WO2001027141A1 - Inhibitors of factor xa having an arginine or arginine aldehyde mimic - Google Patents
Inhibitors of factor xa having an arginine or arginine aldehyde mimic Download PDFInfo
- Publication number
- WO2001027141A1 WO2001027141A1 PCT/US2000/027615 US0027615W WO0127141A1 WO 2001027141 A1 WO2001027141 A1 WO 2001027141A1 US 0027615 W US0027615 W US 0027615W WO 0127141 A1 WO0127141 A1 WO 0127141A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- carbon atoms
- ring
- group
- substituted
- atoms
- Prior art date
Links
- 0 CCOC(CNC([C@@](CC(CCC1)CN1C(N*)N(C)OC(c1ccccc1)=O)NC(OC(C)(C)C)=O)=O)=O Chemical compound CCOC(CNC([C@@](CC(CCC1)CN1C(N*)N(C)OC(c1ccccc1)=O)NC(OC(C)(C)C)=O)=O)=O 0.000 description 2
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06139—Dipeptides with the first amino acid being heterocyclic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06086—Dipeptides with the first amino acid being basic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Peptidyl aldehydes having an arginine or arginine mimic at P3 which are selective inhibitors of certain serine proteases, including factor Xa, are described. These compounds are useful in prevention and treatment of conditions characterized by abnormal thrombosis in mammals.
Description
NOVEL INHIBITORS OF FACTOR XA HAVING AN ARGININE OR
ARGININE ALDEHYDE MIMIC
TECHNICAL FIELDS
In one aspect, the present invention relates to compounds which are potent inhibitors of Factor Xa. In another aspect, the present invention relates to novel peptide aldehydes, their pharmaceutically acceptable salts, and pharmaceutically acceptable compositions thereof which are useful as potent inhibitors of blood coagulation in vitro and in vivo in mammals. In yet another aspect, the invention relates to methods of using these inhibitors as therapeutic agents for disease states in mammals characterized by abnormal thrombosis.
In a further aspect, the present invention relates to methods of using these inhibitors as in vitro diagnostic agents.
BACKGROUND AND INTRODUCTION TO THE INVENTION
Normal hemostasis is the result of a complex balance between the process, es of clot formation (blood coagulation) and clot dissolution (fibrinolysis). The complex interactions between blood cells, specific plasma proteins and the vascular surface, maintain the fluidity of blood unless injury occurs. Damage to the endothelial barrier lining the vascular wall exposes underlying tissue to these blood components. This in turn triggers a series of biochemical reactions altering the hemostatic balance in favor of blood coagulation which can either result in the desired formation of a hemostatic plug stemming the loss of blood or the undesirable formation of an occlusive intravascular thrombus resulting in reduced or complete lack of blood flow to the affected organ.
The blood coagulation response is the culmination of a series of amplified reactions in which several specific zymogens of serine proteases in plasma are activated by limited proteolysis. Nemerson, Y. and
Nossel, H. L., Ann. Rev. Med., 33: 479 (1982). This series of reactions results in the formation of an insoluble fibrin matrix composed of fibrin and cellular components which is required for the stabilization of the primary hemostatic plug or thrombus. The initiation and propagation of the proteolytic activation reactions occurs through a series of amplified pathways which are localized to membranous surfaces at the site of vascular injury (Mann, K. G., Nesheim, M. E., Church, W. R., Haley,
P. and Krishnaswamy, S. (1990) Blood 76: 1-16. and
Lawson, J.
H., Kalafatis, M., Stram, S., and Mann, K. G.
(1994) J. Biol. Chem. 269: 23357-23366).
Initiation of the blood coagulation response to vascular injury follows the formation of a catalytic complex composed of serine protease factor VIIa and the non-enzymatic co-factor, tissue factor (TF) (Rappaport,
S. I. and Rao, L. V. M. (1992) Arteriosclerosis and
Thrombosis 12: 1112-1121). This response appears to be exclusively regulated by the exposure of subendothelial
TF to trace circulating levels of factor VIIa and its zymogen factor VII, following a focal breakdown in vascular integrity. Autoactivation results in an increase in the number of factor VIIa/TF complexes which are responsible for the formation of the serine protease factor Xa.
It is believed that in addition to the factor VIIa/TF complex, the small amount of factor Xa which is formed primes the coagulation response through the proteolytic modification of factor IX to factor
IXalpha which in turn is converted to the active serine protease factor IXab by the factor VIIa/TF complex (Mann,
K. G., Krishnaswamy, S. and Lawson, J. H. (1992) Sem.
Hematology 29: 213-226.). It is factor IXab in complex with activated factor VIIIa, which appears to be responsible for the production of significant quantities of factor Xa which subsequently catalyzes the penultimate step in the blood coagulation cascade; the formation of the serine protease thrombin.
Factor Xa catalyzes the formation of thrombin following the assembly of the prothrombinase complex which is composed of factor Xa, the non-enzymatic cofactor Va and the substrate prothrombin (factor II) assembled in most cases, on the surface of activated platelets which are adhered at the site of injury (Fuster, V., Badimon, L., Badimon, J. J. and Chesebro,
J. H. (1992) New Engl. J. Med. 326: 310-318). In the arterial vasculature, the resulting amplified"burst"of thrombin generation catalyzed by prothrombinase results locally high levels of this protease which is responsible for the formation of fibrin and the further recruitment of additional platelets as well as the covalent stabilization of the clot through the activation of the transglutaminase zymogen factor XIII.
In addition, the coagulation response is further propagated through the thrombin-mediated proteolylic feedback activation of the non-enzymatic co-factors V and VIII resulting in more prothrombinase formation and subsequent thrombin generation (Hemker, H. C. and
Kessels, H. (1991) Haemostasis 21: 189-196).
Substances which interfere in the process of blood coagulation (anticoagulants) have been demonstrated to be important therapeutic agents in the treatment and prevention of thrombotic disorders (Kessler, C. M. (1991)
Chest 99: 97S-112S and Cairns, J. A., Hirsh, J., Lewis,
H. D., Resnekov, L., and Theroux, P. (1992) Chest 102: 456S-481S). The currently approved clinical anticoagulants have been associated with a number of adverse effects owing to the relatively non-specific nature of their effect on the blood coagulation cascade (Levine, M. N., Hirsh, J., Landefeld, S., and Raskob, G.
(1992) Chest 102: 352S-363S). This has stimulated the search for more effective anticoagulant agents which can more effectively control the activity of the coagulation cascade by selectively interfering with specific reactions in this process which may have a positive effect in reducing the complications of anticoagulant therapy (Weitz, J., and Hirsh, J. (1993) J. Lab. Clin.
Med. 122 : 364-373). In another aspect, this search has focused on normal human proteins which serve as endogenous anticoagulants in controlling the activity of the blood coagulation cascade. In addition, various hematophageous organisms have been investigated because of their ability to effectively anticoagulate the blood meal during and following feeding on their hosts suggesting that they have evolved effective anticoagulant strategies which may be useful as therapeutic agents.
A plasma protein, Lipoprotein-Associated
Coagulation Inhibitor (LACI) or recently termed Tissue
Factor Pathway Inhibitor (TFPI), containing three consecutive Kunitz domains has been reported to inhibit the enzyme activity of factor Xa directly and, in a factor Xa-dependent manner, inhibit the enzyme activity the factor VIIa-tissue factor complex. Salvensen, G., and Pizzo, S. V.,"Proteinase Inhibitors: a
Macroglobulines, Serpins, and Kunis","Hemostasis and
Thrombosis, Third Edition, pp. 251-253, J. B. Lippincott
Company (Edit.'R. W. Colman et al. 1994). A cDNA sequence encoding TFPI has been reported, and the cloned protein was reported to have a molecular weight of 31,950 daltons and contain 276 amino acids. Broze, G. J. and Girad, T. J., U. S. Patent No. 5,106,833, col. 1, (1992).
Various recombinant proteins derived from TFPI have been reported. Girad, T. J. and Broze, G. J., EP 439,442 (1991); Rasmussen, J. S. and Nordfand, O. J., WO 91/02753 (1991); and Broze, G. J. and Girad, T. J., U. S.
Patent No. 5,106,833, col. 1, (1992).
Antistasin, a protein comprised of 119 amino acids and found in the salivary gland of the Mexican leech,
Haementeria officinalis, has been reported to inhibit the enzyme activity of factor Xa. Tuszynski et al.
(1987) J. Biol. Chem, 262: 9718; Nutt, et al. (1988) J.
Biol. Chem, 263: 10162. A 6,000 dalton recombinant protein containing 58 amino acids with a high degree homology to antistasin's amino-terminus amino acids 1 through 58 has been reported to inhibit the enzyme activity of factor Xa. Tung, J. et al., EP 454,372 (1991); Tung, J. et al., U. S. Patent No. 5,189,019 (1993).
Tick Anticoagulant Protein (TAP), a protein comprised of 60 amino acids and isolated from the soft tick, Ornithodoros moubata, has been reported to inhibit the enzyme activity of factor Xa but not factor VIIa.
Waxman, L. et al. (1990) Science, 248: 593. TAP made by recombinant methods has been reported. Vlasuk, G. P. et al., EP 419,099 (1991) and Vlasuk, G. P. et al., U. S.
Patent No 5,239,058 (1993).
The dog hookworm, Ancylostoma caninum, which can also infect humans, has been reported to contain a potent anticoagulant substance. A. caninum was reported to contain a substance which inhibited coagulation of blood in vitro. Loeb, L. and Smith, A. J. (1904) Proc.
Pathol. Soc. Philadelphia, 7 : 173-178. Extracts of A. caninum were reported to prolong prothrombin time and partial thromboplastin time in human plasma with the anticoagulant effect being reported attributable to inhibition of factor Xa but not thrombin. Spellman,
Jr., J. J. and Nossel, H. L. (1971) Am. J. Physiol., 220: 922-927. More recently, soluble protein extracts of
A. caninum were reported to prolong prothrombin time and partial thromboplastin time in human plasma in vitro.
The anticoagulant effect was reported to be attributable to inhibition of human factor Xa but not thrombin.
Cappello, M, et al. (1993) J. Infect. Diseases, 167: 1474-1477. See, also United States Patent No.
5,427,937. Other proteins having anticoagulant and/or serine protease activity have been reported. See, e. g. commonly assigned United States Patent Nos. 5,863,894; 5,864,009; 5,866,542; 5,866,543; 5,872,098; 5,945,275; and 5,955,294.
Other compounds are said to have activity against mammalian factor Xa. See, e. g., United States Patent
Nos. 5,721,214 and 5,919,765. See also, United States
Patent No. 5,696,231.
SUMMARY OF THE INVENTION
The present invention is directed to novel peptide aldehyde compounds having an arginine or arginine mimic at PI and arginine mimic at P3. These compounds are potent inhibitors of Factor Xa in vivo and in vitro.
Certain of these compounds exhibit advantageous selectibity for inhibition of Factor Xa in comparison to inhibition of other serine proteases.
Thus, in one aspect, the present invention is directed to compounds of the formula (I):
EMI8.1
wherein
(a) X is selected from the group consisting of -S (O) 2-,-N (R')-S (O) 2-,- (C=O)-,-OC (=O)-,-NH-C (=O)-, and a direct link, wherein R'is hydrogen, alkyl of 1 to about 4 carbon atoms, aryl of about 6 to about 14 carbon atoms or aralkyl of about 7 to 16 carbon atoms;
(b) Ri is selected from the group consisting of:
(1) alkyl of 1 to about 12 carbon atoms which is optionally substituted with Y1,
(2) alkyl of 1 to about 3 carbon atoms substituted with cycloalkyl of about 5 to about 8 carbon atoms which is optionally mono-, di-i or tri-substituted on the ring with Yl, Yz and/or Y3,
(3) cycloalkyl of 3 to about 15 carbon atoms, which is optionally mono-, di-, or tri-substituted on the ring with Yl, Y2 and/or Y3,
(4) heterocycloalkyl of 4 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen, and S (O) i, wherein i is 0,1 or 2, which is optionally mono-, di-, or tri-substituted on the ring with Yi, Yz and/or Y3,
(5) heterocyclo of 4 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen, and S (O) i, including
EMI9.1
, wherein
EMI9.2
is a 5 to 7 member heterocycle of 3 to 6 ring carbon atoms, where V is-CH2-,-O-,-S (=O)-,-S (O) 2-or-S-, which is optionally mono-, di-, or tri-substituted on the ring carbons with Yl, Y2 and/or Y3,
(6) alkenyl of about 2 to about 6 carbon atoms which is optionally substituted with cycloalkyl of about 5 to about 8 carbon atoms, which is optionally mono-, di-, or tri-substituted on the ring carbons with
Yl, Y2 and/or Y3,
(7) aryl of about 6 to about 14 carbon atoms which is optionally mono-,
di-or tri-substituted with Yl, Y2, and/or Y3,
(8)'heteroaryl of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-, di-, or tri-substituted with Yl, Y2, and/or Y3,
(9) aralkyl of about 7 to about 15 carbon atoms which is optionally substituted on the alkyl chain with.
