteritis, Goodpasture's syndrome, hypersensitiveness angiitis, polymyositis
and
dermatomyositis, progressive system sclerosis, M. Behcet, Reiter syndrome
(arthtritis +
urethritis + conjunctivitis), mixed connective tissue disease (Sharp's
syndrome),
spondylitis ankylopoetica (M. Bechterew).
The compounds of the present invention may be administered by any suitable
route,
preferably in the form of a pharmaceutical composition adapted to such a route
and in
dose effective for the treatment intended. Therapeutically effective doses of
the
compounds of the present invention required to prevent or arrest the progress
of the
medical condition are readily ascertained by one of ordinary skill in the art.
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Accordingly, the invention provides a class of novel pharmaceutical
compositions
comprising one or more compounds of the present invention, in association with
one or
more non-toxic pharmaceutically acceptable carriers and/or adjuvants
(collectively
referred to herein as "carrier materials") and, if desired, other active
ingredients. the
compounds and compositions may, for example, be administered intravascularly,
intraperitoneally, subcutaneously, intramuscularly or topically.
For all administrations, the pharmaceutical composition may be in the form of,
for
example, a tablet, capsule, suspension or liquid. The pharmaceutical
composition is
preferably made in the form of a dosage unit contained in a particular amount
of the
active ingredient. Examples of such dosage units are tablets or capsules. A
suitable daily
dose for a mammal may vary widely depending on the condition of the patient
and other
factors. However, a dose from about 0.1 to 300 mg/kg body weight, particularly
from
about 1 to 30 mg/kg body weight may be appropriate. The active ingredient may
also be
administered by injection.
The dose regimen for treating a disease condition with the compounds and/or
compositions of this invention is selected in accordance with a variety of
factors,
including the type, age, weight, sex and medical conditions of the patient.
Severity of the
infection and the role of administration and the particular compound employed
and thus
may vary widely.
For therapeutic purposes, the compounds of the invention are ordinarily
combined with
one ore more adjuvants appropriate to the indicated route of administation. If
per os, the
compounds may be admixed with lactose, sucrose, starch powder, cellulose
esters of
alkanoic acids, cellulose alkyl ester, talc, stearic acid, magnesium stearat,
magnesium
oxide, sodium and calcium salts of phosphoric and sulphuric acids, gelatine,
acacia,
sodium alginate, polyvinyl-pyrrolidone and/or polyvinyl alcohol, and thus
tabletted or
encapsulated for convenient administration. Alternatively, the compounds may
be
dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil,
cotton seed
oil, peanut oil, sesam oil, benryl alcohol, sodium chloride and/or various
buffers. Other
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adjuvants and modes of administration are well and widely known in the
pharmaceutical
art. Appropriate dosages in any given instance, of course, depend upon the
nature and
severity of the condition treated, the route of administration and the species
of mammal
involved, including its size and any individual idiosyncrasies.
Representative carriers, dilutions and adjuvants include, for example, water,
lactose,
gelatine starch, magnesium stearate, talc, vegetable oils, gums, polyalkylene
glycols,
petroleum gelly, etc. The pharmaceutical compositions may be made up in a
solid form,
such as granules, powders or suppositories, or in liquid form, such as
solutions,
suspensions or emulsions. The pharmaceutical compositions may be subjected to
conventional pharmaceutical adjuvants, such as preservatives, stabilizers,
wetting agents,
emulsifiers, buffers, etc.
For use in the treatment of rheumatoid arthritis, the compounds of this
invention can be
administered by any convenient route, preferably in the form of a
pharmaceutical
composition adpted to such route and in a dose effective for the intended
treatment. In
the treatment of arthritis, administration may be conveniently be by the oral
route or by
injection intra-articularly into the affected joint.
As indicated, the dose administered and the treatment regimen will be
dependent, for
example, on the disease, the severity thereof, on the patient being treated
and his
response to treatment and, therefore, may be widely varied.
