U.S. patent application number 11/652675 was filed with the patent office on 2007-06-14 for novel benzamidine derivatives having anti-inflammatory and immunosuppressive activity.
This patent application is currently assigned to ROTTA RESEARCH LABORATORIUM S.p.A. Invention is credited to Roberto Artusi, Francesco Makovec, Lucio Claudio Rovati, Simona Zanzola.
Application Number | 20070135523 11/652675 |
Document ID | / |
Family ID | 11458519 |
Filed Date | 2007-06-14 |
United States Patent
Application |
20070135523 |
Kind Code |
A1 |
Makovec; Francesco ; et
al. |
June 14, 2007 |
Novel benzamidine derivatives having anti-inflammatory and
immunosuppressive activity
Abstract
Compounds which can be represented by the general formula (I)
indicated below: ##STR1## and in which: A is selected independently
from the carboxamide group, the thiocarboxamide group, and the
carbonyl group, R.sub.1 is selected from an alkyl group having from
1 to 3 carbon atoms and the amino group, unsubstituted or
substituted with the nitro group or the methyl group, R.sub.2 is
selected independently from hydrogen, an alkyl group having from 1
to 4 carbon atoms, the methoxy, ethoxy, propoxy group, a mono-, bi-
or tricyclic cycloalkane residue having from 5 to 12 carbon atoms,
the adamantyl group, an aryl, naphthyl or heterocyclic group,
unsubstituted or substituted with methyl, methoxy, hydroxy, amino
or halogen groups, R.sub.3 and R.sub.4 are selected independently
from hydrogen and an alkyl group having from 1 to 3 carbon atoms,
R.sub.5 represents one or two substituents independently selected
from hydrogen and the methyl, methoxyl and hydroxyl groups, n is a
whole number from 0 to 6, and the amidine group is in the para or
meta position relative to the "-A-NH--" group.
Inventors: |
Makovec; Francesco; (Monza
(Milano), IT) ; Zanzola; Simona; (Milano, IT)
; Artusi; Roberto; (Rho (Milano), IT) ; Rovati;
Lucio Claudio; (Monza (Milano), IT) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
ROTTA RESEARCH LABORATORIUM
S.p.A
|
Family ID: |
11458519 |
Appl. No.: |
11/652675 |
Filed: |
January 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10467624 |
Jan 6, 2004 |
7202277 |
|
|
PCT/EP02/01201 |
Feb 6, 2002 |
|
|
|
11652675 |
Jan 12, 2007 |
|
|
|
Current U.S.
Class: |
514/599 ;
514/615; 514/620; 564/147; 564/74 |
Current CPC
Class: |
C07C 257/14 20130101;
C07C 279/36 20130101; A61P 43/00 20180101; A61P 19/02 20180101;
A61P 29/00 20180101; A61P 37/02 20180101; C07C 2601/14 20170501;
C07D 209/40 20130101; C07C 335/20 20130101; C07C 279/18 20130101;
A61P 1/04 20180101; A61P 35/00 20180101; C07D 213/40 20130101; C07C
2603/74 20170501; C07D 277/48 20130101; C07D 209/14 20130101 |
Class at
Publication: |
514/599 ;
514/615; 514/620; 564/074; 564/147 |
International
Class: |
A61K 31/165 20060101
A61K031/165; A61K 31/16 20060101 A61K031/16; C07C 275/42 20060101
C07C275/42 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 8, 2001 |
IT |
T02001A000110 |
Claims
1. A compound of formula (I) below: ##STR7## wherein A is selected
independently from a carboxamide group, a thiocarboxamide group,
and a carbonyl group; R.sub.1 is selected from an alkyl group
having from 1 to 3 carbon atoms and an amino group substituted with
a nitro group when A is the thiocarboxamide group; R.sub.1 is
selected from an alkyl group having from 1 to 3 carbon atoms when A
is selected independently from a carboxamide group and a carbonyl
group; R.sub.2 is selected independently from hydrogen, an alkyl
group having from 1 to 4 carbon atoms, a methoxy group, an ethoxy
group, or a propoxy group, a mono-, bi- or tricyclic cycloalkane
group having from 5 to 12 carbon atoms, an adamantyl group, and an
aryl, naphthyl or heterocyclic group, unsubstituted or substituted
with a methyl group, a methoxy group, a hydroxy group, an amino
group or a halogen group; R.sub.3 and R.sub.4 are selected
independently from hydrogen and an alkyl group having from 1 to 3
carbon atoms; R.sub.5 represents one or two substituents
independently selected from hydrogen and a methyl group, a methoxyl
group and a hydroxyl group; n is a whole number from 0 to 6; and an
amidine group is in the para or meta position relative to the
-A-NH-- group, or a pharmaceutically acceptable salt thereof.
2. The compound of according to claim 1, wherein A is a
thiocarboxamide group; R.sub.1 is an alkyl group having from 1 to 3
carbon atoms; R.sub.2 is a methyl group or a mono-, bi- or
tricyclic cycloalkane group having from 5 to 12 carbon atoms;
R.sub.3, R.sub.4 and R.sub.5 are hydrogen; n is a whole number
between 0 and 6; and the amidine group is in the para position
relative to the -A-NH-- group, or a pharmaceutically acceptable
salt thereof.
3. The compound according to claim 1 wherein A is selected
independently from a carboxamide group and a carbonyl group;
R.sub.1 is an alkyl group having from 1 to 3 carbon atoms; R.sub.2
is a methyl group or a mono-, bi- or tricyclic cycloalkane group
having from 5 to 12 carbon atoms; R.sub.3, R.sub.4 and R.sub.5 are
hydrogen; n is a whole number between 0 and 6; and the amidine
group is in the para position relative to the -A-NH-- group, or a
pharmaceutically acceptable salt thereof.
4. The compound according to claim 1 wherein A is a thiocarboxamide
group; R.sub.1 is selected from an amino group substituted with a
nitro group; R.sub.2 is a methyl group or a mono-, bi- or tricyclic
cycloalkane group having from 5 to 12 carbon atoms; R.sub.3,
R.sub.4 and R.sub.5 are hydrogen; n is a whole number between 0 and
6; and the amidine group is in the para position relative to the
-A-NH-- group, or a pharmaceutically acceptable salt thereof.
5. The compound according to claim 1 wherein A is a thiocarboxamide
group; R.sub.1 is an alkyl group having from 1 to 3 carbon atoms;
R.sub.2 is selected from an aryl group, unsubstituted or
substituted with halogen groups; R.sub.3, R.sub.4 and R.sub.5 are
hydrogen; n is a whole number between 0 and 6, and the amidine
group is in the para position relative to the -A-NH-- group, or a
pharmaceutically acceptable salt thereof.
6. The compound according to claim 1 wherein A is a thiocarboxamide
group; R.sub.1 is selected independently from a methyl group, or a
nitro-amino group; R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are
hydrogen; n is 5; and the amidine group is in the para position
relative to the -A-NH-- group.
7. A pharmaceutical preparation comprising a compound according to
claim 1, or a pharmaceutically-acceptable salt thereof, as an
active ingredient and a pharmaceutically acceptable carrier.
8. The pharmaceutical preparation according to claim 7 further
comprising pharmaceutically-acceptable inactive ingredients
selected from the group consisting of vehicles, binders,
flavourings, sweeteners, disaggregants, preservatives, humectants
and mixtures thereof, ingredients which facilitate rectal,
transdermal or transmucosal absorption, and ingredients that permit
the controlled release of the active ingredient over time.