hydroxy or halogen and mono-, di-, or trisubstituted in the aryl ring with Yl, Y2, and/or Y3,
(10) heteroaralkyl of 5 to 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally substituted on the alkyl chain with hydroxy or halogen and optionally mono-, di-or tri-substituted on the ring with Yl, Y2, and/or Y3,
(11) aralkenyl of about 8 to about 16 carbon atoms which is optionally mono-, di-, or tri-substituted on the aryl ring with Yl, Y2, and/or Y3,
(12) heteroaralkenyl of 5 to 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-,
di-or tri-substituted on the ring with Yl, Y2, and/or Y3,
EMI10.1
EMI10.2
EMI11.1
EMI11.2
(17) fused carbocyclic alkyl of about 5 to about 15 carbon atoms,
(18) difluoromethyl or perfluoroalkyl of 1 to about 12 carbon atoms,
(19) perfluoroaryl of about 6 to about 14 carbon atoms,
(20) perfluoraralkyl of about 7 to about 15 carbon atoms, and
(21) hydrogen when X is a direct link; (c) R2 is
EMI11.3
wherein x is 0 or 1, xl is an integer from 0 to 6 and Ra is hydrogen or alkyl of 1 to about 3 carbon atoms;
(d) T is a divalent radical selected from:
(i) a divalent cycloalkyl group of about 3 to about 8 carbon atoms;
(ii) a divalent aryl group of about 6 to about 14 carbon atoms which is optionally substituted with Y1 ;
(iii) a divalent heteroaryl group of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen and sulfur and which is optionally substituted with Yl ;
(iv) a divalent heterocyclo group of 4 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen and S (O) i wherein i is O, 1 or 2, which is optionally substituted with 1 to 2 substituents independently selected from an oxo group, Yl and Y2;
and
(v) a divalent unsaturated heterocyclo group of about 5 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteratoms are selected from the group consisting of oxygen, nitrogen and S (O) i, wherein i is 0,1, or 2, optionally substituted with Y1 ;
(e) J is-C (=E)-D or-NH-C (=E)-D, wherein D is R6 or NR6R, wherein R6 and R are independently selected from
H, aryl of about 6 to about 10 carbon atoms and lower alkyl of 1 to about 6 carbon atoms, provided that D is not H, and E is O, S or NR6 ;
(f) R3 is selected from the group consisting of
(1) hydrogen;
(2) alkyl of 1 to about 8 carbon atoms optionally substituted with Y4;
(3) alkyl of 1 to about 3 carbon atoms substituted with cycloalkyl of about 5 to about 9 carbon atoms optionally substituted on the ring with Y4;
(4) cycloalkyl of 3 to about 15 carbon atoms, which optionally is substituted on the ring with Y4;
(5) alkenyl of about 3 to about 6 carbon atoms which is optionally substituted with cycloalkyl of about 5 to about 8 carbon atoms, and which optionally is substituted on a ring carbon with Y4;
(6) aryl of about 6 to 14 carbon atoms which is optionally mono or di-substituted with Y1 and/or Y2 ;
(7) aralkyl of about 7 to about 15 carbon atoms which is optionally mono or di-substituted on the aryl ring with Yl and/or Y2 ;
and
(8) heteroaralkyl of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-or di-substituted on the ring with Yl and/or Y2 ;
(g) R4 is selected from the group consisting of hydrogen and alkyl of 1 to about 7 carbon atoms;
(h) alternatively R3 and R4 taken together are -(CH2) q-, where q is 2,3, or 4, to form a cyclic amino acid residue;
(i) Rs is selected from
EMI14.1
wherein d is an integer from 0 to 5 and W is-N-or -CH- ;
and
(j) each Yl, Y2, Y3 and Y4 is
(1) independently selected from the group consisting of halogen, cyano, nitro, tetrazolyl optionally substituted with alkyl of 1 to about 6 carbon atoms, guanidino, amidino, methylamino, methylguanidino, -CF3,-CF2CF3,-CH (CF3) 2,-C (OH) (CF3) 2,-OCF3,-OCF2CF3, -OCF2H, -OC (O) NH2,-OC (O) NHZ,,-OC (O) NZlZ2,-NHC (O) Z1, -NHC (O) NH2,-NHC (O) NHZ1,-NHC (O) NZlZ2,-C (O) OH,-C (O) Zl, -C (O) NH2,-C (O) NHZ1 -C (O) NZlZ2,-P (O) 3H2,-P (O) 3 (Z1) 2, -S (O) 3H,-S (O) mZ,,
-Zl,-OZ,,-OH,-NH2,-NHZ1,-NZlZ2,
N-morpholino, and-S (O) m (CF2) qCF3, wherein m is 0,1 or 2, q is an integer from 0 to 5, and Z1 and Z2 are independently selected from the group consisting of alkyl-of 1 to about 12 carbon atoms, aryl of about 6 to about 14 carbon atoms, heteroaryl of about 5 to about 14 atoms having 1 to about 9 carbon atoms, aralkyl of about 7 to about 15 carbon atoms, and heteroaralkyl of about 5 to about 14 ring atoms having about 3 to about 9 ring carbon atoms, or
(2) Yl and Y2 are selected together to be -O [C (Z3) (Z4)] ru-,
wherein r is an integer from 1 to 4 and
Z3 and Z4 are independently selected from the group consisting of hydrogen, alkyl or 1 to about 12 carbon atoms, aryl of about 6 to about 14 carbon atoms, heteroaryl of about 5 to about 14 ring atoms having 1 to about 9 ring carbon atoms, aralkyl of about 7 to about 15 carbon atoms, and heteroaralkyl of about 5 to about 14 ring atoms having about 3 to about 9 ring carbon atoms, and pharmaceutically acceptable salts thereof with the proviso that when R, X is benzylsulfonyl or Boc, the sum of x and xl is 1, T is unsubstituted phenyl, J is-C (=NH) NH2 or-NHC (=NH) NH2, R3 is H, R4 is H, and R8 is
H,
then Rs is not
EMI15.1
Peptidyl arginine aldehydes have been reported to exist in equilibrium structures in aqueous solutions.
Bajusz, S., et al., J. Med. Chem., 33: 1729 (1990).
These structures, as shown below, include the arginine aldehyde, A, aldehyde hydrate, B, and two amino cyclol forms, C and D. The R group would represent the remainder of a given compound embodied in the present invention. The peptide aldehydes of the present invention include within their definition all the equilibrium forms.
EMI16.1
EMI16.2
EMI16.3
Among other factors, the present invention is based on our finding that the novel compounds of our invention are active as selective inhibitors of Factor Xa. In particular, we have found that certain of the preferred compounds of the present invention exhibit advantageous selectivity in that they are very potent inhibitors of
Factor Xa but are inactive or significantly less active, (several orders of magnitude less) in inhibiting thrombin and plasmin and are significantly less active in inhibiting trypsin. This selectivity for inhibition of Factor Xa gives these compounds a therapeutic advantage in treating or preventing thrombosis in a mammal suspected of having a condition characterized by abnormal thrombosis.
In another aspect, the present invention is directed to pharmaceutical compositions comprising a therapeutically effective amount of a compound of the present invention and a pharmaceutically acceptable carrier.
-In yet another aspect, the present invention is directed to methods of using the compounds and pharmaceutical compositions of the present invention for the prevention of thrombosis in a mammal suspected of having a condition characterized by abnormal thrombosis, comprising administering to said mammal a therapeutically effective amount of a compound of the present invention or pharmaceutical composition. comprising such a compound.
DEFINITIONS
In accordance with the present invention and as used herein, the following terms are defined to have following meanings, unless explicitly stated otherwise:
The term"alkenyl"refers to unsaturated aliphatic groups having at least one double bond.
The term"alkynyl"refers to unsaturated aliphatic groups having at least one triple bond.
The term"alkyl"refers to saturated aliphatic groups including straight-chain, branched-chain and cyclic groups.
The terms'"alkoxy"and"alkoxyl"refer to a group having the formula, R-O-, wherein R is an alkyl group.
The term"alkoxycarbonyl"refers to-C (O) OR wherein R is alkyl.
The term"aralkenyl"refers to an alkenyl group substituted with an aryl group. Preferably the alkenyl group has from 2 to about 6 carbon atoms.
The term"aralkyl"refers to an alkyl group substituted with an aryl group. Suitable aralkyl groups include benzyl, picolyl, and the like, all of which may be optionally substituted. Preferably the alkyl group has from 1 to about 5 carbon atoms.
The term"aryl"refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes a carbocyclic aryl, heterocyclic . aryl and biaryl groups, all of which may be optionally substituted.
The term"aryloxy"refers to a group having the formula, R-O-, wherein R is an aryl group.
The term"aralkoxy"refers to a group having the formula, R-O-, wherein R is an aralkyl group.
The term"amino acid"refers to both natural and unnatural amino acids in their D and L stereoisomers, if their structures allow such stereoisomeric forms, and their analogs. Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val).
Unnatural amino acids include, but are not limited to, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6aminocaproic acid, 2-aminoheptanoic acid, 2 aminoisobutyric acid, 3-aminoisobutyric acid, 2aminopimelic acid, 2,4 diaminoisobutyric acid, demosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, Nethylglycine, N-ethylasparagine, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine, Nmethylisoleucine, N-methylvaline, norvaline, norleucine, ornithine and pipecolic acid.
Amino acid analogs include the natural and unnatural amino acids which are chemically blocked, reversibly or irreversibly, or modified on their N-terminal amino group or their sidechain groups, as for example, methionine sulfoxide, methionine sulfone, S- (carboxymethyl)-cysteine, S (carboxymethyl)-cysteine sulfoxide and S (carboxymethyl)-cysteine sulfone.
The term"amino acid residue"refers to radicals having the structure : (1)-C (O)-R-NH-, wherein R typically is-CH (R')-, wherein R'is H or a carbon containing substituent ; or (2)
EMI19.1
wherein p is 1', 2 or 3 representing the azetidinecarboxylic acid, proline or pipecolic acid residues, respectively.
The term"amino acid analog"refers to an amino acid wherein either the C-terminal carboxy group, the Nterminal amino group or side-chain functional group has been chemically modified to another functional group.
For example, aspartic acid- (beta-methyl ester) is an amino acid analog of aspartic acid; N-ethylglycine is an amino acid analog of glycerine; or alanine carboxamide is an amino acid analog of alanine.
"Arginine mimic side chain"or"side chain of an arginine mimic"refers to a group of atoms which spatially and electronically ressemble or mimic the normal arginine side chain. These groups include the cyclic R5 groups defined in connection with formula (I).
"Biaryl"refers to phenyl substituted by carbocyclic or heterocyclic aryl as defined herein, ortho, meta or para to the point of attachment of the phenyl ring.
"Brine"refers to an aqueous saturated solution of sodium chloride.
"Camphor derivative"refers to the groups:
EMI20.1
"Carbocyclic"refers to a group having one or more rings wherein the ring atoms are all carbon atoms and includes groups having aryl, cycloalkyl, and unsaturated cycloalkyl or a combination of such rings. Such groups include cyclohexyl, cycloheptenyl, tetrahydronaphthyl, phenyl, naphthyl, and the like.
"Carbocyclic aryl"refers to aromatic groups wherein the ring atoms on the aromatic ring are carbon atoms. Carbocyclic aryl groups include monocyclic carbocyclic aryl groups and naphthyl groups, all of which may be optionally substituted. Suitable carbocyclic aryl groups include phenyl and naphthyl.
Suitable substituted carbocyclic aryl groups include indene and phenyl substituted by one to two substituents such being advantageously lower alkyl, hydroxy, lower alkoxy, lower alkoxycarbonyl, halogen, trifluoromethyl, nitro, and cyano. Substituted naphthyl refers to 1-or 2-naphthyl substituted by lower alkyl, lower alkoxy, or halogen.
"Carboxylate mimic"or"carboxylic acid mimic" refers to a group which spatially and electronically mimics a carboxylic acid and provides a net negative charge, i. e., an anion, and also has a pKa value similar to that of a corresponding carboxylic acid, preferably having a pKa of about 4 to 5.
"Cycloalkenyl"or"unsaturated cycloalkyl"refers to a cyclic alkenyl group, that is, a cycloalkyl group modified by having at least one double band. Suitable cycloalkenyl groups include, for example, cyclopentenyl and cyclohexenyl.
"Cycloalkyl"refers to a cyclic alkyl group.
Suitable cycloalkyl groups include, for example, cyclohexyl, cyclopropyl, cyclopentyl, and cycloheptyl.
"Cyclohexylmethyl"refers to a cyclohexyl group attached to CH2.
"Fused carbocyclic"refers to a group having multiple rings which are fused, including multicyclic fused carbocyclic rings having both aromatic and non aromatic rings. Suitable fused carbocyclic rings include fluorenyl, tetralin and the like.
"Fused carbocyclic alkyl"refers to an alkyl group substituted with a fused carbocyclic ring moiety, preferably a multicyclic fused carbocyclic ring having both aromatic and nonaromatic rings. Suitable fused carbocyclic alkyl groups include fluorenyl methyl and the like.
The term"halogen"refers to fluorine, chlorine, bromine and iodine.
"Heterocyclic"refers to a group having 1 or more rings wherein the ring atoms are carbon atoms or heteroatoms, and includes rings that are reduced, saturated, unsaturated and aromatic and, if the group has more than one ring, includes a combination of such rings. Suitabl
Compounds, B. Fundamental Heterocyclic Systems.
Suitable heteroatoms include oxygen, nitrogen, and sulfur. Typical heteroaryl groups include furanyl, thienyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl and the like.
"Heteroaralkenyl"refers to an alkenyl group substituted with a heteroaryl group. Preferably the alkenyl group has from 2 to about 6 carbon atoms.
"Heteroaralkyl"refers to an alkyl group substituted with a heteroaryl group. Preferably the alkyl group has from 1 to about 6 carbon atoms.
"Heterocyclo"refers to a reduced heterocyclic ring system comprised of carbon, nitrogen, oxygen and/or sulfur atoms, and includes such heterocyclic systems described in"Handbook of Chemistry and Physics", 49th edition, 1968, R. C. Weast, editor; The Chemical Rubber
Co., Cleveland, OH. See particularly Section C, Rules for Naming Organic Compounds, B. Fundamental
Heterocyclic Systems.