Enzyme assay
The catalytic domain of MMP8 (Phe'9-MMP8) and MMP3 were used for the
inhibition
experiments. Enzyme assays were performed at 25° C in 10 mM CaClz, 100
mM NaCI,
50 mM Tris/HCl {pH 7.6) using 8 nM enzyme concentrations and the fluorogenic
substrates Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NHZ (Bachem M-1855, 1.10-5 M) for
MMP8 and Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp~NH2 (Bachem M-
2110, 4.10-6 M) for MMP3: Measurements were performed essentially as described
by
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Stack et al. (3) for MIvv~8 and by Nagase et al. (4) for NiNIP3. The increase
in
fluorescence at 350 nm (~8) or 390 nm (M1VB~3) was monitored over a period of
100 sec to determine initial rates of hydrolysis. Evaluation of the kinetic
data was
performed as reported by Copeland et al. (5).
5
Table I. Examples for inhibition of HiIvIPB and M1VVIP3 with non-peptidic L-
cysteine
derivatives of the general formula I
Compound R,-A R K;; Mr~IPB I~; ~3
fpMl fNM1
10 I \ o~ ~ ~ 0.71 4.9
,N
I
14 ~H, ~_~ s_ ~ ~ 1.0 9.7
0
~ o~ I ~ 0.16 i2.3
i
15 Synthesis
General methods:
A) Amidation: 1 mmole N,N'-urethanyl-cystine, 2 mmo! HOBT
(hydroxybenzotriazole)
and 2.08 mmole EDCI (N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride) are dissolved or suspended in 10 ml THF. The amine is added in
excess
{2.5-5 mmoi) and in the case of the hydrochloride equivalent amounts of N-
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methylmorpholine are added. The reaction mixture is stirred overnight at room
temperature, concentrated to a small volume and distributed between ethyl
acetate and
water. The organic layer is washed twice with 5 % NaHC03, 5 % KHS04 and water
and
dried over MgS04. The solvent is evaporated and the residue precipitated from
ethyl
acetate with suitable solvents like petroleum ether, diisopropylether, tert-
butyl methyl
ether or pentane.
B) Reduction: the cystine compound ( 1 mmol) is reacted in 10 ml 95 %
trifluorethanol
with 1. S mmol tributylphosphine. The reaction mixture is stirred overnight at
room
temperature, evaporated to small volume and upon dilution with ethyl acetate
the
product is precipitated with suitable solvents like petroleum ether,
diisopropylether, tert-
butyl methyl ether or pentane.
i 5 N,N'-Bis-benryloxycarbonyl-L-cystine-bis-hydrox~amate (Sa)
Prepared from N,N'-bis-benzyloxycarbonyl-L-cystine (2) and hydroxylamine
according
to procedure (A). Yield: 22 %; homogeneous on TLC (solvent system:
chloroform/methanol; 4 : 1, Rf= 0.5).
FAB-MS: m/z = 539.2 [M+H+]; Mf= 538.2 calculated for C2=H26N4OgS2
Berrryloxycarborryl-L-cysteine-hydroxamate (S)
Prepared according to procedure (B) from Sa. Purified by flash chromatography
(eluent:
CHZC12/MeOH, 95:5 followed by 45:5). Yield: 18 %.
'H-NMR (d6-DMSO): 10.7 (bs, 1H, NHOH); 8.89 (bs, 1H, OH); 7.50 (d, 1H, NH
ureth.); 7.38 (m, SH, arom. H's); 5.03 (s, 2H, CH2 v. Z); 3.99 (m, IH, H-
C(a));
2.75/2.66 (2xm, 2H, ~i-CH2); 2.29 (bs, 1H, SH).
Bis-tert-butyloxycarbonyl-L-cystine-bis-bensylamide (6a)
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Prepared from N,N'-bis-tert-butyloxycarbonyl-L-cystine ( 12) and benzylamine
according
to (A). Yield: 77 %. Homogeneous on TLC (solvent system:
cyclohexane/chloroform/acetic acid, 45:45:10, Rf = 0.7).
L-Cystine-bis-benrylamide hydrochloride (6b)
13.87 g (22.4 mmol) 6a in 100 ml 4.6 M HCl in dioxane was stirred overnight at
room
temperature: The precipitate was collected and washed extensively with ether.
Yield: 11
g (quantitative).
N,N =Bis~rcetyl-L-cystine-bis benzylamide (6c)
300 mg (0.61 mmol) 6b was distributed between ethyl acetate and 40 ml NaHC03
(5 %)
and then reacted with acetic anhydride (0.27 g, 2.6 mmol). The organic layer
was
washed with water, dried over MgSOa and evaporated to dryness. Yield: 94 %;
homogeneous on TLC (solvent system: cyclohexane/chloroform/acetic acid:
45:45:10, Rf
= 0.4).