9. A method for the preparation of a compound of formula (I)
##STR8## wherein A is selected independently from a carboxamide
group, a thiocarboxamide group, and a carbonyl group; R.sub.1 is
selected from an alkyl group having from 1 to 3 carbon atoms and an
amino group substituted with a nitro group or a methyl group;
R.sub.2 is selected independently from hydrogen, an alkyl group
having from 1 to 4 carbon atoms, a methoxy group, an ethoxy group,
or a propoxy group, a mono-, bi- or tricyclic cycloalkane group
having from 5 to 12 carbon atoms, an adamantyl group, and an aryl,
naphthyl or heterocyclic group, unsubstituted or substituted with a
methyl group, a methoxy group, a hydroxy group, an amino group or a
halogen group; R.sub.3 and R.sub.4 are selected independently from
hydrogen and an alkyl group having from 1 to 3 carbon atoms;
R.sub.5 represents one or two substituents independently selected
from hydrogen and a methyl group, a methoxyl group and a hydroxyl
group; n is a whole number from 0 to 6; and an amidine group is in
the para or meta position relative to the -A-NH-- group, or a
pharmaceutically acceptable salt thereof which comprises the steps
of: a) reacting a 1,3 or 1,4-phenylenediamine of formula (IV)
##STR9## in which R.sub.5 has the meaning given above, with the
isothiocyanate of formula (V A), with the acyl chloride of formula
(V B), or with the isocyanate of formula (V C), ##STR10## in which
R.sub.2, R.sub.3, and n have the meanings given above, with an
excess of phenylenediamine, in a neutral solvent and at a
temperature of between 4.degree. C. and the reflux temperature of
the solvent used, to give the corresponding anilines of formula
(III) ##STR11## in which R.sub.2, R.sub.3, R.sub.4, A and n have
the meanings given above, and in which the amine group is in the
meta or para position relative to the chain adjacent to the -A-NH--
group; b) reacting the anilines of formula (III) with an
appropriate imidate hydrochloride of formula (II) ##STR12## in
which R.sub.1 has the meaning given above, in the presence of an
excess of formula (II) and of the corresponding stoichiometric
quantity of a tertiary base, in an inert anhydrous solvent, at a
temperature of between 4.degree. C. and the boiling point of the
solvent to give the corresponding derivatives of formula (I)
##STR13## in which A, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5
and n have the meanings given above and in which the amidine group
is in the para or meta position relative to the -A-NH-- group,
recovering the compound of formula (I) or a
pharmaceutically-acceptable salt thereof from the reaction mass and
purifying; c) alternatively, if R.sub.1 is NH--NO.sub.2, the
corresponding compound of formula (I) in which A is the
thiocarboxamide group and R.sub.2, R.sub.3, R.sub.4, R.sub.5 and n
have the meanings given above is prepared by reacting the anilines
of formula (III) with N-methyl-N-nitroso-N'-nitroguanidine.
Description
[0001] This is a continuation of application Ser. No. 10/467,624
filed Jan. 6, 2004, which is a National Stage Application filed
under .sctn.371 of PCT Application No. PCT/EP02/01201 filed Feb. 6,
2002. The entire disclosure of the prior application, application
Ser. No. 10/467,624 is considered part of the disclosure of the
accompanying continuation application and is hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] The subject of the present invention is novel amidine
derivatives of phenylenediamine which can be represented by the
general formula (I) indicated below: ##STR2## and in which: [0003]
A is selected independently from the carboxamide group, the
thiocarboxamide group, and the carbonyl group, [0004] R.sub.1 is
selected from an alkyl group having from 1 to 3 carbon atoms and
the amino group, unsubstituted or substituted with the nitro group
or the methyl group, [0005] R.sub.2 is selected independently from
hydrogen, an alkyl group having from 1 to 4 carbon atoms, the
methoxy, ethoxy, or propoxy group, a mono-, bi- or tricyclic
cycloalkane residue having from 5 to 12 carbon atoms, the adamantyl
group, an aryl, naphthyl or heterocyclic group, unsubstituted or
substituted with methyl, methoxy, hydroxy, amino or halogen groups,
[0006] R.sub.3 and R.sub.4 are selected independently from hydrogen
and an alkyl group having from 1 to 3 carbon atoms, [0007] R.sub.5
represents one or two substituents independently selected from
hydrogen and the methyl, methoxyl and hydroxyl groups, [0008] n is
a whole number from 0 to 6, and [0009] the amidine group is in the
para or meta position relative to the "-A-NH--" group.
[0010] In the compounds of the invention, R.sub.2 is directly
linked to the A group (n=o) or is linked to A through an alkaline
group, having from 1 to 6 carbon atoms, optionally substituted with
one or more alkyl groups having from 1 to 3 carbon atoms.
SUMMARY OF THE INVENTION
[0011] The compounds of the present invention have been found to be
potent antagonists of various mediators of inflammation and also
have immunosuppressive properties. In vitro, they have been found
to be inhibitors of inducible nitric oxide synthase (iNOS) and of
the enzyme cyclooxygenase (COX). In vivo, they have been found to
be potent inhibitors of the cytokine "tumour necrosis factor"
(TNF.alpha.). Moreover, many of the products of the invention can
antagonise the collagenase activity of the metalloproteases.
[0012] Nitric oxide (NO) is formed at cell level by L-arginine, by
means of the enzyme NOS. There are three subtypes of this enzyme.
The enzyme (iNOS) which can be induced in the presence of
pro-inflammatory cytokines or of endotoxins is expressed in cells
of many types, amongst which are macrophages and neutrophils.
[0013] Vasodilatation, which is a characteristic of acute
inflammation, depends, for many mediators of the inflammatory
process, such as, for example, histamine, brakykinin, substance P,
PAF, etc., on the release of NO. In general, NO increases the
inflammatory responses in many experimental models, both acute and
chronic.
[0014] It should be noted that NO can be produced massively in
response to a stimulus induced by cytokines in the inflamed joints
of patients with rheumatoid arthritis and osteoarthritis, and that
the plasma concentrations of NO in the synovial fluid in these
patients are generally very high.
[0015] The fact that the activity of iNOS is also very high in the
colons of patients with ulcerative colitis is also interesting.
[0016] The prostaglandins (PGE) are mediators of inflammation
generated by the enzyme cyclooxygenase (COX). The inducible isoform
(COX-2) is overproduced ("upregulated") in the inflamed tissues and
this leads to increased synthesis of PGE.
[0017] There are interactions between the NOS and COX systems and
the role of NO in inflammation may therefore depend not only on its
direct effect, but also on its modulatory effect on the
bio-synthesis of PGE.
[0018] TNF.alpha. is a primary cytokine which initiates the cascade
of events that characterise an inflammatory process, inducing the
synthesis and release of secondary cytokines and enzymes such as
metalloproteases (MMP, amongst which is collagenase), iNOS and
COX-2. As already mentioned, the intestinal mucosa is one of the
most important sites of pro-inflammatory cytokine production, as
observed in pathological conditions such as chronic inflammation of
the colon (IBS) and ulcerative colitis.
[0019] It can therefore be considered that the compounds of the
present invention may be used with advantage in the treatment of
various diseases in man which are characterised by nonspecific
inflammation such as, for example, rheumatoid arthritis which is a
syndrome with a chronic course which can develop into progressive
destruction of the joint and periarticular structures,
osteoarthrosis which is a disease characterised by the degeneration
of the joint cartilage, often accompanied by secondary inflammation
of the sinovial membrane, or in other pathological conditions, for
example, in the gastrointestinal system, ulcerative colitis,
Crohn's disease, IBS or food allergies and intolerance.
[0020] Advantageous use of the compounds of the invention can also
be predicted in other areas and systems, for example, in the
treatment of pathological conditions of the cardiovascular system
with an inflammatory or atherosclerotic basis which are sensitive
to treatment of iNOS inhibitors.
[0021] Moreover, for the compounds of the invention which have
MMP-inhibiting activity, advantageous use can be predicted in the
treatment of tumoral conditions, since they potentially prevent
localised or metastatic invasion of tumoral cells, both by
inhibiting the activation of various growth factors and by blocking
angiogenesis.