"Unsaturated heterocyclo"refers to a heterocyclo group which is modified by having at least one double bond, but which is not aromatic.
"Heterocycloalkyl"refers to an alkyl group substituted with a heterocyclo group. Preferably the alkyl group has from 1 to about 6 carbon atoms.
The term"lower"referred to herein in connection with organic radicals or compounds defines such with up to and including 6, preferably up to and including 4 and advantageously one or two carbon atoms. Such groups may be straight chain or branched chain.
"Perfluoroalkyl"refers to an alkyl group which has every hydrogen replaced with fluorine.
"Perfluoroaryl"refers to an aryl group which has every hydrogen replaced with fluorine.
"Perfluoroarylalkyl"or"Perfluoroaralkyl"refers an aralkyl group in which every hydrogen on the aralkyl moiety is replaced with fluorine.
"Pharmaceutically acceptable salt"includes salts of the compounds of the present invention derived from the combination of such compounds and an organic or inorganic acid. In practice the use of the salt form amounts to use of the base form. The compounds of the present invention are useful in both free base and salt form, with both forms being considered as being within the scope of the present invention.
The term"quaternary ammonium salt"refers to compounds produced by reaction between a basic nitrogen in an R substituent and an alkylhalide, arylhalide, and aralkylhalide. Other reactants with good leaving groups may also be used, such as alkyl trifluoromethanesulfonates, alkyl methanesulfonates, and alkyl p-toluenesulfonates. A quaternary ammonium salt has a positively charged nitrogen in the R substituent.
Pharmaceutically acceptable counterions include C1-, Br',
I-, CF3C (O) O-and CH3C (O) 0-. The counterion of choice can be made using ion exchange resin columns. R groups with basic nitrogens include-CH2CH2CH2NHC (=NH) NH2,
EMI25.1
- (CH2) pNH2, wherein p is an integer from 1 to 6. For example, the following R groups contain basic nitrogens: 3- (R)-quinuclidine, 3- (S)-quinuclidine, 3-yl2-ethyl-4 (3H)-quinazolinone, ethyl morpholine, ethyl piperidine, 2- (2-ethyl) pyridine, and 4- (methyl)-5- hydroxy-6-methyl-3-pyridine methanol.
The term"Arg-al"refers to the residue of Largininal which has the formula:
EMI25.2
The term"argininal mimic"refers to an argininal group wherein the arginine side chain is replaced with an arginine mimic side chain.
The term"N-alpha-t-butoxycarbonyl-Ng-nitro-L- arginine"refers to the compound which has the formula:
EMI25.3
The term"terminal carbon"refers to the carbon atom of a straight chain alkyl which is furthest from the parent structure.
In addition, the following abbreviations stand for the following:
-"Bn"refers to benzyl.
"Boc"or"BOC"refers to t-butoxycarbonyl.
"BOP"refers to benzotriazol-1-yl-oxy-tris (dimethylamino)-phosphonium hexafluorophosphate.
"BnS02"or"BzlS02"refers to benzylsulfonyl.
"Cbz"or"CBz"refers to benzyloxycarbonyl.
"DCA"refers to dichloroacetic acid.
"DCC"refers to N, N'-dicyclohexylcarbodiimide.
"DCM"refers to dichloromethane (also called methylene chloride).
"DMF"refers to N, N-dimethylformamide.
"DMSO"refers to dimethyl sulfoxide.
"DMAP"refers to 4-N, N-dimethylamino-pyridine.
"EDAC"or"EDC"refers to 1-ethyl-3- (3- dimethylamino-propyl) carbodiimide hydrochloride salt.
"Et3N"refers to triethylamine.
"EtOH"refers to ethanol.
"HATU"refers to 0- (7-azabenzotriazol-1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate.
"HBTU" refers to 2-(lH-benzotriazol-1-yl)-1, 1, 3,3tetramethyluronium hexafluorophosphate.
"HCl"refers to hydrochloric acid.
"HOAc"refers to acetic acid.
"HOAt"refers to 1-hydroxy-7-azabenzotriazole.
"HOBt"refers to 1-hydroxybenzotriazole monohydrate.
"HPLC"refers to high pressure liquid chromatography.
"i-BuOCOCl"refers to isobutylchloroformate.
."LiAlH4"refers to lithium aluminum hydride.
"LiAlH2 (OEt) 2" refers to lithium aluminum dihydride diethoxide.
"Me"refers to methyl.
"NaOH"refers to sodium hydroxide.
"NMM"refers to N-methylmorpholine.
"2-PrPen"refers to 2-propylpentanoyl.
"TBTU"refers to 2-(lH-benzotriazol-1-yl)-1, 1, 3,3tetramethyluronium tetrafluoroborate.
"TFA"refers to trifluoroacetic acid.
"THF"refers to tetrahydrofuran.
"THF"refers to tetrahydrofuran.
"TLC"refers to thin layer chromatography.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a reaction scheme for the synthesis of compounds of the presentinvention having an argininal at P1. In this figure,"i"through"xii" are defined as: i) EDC, HOBt, NMM, gly-OMetHCl, acetonitrile; ii) H2, PtO2, MeOH, HOAc ; iii) Fmoc-OSu, NaHC03, aqueous dioxane; iv) HC1, dioxane; v) collidine, BnSO2Cl, acetonitrile; vi) piperidine, THF; vii) Bis-Boc
S-Me-isothiourea, 3 days, room temperature; viii) NaOH, aqueous THF;
ix) EDC, HOAt, NMM, cycloArg (N02) OEtcHCl, acetonitrile, DMF; x) 10% Pd/C, H2, HOAc, H2O, EtOH ; xi) HC1, aqueous acetonitrile; and xii) preparative HPLC.
Figure 2 depicts a reaction scheme for the synthesis of compounds of the present invention having an argininal at P1. In this figure,"i"through"x"are defined as; i) H2, PtO2, MeOH, HOAc ; ii) bis-Cbz-S-Me- isothiourea, NMM, THF, reflux, 12 hours; iii) Gly-OEtwHCl, EDC, HOBt, DIEA, acetonitrile; iv) HC1,
EtOH ; v) BnSOzCl, NMM, acetonitrile; vi) LiOH, aqueous EtOH, vii) EDC, HOBt, NMM, cycloArg (NO2) OEt*HCl, acetonitrile, DMF; viii) 10% Pd/C, H2, HOAc, H20, EtOH ;
ix) HC1, aqueous acetonitrile; and x) separation of isomers by preparative HPLC.
Figure 3 depicts a reaction scheme for the synthesis of compounds of the present invention having an argininal at PI. In this figure,"i"through"x"are defined as : i) EDC, HOBt, NMM, Gly-OMetHCl, acetonitrile ; ii) H2, Pt02, EtOH, HOAc ; iii) bisOCbz-S
Me-isothiourea, NMM, THF, 50 C, 4 hours; iv) HC1, EtOAc; v) BnSO2Cl, NMM, CH2Cl2, DMF; vi) LiOH, aqueous EtOH ; vii) EDC, HOBt, NMM, cycloArg (NO2) OEt*HC1, acetonitrile,
DMF;
viii) 10% Pd/C, H2, HOAc, H2O, EtOH ; ix) HC1, aqueous acetonitrile; and x) preparative HPLC.
Figure 4 depicts a reaction scheme for the synthesis of compounds of the present invention having an argininal at PI. In this figure,"i"through"ix" are defined as' : i) EDC, HOBt, NMM, Gly-OMetHCl, DMF; ii)
TFA, CH2C12 ; iii) BnSO2Cl, DIEA, DMF; iv) NaOH, aqueous MeOH ; v) EDC, HOBt, NMM, cycloArg (N02) OEt*HCl, DMF; vi) NH20H, NaOAc, EtOH ; vii) Pd (OH) 2, H2, HOAc, HzO, EtOH ; viii) HC1, aqueous acetonitrile; and ix) preparative
HPLC.
Figure 5 depicts a reaction scheme for the synthesis of compounds of the present invention having
an argininal at P1. In this figure,"i"through"viii"
are defined as: i) EDC, HOBt, NMM, Sar-OBncHCl ; DMF; ii)
HC1, dioxane; iii) BnSOzCl, DIEA, DMF; iv) H2, 10% Pd/C,
CH2Cl2, MeOH ; v) EDC, HOBt, NMM, cyclo-Arg (NO2) OEtcHCl,
DMF; vi) HC1, aqueous acetonitile; and viii) preparative
HPLC.
Figure 6 depicts a reaction scheme for the
synthesis of compounds of the present invention having
an argininal at P1. In this figure,"i"through"xii"
are defined as: i) 2,3,4,6-tetra-O-Piv-beta-Dgalactopyranosylamine, ethyl isocyanoacetate, formic acid, THF, ZnCl2 ; ii) HC1, EtOH ; iii) Boc2O, Et3N,
dioxane; iv) H2, PtO2, EtOH, H2O, HOAc ; v) bis-Cbz-S-Me
isothiourea, NMM, THF; vi) HC1, EtOH ; vii) BnSO2Cl, NMM,
acetonitrile; viii) LiOH, aqueous EtOH ;
ix) EDC, HOBt,
NMM, cycloArg (NO2) OEtcHCl, acetonitrile, DMF; x) 10%
Pd/C, H2, HOAc, H20, EtOH ; xi) HC1, aqueous acetonitrile; xii) preparative HPLC.
Figure 7 depicts a reaction scheme for preparation of an intermediate used for the synthesis of compounds
of the present invention having a 3-piperidinyl- (N- guanidino) alariinal at P1. In this figure,"i"through
"vi"are defined as: i) thionyl chloride, methanol; ii) di-tert-butyl dicarbonate, pH 7 to 8; iii) hydrogen gas, platinum oxide in ethanol, water and acetic acid; iv) bis-benzyloxycarbonyl S-methylisothiourea, base,
tetrahydrofuran; v) calcium chloride, sodium borohydride
in tetrahydrofuran and ethanol; vi) HC1, ethyl acetate.
"*". indicates the position of an asymmetric carbon atom.
See also Examples 54 to 58.
Figure 8 depicts a reaction scheme for preparation of an intermediate used for the synthesis of compounds of the present invention having a 3amidinophenylalaninal at P1. In this figure,"i" through"xi"are defined as i) potassium iodide, dioxane; 2.5 M sodium ethoxide in ethanol, argon atmosphere, reflux 6 hours; yield after work up 60% ; ii) pyridine, triethylamine; HzS (g), stirred at room temperature 16 hours, yield after work up 98% ; iii) acetone, iodomethane, reflux 30 minutes, filtration, methanol; iv) ammonium acetate, reflux 1 hour, filter and dry; v) concentrated HC1, reflux 3 hours, yield after workup 30% ;
vi) dioxane, sodium bicarbonate, di-tbutyl dicarbonate, stir 18 hours at room temperature; vii) 4 C, 4. ON NaOH to pH 12; 4-methoxy-2,3,6trimethylbenzene sulfonyl chloride in dioxane; 1. ON HCl to pH 7 to 8, water, yield after work up 68%, viii) 0,
N-dimethylhydroxylamine hydrochloride, EDC, hydroxybenzotriazole hydrate, 4-methylmorpholine, THF, stir 2 hours, yield after workup 69% ; ix) LiAlH4, THF, -78 C ; aqueous potassium bisulfate, yield after work-up 86% ; x) 4-benzhydrylsemicarbazide trifluoroacetate salt (the compound of Example 65), sodium acetate trihydrate in ethanol, reflux, yield after workup 89% ;
and xi) 50%
TFA/DCM, add to ether, yield after workup 79%. See also
Examples 59 to 66.
Figure 9 depicts a reaction scheme for the preparation of an intermediate which is used in the synthesis of compounds of the present invention having a 4-piperidinyl- (N-guanidino) alaninal at P1. In this figure,"i"through"vi"are defined as i) thionyl chloride, methanol; ii) di-tert-butyl carbonate, pH 7 to 8; iii) hydrogen gas, platinum oxide in ethanol, water and acetic acid; iv) bis-benzyloxycarbonyl Smethylisothiourea, base, tetrahydrofuran; v) calcium chloride, sodium borohydride in tetrahydrofuran and ethanol; and vi) HCl in ethyl acetate. See Example 67.
Figures 10A to 10D depict compounds A1 to A19 which are preferred compounds according to one embodiment of the present invention.
Figures 11A to 11G depict certain preferred compounds of the present invention.