N Acetyl L-cysteirre-berrrylamide (6)
6c was reduced according to procedure (B). Yield: 65 %; homogeneous on TLC
(solvent system: CHCI3MIeOH; 4 : 1, Rr= 0.7); m.p. 186 - 189 °C;
'H-NMR (d6-DMSO): 8.56 (t, 1H, NH, amide); 8.23 (d, 1H, NH ureth.); 7.32-7.09
(m,
SH, arom. H's); 4.56, or 4.38 (m, 1H, H-CQ ); 4.28 (m, 2H, CH2-Bn); 3.12/2.89,
oder
2.79/2.69 (2xm, 2H, ~i-CHZ); 2.30 (bs, 1H, SH); 1.87 (d, 3H, CH3).
FAB-MS: m/z = 253.1 [M+H+]; M~= 252.1 calculated for C12H~6N2O2S
N,N -Bis formyl-L-cysti»e-bis-benrylamide (7a)
Prepared from 6b and formic acid according to procedure (A}. Yield: 59 %;
homogeneous on TLC (solvent system: cyclohexane/CHCi3/acetic acid, 45:45:10;
Rr =
0.1).
N Formyl-L-cysteine-benrylamide (7)
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Reduction of 7a according to procedure (B). Yield: 62 %; homogeneous on TLC
(solvent system: cyclohexane/CHCl3/acetic acid; 45:45:10; Rf = 0.45); m.p. 180
- 183
°C.
'H-NMR (d6-DMSO): 8.58 (t, 1H, NH, amide); 8.35 (d, 1H, NH ureth.); 8.10 (s,
1H,
formyl-H); 7.33-7.21 (m, 5H, atom. H's); 4.49, (m, 1H, H-Ca ); 4.30 (d, 2H,
CHZ-Bn);
2.82/2.72 (2xm, 2H, ~i-CH2); 2.26 (bs, 1H, SH).
FAB-MS: m/z = 239.0 [M+H+]; Ivi~= 238.3 calculated for C"H,4NZOZS
tert-Butyloxycarborryl-L-cysteine-berrzylamide (8)
Reduction of 6a according to procedure (B). Yield: 38 %; homogeneous on TLC
(solvent system: heptanelt-butanoUacetic acid 5:1:1, Rf= 0.7); m.p. 97 - 100
°C;
'H-NMR (d6-DMSO): 8.38 (m, 1H, NH, amide); 7.32-7.21 (m, 5H, atom. H's); 6.95
(d,
1H, NH-ureth.); 4.29 (d, 2H, CH2-Bn); 4.08 (m, 1H, H-Ca ); 2.81/2.68 (2xm, 2H,
~3-
CH2); 2.29 (bs, 1H, SH); 1.40 (s, 9H, t-Bu).
FAB-MS: m/z = 311.1 [M+H+]; Mt= 310.1 calculated for C,sHZZN203S
N,N =Bis-benzyloxycarborryl-L-cystine-bis-benzylamide (9a)
From N,N'-bis-benzyloxycarbonyl-cystine and benzylamine according to (A).
Yield: 90
N Benzyloxycarbonyl-L-cysteine-benzylamide (9)
Reduction of 9a according to (B). Yield: 41 %; homogeneous on TLC (solvent
system:
cyclohexaneJCHCI~/acetic acid, 45:45:10, Rf= 0.4); m.p. 148 - 152 °C;
. 'H-NMR (d6-DMSO): 8.50 (m, 1H, NH, amide); 7.49 (d, 1H, NH ureth.); 7.37-
7.23 (m,
IOH, atom.. H's); 5.06 (dd, 2H, CH2 v. Z); 4.30 (d, 2H, CHZ-Bn); 4.16 (m, 1H,
H-Ca );
2.84/2.70 (2xm, 2H, ~i-CH2); 2.33 (bs, 1H, SH).
FAB-MS: m/z = 345.0 [M+H+]; Mr= 344.4 calculated for C'8H2oN203S
N,N'-Bis-benryloxycarbonyl-L-cystine-bis-4 pyridylmethylamide (l0a)
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14
From N,N'-bis-benzyloxycarbonyl-L-cystine and 4-(aminomethyl)pyridine
according to
procedure (A). Yield. 59 %; homogeneous on TLC (solvent system: CHCIs/MeOH,
4:1,
Rr= 0.65).