[0022] An enormous number of studies have been performed in the
search for drugs with anti-inflammatory activity which can perform
an inhibiting action on pro-inflammatory cytokines and which are
free of the side effects of conventional antiinflammatory drugs
(COX inhibitors).
[0023] Chiou et al [Exp. Opin, Ther. Patents 6(1), 41-56 (1996)]
have recently reviewed, in a monographic work, a large number of
publications and patents in which various classes of compounds
which inhibit the production of cytokines by blocking their
release, their receptors, or their converting enzymes, are
described. Many monographic works have also been published on the
inhibitors of MMPs, such as, for example, that of Summers et al
[Annual Reports in Med. Chemistry 33, 131-140 (1998)] in which
various chemical classes of MMP inhibitors are examined and their
therapeutic potential is discussed. Amongst others, patents in
which the NO-synthase inhibitory activity of various amidines is
described, such as, for example, the patent PCT/GB/92/02387 and the
patent PCT/GB/94/01325, have been published.
[0024] However, the compounds described are amidine derivatives of
amino-acids which are structurally very similar to analogous
derivatives of L-arginine, such as L-N-monomethyl arginine which is
the subject of the patent WO91/04024, but are different from the
benzamidines of the present invention, both structurally and with
regard to their pharmacological activity as a whole.
[0025] All of these publications and researches show that there is
a great therapeutic need to find ever more potent and
better-tolerated novel anti-inflammatory drugs. In accordance with
this need, the object of the present invention is to provide, for
treatment, novel drugs which, simultaneously, have antiinflammatory
and immunosuppressive activity, expressed by their combined iNOS
and COX antagonistic activities, their MMP-inhibiting activity, and
their activity in inhibiting the production of TNF-.alpha., and
which can thus be used advantageously in the treatment of
pathological conditions in man which are characterised by
non-specific or autoimmune-based inflammation.
[0026] Pharmaceutical forms of the compounds of the invention can
be prepared by conventional techniques, for example, as tablets,
pills, capsules, suppositories, suspensions, solutions, patches,
creams or ointments, and can be administered by oral, parenteral,
rectal, transdermal, or transmucosal routes, or in other forms
suitable for achieving the therapeutic effect such as, for example,
solid preparations for oral use with delayed action which permit
the controlled release of the active substance over time.
[0027] The active ingredient is usually administered to the patient
with a reference dose variable from 0.1 to 10 mg/kg of body weight
per dose.
[0028] For parenteral administration, the use of a water-soluble
salt of the compounds of the invention such as the hydrochloride or
another salt derived from a non-toxic and
pharmaceutically-acceptable inorganic or organic acid is preferred.
For the derivatives of the invention with a slightly acid
character, such as the derivatives in which R.sub.1 is the
nitro-amino group, the corresponding sodium salts or equivalent
salts can be prepared by conventional methods.
[0029] Substances commonly used in pharmaceuticals such as
excipients, binders, flavourings, disaggregants, substances for
stimulating transdermal and transmucosal absorption, colourings,
humectants, etc., may be used as inactive ingredients.
[0030] The method for the preparation of the derivatives of the
invention consists of a series of reactions which comprises:
[0031] a) reacting the 1,3 or 1,4-phenylenediamine of formula (IV),
suitably substituted and in which R.sub.5 has the meaning given
above, with the appropriate isothiocyanate (V A), acyl chloride (V
B), or isocyanate (V C), in which R.sub.2, R.sub.3, R.sub.4 and n
have the meanings given above, in the presence of an excess of
phenylenediamine, in an inert solvent and at a temperature of
between 4.degree. C. and the reflux temperature of the solvent
used, to give the corresponding anilines of formula (III) in which
R.sub.2, R.sub.3, R.sub.4, R.sub.5, A and n have the meanings given
above, and in which the amine group is in the meta or para position
relative to the chain with the "A-NH" group (see General Synthesis
Scheme, Step 1), and
b) reacting the anilines of formula (III), in which R.sub.2,
R.sub.3, R.sub.4, R.sub.5, A and n have the meanings given above,
with the appropriate imidate of formula II, generally salified in
hydrochloride form, in which R.sub.1 has the meaning given
above.
[0032] The reaction takes place in the presence of an excess of
(II) relative to (III) (preferably of 2 moles to 1) and in the
presence of a stoichiometric quantity, relative to (II), of a
tertiary base, preferably triethylamine, in an inert anhydrous
solvent, such as, for example tetrahydrofuran, at a temperature of
between 4.degree. C. and the boiling point of the solvent, for a
period of between 2 and 48 hours, to give the corresponding final
derivatives of formula (I) in which A, R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5 and n have the meanings given above and in which
the amidine group is in the para or meta position relative to the
"A-NH" group.
[0033] The compounds of formula (I) described in Table 2 (I-30 and
I-31) were obtained with the use of reagents other than those of
the general formula (II) and, in particular,
benzotriazolo-1-carboxamidinium tosylate (see Example 5) and
N-methyl-N-nitroso-N'-nitroguanidine (see Example 6),
respectively.
[0034] The starting phenylenediamines, as well as the
isothiocyanates (V A), the acyl chlorides (V B), the isocyanates (V
C), and the imidates of formula (II) are commercially available or
were prepared by conventional methods in accordance with existing
literature. ##STR3##
[0035] The following examples are given below as further
illustration of the invention.
EXAMPLE 1
Preparation of N-(4-aminophenyl)-N'-pentyl thiourea (Compound III-4
of Table 1)
[0036] 71.8 g of 1,4-phenylenediamine (0.66 moles) was suspended in
300 ml of tetrahydrofuran, and 43 g of pentyl isothiocyanate (0.33
moles), dissolved in 50 ml of tetrahydrofuran, was added slowly
dropwise, with stirring and at ambient temperature. After 24 hours,
the solvent was evaporated under vacuum and the residue, taken up
with ethyl acetate, was washed with water, 0.1N citric acid,
saturated sodium bicarbonate, and water to neutral pH. The solvent
was rendered anhydrous with anhydrous sodium sulphate and
evaporated under vacuum, to give 75 g of crude product which was
re-crystallised from toluene. 60 g was obtained.
[0037] Formula: C.sub.12H.sub.19N.sub.3S (M.W. 237.46). Yield
77%.
[0038] TLC: (chloroform/methanol 9/1) rf 0.7. M.P. 125.degree.
C.
[0039] HPLC: retention time (rt) 4.60 minutes.
[0040] HPLC conditions: Supelcosil LC-DP column, 100.times.4.6 mm,
eluent
[0041] KH.sub.2PO.sub.4 0.01M 25/MetOH 27/MetCN 48 (pH 2.1), flow
0.4 ml/min, UV detector at 248 nm.
[0042] .sup.1HNMR (DMSO-d.sub.6), ppm: 2.21 (bt, 3H, J=5.60 Hz);
0.98-1.71 (m, 6H); 3.33 (q, 2H, J=6.68 Hz); 4.93 (bs, 2H); 6.45 (d,
2H, J=8.59 Hz); 6.81 (d, 2H, J=8.59 Hz); 7.05 (m, 1H); 8.88 (s,
1H).
[0043] All of the intermediate compounds of formula (III) according
to the invention in which A is the thiocarboxamide group were
synthesised with the use of the same method (see Scheme 1, step
1a).
EXAMPLE 2
Preparation of N-(4-aminophenyl)-N'-pentylamide (Compound III-22 of
Table 1)
[0044] 20.2 g di 1,4-phenylenediamine (0.181 moles) was dissolved
in 200 ml of tetrahydrofuran, together with 11.1 ml of
triethylamine (0.080 moles). The solution was cooled to 0.degree.