DETAILED DESCRIPTION OF THE INVENTION 1. Preferred Compounds
According to one aspect, the present invention is directed to compounds having the formula (I):
EMI31.1
wherein
- (a) X is selected from the group consisting of -S (O) 2-,-N (R')-S (0) 2-,- (C=O)-,-OC (=O)-,-NH-C (=O)-, and a direct link, wherein R'is hydrogen, alkyl of 1 to about 4 carbon atoms, aryl of about 6 to about 14 carbon atoms or aralkyl of about 7 to 16 carbon atoms;
(b) Ri is selected from the group consisting of:
(1) alkyl of 1 to about 12 carbon atoms which is optionally substituted with Y1,
(2) alkyl of 1 to about 3 carbon atoms substituted with cycloalkyl of about 5 to about 8 carbon atoms which is optionally mono-, di-, or tri-substituted on the ring with Yl, Y2 and/or Y3,
(3) cycloalkyl of 3 to about 15 carbon atoms, which is optionally mono-, di-, or tri-substituted on the ring with Y1, Y2 and/or Y3,
(4) heterocycloalkyl of 4 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen, and S (O) i, wherein i is 0,1 or 2, which is optionally mono-, di-,
or tri-substituted on the ring with Yi, Y2 and/or Y3,
(5) heterocyclo of 4 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen, and S (O) i, including
EMI32.1
wherein
EMI32.2
is a 5 to 7 member heterocycle of 3 to 6 ring carbon atoms, where V is -CH20-, -O-,
S (=0.)-,-S (O) 2- or-S-, which is optionally mono-, di-, or tri-substituted on the ring carbons with Y1, Y2 and/or
(6) alkenyl of about 2 to about 6 carbon atoms which is optionally substituted with cycloalkyl of about 5 to about 8 carbon atoms, which is optionally mono-, di-,
or tri-substituted on the ring carbons with Y1 Y2 and/or Y3,
(7) aryl of about 6 to about 14 carbon atoms which is optionally mono-, di-or tri-substituted with Y1, Y2, and/or Y3,
(8) heteroaryl of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-, di-, or tri-substituted with Y1, Y2, and/or Y3,
(9) aralkyl of about 7 to about 15 carbon atoms which is optionally substituted on the alkyl chain with hydroxy or halogen and mono-, di-, or trisubstituted in the aryl ring with Yl, Y2, and/or Y3,
(10)
heteroaralkyl of 5 to 14 ring atoms with the ring atoms'selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally substituted on the alkyl chain with hydroxy or halogen and optionally mono-, di-or tri-substituted on the ring with Y1, Y2, and/or Y3,
(11) aralkenyl of about 8 to about 16 carbon atoms which is optionally mono-, di-, or tri-substituted on the aryl ring with Y1, Y2, and/or Y3,
(12) heteroaralkenyl of 5 to 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-,
di-or tri-substituted on the ring with Y1, Y2, and/or Y3,
EMI34.1
EMI34.2
EMI34.3
EMI34.4
(17) fused carbocyclic alkyl of about 5 to about 15 carbon atoms,
(18) difluoromethyl or perfluoroalkyl of 1 to about 12 carbon atoms,
(19) perfluoroaryl of about 6 to about 14 carbon atoms,
(20) perfluoraralkyl of about 7 to about 15 carbon atoms, and
(21) hydrogen when X is a direct link;
(c) R2 is
EMI35.1
wherein x is 0 or 1, xl is an integer from 0 to 6 and Ra is hydrogen or alkyl of 1 to about 3 carbon atoms;
(d) T is a divalent radical selected from:
(i) a divalent cycloalkyl group of about 3 to about 8 carbon atoms;
(ii) a divalent aryl group of about 6 to-about 14 carbon atoms which is optionally substituted with Y1 ;
(iii) a divalent heteroaryl group of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen and sulfur and which is optionally substituted with Y1 ;
(iv) a divalent heterocyclo group of 4 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen and-S (O) i wherein i is O, 1 or 2, which is optionally substituted with 1 to 2 substituents independently selected from an oxo group, Y1 and Y2 ;
and
(v) a divalent unsaturated heterocyclo group of about 5 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteratoms are selected from the group consisting of oxygen, nitrogen and S (0) i, wherein i is 0,1, or 2, . optionally substituted with Y1 ;
(e) J is-C (=E)-D or-NH-C (=E)-D, wherein D is R6 or NRgR wherein R6 and R7 are independently selected from
H, aryl of about 6 to about 10 carbon atoms and lower alkyl of 1 to about 6 carbon atoms, provided that D is not H, and E is O, S or NR6 ;
(f) R3 is selected from the group consisting of
(1) hydrogen;
(2) alkyl of 1 to about 8 carbon atoms optionally substituted with Y4;
(3) alkyl of 1 to about 3 carbon atoms substituted with cycloalkyl of about 5 to about 9 carbon atoms optionally substituted on the ring with Y4;
(4)'cycloalkyl of 3 to about 15 carbon atoms, which optionally is substituted on the ring with Y4;
(5) alkenyl of about 3 to about 6 carbon atoms which is optionally substituted with cycloalkyl of about 5 to about 8 carbon atoms, and which optionally is substituted on a ring carbon with Y4;
(6) aryl of about 6 to 14 carbon atoms which is optionally mono or di-substituted with Y1 and/or Y2 ;
(7) aralkyl of about 7 to about 15 carbon atoms which is optionally mono or di-substituted on the aryl ring with Y1 and/or Y2 ;
and
(8) heteroaralkyl of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-or di-substituted on the ring with Y1 and/or Y2 ;
(g) R4 is selected from the group consisting of hydrogen and alkyl of 1 to about 7 carbon atoms;
(h) alternatively R3 and R4 taken together are - (CH2) q-, where q is 2,3, or 4, to form a cyclic amino acid residue;
(i) Rs is selected from
EMI37.1
wherein d is an integer from 0 to 5 and W is-N-or CH- ;
and
(j) each Yl, Y2, Y3 and Y4 is
(1) independently selected from the group consisting of halogen, cyano, nitro, tetrazolyl optionally substituted with alkyl of 1 to about 6 carbon atoms, guanidino, amidino, methylamino, methylguanidino, -CF3,-CF2CF3,-CH (CF3) 2,-C (OH) (CF3) 2,-OCF3,-OCF2CF3, -OCF2H,-OC (O) NH2,-OC (O) NHZ1,-OC (O) NZlZ2,-NHC (O) Z1, -NHC (O) NH2,-NHC (O) NHZ1,-NHC (O) NZ1Z2,-C (O) OH,-C (O) Z1, -C (O) NH2,-C (O) NZ1Z2,
-P (0) 3H2,-P (O) 3 (Z1) 2,-S (O) 3H, -S (O) mZl,-Zi,-OZ1,-OH,-NH2,-NHZ1,-NZ1Z2,
N-morpholino, and-S (O) m (CF2) qCF3, wherein m is 0,1 or 2, q is an integer from 0 to 5, and Z1 and Z2 are independently selected from the group consisting of alkyl of 1 to about 12 carbon atoms, aryl of about 6 to about 14 carbon atoms, heteroaryl of about 5 to about 14 atoms having 1 to about 9 carbon atoms, aralkyl of about 7 to about 15 carbon atoms, and heteroaralkyl of about 5 to about 14 ring atoms having about 3 to about 9 carbon atoms, or
(2) Yl and Y2 are selected together to be -O [C (Z3)
(Z4)] ru-, wherein r is an integer from 1 to 4 and
Z3 and Z4 are independently selected from the group consisting of hydrogen, alkyl or 1 to about 12 carbon atoms, aryl of about 6 to about 14 carbon atoms, heteroaryl of about 5 to about 14 ring atoms having 1 to about 9 carbon atoms, aralkyl of about 7 to about 15 carbon atoms, and heteroaralkyl of about 5 to about 14 ring atoms having about 3 to about 9 carbon atoms, and pharmaceutically acceptable salts thereof with the proviso that when R1X is benzylsulfonyl or Boc, the sum of x and xl is 1, T is unsubstituted phenyl, J is -C (=NH) NH2 or-NHC (=NH) NH2, R3 is H, R4 is H, and R8 is H then Rs is not
EMI39.1
In compounds of formula (I),
the portions termed P1,
P2, P3 and P4 are as follows:
EMI39.2
(IA)
Preferred X groups include-S02-,-NH-S (O) 2-, -OC (=O)- and-N (R')-S (0) 2-. Especially preferred X groups include-SO2-and-OC (=O)-.
For compounds of formula (I), preferred R1 groups include alkyl, cycloalkyl, aralkyl, and aryl groups.
Suitable aralkyl and aryl groups include substituted or unsubstituted benzyl, phenyl and naphthyl. Preferred Ri aryl groups include substituted or unsubstituted phenyl and naphthyl. Preferred substitutions include-C (0)OH, -C (O) OZ1,-S (O) mol, methyl, methoxy, fluoro, chloro, trifluoromethyl, tetrazolyl,-P (O) 3H2,-P (0) 3 (Z1) 2, and -OCF3. Particularly preferred substitutions include -C (O) OH,-C (O) OZ1, -P (O) 3H2,-P (O) 3 (Z1) 2 and tetrazolyl.
. Particularly preferred R1 groups include aralkyl groups. Especially preferred R1 groups include substituted or unsubstituted benzyl and naphthyl groups.
Cyclohexyl and cyclohexylmethyl are other especially preferred R1 groups.
Preferred R2 groups include groups wherein x is 0 or 1.
It is preferred that R2 is selected so as to be in the D-stereoconfiguration in relation to the amide (peptide) backbone.
Preferred T groups include
EMI40.1
Preferred compounds having these T groups include those where X is-S (O) 2-and R1 is benzyl optionally substituted with Y1. According to one aspect of this embodiment, P1 is an argininal mimic; alternatively, Pi is an argininal group. Also according to this embodiment, R1 is substituted benzyl. According to an alternate embodiment of compounds having these T groups,
X is-OC(=0)-andRi is phenethyl optionally substituted with Y1. According to this other aspect, preferably P1 is an argininal mimic; alternatively, P1 is an argininal group. In a further group of preferred compounds having these T groups, x is 1, xl is 0 or 1 and Ra is methyl.
Other preferred T groups include
EMI40.2
Preferred compounds having these T groups include those where the sum of x and xl is 0 to 2. According to one aspect of this embodiment, x is 1, xl is 0 or 1 and R8 is methyl.
According to this aspect, P1 is an argininal mimic; alternatively, P1 is an argininal group.
Alternate preferred T groups include
EMI41.1
Preferred compounds . having these T groups include those where the sum of x and xl is 0 to 2. According to one aspect of this embodiment, x is 1, xl is 0 or 1 and R8 is methyl.
According to this aspect, P1 is an argininal mimic; alternatively, P1 is an argininal group.
When T is a divalent heteroaryl group, preferred T groups include pyrazinyl, pyridazinyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, thiophenyl, and furanyl.
Preferred J groups include-C (=NH)-R6,
-NH-C (=NH)-NH2,-NH-C (=NH)-R6, and-C (=NH)-NR6R,.
When T is
EMI41.2
preferred J groups include-NH-C (=NH)-NH2,-NHC (=NH)-R6, and -C (=NH) NR6R7 ; especially preferred J groups include -C (=NH) NH2 and-NHC (=NH) NH2. When T is
EMI41.3
EMI41.4
preferred J groups include-C (=NH)-R6, -NH-C (=NH)-R6 and-C (=NH) NR6R"especially preferred is
C (=NH) NH2-
-Preferred R3 groups include hydrogen and alkyl groups of 1 to about 7 carbon atoms optionally substituted with carboxylate, a carboxylate mimic or hydroxy on a terminal carbon atom.
Carboxylate mimics include functions which spatially and electronically mimic a carboxylic acid and provide a net negative charge, i. e., an anion, and also has a pKa value similar to that of a carboxylic acid, preferably having a pKa of about 4 to 5. Other preferred R3 groups include hydrogen, methyl, cyclohexylmethyl, optionally substituted phenyl and optionally substituted benzyl.
Preferred substitutions for phenyl and benzyl include -C (O) OH,-C (O) OZ1,-P (0) 3H2,-P (0) 3 (Zl) 2 and tetrazolyl.
Particularly preferred R3 groups are hydrogen, methyl, cyclohexyl and benzyl or phenyl optionally substituted with carboxylic or acidic substituents. Especially preferred are hydrogen and methyl.
Preferred R4 groups include hydrogen and alkyl groups of 1 to about 7 carbon atoms, more preferably 1 to 3 carbon atoms, optionally substituted with hydroxy on a terminal carbon atom. Particularly preferred for R4 is hydrogen.
According to one aspect of the present invention are compounds where R3 and R4 taken together are- (CH2) 3- to give a proline residue at Pz.
Preferred compounds include those where the R4 substituent is in the L-stereochemical configuration in relation to the compound's amide (peptide) backbone.
Preferred Rs groups include
EMI43.1
Preferred compounds include those where the Rs substituent is in the L-stereochemical configuration in relation to the compound's amide (peptide) backbone.
Especially preferred compounds are having P1 in the
L-configuration, P2 in the L-configuration and P3 in the
D-configuration.
According to an alternate preferred embodiment, provided are compounds of formula (I) wherein T is divalent cycloalkyl, azetidyl, pyrrolidinyl or piperidinyl. Preferably x is 0,1 or 2. Preferred compounds of this alternate embodiment include those depicted in Figures 10A to 10D and 11A to 11G.
2. Preparation of Preferred Compounds
Figures 1 to 6 depict synthetic schemes for preparation of certain compounds of the present invention.
Figure 1 depicts synthesis of a compound of the present invention having an argininal at P1 and a 4amidino-cyclohexylalanyl at P3. Examples 1 to 8 describe the synthesis in further detail.
Figure 2 depicts synthesis of a compound of the present invention having an argininal at P1 and a 3- [N- (guanidino) piperidinyl]-alanyl at P3. Examples 9 to 17 describe the synthesis in further detail.
Figure 3 depicts synthesis of a compound of the present invention having an argininal at P1 and a 4- [N- (guanidino)-piperidinyl] alanyl at P3. Examples 24 to 29 describe the synthesis in further detail.
Figure 4 depicts synthesis of a compound of the present invention having an argininal at P1 and a 4 (amidino) phenylalanyl group at P3. Examples 31 to 37 describe the synthesis in further detail.
Figure 5 depicts synthesis of a compound of the present invention having an argininal at P1 and a 4aminophenylalanyl at P3. Examples 38 to 43 describe the synthesis in further detail.