N Benryloxycarbonyl-L-cysteine-4 pyridylmethylamide (10)
From l0a according to procedure (B). Yield: 65 %; homogeneous on TLC (solvent
system: CHCI3/MeOH, 4:1, Rf= 0.8); m.p. 122 - 125 °C;
'H-NMR (d6-DMSO): 8.61 (t, 1H, NH, amide); 8.48/7.37/7.25 (m, respectively,
9H,
arom. H's); 7.56 (d, 1H, NH-ureth.); 5.08 (dd, 2H, CHz, Z}; 4.32 (d, 2H, CHZ-
Bn); 4.18
(m, 1H, H-Ca ); 2.87/2.72 (2xm, 2H, ~i-CHZ); 2.40 (bs, 1H, SH).
FAB-MS: m/z = 346.2 [M+H+]; Mf= 345.1 calculated for C,~H,9N3O3S
N,N =Bis benzyloxycarbonyl L-cystine-bis-3 pyridylmethylamide (ll a)
From N,N'-bis-benzyloxycarbonyl-cystine and 3-(aminomethyl)pyridine according
to
procedure (A). Yield: 69 %; homogeneous on TLC (solvent system: CHCI3/MeOH,
4:1,
Rr= 0.2).
N Benryloxycarborryl-L-cysteine-3 pyridylmethylamide (ll)
Reduction of lla according to procedure (B). Yield: 14 %; homogeneous on TLC
(solvent system: CHCI3lMeOH, 4:1, Rf= 0.8); m.p. 125 - 127 °C;
1H-NMR (d6-DMSO): 8.58 (t, 1H, NH, amide); 8.50/8.45/7.65/7.36 (m,
respectively,
9H, arom. H's); 7.52 (d, 1H, NH-ureth.); 5.07 (dd, 2H, CHz v. Z); 4.42 (d, 2H,
CHZ-
Bn); 4.15 (m, 1H, H-CQ ); 2.82/2.?1 (2xm, 2H, ~i-CH2}; 2.36 (bs, 1H, SH).
FAB-MS: m/z = 346.1 [M+H+]; Mf= 345.1 calculated for C,7H,gN;O;S
N,N'-Bis-berrryloxycarbonyl-L-cystirte-bis-2 pyridylmethylamide (12a)
From N,N'-bis-benzyloxycarbonyl-cystine and 2-(aminomethyl)pyridine according
to
(A). Yield: 96 %; homogeneous on TLC (solvent system: CHCl3/MeOH, 4:1, Rf=
0.7).
N Benryloxycarborryl L-cysteine-2 pyridylmethylamide (12)
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Reduction of 12a according to (B). Yield: 33 %; homogeneous on TLC (solvent
system:
CHC13/MeOH, 4:1, Rf= 0.8); m.p. 129 - 131 °C;
'H-NMR (d6-DMSO): 8.59 (t, 1H, NH, amide); 8.48/7.72/7.36-7.22
(m,respectively,
9H, arom. H's); 7.52 (d, IH, NH-ureth.); 5.07 (dd, 2H, CHZ v. Z); 4.49 (d, 2H,
CHZ-
S Bn); 4.20 (m, 1H, H-CQ ); 2.85/2.72 (2xm, 2H, ~i-CH2); 2.42 (bs, 1H, SH).
FAB-MS: m/z = 346.1 [M+H+]; Mf= 345.1 calculated far C,~H19N3O3S
N,N =Bis-benzoyl L-cystine-bis-benrylamide (13a)
10 From 6b and benzoic acid according to (A). Yield: 78 %; homogeneous on TLC
(solvent
system: cyclohexane/CHCI~/acetic acid; 45:45:10, Rr= 0.65).
N Be~uoyl L-cysteine-benrylamide (13)
By reduction of 13a according to (B). Yield: 57 %; homogeneous on TLC (solvent
15 system: cyclohexanelCHCI~/acetic acid; 45:45:10, Rf= 0.55); m.p. 174 - 176
°C;
1H-NMR {d6-DMSO): 8.56 (t, 1H, NH, amide); 7.92 {d, 1H, NH ureth.); 7.57-7.22
(m,
lOH, arom. H's); 4.59, (m, 1H, H-CQ ); 4.31 (d, 2H, CH2-Bn); 2.98/2.89 (2xm,
2H, (i-
CH2); 2.41 (t, 1H, SH).