C. and 10 ml of caproyl chloride (0.0724 moles) was added slowly
dropwise so that the temperature did not exceed 5.degree. C. Upon
completion of the addition, the temperature was increased to
ambient temperature. After reaction for 24 hours, the solvent was
evaporated under vacuum and the residue, taken up with ethyl
acetate, was washed with water. The solvent was rendered anhydrous
with anhydrous sodium sulphate and evaporated under vacuum to give
12 g of crude product which was recrystallised from toluene. 7.5 g
was obtained.
[0045] Formula: C.sub.12H.sub.18N.sub.2O (M.W. 206.28). Yield
51%
[0046] TLC: (chloroform/methanol 9/1) rf 0.51. M.P. 88.5-89.degree.
C.
[0047] HPLC: retention time (rt) 5.54 minutes.
[0048] HPLC conditions: see Example 1.
[0049] .sup.1HNMR (DMSO-d.sub.6), ppm: 0.86 (bt, 3H, J=5.59 Hz);
1.03-1.79 (m, 6H); 2.18 (t, 2H, J=6.99 Hz); 4.69 (s, 2H); 6.43 (d,
2H, J=8.39 Hz); 7.15 (d, 2H, J=8.39 Hz); 9.28 (s, 1H).
EXAMPLE 3
Preparation of N-(4-aminophenyl)-N'-cyclohexyl urea (Compound
III-28 of Table 1)
[0050] 10.4 g of 1,4-phenylenediamine (0.095 moles) was suspended
in 100 ml of tetrahydrofuran and 5 ml of cyclohexyl isocyanate
(0.038 moles), dissolved in 20 ml of tetrahydrofuran, was added
slowly dropwise, with stirring and at ambient temperature. After 24
hours, the solid formed was filtered out and washed with cold
tetrahydrofuran, water, and ethyl ether. 8.8 g was obtained.
[0051] Formula: C.sub.13H.sub.19N.sub.3O (M.W. 233.31). Yield
98%
[0052] TLC: (chloroform/methanol 9/1) rf 0.40. M.P.
199.8-202.4.degree. C.
[0053] HPLC: retention time (rt) 6.39 minutes.
[0054] HPLC conditions: see Example 1.
[0055] .sup.1HNMR (DMSO-d.sub.6), ppm: 0.75-2.00 (m, 10H); 3.47 (m,
1H); 4.62 (bs, 2H); 5.75 (d, 2H, J=7.50 Hz); 6.49 (d, 2H, J=8.75
Hz); 7.00 (d, 2H, J=8.75 Hz); 7.75 (bs, 1H).
[0056] Some derivatives of formula (III) obtained as described
above are given in Table 1 below, with some identifying chemical
and physical characteristics. TABLE-US-00001 TABLE 1 Compounds of
formula (III) (III) ##STR4## M.P Compound R.sub.2 R.sub.3 n A Crude
Formula (crystallisation solvent).sup.d TLC(R.sub.f).sup.e III-1
CH.sub.3 -- 0 NH--CS C.sub.8H.sub.11N.sub.3S 173.5-174.2(A) 0.65(I)
III-2 CH.sub.3 H 2 NH--CS C.sub.10H.sub.15N.sub.3S 118.6-120.2
0.58(I) III-3 CH.sub.3 H 3 NH--CS C.sub.11H.sub.17N.sub.3S
124.5-127 0.67(I) III-4 CH.sub.3 H 4 NH--CS
C.sub.12H.sub.19N.sub.3S 113-115(B) 0.70(I) III-5 CH.sub.3 H 5
NH--CS C.sub.13H.sub.21N.sub.3S 119.5-120.3 0.75(I) III-6 CH.sub.3
H 6 NH--CS C.sub.14H.sub.23N.sub.3S 107.0-107.4 0.75(I) III-7
Isopropyl H 2 NH--CS C.sub.12H.sub.19N.sub.3S 153.5-154.6(B)
0.60(I) III-8 CH.sub.3 CH.sub.3 1 NH--CS C.sub.10H.sub.15N.sub.3S
137.2-137.8(B) 0.63(I) III-9 Ethyl CH.sub.3 1 NH--CS
C.sub.12H.sub.19N.sub.3S 165.6-166.8 0.75(I) III-10 CH.sub.3--O H 3
NH--CS C.sub.11H.sub.17N.sub.3OS 104.6-105.2 0.65(I) III-11
Cyclohexyl -- 0 NH--CS C.sub.13H.sub.19N.sub.3S 168.6-169.4 0.70(1)
III-12 Phenyl -- 0 NH--CS C.sub.13H.sub.13N.sub.3S 263.0-264.0(A)
0.79(II) III-13 Phenyl H 2 NH--CS C.sub.15H.sub.17N.sub.3S
168.2-169.0 0.83(II) III-14 Phenyl H 3 NH--CS
C.sub.16H.sub.19N.sub.3S 117.3-117.9 0.83(II) III-15 4-F-Phenyl H 2
NH--CS C.sub.15H.sub.17ClFN.sub.3S 190.2-191.6 0.85(II) III-16
4-Cl-Phenyl H 2 NH--CS C.sub.15H.sub.16ClN.sub.3S 116.6-1 16.7
0.83(II) III-17 2,6-diF-Phenyl H 2 NH--CS
C.sub.15H.sub.15F.sub.2N.sub.3S 147.7-149.7 0.50(I) III-18
2-Piridyl H 1 NH--CS C.sub.13H.sub.14N.sub.4S 106.0-108.0 0.41(I)
III-19 2-Piridyl H 2 NH--CS C.sub.14H.sub.16N.sub.4S 82.0-84.0
0.38(I) III-20 5-CH.sub.3-2- -- 0 NH--CS
C.sub.11H.sub.12N.sub.4S.sub.2 157.5-159.0 0.40(I) Thiazolyl III-21
CH.sub.3 H 4 NH--CS C.sub.14H.sub.23N.sub.3S 101.4-102.8 0.75(I)
III-22 CH.sub.3 H 4 CO C.sub.12H.sub.18N.sub.2O 88.5-89.0(B)
0.51(I) III-23 Cyclohexyl -- 0 CO C.sub.13H.sub.18N.sub.2O
176.1-177.0(B) 0.50(I) III-24 1-Adamantyl -- 0 CO
C.sub.17H.sub.22N.sub.2O 169.6-171.2 0.45(I) III-25 2-Indolyl -- 0
CO C.sub.15H.sub.13N.sub.3O 193.8-194.9(A) 0.47(I) III-26 3-Indolyl
H 1 CO C.sub.16H.sub.14N.sub.3O 122.0-122.7(B) 0.40(I) III-27
1-CH.sub.3-2-Indolyl -- 0 CO C.sub.16H.sub.15N.sub.3O 232.6-235.3
0.55(I) III-28 Cyclohexyl -- 0 NH-CO C.sub.13H.sub.19N.sub.3O
199.8-202.4 0.40(I) III-29 Cyclohexyl -- 0 NH--CS
C.sub.13H.sub.19N.sub.3S 101.0-102.8 0.70(I) Note: .sup.aIn all of
the compounds given by way of example, R.sub.4 is H, with the
exception of compound III-9 in which R.sub.4 is CH.sub.3. .sup.bIn
all of the compounds given by way of example, the amino group is in
the para position relative to the "NH-A" group, with the exception
of compound III-29 in which the amino group is in the meta position
relative to the "NH-A" group. .sup.cIn all of the compounds given
by way of example, R.sub.5 is H except for compound III-21 in which
R.sub.5 is 2,5-dimethyl. .sup.dCrystallisation solvent:
A(isopropanol); B(toluene). .sup.eEluent: (I) chloroform/methanol
(9/1)(v/v); (II) chloroform/methanol/water/ammonia
(85/25/2/1)(v/v).