Figure 6 depicts the synthesis of a compound of the present invention having an argininal at P1 and a 4 [N (piperidinyl)] glycyl at P3. Examples 44 to 53 describe the synthesis in further detail.
Figure 7 depicts a synthetic scheme for preparation of an intermediate used in synthesizing compounds of the present invention having an 3-piperidinyl- (N-guanidino)- alaninal group at P1. Examples 54 to 58 describe its preparation in further detail.
Figure 8 depicts a reaction scheme for preparation of an intermediate which is used in the synthesis of compounds of the present invention having a 3amidinophenylalaninal group at P1. Intermediates used in the preparation of compounds of formula (I) having a 4-amidinopherylalaninal group at PI may be prepared according to the reaction scheme depicted in Figure 8 and described in Examples 59 to 66, using the appropriate a-bromo-para-tolunitile starting material.
Figure 9 depicts a reaction scheme for the preparation of an intermediate which is used in the synthesis of compounds of the present invention having a 4-piperidinyl- (N-guanidino) alaninal group at P1. This intermediate is prepared using procedures similar to those described in Examples 54 to 58 and using the appropriate starting materials.
Preferred means of chemically coupling (as for example, amide bond function) include formation of a peptide bond by using conventional coupling reagents known in the art. See Bodanszky, N., Peptide Chemistry, pp. 55-73, Springer-Verlag, New York (1988) and references cited therein. The chemical coupling may be either by means of one-step or two-step coupling. In one-step coupling, the two coupling partners are coupled directly. Preferred coupling reagents for one-step coupling of the include DCC with HOBt, EDC with HOBt,
HBTU or TBTU. In two-step coupling, an activated ester or anhydride of the C-terminal carboxy group of one coupling partner is formed prior to its coupling to the other coupling partner.
For preparation of certain compounds having hydrogenation sensitive substituent groups, it is preferred to avoid the use of hydrogen gas with palladium on carbon. Another preferred method for preparing compounds of the present invention containing
hydrogenation sensitive groups such as alkenyl or aryl
moieties substituted with halogen, cyano, nitro, or
-S-Z1, is to use boron tris (trifluoroacetate),
B (OCOCF3) 3, to cleave the Ng-nitro of the arginine group.
The reagent is prepared by the reaction of BBr3 and CF3COOH. in dichloromethane at 0 C. The reagent is also commercially available. Generally, the Ng-nitro compound
is treated with boron tris (trifluoroacetate) in -trifluoroacetic acid. at 0 C. See, e. g., Fieser, M. and
Fieser, L. F., Reagents for Organic Synthesis, p. 46,
John Wiley & Sons, New York (1974); Pless, J., and
Bauer, W. Angew. Chem., Internat. Ed., 12,147 (1973).
In addition, another preferred reagent for selective nitro group cleavage is titanium trichloride.
This reagent is commercially available. The Ng nitro compound is treated with titanium trichloride in aqueous methanol containing an ammonium acetate buffer followed by exposure of the reaction mixture to air or dimethyl sulfoxide. Freidinger, R. M., Hirschmann, R., and Veber,
D. F., J. Ora. Chem., 43,4800 (1978).
Another preferred method for synthesizing these compounds having an L-argininal moiety is to use the di
N-t-butoxycarbonyl protecting group for the L-argininal moiety for groups incompatible with hydrogenation with palladium on carbon. For example, alpha-Nbenzyloxycarbonyl-omega, omega'-di-N-t- butoxycarbonylarginine is dissolved in acetonitrile and treated with hydroxybenzotriazole and 1-ethyl-3- (3- dimethylamino-propyl) carbodiimide HC1 salt to form alpha-N-benzyloxycarbonyl-omega, omega'-di-N-t- butoxycarbonyl-L-arginine lactam. The lactam is reduced by treatment with LiAlH4 in THF at-70 C to provide alpha-N-benzyloxycarbonyl-omega,omega;-di-N-tbutoxycarbonyl-L-argininal. This aldehyde is protected as the diethyl acetal by treatment with ethanol and HC1.
The N-benzyloxycarbonyl protecting group is removed by treatment with hydrogen gas and palladium on carbon to give omega, omega'-di-N-t-butoxycarbonyl-L-argininal diethyl acetal, HCl salt. This protected L-argininal moiety can then be coupled to a desired carboxylic acid by treatment with N-hydroxybenzotriazole and 1-ethyl-3- (3-dimethylamino-propyl) carbodiimide HCl salt. The diethyl acetal and the di-Boc protecting groups are removed by treatment with hexafluorophosphoric acid in acetonitrile at 0 C. The reaction mixture is quenched by addition of 2.5 M aqueous sodium acetate until pH 4 is reached. The mixture is filtered through a 2 micron filter.
Preparative HPLC using 0.1% CF3COOH in 10-40% aqueous acetonitrile provides the trifluoroacetate salt of the desired substituted L-argininal compound.
3. Selection of Preferred Compounds
The compounds of the present invention are screened for their ability to inhibit some or all of thrombin, factor Xa, plasmin, recombinant tissue plasminogen activator (rt-PA), activated protein C (aPC), chymotrypsin, and trypsin as set forth below. Certain of the preferred compounds are distinguished by their abil. ity to inhibit factor Xa, while not substantially inhibiting some or all of thrombin, plasmin, tissue plasminogen activator (t-PA), activated protein C (aPC), chymotrypsin, and trypsin.
With respect to factor Xa and the other enzymes and as used herein, the term"not substantially inhibiting"means that the IC50 (or Ki) for thrombin, plasmin, t-PA, aPC, chymotrypsin, and trypsin for a given compound is greater than or equal to its ICso (or Ki, respectively) for factor Xa. Preferably the ratio of ICso's for thrombin to ICso for factor Xa will be at least about 25 or greater, more preferably about 100 or greater. It is believed that the ability to selectively inhibit factor Xa will result in therapeutic benefits to patients.
The compounds of the present invention are dissolved in buffer to give solutions containing concentrations such that assay concentrations range from 0 to 100 micromolar. In the assays for factor Xa, thrombin, plasmin, t-PA, aPC, chymotrypsin, and trypsin, a chromogenic synthetic substrate is added to a solution containing test compound and the enzyme of interest, and the residual catalytic activity of that enzyme is determined spectrophotometrically. The ICso of a compound of the present invention is determined from the rate of substrate turnover caused by the specific enzyme being measured. ICso is that concentration of test compound giving 50% inhibition of the rate of substrate turnover.
Likewise, the Ki of a compound of the present invention is determined from the rate of substrate turnover caused by the specific enzyme being measured at various enzyme concentrations. Example A provides an exemple of the in vitro assays used to select the preferred compounds of the present invention.
Certain of the preferred compounds of the present invention have a Ki of about 1 pM to about 200 nM in the factor Xa assay. Especially preferred compounds have a
Ki of about 1 pM to about 50 nM. The more especially preferred compounds have a Ki of about 1 pM to about 10 nM.
Certain of the preferred compounds of the present invention have a ICso for thrombin, plasmin, t-PA, aPC, chymotrypsin, and trypsin which is at least 10 times greater than its ICso for factor Xa. Especially preferred compounds have an ICso for thrombin, plasmin, t-PA, aPC, chymotrypsin, and trypsin which is about 20 to about 100,000 times greater than its ICso for factor
Xa. More especially preferred compounds have an ICso for thrombin, plasmin, t-PA, aPC, chymotrypsin, and trypsin which is about 100 to about 1,000,000 times greater than its IC5o for factor Xa.
In the event that a compound of the present invention has an ICso with respect to thrombin, plasmin, rt-PA, aPC, chymotrypsin, or trypsin which is greater than the highest concentration of compound tested, the ICso is taken to be that highest concentration of compound.
Example A provides a method for identifying and selecting compounds of the present invention that inhibit thrombin, plasmin, t-PA, aPC, chymotrypsin and trypsin to a greater extent than they inhibit factor Xa and, thus, have utility as inhibitors of those proteases.
4. Pharmaceutical Compositions
In another aspect, the present invention encompasses pharmaceutical compositions prepared for storage or administration which comprise a therapeutically effective amount of a compound of the present invention in a pharmaceutically acceptable carrier.
The therapeutically effective amount of a compound of the present invention will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the specific mammal under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy but will depend on such factors as weight, diet, concurrent medication and other factors which'those skilled in the medical arts will recognize.
The therapeutically effective amount of the compound of the present invention can range broadly depending upon the desired affects and the therapeutic indication. Typically, dosages will be between about 0.01 mg/kg and 100 mg/kg body weight, preferably between about 0.01 and 10 mg/kg, body weight.
-Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Reminqton's
Pharmaceutical Sciences, Mack Publishing Co. (A. R.
Gennaro edit. 1985). For example, sterile saline and phosphate-buffered saline at physiological pH may be used. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. Id. at 1449. In addition, antioxidants and suspending agents may be used. Id.
The pharmaceutical compositions of the present invention may be formulated and used as tablets, capsules or elixers for oral administration; suppositories for rectal administration; sterile solutions and suspensions for injectable administration; and the like. The dose and method of administration can be tailored to achieve optimal efficacy but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
When administration is to be parenteral, such as intravenous on a daily basis, injectable pharmaceutical compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, or the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxilliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption enhancing preparations (e. g., liposomes) may be utilized.
5. Utility and Methods
Compounds of the present invention when made and selected as disclosed are useful as potent inhibitors of
Factor Xa in vitro and in vivo. As such, these compounds are useful as in vitro diagnostic reagents to prevent the clotting of blood and as in vivo pharmaceutical agents to prevent, inhibit and/or attenuate thrombosis in mammals suspected of having a condition characterized by abnormal thrombosis.
The compounds of the present invention are useful as in vitro diagnostic reagents for inhibiting clotting in blood drawing tubes. The use of stoppered test tubes having a vaccum therein as a means to draw blood obtained by venipuncture into the tube is well known in the medical arts. Kasten, B. L.,"Specimen Collection",
Laboratory Test Handbook, 2nd Edition, Lexi-Comp Inc.,
Cleveland pp. 16-17 (Edits. Jacobs, D. S. et al. 1990).
Such vacuum tubes may be free of clot-inhibiting additives, in which case, they are useful for the isolation of mammalian serum from the blood. They may alternatively contain clot-inhibiting additives (such as heparin salts, EDTA salts, citrate salts or oxalate salts), in which case, they are useful for the isolation of mammalian plasma from the blood. The compounds of the present invention are potent inhibitors of thrombin, and as such, can be incorporated into blood collection tubes to prevent clotting of the mammalian blood drawn into them.
* The compounds of the present invention are used alone, in combination with other compounds of the present invention, or in combination with other known inhibitors of clotting, in the blood collection tubes.
The amount to be added to such tubes is that amount sufficient to inhibit the formation of a clot when mammalian blood is drawn into the tube. The addition of the compounds to such tubes may be accomplished by methods well known in the art, such as by introduction of a liquid composition thereof, as a solid composition thereof, or liquid composition which is lyophilized to a solid. The compounds of the present invention are added to blood collection tubes in such amounts that, when combined with 2 to 10 mL of mammalian blood, the concentration of such compounds will be sufficient to inhibit clot formation. Typically, the required concentration will be about 1 to 10,000 nM, with 10 to 1000 nM being preferred.
The compounds of the present invention are useful as a pharmaceutical agent for preventing, inhibiting and/or attenuating thrombosis in a mammal suspected of having a condition characterized by abnormal thrombosis.
-Conditions characterized by abnormal thrombosis are well known in the medical arts and include those involving the arterial and venous vasculature of mammals. With respect to the coronary arterial vasculature, abnormal thrombosis (thrombus formation) characterizes the rupture of an established atherosclerotic plaque which is the major cause of acute myocardial infarction and unstable angina, as well as also characterizing the occlusive coronary thrombus formation resulting from either thrombolytic therapy or percutaneous transluminal coronary angioplasty (PTCA).
With respect to the venous vasculature, abnormal thrombosis characterizes the condition observed in patients undergoing major surgery in the lower extremities or the abdominal area who often suffer from thrombus formation in the venous vasculature resulting in reduced blood flow to the affected extremity and a predisposition to pulmonary embolism. Abnormal thrombosis further characterizes disseminated intravascular coagulopathy which commonly occurs within both vascular systems during septic shock, certain viral infections and'cancer, a condition wherein there is rapid consumption of coagulation factors and systemic coagulation which results in the formation of lifethreatening thrombi occurring throughout the microvasculature leading to widespread organ failure.
The present invention includes methods for preventing a condition in a mammal suspected of having a condition characterized by abnormal thrombosis, comprising administering to said mammal a therapeutically effective amount of a compound or a pharmaceutical composition of the present invention.
The compounds or pharmaceutical compositions of the present invention are administered in vivo, ordinarily in a mammal, preferably in a human. In employing them in vivo, the compounds or pharmaceutical compositions can be administered to a mammal in a variety of ways, including orally, parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperitoneally, employing a variety of dosage forms. Administration is preferably parenteral, such as intravenous on a daily basis. Alternatively, administration is preferably oral, such as by tablets capsules or elixers taken on a daily basis.
In practicing the methods of the present invention, the compounds or pharmaceutical compositions of the present invention are administered alone or in combination with one another, or in combination with other therapeutic or in vivo diagnostic agents.
As is apparent to one skilled in the medical art, a "therapeutically effective amount"of the compounds or pharmaceutical compositions of the present invention will vary depending upon the age, weight and mammalian species treated, the particular compounds employed, the particular mode of administration and the desired affects and the therapeutic indication. Because these factors and their relationship to determining this amount are well known in the medical arts, the determination of therapeutically effective dosage levels, the amount necessary to achieve the desired result of preventing thrombosis, will be within the ambit of one skilled in these arts.