FAB-MS: m/z = 315.1 [M+H+]; Mf= 314.1 calculated for C,~H~8N202S
N,N'-Bis-tosyl L-cystine-bis-benzylamide (14a)
390 mg (0.794 mmol) 6b were reacted with 180 mg (0.952 mmol) tosyl chloride in
6 ml
pyridine. After 12 h stirring at room temperature the solid was filtered off
and the filtrate
evaporated to dryness adding toluene and finally tent-butyl methyl ether.
Yield: 81 %;
homogeneous on TLC (solvent system: CHCI3/MeOH, 4:1, Rf= 0.7).
N Tosyl L-cystei»e-benzylamide (14)
By reduction of 14a according to (B). Yield: 54 %; homogeneous on TLC (solvent
system: CHC13/MeOH, 4:1, Rf= 0.6); m.p. 180 - 182 °C;
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'H-NMR (d6-DMSO): 8.41 (t, 1H, NH, amide); 7.98/7.68/7.35-7.14 (m,
respectively,
and d's, IOH, arom. H's, NH ureth.); 4.13 (d, 2H, CHZ-Bn); 3.86 (m, 1H, H-Ca);
2.59
(m, 2H, [i-CH2); 2.38 (s, 3H, CH3); 2.17 (t, 1H, SH).
FAB-MS: m/z = 365.1 [M+H']; Mr= 364.1 calculated for C,~HZON203S
N,N'-Bis-benryloxycarbonyl L-cystine-bis-2 phenylethylamide (1 Sa)
From N,N'-bis-benzyloxycarbonyl-L-cystine and 2-phenylethylamine according to
(A).
Yield: 34 %; homogeneous on TLC (solvent system: CHC13/MeOH, 19:1, Rf= 0.8).
N Benryloxycarbonyl-L-cysteine-2 phenylethylamide (1 S)
Reduction of 15a according to (B). Yield: 61 %; homogeneous on TLC (solvent
system:
cyclohexane/CHCl3/acetic acid, 45:45:10, Rr= 0.6); m.p. 119 - 121 °C;
'H-NMR (d6-DMSO): 8.02 (t, 1H, NH, amide); 7.39-7.18 (m, 11H, arom. H's, NH
ureth.); 5.03 (dd, 2H, CH2 v. Z); 4.06 (m, 1H, H-Ca ); 2.72/2.61 (2xm, 4H, CHz-
CH2);
2.22 (bs, 1H, SH).
FAB-MS: m/z = 359.1 [M+H+];1VI<= 358.1 calculated for C'9HzZN2O3S
N,N'-Bis-benryloxycarbonyl-L-cystine-bis-2-(4-hydroxyphenyl)ethylamide (16a)
From N,N'-bis-benzyloxycarbonyl-cystine and 2-(4-hydroxyphenyl)ethylamine
according
to (A). Yield: 71 %; homogeneous on TLC (solvent system:
cyclohexane/CHC13/acetic
acid, 45:45:10, Rf= 0.5).
N Benryloxycarbonyl-L-cysteine-2-(4-hydroxyphenyl)ethylamid (16)
Reduction of 16a according to (B). Yield: 24 %; homogeneous on TLC (solvent
system:
cyciohexane/CHCl3/acetic acid, 45:45:10, R~= 0.6); m.p. 133 - 135 °C;
1H-NMR {d6-DMSO): 9.11 (s, 1H, phenol. OH); 7.98 (t, 1H, NH, amide); 7.38 (m,
6H,
arom. v. Z, NH ureth.); 6.99/6.68 (2xd, 4H, arom., phenol. H's); 5.04 (dd, 2H,
CH2 v.
Z); 4.05 (m, 1H, H-CQ ); 2.73/2.60 (2xm, 4H, CH2-CH2); 2.26 (bs, 1H, SH).
FAB-MS: m/z = 375.2 [M+H+]; Mr= 374.1 calculated for C'9H22NZO4S
N,N'-Bis-benryloxycarbonyl-L-cystine-bis-.f-chlorobenrylamide (17a)
T.____~ _ _._____ _ __ ._.___.