EXAMPLE 4
Preparation of N-[4-(N-acetamidine)phenyl]-N'-pentyl thiourea
(Compound I-4 of Table 2)
[0057] 55 g di N-(4-aminophenyl)-N'-pentyl thiourea (0.23 moles)
was dissolved in 300 ml of tetrahydrofuran. 64.5 ml of
triethylamine (0.46 moles) and 50.7 g of methyl acetamidate
hydrochloride (0.46 moles) was added, with stirring at ambient
temperature; the pH of the suspension was approximately 9. After 24
hours (the pH fell to 7), the solid was filtered out and washed
with a little tetrahydrofuran and ethyl ether. The residue, taken
up with water, was rendered basic with 4N sodium hydroxide to pH 11
and was left for 1 hour with stirring and then filtered, washed
with water, and ethyl ether and purified hot with acetonitrile. 51
g was obtained.
[0058] Formula: C.sub.14H.sub.22N.sub.4S (M.W. 278.42). Yield
80%.
[0059] TLC: (butanol/acetic acid/water 5/2/2) rf 0.70;
(chloroform/methanol saturated with ammonia 9/1) rf 0.37. M.P.
191.4.degree. C.
[0060] HPLC: retention time (rt) 7.70 minutes.
[0061] HPLC conditions: see Example 1.
[0062] .sup.1HNMR (DMSO-d.sub.6), ppm: 0.88 (bt, 3H, J=5.60 Hz);
1.00-1.55 (m, 6H); 1.82 (s, 3H); 3.35 (m, 2H); 5.91 (bs, 1H); 6.63
(d, 2H, J=8.56 Hz); 7.11 (d, 2H, J=8.56 Hz); 7.28 (bs, 1H); 9.08
(bs, 1H).
[0063] All of the derivatives of formula (I) in which R1 was methyl
were prepared in similar manner with the use of the appropriate
aniline of formula (III) in place of N-(4-aminophenyl)-N'-pentyl
thiourea.
EXAMPLE 5
Preparation of 1-guanidinophenyl-4-cyclohexyl thiourea (Compound
I-30 of Table 2)
[0064] 15 g of N-(4-aminophenyl)-N'-cyclohexyl thiourea (0.06
moles) was suspended in 100 ml of acetonitrile, and 20 g of
benzotriazolo-1-carboxamidinium tosylate [0.06 moles, Katrizky, A.
R. et al. Synth. Comm. 25(8), 1173-1186, (1995)] was added, with
stirring at ambient temperature. After 72 hours, the solvent was
evaporated under vacuum and the residue, taken up with ethyl
acetate, was washed with 0.1N sodium hydroxide and extracted with
0.1N citric acid. The aqueous phase was brought to pH 9 with 2N
sodium hydroxide, and extracted with ethyl acetate and the organic
phase was washed with water. The solvent was rendered anhydrous
over anhydrous sodium sulphate and evaporated under vacuum to give
8 g of crude product which was purified with isopropyl ether. 7.4 g
was obtained.
[0065] Formula: C.sub.14H.sub.21N.sub.5S (M.W. 291.42). Yield
43%.
[0066] TLC: (butanol/acetic acid/water 5/2/2) rf 0.71. M.P.
188.6.degree. C.
[0067] HPLC: retention time (rt) 8.0 minutes.
[0068] HPLC conditions: see Example 1.
[0069] .sup.1HNMR (DMSO-d.sub.6), ppm: 0.67-2.17 (m, 10H); 4.05 (m,
1H); 5.73 (bm, 6H); 6.71 (d, 2H, J=8.43 Hz); 7.17 (d, 2H, J=8.43
Hz).
EXAMPLE 6
Preparation of 1-nitroguanidinophenyl-4-cyclohexyl thiourea
(Compound I-31 of Table 2)
[0070] 9 g of N-(4-aminophenyl)-N'-cyclohexyl thiourea (0.036
moles) was suspended in 140 ml of a 1/1 ethanol/water mixture, and
3 g of N-methyl-N-nitroso-N'-nitroguanidine [0.021 moles, McKay, A.
F. J. Am. Chem. Soc. 71, 1968-1970, (1949)] was added, with
stirring at ambient temperature. After 2 hours at ambient
temperature, the reaction mixture was heated under reflux for 1
hour; the precipitate which formed was filtered hot, washed with
ethyl ether and dried. 3.8 g was obtained.
[0071] Formula: C.sub.14H.sub.20N.sub.6O.sub.2S (M.W. 336.41).
Yield 54%.
[0072] TLC: (butanol/acetic acid/water 5/2/2) rf 0.95. M.P.
215.2.degree. C.
[0073] HPLC: retention time (rt) 4.16 minutes.
[0074] HPLC conditions: see Example 1.
[0075] .sup.1HNMR (DMSO-d.sub.6), ppm: 0.84-1.99 (m, 10H); 4.01 (m,
1H); 7.15 (d, 2H, J=8.76 Hz); 7.42 (d, 2H, J=8.76 Hz); 7.52 (bd,
1H, J=7.64 Hz); 8.03 (bs, 2H); 9.28 (s, 1H); 9.42 (bs, 1H).
EXAMPLE 7
Preparation of N-[4-(N-acetamidino)phenyl]-N'-pentyl thiourea
hydrochloride (the hydrochloride of Compound I-4)
[0076] 3 g of N-[4-(N-acetamidino)phenyl]-N'-pentyl thiourea (0.011
moles) was dissolved in 1N HCl. After 0.5 hours at ambient
temperature, the precipitate which formed was filtered out, washed
with a little water, and with ethyl ether, and dried. 3.2 g was
obtained.
[0077] Formula: C.sub.14H.sub.23ClN.sub.4S (M.W. 314.88). Yield
93%.
[0078] TLC: (butanol/acetic acid/water 5/2/2) rf 0.70. M.P.
180.6.degree. C.
[0079] .sup.1HNMR (DMSO-d.sub.6), ppm: 0.84 (t, 3H, J=5.60 Hz);
1.02-1.78 (m, 6H); 2.28 (s, 3H); 3.43 (bq, 2H, J=6.05 Hz); 7.15 (d,
2H, J=8.56 Hz); 7.73 (d, 2H, J=8.56 Hz); 8.39 (m, 1H); 9.42 (bs,
1H); 10.38 (s, 1H); 11.28 (s, 1H).