Typically, administration of the compounds or pharmaceutical composition of the present invention is commenced at lower dosage levels, with dosage levels being increased until the desired effect of preventing in vivo thrombosis is achieved which would define a therapeutically effective amount. For the compounds of the present invention, alone or as part of a pharmaceutical composition, such doses are between about 0.01 mg/kg and 100 mg/kg body weight, preferably between about 0.01 and 10 mg/kg, body weight.
To assist in understanding, the present invention will now be further illustrated by the following examples. These examples as they relate to this invention should not, of course, be construed as specifically limiting the invention and such variations of the invention, now known or later developed, which wouldbe within the purview of one skilled in the art are considered to fall within the scope of the invention as described herein and hereinafter claimed.
EXAMPLES
Example 1
Preparation of 1-2
EMI57.1
Boc-D-Phe (p-NO2) OH (1-1) (5 g, 16.1 mmol), EDACHCl (3.38 g, 17.7 mmol), and 1-hydroxybenzotriazole (2.39 g, 17.7 mmol) were dissolved with stirring in CH3CN (20 mL) and cooled in an ice-water bath. To this solution was added glycine hydrochloride (2.22 g, 17.7 mmol), then Nmethylmorpholine (4.88 g, 48.3 mmol), and the reaction mixture stirred 18 hours. The reaction mixture was concentrated in vacuo, diluted with ethyl acetate (350 mL), then washed with 1N HC1 (50 mL), saturated NaHC03 (50 mL), and brine (50 mL). The organic fraction was dried over MgSO4 and concentrated in vacuo.
Chromatography (silica, 3% ethanol/CH2Cl2) afforded 5.6 g (91%) of 1-2 as a solid. Rf = 0. 6 (5% ethanol/CH2Cl2).
Example 2
Preparation of 1-3
EMI57.2
-A Parr reaction bottle was charged with the compound of Example 1 (2.8 g, 7.34 mmol), 9/1 methanol/acetic acid (100 mL), and platinum oxide (0.28 g). The apparatus was pressurized to 45 psi H2, and the reaction mixture shaken for 16 hours. The catalyst was removed by filtration, and the filtrate concentrated in vacuo and dried azeotropically with toluene. The resulting solid was precipitated from methanol/hexanes/ diethyl ether to afford 3.2 g (quantitative yield) of 13 as a solid.
Example 3
Preparation of 1-4
EMI58.1
1-4
To the compound of Example 2 (1 ; 25 g, 3 mmol) dissolved in dioxane (6 mL), was added saturated NaHC03 (6 mL) and Fmoc-OSu (1.01 g, 3 mmol). After stirring 16 hours, ethyl acetate (200 mL) was added, and the organic fraction washed with 1N HC1 (50 mL), saturated NaHC03 (50 mL), and brine (50 mL). The organic fraction was dried over MgS04 and concentrated in vacuo. Chromatography (silica, 40/60 ethyl acetate/hexanes) afforded 1.28 g (73.7%) of 1-4 as a white solid. Rf = 0. 6 (40/60 ethyl acetate/hexanes).
Example 4
Preparation of 1-5
EMI59.1
1-5
To the compound of Example 3 (4.3 g, 7.43 mmol) dissolved in CH2Cl2 (8 mL), was added 4M HC1 in dioxane (9.2 mL). The reaction mixture was stirred 2 hours and concentrated in vacuo to a solid. This solid was dissolved with stirring in CH3CN (10 mL) and N, Ndimethylformamide (1.5 mL), and cooled in an ice-water bath. To this solution was added in order, benzylsulfonyl chloride (1.7 g, 8.9 mmol) and 2,4,6collidine (3. 6 g, 29.72 mmol), and the reaction mixture was stirred 18 hours. The reaction mixture was diluted with ethyl acetate (350 mL), and washed with 1N HC1 (50 mL), saturated NaHC03 (50 mL), and brine (50 mL).
The organic fraction was dried over MgS04 and concentrated in vacuo to afford 2.98 g (63%) of 1-5.
Example 5
Preparation of 1-6
EMI60.1
1-6
To a stirred solution of the compound of Example 4 (1.15 g, 2 mmol) dissolved in tetrahydrofuran (12 mL), was added piperidine (1.7 g, 20 mmol). After stirring 1 hour, the reaction mixture was concentrated in vacuo, precipitated with ethyl ether, and the supernatant decanted. The resulting solid was dissolved in tetrahydrofuran (4.5 mL) and N, N-dimethylformamide (0.5 mL). To this solution was added bis-Boc-Smethylisothiourea (0.871 g, 3 mmol) and the reaction mixture was stirred 3 days. The reaction mixture was diluted with ethyl acetate (300 mL), then washed with 1N HC1 (75 mL), saturated NaHC03 (75 mL), and brine (75 mL).
The organic fraction was dried over MgSO4 and concentrated in vacuo. Chromatography (silica, 20% ethyl acetate/CH2Cl2) afforded 0.48 g (41%) of 1-6. = 0.29 (60/40 ethyl acetate/hexanes).
Example 6
Preparation of 1-8
EMI61.1
1-8
The compound of Example 5 (0.44 g, 0.67 mmol) was dissolved with stirring in tetrahydrofuran (4 mL) and cooled in an ice-water bath. To this solution was added 0.979M NaOH (1.36 mL, 1.34 mmol), and the reaction mixture was stirred 20 minutes. The pH was adjusted to 6 with 1N HC1, and the reaction mixture was concentrated from CH3CN to dryness. This salt (1-7), EDAC-HC1 (0.334 g, 1.75 mmol), and HOAt (0.238 g, 1.75 mmol) were dissolved with stirring in CH3CN (1 mL) and N, Ndimethylformamide (0.3 mL), and cooled in an ice-water bath. To this mixture was added HCl-cycloArg (N02) OEt (2.69 g, 1 mmol) and N-methylmorpholine (0.203 g, 2.34 mmol), and the reaction mixture was stirred 2 days.
The reaction mixture was concentrated in vacuo, diluted with ethyl acetate (300 mL), and washed with 1N HCl (50 mL), saturated NaHC03 (50 mL), and brine (50 mL). The organic fraction was dried over MgS04 and concentrated in vacuo.
Chromatography (silica, 4% ethanol/CH2Cl2) </R
Example 7
Preparation of 1-10 and 1-11
EMI62.1
1-10
+ diastereomer 1-11
A Parr reaction bottle was charged with the compound of Example 6, (0.235 g, 0.29 mmol), 20% acetic acid/ethanol (40 mL), and 10% palladium on carbon (120 mg). The apparatus was pressurized to 40 psi H2 and shaken 18 hours. The catalyst was removed by filtration, and the filtrate was concentrated in vacuo and dried azeotropically with toluene. The resulting solid was precipitated from ethanol/diethyl ether/hexanes to afford 0.25 g (quantitative yield) of the denitrosylated compound (1-9). This compound was treated with 0.1% trifluoroacetic acid in H20/CH3CN at 4 C until the mono-deprotection of the di-Boc-guanidine was complete as indicated by HPLC.
The diastereomeric products were separated by preparative HPLC (reverse phase, 115 mL/min, 8-15% CH3CN/H2O, 0.1% TFA, 60 min program). The appropriate fractions were combined and the solvent removed in vacuo. The remaining liquid was frozen and lyophilized to afford 0.06 g of 1-10 as a fluffy, white powder. Mass spectral analysis gave an ion m/e = 707 (calc 706.7).
Example 8
Preparation of 1-12
EMI63.1
1-12
The compound of Example 7 (1-10) was dissolved with stirring in 50% aqueous CH3CN (8 mL) and cooled in an ice-water bath. To this solution was added concentrated HCl (8 mL), and the reaction mixture was allowed to warm to room temperature. After stirring 2 hours, the reaction mixture was cooled in an ice-water bath and neutralized with saturated sodium acetate (50 mL). The product was purified by preparative HPLC (reverse phase, 115 mL/min, 5-12% CH3CN/H20, 0.1% TFA, 60 min program).
The appropriate fractions were combined and the solvent was removed in vacuo. The remaining liquid was frozen and lyophilized to afford 0.0084 g of 1-12 as a fluffy, white powder. Mass spectral analysis gave an ion m/e = 580 (calc 579.7).
Example 9
Preparation of Na-tert-butyloxycarbonyl-d-3- piperidylalanine, 2-1
EMI64.1
2-1
An hydrogenation apparatus was charged with Boc-D- 3-piperidylalanine (5 g, 18.8 mmol) dissolved in methanol (40 mL), water (10 mL), and acetic acid (10 mL), and platinum oxide (500 mg). The apparatus was pressurized to 30 psi H2 and the reaction was run 12 hours. The catalyst was removed by filtration and the solvent removed in vacuo giving a quantitative yield of 2-1 as a clear oil.
Example 10
Preparation of N=-tert-butyloxycarbonyl-d-3-[N- (bis (benzyloxycarbonyl) guanidino) piperidyllalanine, 2-2
EMI65.1
2-2
To a solution of the compound of Example 9 (6.29 g, 19.8 mmol) dissolved in tetrahydrofuran (50 mL), was added 1, 3-bis (benzyloxycarbonyl)-2-methyl-2thiopseudourea (10.62 g, 29.64 mmol), followed by
N-methylmorpholine (2.22 mL, 19.8 mmol). The reaction mixture was refluxed 12 hours, then diluted with ethyl acetate (80 mL) and washed with 1N NaHS04 (2 x 50 mL), saturated NaHCO3 (2 x 50 mL), and brine (2 x 50 mL). The organic fraction was dried over Na2SO4 and concentrated in vacuo to a brown oil.
Flash chromatography [silica, 230-400 mesh, 2.5 x 6 in, 1/1 ethyl acetate/hexane (3 column volumes), 1% acetic acid/ethyl acetate] afforded 4.73 g (41.1%) of 2-2 as a yellowish foam. Rf = 0. 3, 0.39 (1% acetic acid/ethyl acetate), two diastereomers.
Example 11
Preparation of Na-tert-butyloxycarbonyl-D-3- [N- (bis (benzyloxycarbonyl) quanidino) piperidyllalanyl- glycine ethyl ester, 2-3
EMI66.1
2-3
To a solution of the compound of Example 10 (4.70 g, 8.1 mmol) dissolved in CH3CN (32 mL) were added in order, glycine ethyl ester hydrochloride (1.35 mL, 9.7 mmol), 1-hydroxybenzotriazole hydrate (1. 4 g, 8.91 mmol),
EDC-HCl (1.7 g, 8.91 mmol), and diisopropylethylamine (4.23 mL, 24.3 mmol). This solution was stirred at ambient temperature 12 hours, then diluted with ethyl acetate (50 mL). This solution was washed with 1N NaHS04 (2 x 30 mL), saturated NaHC03 (2 x 30 mL), and brine (2 x 30 mL).
The organic fraction was dried over Na2SO4 and concentrated in vacuo. Flash chromatography (silica, 230-400 mesh, 2 x 6 in, ethyl acetate) afforded 2.85 g (53%) of 2-3 as a white foam. Rf = 0.68,0.72 (ethyl acetate), two diastereomers.
Example 12
Preparation of D-3- [N- (bis (benzyloxycarbonyl) quanidino) piperidyllalanyl- glycine ethyl ester, hydrochloride, 2-4
EMI67.1
2-4
The compound of Example 12 (2.8 g, 4.2 mmol) was dissolved in HCl saturated ethanol (30 mL) and stirred 1 hour at ambient temperature. The solution was concentrated in vacuo to afford 2-4 as a beige foam which was used directly for the procedure described in
Example 13 without further purification. Mass spectral analysis confirmed the unit mass (MH+ = 568).
Example 13
Preparation of benzvlsulfonyl-d-3-fN- (bis (benzyloxycarbonyl) quanidino) oiperidyllalanyl- glycine ethyl ester, 2-5
EMI68.1
2-5
The compound of Example 12 (1.01 g, 1.7 mmol) was dissolved in CH3CN (8 mL) and cooled in an ice-water bath. To this solution was added a-toluenesulfonyl chloride (637 mg, 33 mmol) in a single portion, followed by dropwise addition of N-methylmorpholine (407 pL, 3.7 mmol). The reaction mixture was stirred at 0 C for 0.5 hours and then 12 hours at ambient temperature. The solvent was removed in vacuo and the residue dissolved in ethyl acetate (10 mL). This solution was washed with 1N NaHS04 (2 x 5 mL), saturated NaHCO3 (2 x 5 mL), and brine (2 x 5 mL).
The organic fraction was dried over Na2SO4 and concentrated in vacuo. Flash chromatography (silica, 230-400 mesh, 1 x 6 in, 1/9 ethyl acetate/hexane (3 column volumes), 1/1 ethyl acetate/hexane (3 column volumes), ethyl acetate] afforded 313 mg (26%) of 2-5 as a white foam. Rf = 0. 21 (1/1-ethyl acetate/hexane). Mass spectral analysis confirmed the unit mass (MH+ = 721. 9).
Example 14
Preparation of benzylsulfonyl-D-3-N- (bis (benzyloxycarbonyl) quanidino) piperidyllalanyl- glycine, 2-6
EMI69.1
2-6
To a stirred solution of the compound of example 13 (200 mg, 0.28 mmol) in ethanol (6 mL), was added 1N LiOH (3.3 mL). The reaction mixture was stirred 2 hours at ambient temperature and the solvent removed in vacuo.