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From N,N'-bis-benzyloxycarbonyl-cystine and 4-chlorobenzylamine according to
(A).
Yield: 99 %; homogeneous on TLC (solvent system: CHCl3/MeOH, 19:1, Rf= 0.8)
N Benryloxycarbonyl-L-cysteine-4-chlorobenrylamide (17)
Reduction of 17a according to (B). Yield: 71 %; homogeneous on TLC (solvent
system:
cyclohexane/CHC13/acetic acid, 45:45:10, Rr= 0.85); m.p. 137 - 139 °C;
1H-NMR (d6-DMSO): 8.53 (t, 1H, NH, amide); 7.51 (d, 1H, NH ureth.); 7.38-7.26
(m,
9H, arom.. H's); 5.06 (dd, 2H, CH2 v. Z); 4.29 (d, 2H, CH2-Bn); 4.13 (m, 1H, H-
Ca );
2.82/2.70 (2xm, 2H, (3-CHZ); 2.53 (bs, 1H, SH).
FAB-MS: m/z = 379.1 [M+H+]; Mf= 378.1 calculated for C,8H19C1N203S
N,N=Bis-benryloxycarborryl L-cystine-bis-3 phenylpropylamide (18a)
From N,N'-bis-benzyloxycarbonyi-cystine and 3-phenylpropylamine according to
(A).
Yield: 94 %; homogeneous on TLC (solvent system: cyclohexanelCHCl3/acetic
acid,
1 S 45:45:10, Rf = 0.7).
N Benryloxycarbonyl L-cysteine-3 phenylpropylamide (18)
By reduction of 18a according to (B). Yield: 76 %; homogeneous on TLC (solvent
system: cyclohexane/CHCh/acetic acid, 45:45:10, Rf= 0.7); m.p. 104 - 106
°C;
'H-NMR (d6-DMSO): 8.02 (t, 1H, NH, amide); 7.43 (d, 1H, NH ureth.); 7.38-7.15
(m,
IOH, arom.. H's); 5.05 (dd, 2H, CH2 v. Z); 4.09 (m, 1H, H-CQ ); 3.12 (m, 2H, N-
CHZ);
2.70/2.69 (2xm, 2H, ~3-CH2); 2.58 (t, 2H, CH2-Ph); 2.32 (bs, 1H, SH); 1.70 (m,
2H,
CHZ.-CH2-CHZ).
FAB-MS: m/z = 373.2 [M+H+]; Mf= 372.2 calculated for C2oH24N203S
N,N' Bis-benryloxycarbonyl L-cystine-bis-tryptamide (19a)
From N,N'-bis-benzyloxycarbonyl-cystine and tryptamine according to (A).
Yield: 75 %;
homogeneous on TLC (solvent system: cyclohexane/CHCl3/acetic acid, 45:45:10,
Rf =
0.6).
Benryloxycarbonyl-L-cysteine-tryptamide (19)
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Reduction of 19a according to (B): Yield: 62 %; homogeneous on TLC (solvent
system:
cyclohexane/CHCh/acetic acid, 45:45:10, Rf= 0.7); m.p. I50 - 152 °C;
'H-NMR (d6-DMSO): 10.80 (s, 1H, NH-tryptamine); 8.09 (t, IH, NH amide); 7.54 -
6.96 (m, I1H, arom. H's, ureth. NH); 5.05 (dd, 2H, CHZ v. Z); 4.08 (m, 1H, H-
C(cc));
3.32 (m, 2H, NHCH~CHz); 2.82 (t, 2H, NHCHZCH2); 2.77/2.65 (2xm, 2H, ~3-CH2);
2.26
(m, 1H, SH).
FAB-MS: m/z = 398.2 [M+H+]; Mf= 397.2 calculated for C21Hz3NsOsS
N,N'-Bis-hexanoyl-L-cystine-bis-benrylamide (20a)
Prepared from 6b and hexanoic acid according to procedure (A). Yield: 86 %;
homogeneous on TLC (solvent system: cyclohexane/chloroform/acetic acid,
45:45:10; Rf
= 0.6).