[0080] Some derivatives of formula (I) obtained according to the
invention are given in Table 2 below, with some identifying
chemical and physical characteristics, without thereby in any way
limiting the spirit or the scope of the invention. TABLE-US-00002
TABLE 2 Compounds of formula(I) (I) ##STR5## M.P. Com-
(crystallisation TLC pound R.sub.1 R.sub.2 R.sub.3 n A Crude
formula solvent).sup.d (R.sub.f).sup.e I-1 CH.sub.3 CH.sub.3 -- 0
NH--CS C.sub.10H.sub.14N.sub.4S 186.2-187.8 0.50 I-2 CH.sub.3
CH.sub.3 H 2 NH--CS C.sub.12H.sub.18N.sub.4S 188.1-189.1(A) 0.70
I-3 CH.sub.3 CH.sub.3 H 3 NH--CS C.sub.13H.sub.20N.sub.4S
182.3-183.1(A) 0.72 I-4 CH.sub.3 CH.sub.3 H 4 NH--CS
C.sub.14H.sub.22N.sub.4S 190.7-191.4(A) 0.71 I-5 CH.sub.3 CH.sub.3
H 5 NH--CS C.sub.15H.sub.24N.sub.4S 177.6-178.0 0.80 I-6 CH.sub.3
CH.sub.3 H 6 NH--CS C.sub.16H.sub.26N.sub.4S 177.8-178.4 0.80 I-7
CH.sub.3 Isopropyl H 2 NH--CS C.sub.14H.sub.22N.sub.4S
177.7-179.1(A) 0.58 I-8 CH.sub.3 CH.sub.3 CH.sub.3 1 NH--CS
C.sub.12H.sub.18N.sub.4S 163.0-163.9(A) 0.58 I-9 CH.sub.3 Ethyl
CH.sub.3 1 NH--CS C.sub.14H.sub.22N.sub.4S 145.7-147.4(B) 0.61 I-10
CH.sub.3 CH.sub.3--O H 3 NH--CS C.sub.13H.sub.24N.sub.4OS
146.9-149.6(B) 0.54 I-11 CH.sub.3 Cyclohexyl -- 0 NH--CS
C.sub.15H.sub.22N.sub.4S 171.3-171.6(A) 0.60 I-12 Ethyl Cyclohexyl
-- 0 NH--CS C.sub.16H.sub.24N.sub.4S 155.0-156.0 0.66 I-13 CH.sub.3
Phenyl H 0 NH--CS C.sub.15H.sub.16N.sub.4S 118.7-120.5 0.70 I-14
CH.sub.3 Phenyl H 2 NH--CS C.sub.17H.sub.20N.sub.4S 186.6-188 0.63
I-15 CH.sub.3 Phenyl H 3 NH--CS C.sub.18H.sub.22N.sub.4S
146.6-148.0(B) 0.60 I-16 CH.sub.3 4-F-Phenyl H 2 NH--CS
C.sub.17H.sub.19FN.sub.4S 179.1-181.6 0.57 I-17 CH.sub.3
4-Cl-Phenyl H 2 NH--CS C.sub.17H.sub.19ClN.sub.4S 174.0-176.0(A)
0.60 I-18 CH.sub.3 2,6-diF-Phenyl H 2 NH--CS
C.sub.17H.sub.18F.sub.2N.sub.4S 158.8-160.4 0.58 I-19 CH.sub.3
2-Piridyl H 1 NH--CS C.sub.15H.sub.17N.sub.5S 159.0-16 1.0(A) 0.48
I-20 CH.sub.3 2-Piridyl H 2 NH--CS C.sub.16H.sub.19N.sub.5S
171.5-173.0(A) 0.42 I-21 CH.sub.3 5-CH.sub.3-2-Thiazolyl -- 0
NH--CS C.sub.13H.sub.15N.sub.5S.sub.2 107.8-110.1 0.45 I-22
CH.sub.3 CH.sub.3 H 4 NH--CS C.sub.16H.sub.26N.sub.4S 118-121.7(B)
0.61 I-23 CH.sub.3 CH.sub.3 H 4 CO C.sub.14H.sub.21N.sub.3O
160.4-161.6 0.60 I-24 CH.sub.3 Cyclohexyl -- 0 CO
C.sub.15H.sub.21N.sub.3O 194.6-195.8(B) 0.60 I-25 CH.sub.3
1-Adamantyl -- 0 CO C.sub.19H.sub.25N.sub.3O 224.4-224.7 0.61 I-26
CH.sub.3 2-Indolyl -- 0 CO C.sub.17H.sub.16N.sub.4O 192.0-193.5
0.61 I-27 CH.sub.3 3-Indolyl H 1 CO C.sub.18H.sub.18N.sub.4O
159.0-160.0 0.51 I-28 CH.sub.3 1-CH.sub.3-2-Indolyl -- 0 CO
C.sub.18H.sub.18N.sub.4O 195.5-196.5 0.68 I-29 CH.sub.3 Cyclohexyl
-- 0 NH-CO C.sub.15H.sub.22N.sub.4O 220.2-224.6 0.50 I-30 NH.sub.2
Cyclohexyl -- 0 NH--CS C.sub.14H.sub.21N.sub.5S 176.5-179.4 0.71
I-31 NO.sub.2--NH Cyclohexyl -- 0 NH--CS
C.sub.14H.sub.20N.sub.6O.sub.2S 213.9-215.2 0.95 I-32 CH.sub.3
Cyclohexyl -- 0 NH--CS C.sub.15H.sub.22N.sub.4S 171.4-173.2 0.66
Note: .sup.aIn all of the compounds given by example, R.sub.4 is H,
with the exception of compound 1-9 in which R.sub.4 is CH.sub.3.
.sup.bIn all of the compounds given by way of example, the amino
group is in the para position relative to the "NH--A" group, with
the exception of compound 1-32 in which the amino group is in the
meta position relative to the "NH--A" group. .sup.cIn all of the
compounds given by way of example, R.sub.5 is H, except for
compound 1-22 in which R.sub.5 is 2,5-dimethyl.
.sup.dCrystallisation solvent: A(acetonitrile); B(toluene).
.sup.eEluent: butanol/acetic acid/water (5/2/2)(v/v).
Pharmacological Activity
[0081] a) The activity in inhibiting the formation of NO, measured
as NO.sub.2.sup.- (nitrites), PGE.sub.2, and neutral protease, was
investigated in vitro on culture broths of rabbit joint
chondrocytes stimulated by cytokine IL-1.beta. (1 ng/ml) for 48
hours. For the preparation of the chondrocytes, the method
described by Berenbaum et al [FEBS Letters 340, 51-55 (1994)] was
followed. Briefly, fragments of cartilage removed under sterile
conditions from the heads of rabbit shoulder, hip and knee joints
were chopped finely and digested at 37.degree. C. by hyaluronidase,
trypsin and collagenase solutions, giving rise, after filtration on
sterile gauze and centrifuging at 600.times.g and suitable dilution
with 10% DMEM-FCS, to a concentration of approximately
1.times.10.sup.5 cells per well. The cells were kept in these
conditions until confluence (about 15 days), the broth being
changed every 3 days. At this point, the products under test,
dissolved in the medium, were added to each test sample and, after
20 minutes, 350 .mu.l IL-1.beta. was added in order to have a final
concentration of 1 ng/ml. The duration of the stimulation was 48
hours at 37.degree. C. (incubation air-CO.sub.2 7%). Measurement of
the nitrites, as described by Green et al. [Anal. Biochem. 126,
131-138 (1982)], and of the PGE.sub.2s by RIA measurement, was then
performed on the cell supernatant fluid. The measurement of the
neutral proteases was performed in the cell supernatant fluid
containing the p-aminophenyl mercury acetate (APMA) activator with
the use of azocoll as the substrate and with incubation at
37.degree. C. for 17 h as described by Chavira et al. [Anal.
Biochem. 136, 446-450 (1984)]. In order to evaluate the direct
inhibitory effect of the compounds under test on the hydrolytic
activity of the cell supernatant fluid, they were added to the
supernatant fluid containing the proteases induced by IL-1.beta.
and already activated by AMPA.
[0082] The results obtained are shown in Table 3, in which the
IC.sub.50, that is, the concentration (micromolar) of antagonist
which can inhibit the formation of nitrites, PGE.sub.2s, and
neutral proteases, respectively, by 50% relative to the control
group, that is, to the cells stimulated with IL-1.beta. but without
the addition of antagonists, is given for some of the compounds of
the invention already given by way of example in Table 2.