The residue was diluted with water (10 mL) and extracted with diethyl ether (2 x 5 mL). The aqueous fraction was adjusted to pH 1 with 6N HCl and extracted with ethyl acetate (3 x 5 mL). The combined organic fractions were dried over Na2SO4 and concentrated in vacuo to afford 195 mg (99%) of 2-6 as an oil. Mass spectral analysis confirmed the unit mass (MH+ = 694).
Example 15
Preparation of benzylsulfonyl-d-3-fN- (bis (benzyloxycarbonyl) quanidino) piperidyllalanyl- qlvcyl-arqinine (nitro)-ethylaminal, 2-7
EMI70.1
2-7
To a solution of the compound of Example 14 (250 mg, 0.36 mmol) in N, N-dimethylformamide (2 mL), was added in order,'arginine (nitro)-ethylaminal hydrochloride (96 mg, 0.36 mmol), 1-hydroxybenzotriazole hydrate (58 mg, 0.378 mmol), EDC-HCl (69 mg, 0.36 mmol), and diisopropylethylamine (313 pL, 1.8 mmol). The reaction was stirred 12 hours at ambient temperature and concentrated in vacuo.
The residue was dissolved in ethyl acetate (5 mL) and washed with 1N NaHS04 (2 x 5 mL), saturated NaHC03 (2 x 5 mL), and brine (2 x 5 mL).
Flash chromatography [silica, 230-400 mesh, 1 x 6 in, ethyl acetate (3 column volumes), 9/1 CHzCl2/methanol] afforded 140 mg (43%) of 2-7 as a white foam. Rf = 0. 22 (ethyl acetate). Mass spectral analysis confirmed the unit mass (MH+ = 907, 5).
Example 16
Preparation of benzylsulfonyl-D-3- (N- quanidinopuiperidyl)alanyl-glycyl-arginine-ethylaminal 2-8
EMI71.1
2-8
A hydrogenation apparatus was charged with the compound of Example 15 (140 mg, 0.15 mmol), ethanol (4 mL), water (1 mL), acetic acid (1 mL), and 10% palladium on carbon (15 mg). The apparatus was pressurized to 30 psi H2 and the reaction run 12 hours. The catalyst was removed by filtration and the filtrate concentrated in vacuo affording quantitatively, 2-8 as a clear oil.
Analytical HPLC (4.6 x 250 mm column, C-18 reverse phase, 5 ym particles, 300 A pores, 5-50% CH3CN/H2O, 0. 1%
TFA) gave a single peak (12 minutes). Mass spectral analysis confirmed the half-unit mass [(M + 2)/2 = 297.6].
Example 17A and 17 B
Preparation of benzylsulfonyl-D-3- (N- quanidinopiperidyl) alanyl-qlycyl-argininal (CVS 2763 and 2764), 2-9 and 2-10
EMI72.1
2-9 (17A)
2-10 (17B)
To a solution of compound of Example 16 (140 mg, 0.21 mmol) dissolved in CH3CN (2 mL), was added 6N HC1 (14 mL), and the reaction mixture was stirred for 1 hour.
The reaction mixture was diluted with water (25 mL) and neutralized with solid NH40Ac. The products were purified by preparative HPLC (2.2 x 25 cm column, C-18 reverse phase, 10 ym particles, 300 A pores, 5-25% CH3CNlH2O, 0.1% TFA) affording two diastereomers which were isolated and lyophilized separately (17 mg, 14% overall). Analytical HPLC of the faster eluting diastereomer (CVS 2763, 2-9) gave two peaks (13 minutes, 13.8 minutes). Analytical HPLC of the slower eluting diastereomer (CVS 2764,2-10) gave two peaks (15.8 minutes, 16.1 minutes). Mass spectral analysis of both diastereomers confirmed the half-unit-mass [(M + 2)/2 = 283.8].
Example 18
Preparation of 2- (carbomethoxy) benzvlsulfonyl-D-3- N- (bis (benzyloxycarbonyl) quanidino) piperidyllalanyl- glycine ethyl ester, 2-11
EMI73.1
2-11
The compound of Example 12 (1.6 g, 2.6 mmol) was dissolved in CH3CN (10 mL) and cooled in an ice-water bath. To this solution was added 2 (carbomethoxy) benzylsulfonyl chloride (1.3 g, 5.3 mmol) in a single portion, followed by dropwise addition of Nmethylmorpholine (629 pL, 5.72 mmol). The reaction mixture was stirred for 0.5 hour at 0 C, then 12 hours at ambient temperature. The solvent was removed in vacuo and the residue dissolved in ethyl acetate (20 mL).
This solution was washed with 1N NaHS04 (2 x 10 mL), saturated NaHC03 (2 x 10 mL), and brine (2 x 10 mL). The organic fraction was dried over Na2SO4 and concentrated in vacuo. Flash chromatography [silica, 230-400 mesh, 1.5 x 6 in, 1/1 ethyl acetate/hexane (3 column volumes), ethyl acetate] afforded 330 mg (16%) of a white foam. Rf = 0 : 6 (ethyl acetate). Mass spectral analysis confirmed the unit mass (MH-780).
Example 19
Preparation of 2- (carbomethoxy) benzylsulfonyl-D-3-fN- (bis (benzyloxycarbonyl) cfuanidino) piperidynalanyl- glycine, 2-12
EMI74.1
2-12
To the compound of Example 18 (400 mg, 0.51 mmol) dissolved in methanol (5 mL), was added 1N LiOH (1.02 mL). After stirring 2 hours, the solvent was removed in vacuo, the residue dissolved in water (5 mL), and extracted with diethyl ether (2 x 5 mL). The aqueous fraction was adjusted to pH 1 with 6N HCl and extracted with ethyl acetate (3 x 5 mL). The organic fraction was dried over Na2SO4 and concentrated in vacuo to afford 270 mg (71%) of 2-12 as a white foam. Mass spectral analysis confirmed the unit mass (MH+ = 752).
Example 20
Preparation of 2- (carbomethoxy) benzvlsulfonyl-D-3-fN- (bis (benzyloxycarbonyl) guanidino) piperidyllalanyl- glycyl-arqinine (nitro)-ethylaminal, 2-13
EMI75.1
2-13
To a solution of the compound of Example 19 (270 mg, 0.36 mmol) in N, N-dimethylformamide (2 mL), were added in order,. arginine (nitro)-ethylaminal hydrochloride (191 mg, 0.72 mmol), 1hydroxybenzotriazole hydrate (58 mg 0.38 mmol), EDC#HCl (69 mg, 0.36 mmol)., and diisopropylethylamine (215 L, 1.44 mmol). The resulting solution was stirred 12 hours'. The solvent was removed in vacuo and the residue dissolved in ethyl acetate (5 mL).
This solution was washed with 1N NaHS04 (2 x 5 mL), saturated NaHCO3 (2 x 5 mL), and brine (2 x 5 mL). The organic fraction was. dried over Na2SO4 and concentrated in vacuo to give 306 mg (88%) of 2-13 as a white foam. Rf = 0.23 (9/1 CH2C12/methanol). Analytical HPLC (4.6 x 250 mm column,
C-18 reverse phase, 5 Um particles, 300 A pores, 5-75% CH3CN/H20, 0.1% TFA) gave a major peak (14.5 minutes).
Mass spectral analysis confirmed the unit mass (MHE = 965.5).
Example 21
Preparation of 2-(carbomethoxy) benzylsulfonyl-D-3-EN- (quanidino) piperidyllalanyl-qlvcvl-arginine-ethylamin 2-14
EMI76.1
2-14
A hydrogenation apparatus was charged with compound of Example 20 (300 mg, 0.31 mmol), ethanol (18 mL), water (2 mL), acetic acid (2 mL), and 10% palladium on carbon (50 mg). The apparatus was pressurized to 30 psi
H2 and the reaction run 12 hours. The catalyst was removed by filtration and the filtrate concentrated in vacuo to afford 2-14 as a clear oil in quantitative yield. Analytical HPLC (4.6 x 250 mm column, C-18 reverse phase, 5 Um particles, 300 A pores, 5-75% CH3CN/H20, 0.1% TFA) gave two peaks corresponding to two diastereomers (17.3 minutes, 17.5 minutes).
Mass spectral analysis confirmed the half-unit mass [ (M + 2)/2 = 326. 5].
Example 22A and 22B
Preparation of 2- (carbomethoxy) benzvlsulfonyl-D-3-fN- (guanidino) piperidyllalanyl-qlycvl-arqininal (A6 & A7), 2-15 and 2-16
EMI77.1
2-15 (22A)
2-16 (22B)
To a solution of the compound of Example 21 (118 mg, 0.15 mmol) dissolved in CH3CN (1 mL), was added 6N HCl (10 mL). The reaction mixture was stirred 1.5 hours-, diluted with water (25 mL), and neutralized with solid NH40Ac. The product was purified by preparative
HPLC (2.2 x 25 cm column, C-18 reverse phase, 10 pm particles, 300 A pores, 5-25% CH3CN/H20, 0.1% TFA) affording two diastereomers which were isolated and lyophilized separately (22.5 mg, 24% overall).
Analytical HPLC of the faster eluting diastereomer (CVS 2792,2-15 (22A)) gave three peaks (15.74 minutes, 17.28 minutes, 17.91 minutes). Analytical HPLC of the slower eluting diastereomer (CVS 2793,2-16 (22B)) gave three peaks (16.04 minutes, 17.58 minutes, 18.26 minutes)
Mass spectral analyses of both diastereomers confirmed the half-unit mass [(M + 2)/2 = 312. 5].
Example 23A and 23B
Preparation of 2-(carboxy) benzylsulfonyl-D-3-[N- (uanidino) piperidyllalanyl-glycyl-argininal (CVS A8 & A9), 2-17 and 2-18
EMI78.1
2-17 (23A)
2-18 (23B)
To the compound of Example 21 (110 mg, 0.14 mmol) dissolved in CH3CN (0.3 mL), was added 1N LiOH (0.42 mL).
The reaction mixture was stirred 96 hours. The reaction mixture was concentrated in vacuo and used for the next step without further purification. Analytical HPLC (4.6 x 250 mm column, C-18 reverse phase, 5 Hm particles, 300
A pores, 5-75% CH3CN/H2O, 0. 1% TFA) gave two peaks corresponding to two diastereomers (9 minutes, 9.1 minutes).
This residue was suspended in CH3CN (2 mL) and treated with 6N HC1 (4 mL) for 2 hours. The volume was brought to 20 mL with water, and the reaction mixture was neutralized with solid NH40Ac. The product was purified by preparative HPLC (2.2 x 25 cm column, C-18 reverse phase, 10 ym particles, 300 A pores, 0-20%
CH3CN/H20, 0.1% TFA), affording two diastereomers which were isolated and lyophilized separately (23 mg, 28% overall). Analytical HPLC (5-20% CH3CN/H2O) of the faster eluting diastereomer (CVS 2836,2-17 (23A)) gave . three peaks (14.57 minutes, 16.36 minutes, 17.09 minutes).
Analytical HPLC of the slower eluting diastereomer (CVS 2837,2-18 (23B)) gave three peaks
(14. 94 minutes, 16.64 minutes, 17.44 minutes) Mass spectral analyses of both diastereomers confirmed the half-unit mass [ (M + 2)/2 = 306].
Example 24
Preparation of N-tert-butyloxycarbonyl-D-4- p, vridylalanyl-clycine methyl ester, 3-2
EMI79.1
3-2
To a solution of Boc-D-4-pyridylalanine (25 g, 93.9 mmol) 3-1 in CH3CN (400 mL), were added in order, glycine methyl ester hydrochloride (14.2 g, 113 mmol), EDC-HC1 (27 g, 141 mmol), 1-hydroxybenzotriazole hydrate (19 g, 14l-mmol) and N-methylmorpholine (26 mL, 235 mmol). The solution was stirred at ambient temperature for 29 hours, then concentrated in vacuo. The residue was diluted in ethyl acetate and washed twice each with saturated NaHC03 and brine. The organic fraction was dried over MgSO4 and concentrated in vacuo affording 3-2 as a foam.
Rf = 0.20 (5% methanol/CH2Cl2).
Example 25
Preparation of N-tert-butyloxycarbonyl-D-4- piperidylalanyl-cflycine methyl ester, 3-3
EMI80.1
3-3
A hydrogenation apparatus was charged with the compound of Example 24 (15.83 g, 46.94 mmol) dissolved in 4/1/1 ethanol/acetic acid/water (70 mL), and platinum oxide (1.6 g). The apparatus was pressurized to 30 to 42 psi H2 and the reaction run 34 hours. Celite was added to the reaction mixture and the mixture filtered through a pad of Celite. The hydrogenation of the compound of Example 24 was repeated on an identical scale and the combined filtrates concentrated in vacuo.
The residue was dissolved in toluene, and the solvent removed in vacuo to afford 40.5 g of 3-3 in quantitative yield. Rf = 0.19 (10% i-propanol/CH2Cl2).
Example 26
Preparation of N-tert-butyloxycarbonyl-D-4-[N- [bis (benzyloxycarbonyl) guanidinolpiperidyllalanyl- glycine methyl ester, 3-4
EMI81.1
3-4
To a solution of the compound of Example 25 (20.25 g, 46.9 mmol) dissolved in tetrahydrofuran (120 mL), were added in order, 1, 3-bis (benzyloxycarbonyl)-2methyl-2-thiopseudourea (25.23 g, 70.41 mmol) and Nmethylmorpholine (5.2 mL, 47 mmol). The reaction mixture was heated 4 hours in a 50 C oil bath, then concentrated in vacuo. The residue was diluted with ethyl acetate (1 L), and washed with 1N HC1 (500 mL) and brine (500 mL).