'H-NMR (d6-DMSO): 8.48 (t, 1H, NH, amide); 8.02 (d, 1H, NH-ureth.); 7.3-7.2
(m,
SH, arom.. H's); 4.41 (m, 1H, H-Ca ); 4.28 (d, 2H, CH2-Bn); 2.80/2.70 (2xm,
2H, (3-
i 5 CH2); 2.25 (t, I H, SH); 2.17 (m, 2H, -CHrCO-); 1.49-1.21 (m, 1 OH,
alkyl), 0.87 (t, 3H,
_CH3).
N Hexanoyl L-cysteine-benrylamide (20)
Reduction of 20a according to procedure (B). Yield: 69 %; m.p. 141 - 143
°C;
'H-NMR (d6-DMSO): 'H-NMR (d6-DMSO): 8.48 (t, 1H, NH, amide); 8.02 (d, 1H, NH-
ureth.); 7.31-7.2 (m, SH, arom.. H's); 4.40 (m, IH, H-Ca ); 4.22 (d, 2H, CH2-
Bn);
2.80/2.70 (2xm, 2H, (3-CH2); 2.3 (bs, 1H, SH); 2.12 (m, 2H, -CHI-CO-); 1.50-
1.19 {m,
6H, alkyl), 0.85 (t, 3H, -CH3).
FAB-MS: m/z = 309.2 [M+H+]; Mr= 308.2 calculated for C,6Fi24N2O2S
N,N =Bis-octanoyl-L-cystine-bis-benrylamide (21 a)
Prepared from 6b and octanoic acid according to procedure (A). Yield: 86 %;
homogeneous on TLC (solvent system: cyclohexanelchloroform/acetic acid,
45:45:10; Rf
= 0.6).
N-0ctanoyl-L-cysteine-benrylamide (21)
Reduction of 21a according to procedure (B). Yield: 73 %; m.p. 137 - 139
°C;
__.___ T ___ _ __. _ ._____ _____._T_
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~H-NMR (d6-DMSO): 1H-NMR (d6-DMSO): 8.48 (t, 1H, NH, amide); 8.02 (d, IH, NH-
ureth.); 7.3-?.2 (m, SH, atom.. H's); 4.41 (m, 1H, H-CQ ); 4.28 (d, 2H, CH2-
Bn);
2.80/2.70 (2xm, 2H, ~i-CH2); 2.25 (t, 1H, SH); 2.17 (m, 2H, -CHz-CO-); 1.49-
I.21 (m,
IOH, alkyl), 0.87 (t, 3H, -CH3).
S FAB-MS: m/z = 337.2 [M+H+]; Mr= 336.2 calculated for C,gH2gN2OZS
N,N' Bis~decanoyl-L-cystine-bis-benrylamide (ZZa)
Prepared from 6b and decanoic acid according to procedure (A). Yield:
quantitative;
homogeneous on TLC (solvent system: cyclohexane/chloroform/acetic acid,
45:45:10; Rf
= 0.9).
N Decanoic-L-cysteine-benrylamide (ZZ)
Reduction of 22a according to procedure (B). Yield: 33 %; Rf= 0.7); m.p. 138 -
140 °C;
'H-NMR (d6-DMSO): 8.46 (t, 1H, NH, amide); 8.02 (d, 1H, NH-ureth.); 7.3-7.2
(m,
SH, atom.. H's); 4.4 (m, IH, H-CQ ); 4.29 (d, 2H, CHZ-Bn); 2.80/2.70 (2xm, 2H,
[i-
CHZ); 2.25 (t, 1H, SH); 2.18 (m, 2H, -CH2-CO-); 1.49-1.19 (m, 14H, alkyl),
0.85 (t, 3H,
_CH3).
FAB-MS: m/z = 365.2 [M+H+]; M~= 364.2 calculated for C2d-i32N202S
1. Beckett, R. P.; Davidson, A. H.; Drummond, A. H.; Huxley, P.; Whittaker, M.
Drug Disc. Today 1996
2. Wiinsch, E. in Houben-Weyl, Methoden der Organischen Chemie, Vol. 15/I,
Springer Verlag, Stuttgart, 1974.
3. Stack, M. S.; Gray, R. D. J. Bio1 Chem. 1989, 264, 4277.
4. Nagase, H.; Fields, C. G.; Fields, G. B. J. Biol. Chem. 1994, 269, 20952.
S. Copeland, R. A.; Lombardo, D.; Giannaras, J.; Decicco, C. P. Bioorg. Med
Chem.
Lett. 1995, 5, 1947.