TABLE-US-00003 TABLE 3 Compounds of formula(I) (I) ##STR6## Rabbit
joint chondrocytes NO PGE2 MP Compound R.sub.1 R.sub.2 R.sub.3 n A
IC.sub.50(.+-.10.sup.-6M) I-1 CH.sub.3 CH.sub.3 -- 0 NH--CS IN IN
30 I-2 CH.sub.3 CH.sub.3 H 2 NH--CS IN IN 30 I-3 CH.sub.3 CH.sub.3
H 3 NH--CS 190 250 13.3 I-4 CH.sub.3 CH.sub.3 H 4 NH--CS 6.6 3.3
6.6 I-5 CH.sub.3 CH.sub.3 H 5 NH--CS 10.0 10.0 3.3 I-6 CH.sub.3
CH.sub.3 H 6 NH--CS 30.0 20.0 20.0 I-7 CH.sub.3 Isopropyl H 2
NH--CS 3.3 6.6 13.3 I-8 CH.sub.3 CH.sub.3 CH.sub.3 1 NH--CS 30.0
3.3 3.3 I-9 CH.sub.3 C.sub.2H.sub.5 CH.sub.3 1 NH--CS 10.0 10.0 6.6
I-10 CH.sub.3 CH.sub.3--O H 3 NH--CS 50.0 50.0 6.6 I-11 CH.sub.3
Cyclohexyl -- 0 NH--CS 110 16.6 30 I-12 Ethyl Cyclohexyl -- 0
NH--CS IN 13.3 100 I-13 CH.sub.3 Phenyl H 0 NH--CS 100 10 6.6 I-14
CH.sub.3 Phenyl H 2 NH--CS 100 33 6.6 I-15 CH.sub.3 Phenyl H 3
NH--CS 33 16.6 6.6 I-16 CH.sub.3 4-F-Phenyl H 2 NH--CS 13.3 26.6
6.6 I-17 CH.sub.3 4-Cl-Phenyl H 2 NH--CS 10.0 10 10 I-18 CH.sub.3
2,6-diF-Phenyl H 2 NH--CS IN 6.6 6.6 I-19 CH.sub.3 2-Piridyl H 1
NH--CS 300 IN 13.3 I-20 CH.sub.3 2-Piridyl H 2 NH--CS 16.6 16.6 6.6
I-21 CH.sub.3 5-CH.sub.3-2-Thiazolyl -- 0 NH--CS 66.6 IN IN I-22
CH.sub.3 CH.sub.3 H 4 NH--CS 16.6 10.0 20.0 I-23 CH.sub.3 CH.sub.3
H 4 CO 100 IN IN I-24 CH.sub.3 Cyclohexyl -- 0 CO 33.3 33.3 IN I-25
CH.sub.3 1-Adamantyl -- 0 CO IN 16.6 IN I-26 CH.sub.3 2-Indolyl --
0 CO 300 IN IN I-27 CH.sub.3 3-Indolyl H 1 CO 10 IN IN I-28
CH.sub.3 1-CH.sub.3-2-Indolyl -- 0 CO IN 100 IN I-29 CH.sub.3
Cyclohexyl -- 0 NH-CO 25.0 6.6 25.0 I-30 NH.sub.2 Cyclohexyl -- 0
NH--CS 6.6 10 13.3 I-31 NO.sub.2--NH Cyclohexyl -- 0 NH--CS 46.6 30
6.6 I-32 CH.sub.3 Cyclohexyl -- 0 NH--CS 300 300 IN L-NAME -- -- --
-- -- -- IN 3(mM) IN Note: .sup.afor the structural identification,
see Notes a, b and c of Table 2 .sup.bNO determined as nitrites
.sup.cMP: metalloproteases
[0083] It can be seen from the data given in Table 3 that some of
the compounds which were tested and which are subjects of the
invention have a potent inhibitory effect, at micromolar level, on
the production of nitrites, PGE.sub.2s and metalloproteases induced
by IL-1.beta. cytokine in rabbit chondrocyte cultures. The best
compounds were those in which R.sub.1 was CH.sub.3, R.sub.3 and
R.sub.4 were H, R.sub.2 was CH.sub.3 if n was 4 or 5, isopropyl if
n was 2, or the 2,6-difluoro-phenyl group if n was 2, and in which
A was NH--CS (compounds I-4, I-5, I-7 and I-18, respectively).
Compound I-31 in which R.sub.1 was the NH--NO.sub.2 group, R.sub.2
was cyclohexyl, and A was the NH--CS group was also very active. It
is interesting to note that the inhibitory activity on the
metalloproteases was expressed solely by the compounds in which A
was the NH--CS group. It should also be noted that the reference
NO-synthase inhibitor compound L-NAME generally had an activity
about 30-100 times less potent than the best compounds of the
invention, such as the compound I-4, and was completely inactive in
inhibiting the metalloproteases.
[0084] b) Some of the compounds of the invention, such as compounds
I-4, I-11 and I-31, were evaluated in vivo in a series of
experimental extravasation models in which 5 .mu.l of the
preselected phlogogenic agent dissolved in physiological solution
was injected intradermally into the ears of mice as described by
Erdo et al., with slight modifications [Agents and Actions 39,
137-142 (1993)].
[0085] The products under test were administered orally 1 hour
before the challenge and, 30 minutes before the challenge, the dye
Evans Blu was injected intravenously in a dose of 100 mg/kg. The
animals were killed at a time predetermined according to the test,
30 min.-2 hours after the challenge. The extravasation was
evaluated by determining the quantity of dye present in the ear,
extracted after homogenisation of the tissue in 2 ml of formamide
and incubation at 50.degree. C. for 2 hours. After centrifuging,
the amount of dye was determined by measuring the absorption at 620
nm. The maximum % effect (% MPE) was calculated by the following
formula: % .times. .times. MPE = ( E V - E D ) E V - E B .times.
100 ##EQU1## in which E.sub.V is the mean absorption observed in
the group of animals treated solely with the phlogogenic agent,
E.sub.D is the group treated with the phlogogenic agent and the
drug, and E.sub.B is the base value, that is, the value for the
animals injected with physiological solution alone.
[0086] The phlogogenic agents used were:
Arachidonic acid (dissolved in EtOH); (1 mg/mouse; killed+30 min);
histamine (3 nmoli/mouse; killed+30 min); PAF (30 pmoli/mouse;
killed+60 min); Zymosan (10 .mu.g/mouse; killed+120 min);
bradykinin (0.6 nmoli/mouse; killed+30 min).
[0087] The results obtained with Compound I-4 are summarised in
Table 4. TABLE-US-00004 TABLE 4 Activity of Compound I-4 on
extravasation induced by algogenic agents in the ears of mice %
inhibition effects Arachidonic Hista- Zymo- Brady- Acid mine PAF
san kinin Dose mg/ Duration kg (OS) 30 min 30 min 1 h 2 h 30 min
2.5 mg/kg -- -- 41.9 40.0 -- 5 mg/kg 37.8 17.8 49.9 66.6 30.1 10
mg/kg 40.0 27.7 55.1 65.8 30.7 20 mg/kg 52.6 38.8 56.4 83.9 49.1 40
mg/kg 55.5 52.5 66.2 89.8 51.3 80 mg/kg -- -- -- 95.5 --
[0088] Compound I-4 was found to be particularly effective in
extravasation induced by Zymosan (ED.sub.50 3.1 mg/kg); however, in
the other extravasation models investigated, a dose of only 5 mg/kg
also produced a mean inhibitory effect of approximately 35%. A
non-selective NO-synthase inhibitor, N-nitro-L-arginine methyl
ester (L-NAME), a COX.sub.1 inhibitor (Piroxicam) and a COX.sub.2
inhibitor (Nimesulide) were used as comparison drugs.
[0089] The results obtained are given in Table 5 below.
TABLE-US-00005 TABLE 5 Inhibition of extravasation in the ears of
mice induced by algogenic agents (ED.sub.50 mg/kg OS) Hista- AA
mine PAF Zymosan Bradykinin Compound I-4 20.5 37.0 6.1 3.1 32.6
Compound I-31 30.5 -- 10.8 10.5 -- L-NAME 55.1 36.4 40.0 IN
(>100) 39.4 Piroxicam -- -- 21.2 6.1 -- Nimesulide 41.0 -- IN
(>40) -- -- Note: AA = Arachidonic acid --: not tested
[0090] In general, Compound I-4 was the most active of the
compounds investigated. In fact, of the reference compounds, L-NAME
was approximately as active as Compound I-4 in extravasation
induced by histamine and bradykinin but was two times less active
in extravasation by AA, much less active in antagonising PAF, and
completely inactive in extravasation induced by Zymosan.
[0091] Piroxicam was 2-3 times less active than Compound I-4 in the
models in which PAF and Zymosan were used, and Nimesulide was less
active (2 times) in antagonising AA and was inactive in
antagonising PAF. Compound I-31 had an activity profile similar to
that of compound I-4 but was generally 2-3 times less active.
[0092] c) The immunosuppressive activity of Compound I-4 was
evaluated in an in vivo test in the rat, in which a
lipopolysaccharide (LPS) of bacterial origin, injected i.p. at a
dose of 6 mg/kg, induced a shock characterised by urgent diarrhea
accompanied by a large increase in the plasma TNF.alpha.
concentration.
[0093] Blood was taken from the animals which were killed 90
minutes after the challenge and the concentration of TNF.alpha. in
the plasma was determined by Elisa (Amersham Kit cod. RPN2734). The
results thus obtained are given in Table 6. TABLE-US-00006 TABLE 6
Effects of Compound I-4 on plasma TNF.alpha. concentration in rats
after stimulation with LPS (6 mg/kg/ I.P.) Inhibition TNF.alpha.
(ng/ml .+-. SD) (%) Control (SHAM) <1 (n = 4) -- Control LPS 61
.+-. 13.4 (n = 8) -- Compound I-4 2.3 .+-. 1.2 (n = 6) 96.2 (50
.mu.g/kg ICV) + LPS Compound I-4 12.9 .+-. 7.2 (n = 6) 78.9 (10
mg/kg IV) + LPS Compound I-4 30.6 .+-. 1.0 (n = 6) 49.8 (20 mg/kg
OS) + LPS
[0094] It is clear from the data given above that compound I-4 is
very active in inhibiting the increase in plasma TNF.alpha. in the
course of endotoxic shock induced by LPS. For example, the oral
dose of 20 mg/kg inhibited the effect of the LPS by about 50%.
[0095] d) Intestinal anti-inflammatory activity.
[0096] TNF.alpha. is a cytokine implicated in the pathogenesis of a
variety of immunology-based inflammatory diseases, amongst which is
inflammation of the intestine. It has been shown [Bertrand et al.
Br. J. Pharmacol. 124, 1385-1394 (1998)] that inducing an
overproduction of TNF.alpha., induced by COX.sub.1-type
antiinflammatory drugs, brings about activation of the neutrophiles
and an increase in the production of nitrites due to the activation
of the tissue iNOS, which together contribute to the
toxic-ulcerative effects on the intestinal mucosa.
[0097] The combined capability of some of the compounds of the
invention to inhibit both iNOS and the synthesis of TNF.alpha. has
led to their activity being checked in an experimental model of
colitis caused by a chemical hapten, trinitrobenzenesulphonic acid
(TNBS), which can bind to the tissue proteins and stimulate
cell-mediated immunity. The intrarectal administration of TNBS in
association with ethanol causes acute inflammation characterised by
extensive ulceration and necrosis.
[0098] A distal colitis was therefore induced in the rat by
intrarectal instillation of TNBS (40 mg/kg) dissolved in 50%
ethanol (0.5 ml/rat). The drugs were administered orally twice a
day on days -2; -1; 0; +1 and the animals were killed 48 hours
after the administration of TNBS.
[0099] The parameters examined were: total weight of the colon (g),
macroscopic tissue damage "score", measurement of the tissue
myeloperoxidase (MPO) activity, which is a marker of the
infiltration of the neutrophiles, and measurement of the iNOS
activity (pmoles/g tissue/min.).
[0100] The macroscopic score (MDS) was taken on about 10 cm of
colon in accordance with the following arbitrary scale:
TABLE-US-00007 0 No damage 1 Hyperaemia (no ulcers) 2 1 small ulcer
or erosion 3 1 ulcer with inflammation 4 Two ulceration sites 5
More than two ulceration sites or 1 ulcer >1 cm 6-10 If the
damage was >2 cm, the score was increased by 1 for each cm
[0101] The tissue MPO measurement was performed according to
Velgara et al., JPET 1994. The measurement of the tissue nitrites
was performed by applying the method described above for the
chondrocytes to the supernatant fluid of the tissue homogenate.
[0102] Compound I-4, administered in doses of 5, 10 and 20 mg/kg,
and compound I-11 were examined, by administering them orally, in
comparison with sulphasalazine (250 mg/kg) and 5-aminosalicylic
acid (5-ASA) (100 mg/kg), 2 drugs which are widely used in the
treatment of ulcerative colitis.
[0103] The results thus obtained are given in the following table.
TABLE-US-00008 TABLE 7 Effects of compound I-4, I-11,
sulphasalazine and 5-ASA on colitis induced by TNBS in rats Weight
of Macroscopic Myeloperoxidase Treatment groups the colon damage
activity iNOS activity [mg/kg; number (n)] (g) (MDS) (UMPO/g/min)
(pmoles/g/min) Control (SHAM) (n = 6) 1.5 .+-. 0.2 -- 0.9 .+-. 0.3
6.6 .+-. 4.4 Control (TNBS) (n = 8) 2.4 .+-. 0.3 7.4 .+-. 1.4 7.7
.+-. 1.1 190.9 .+-. 98.6 I-4 (5 mg/kg; n = 8) 2.2 .+-. 0.2 5.0 .+-.
0.9 5.5 .+-. 1.4 131.7 .+-. 91.9 I-4 (10 mg/kg; n = 8) 2.1 .+-. 0.3
4.4 .+-. 0.8(*) 5.0 .+-. 1.7(*) 98.6 .+-. 60.0 I-4 (20 mg/kg; n =
8) 2.0 .+-. 0.2(*) 3.5 .+-. 0.8(*) 5.1 .+-. 1.2(*) 61.5 .+-.
31.4(*) I-11 (20 mg/kg; n = 7) 2.2 .+-. 0.3 4.3 .+-. 0.9(*) 6.5
.+-. 1.8 90.6 .+-. 45.6(*) 5-ASA (100 mg/kg; n = 7) 2.4 .+-. 0.4
6.9 .+-. 1.1 7.9 .+-. 2.3 167.3 .+-. 106.5 Sulphasalazine (250
mg/kg; n = 7) 2.5 .+-. 0.3 6.0 .+-. 2.9 7.5 .+-. 5.5 100.8 .+-.
86.8 The values given are means .+-. Standard Deviation (*)value
significantly different from the TNBS control group (Duncan's
test)
[0104] It is clear from the data given in the table that compound
I-4 had a strong protective effect in the experimental model of
colitis induced in the rat by TNBS. In fact I-4 reduces both
macroscopic damage and all of the other inflammatory parameters
taken into consideration in a dose-dependent and significant manner
at medium and high dose (10 and 20 mg/kg). In particular, it
reduces the increase in the weight of the colon induced by
treatment with TNBS, reduces macroscopic damage by about 50% at
doses of 10 and 20 mg/kg, and reduces the increase in tissue MPO
activity and iNOS activity induced by TNBS by 40% and 50%,
respectively, at the same doses. In contrast, of the two reference
drugs selected, 5-ASA (100 mg/kg) was inactive on all of the
parameters, and sulphasalazine was slightly active at the very high
dose of 250 mg/kg and in a non-statistically significant manner
solely on macroscopic damage (about 20% effect) and on iNOS
activity (about 50% effect). The other compound of the invention
which was tested (Compound I-11) was also active at the dose of 20
mg/kg although the effects in reducing damage, on the parameters of
increase of the weight of the colon, and MPO activity were less
clear than those of the compound I-4.
[0105] Finally, in an experimental model of colitis in the rat
which imitates as closely as possible a pathological condition
which can be correlated with ulcerative colitis in man [Morris et
al. Gastroenterology 96, 795-803, (1989)] some of the compounds of
the invention have a protective effect much greater, and at lower
doses, than that of sulphasalazine which is a drug widely used in
the treatment of ulcerative colitis and Crohn's disease.
* * * * *