The aqueous fraction was back-extracted with ethyl acetate (500 mL), and the combined organic fractions dried over MgS04 and concentrated in vacuo to a yellow oil. Flash chromatography (silica, 230-400 mesh, 1/1 ethyl acetate/hexanes, 2/1 ethyl acetate/hexanes, loaded as a CH2Cl2 solution) afforded 17.0 g (55%) of 3-4 as a white foam which collapsed into an oil and crystallized upon standing. Rf = 0. 08 (50% ethyl acetate/hexanes).
Example 27
Preparation of D-4-fN- (bis (benzvloxycarbonvl) quanidino) piperidyllalanyl- glycine methyl ester hydrochloride, 3-5
EMI82.1
3-5
The compound of Example 26 (29.13 g, 44.6 mmol) was dissolved in ethyl acetate (400 mL) and cooled in an ice-water bath. To this solution was added 5MHC1 in ethyl acetate (380 mL). The reaction mixture was stirred 0.5 hour at 0 C, then allowed to warm to ambient temperature over 3 hours. The reaction mixture was poured into diethyl ether (12 L) whereupon a white precipitate formed. The precipitate was collected by filtration under a nitrogen atmosphere and dried in. vacuo to 20.14 g of 3-5. Additional material precipitated from the filtrate and oiled.
The oil was collected, dissolved in methanol, and concentrated in vacuo to an additional 8.0 g of 3-5 as a pale green foam. The combined yield was quantitative. Rf = 0. 55 (1/3/27 conc ammonia/methanol/CH2C12).
Examples 28
Preparation of benzylsulfonyl-D-4-[N- (bis (benzyloxycarbonyl) quanidino) piperidyllalanyl- glycine methyl ester, 3-6
EMI83.1
3-6
The compound of Example 27 (6.29 g, 10.67 mmol) was dissolved in CH2Cl2 (50 mL) and N, N-dimethylformamide (5 mL), and cooled in an ice-water bath. To this solution was added in order, a-toluenesulfonyl chloride (4.07 g, 21.3 mmol) in a single portion and N-methylmorpholine (8.75 mL, 79.5 mmol) in 1 mL portions. The reaction mixture was stirred at 0 C for 5 minutes and ambient temperature for 21 hours. Additional a-toluenesulfonyl chloride (2.0 g, 11 mmol) was added in a single portion followed by N-methylmorpholine (2.0 mL, 18 mmol).
The solvent was removed in vacuo and the residue dissolved in ethyl acetate (400 mL). This solution was washed with 1N HC1 (100 mL), saturated NaHC03 (100 mL), and brine (100 mL). The HC1 wash was back-extracted with ethyl acetate (100 mL), and this organic fraction used to back-extract the NaHC03 and brine washes. The combined organic fractions were dried over MgSO4 and concentrated in vacuo. Flash chromatography (silica, 230-400 mesh, 60% ethyl acetate/hexanes) afforded 6.7 g
(85%) o. 3-6 as a white foam.
Rf = 0.75 (1/3/27 conc ammoniatmethanol/cH2cl2)-
Example 29
Preparation of benzylsulfonyl-D-4- (N- quanidinopiperidyl) alanyl-qlycyl-L-arqininal, 3-7
EMI84.1
3-7
Following the four-step protocol outlined in
Examples 14 to 17,3-7 was prepared from the compound of
Example 28, 3-6.
Examples 30A to 30D
General Procedure for the Preparation of the Compounds of the Present Invention
Following the six-step protocol outlined in
Examples 18 to 23, the intermediate of Example 27,3-5, was used to synthesize the following compounds of the present invention:
EMI85.1
2- (carbomethoxy) benzylsulfonyl-D-4- (N guanidinopiperidinyl) alanyl-glycyl-L-argininal (30A) ;
EMI85.2
2-carboxybenzylsulfonyl-D-4- (N- guanidinopiperidinyl) alanyl-glycyl-L-argininal (30B) ;
EMI85.3
3- (carbomethoxy) benzylsulfonyl-D-4- (N guanidinopiperidinyl) alanyl-glycyl-L-argininal (30C);
and
EMI86.1
3-carboxybenzylsulfonyl-D-4- (N- guanidinopiperidinyl) alanyl-glycyl-L-argininal (30D),
Example 31
Preparation of 4-2
EMI86.2
4-2
To a stirred solution of Boc-D-p-cyanophenylalanine (5 g, 17.28 mmol) 4-1 and glycine methyl ester hydrochloride (2.59 g, 20.6 mmol) in N, Ndimethylformamide (90 mL), were added in order, EDAC-HC1 (3.96 g, 20.6 mmol), 1-hydroxybenzotriazole (2.8 g, 20.6 mmol), and N-methylmorpholine (9.4 mL, 86 mmol). The reaction mixture was stirred 3 days and then diluted with ethyl acetate (700 mL). This solution was washed with water (200 mL), 1N HC1 (200 mL), saturated NaHCO3 (200 mL), and brine (200 mL).
The organic fraction was dried over MgS04 and concentrated in vacuo to an oil. To a stirred solution of this oil in CH2C12 (50 mL), was added trifluoroacetic acid (50 mL). After stirring 16 hours, the reaction mixture was concentrated in vacuo and redissolved in CH2Cl2 (25 mL). Addition of diethyl ether (500 mL) precipitated the product which was filtered and dried in vacuo to afford 5.39 g (81%) of 42 as a white foam.
Example 32
Preparation of 4-3
EMI87.1
4-3
To a stirred solution of the compound of Example 31 (5.39 g, 13.9 mmol) dissolved in N, N-dimethylformamide (35 mL), were added in order, benzylsulfonyl chloride (2.7 g, 13. 9 mmol) and diisopropylethylamine (12 mL, 69.5'mmol). The reaction mixture was stirred 16 hours and diluted with ethyl acetate (700 mL). This solution was washed with water (100 mL), 1N HC1 (100 mL), saturated NaHC03 (100 mL), and brine (100 mL). The organic fraction was dried over MgS04 and concentrated in vacuo to an solid which was redissolved in ethyl acetate and precipitated with diethyl ether. The precipitate was-collected by filtration and dried in vacuo to afford 5.45 g (73%) of 4-3 as a white foam.
Example 33
Preparation of 4-4
EMI88.1
4-4
To a stirred solution of the compound of Example 32 (3 g, 7.2 mmol) dissolved in methanol (200 mL), was added 1N NaOH (20 mL, 20 mmol). After stirring 2 hours, the solution was adjusted to pH 7 with 1N HC1 and the mixture concentrated to dryness. The resulting oil was dissolved in saturated NaHC03 (250 mL) and the solution washed with ethyl acetate (300 mL). The aqueous fraction was acidified with HC1 and extracted with ethyl acetate (2 x 300 mL).
The combined organic fractions were washed with brine (200 mL), dried over MgS04, and concentrated in vacuo to afford 1.88 g (65%) of 4-4 as a white foam which was used for the next reaction without further purification.,
Example 34
Preparation of 4-5
EMI89.1
4-5
To a stirred solution of the compound of Example 33 (1.88 g, 4.6 mmol) and HC1-cycloArg (NO2) OEt (1.5 g, 5.52 mmol) in N, N-dimethylformamide (12 mL), were added in order, EDAC-HC1 (1 g, 5.52 mmol), 1-hydroxybenzotriazole (0.8 g, 5.52 mmol), and N-methylmorpholine (2.5 mL, 23 mmol). The reaction mixture was stirred 2 days and diluted with ethyl acetate (500 mL). This solution was was
Example 35
Preparation of 4-6
EMI90.1
4-6
To a stirred solution of the compound of Example 34 (2.17 g, 3.5 mmol) dissolved in ethanol (20 mL), were added hydroxylamine hydrochloride (0.36 g, 5.25 mmol) and sodium acetate (0.34 g, 4.2 mmol). The reaction mixture was heated to 80 C for 16 hours, and was 84% complete as indicated by HPLC (reverse phase, 1 mL/min, 10-70% CH3CN/H2O, 0.1% TFA, 15 minute program, 8.77 minute retention time).
The reaction mixture was concentrated and the products purified by preparative
HPLC (reverse phase, 115 mL/min, 7 to 25% CH3CN/H2O, 0.1% TFA,-120 minute program) to afford two isomeric products, 0.9 g (41%) and 0.39 g (18%).
Examples 36
Preparation of 4-7
EMI91.1
4-7
To a stirred solution of the compound of Example 35 (0.39 g, 0.6 mmol) dissolved in 4/1 ethanol/acetic acid (150 mL), was added 20% Pearlman's catalyst (0.5 g).
The reaction mixture was placed under H2 (1 atm) for 3 days, whereupon no starting material was detected by
HPLC (reverse phase, 1 mL/min, 10 to 70% CH3CN/H2O, 0. 1%
TFA, 15 minute program, 7.2 minute retention time). The catalyst was removed by filtration and the filtrate concentrated in vacuo to afford 0.375 g (quant) of 4-7 as an off-white foam which was used for the next reaction as described in Example 37 without further purification.
Example 37
Preparation of 4-8
EMI92.1
4-8
The compound of Example 36 (0.375 g) was dissolved with stirring in 50% aqueous CH3CN (20 mL) and cooled in an ice-water bath. To this solution was added concentrated HC1 (20 mL) and the reaction mixture was stirred 2 hours. The pH of the reaction mixture was adjusted to 5 with saturated sodium acetate and the product purified by preparative HPLC (reverse phase, 25 mL/min, 3-7% CH3CN/H20, 0.1% TFA, 30 minute program).
Appropriate fractions were combined and concentrated in vacuo, and the remaining liquid was frozen and lyophilized to afford 4-8 as a fluffy white powder.
Mass-spectral,. analysis gave an ion at m/e = 280 [(M +
H)/2 (calc 558.66)].
Example 38
Preparation of 5-2
EMI93.1
5-2
To a stirred solution of Boc-D-p-nitrophenylalanine (4 g, 12.9 mmol) 5-1 and sarcosine benzyl ester ptoluenesulfonate (5.4 g, 15.5 mmol) in N, Ndimethylformamide (30 mL), were added in order, EDACHC1 (3.7 g, 19.3 mmol), 1-hydroxybenzotriazole (2.6 g, 19.3 mmol), and N-methylmorpholine (6.5 mL, 64.6 mmol). The reaction mixture was stirred 16 hours and diluted with ethyl acetate (600 mL). This solution was washed with water (75 mL), 1N HC1 (75 mL), saturated NaHC03 (75 mL), and brine (75 mL). The organic fraction was dried over MgSO4 and concentrated in vacuo to an oil.
To this oil dissolved in CH2C12 (100 mL), was added 4N HC1 in dioxane (15 mL, 60 mmol). After stirring 2 hours, diethyl ether (1 L) was added to precipitate the product which was filtered and dried to afford 5 g (94%) of 5-2 as a white powder which was used for the next reaction described in
Example 39 without further purification.
Example 39
Preparation of 5-3
EMI94.1
5-3
To a stirred solution of the compound of Example 38 (2 g, 12.2 mmol) in N, N-dimethylformamide (30 mL), was added in order, benzylsulfonyl chloride (2.3 g, 12.2 mmol) and diisopropylethylamine (10.6 mL, 61 mmol). The reaction mixture was stirred 3 days and then diluted with ethyl acetate (600 mL). This solution was washed with water (100 mL), 1N HC1 (100 mL), water (100 mL), saturated NaHC03 (100 mL), and brine (100 mL). The organic fraction was dried over MgS04 and concentrated in vacuo to afford 5.78 g (89%) of 5-3 as an off-white foam. Rf = 0.86 (10% methanol/CH2Cl2).
Example 40
Preparation of 5-4
EMI94.2
A mixture of the compound of Example 39 (5.78 g, 10.9 mmol) and 10% palladium on carbon (2 g) was moistened with CHzClz (10 mL) and then suspended in methanol (250 mL). This mixture was placed under H2 (1 atm) and stirred 16 hours. The catalyst was removed by filtration and concentrated in vacuo to a yellow oil which was purified by preparative HPLC (reverse phase, 115 mL/min, 7 to 17% CH3CN/H20, 0.1% TFA, 70 minute program). Appropriate fractions were combined and concentrated in vacuo. The remaining liquid was frozen and lyophilized to afford 2 g (45%) of 5-4 as a fluffy, white powder.
Example 41
Preparation of 5-5
EMI95.1
5-5
To a stirred solution of the compound of Example 40 (2.04 g, 5.03 mmol) and HC1-cycloArg (NO2) OEt (1.48 g, 5.5 mmol) dissolved in N, N-dimethylformamide (25 mL), were added in order, EDACHCl (1.15 g, 6 mmol). 1hydroxybenzotriazole (0.75 g, 6 mmol), and Nmethylmorpholine (3 mL, 15 mmol). The reaction mixture was-stirred 16 hours and then diluted with ethyl acetate (800 mL). This solution was washed with water (100 mL), saturated NaHC03 (100 mL), and brine (100 mL). The organic fraction was dried over MgSO4 and concentrated in vacuo. Chromatography (silica, 5% methanol/CH2Cl2) afforded 1.13 g (30%) of 5-5.
Rf = 0.33 (10% methanol/CH2Clz).
Example 42
Preparation of 5-6
EMI96.1
5-6
To a stirred solution of the compound of Example 41 (1 g, 1.6 mmol) dissolved in 1/4 acetic acid/ethanol (150 mL), was added 20% palladium hydroxide on carbon (1 g). The mixture was placed under H2 (1 atm) and stirred 16 hours. The catalyst was removed by filtration and the filtrate concentrated in vacuo to an oil which was purified by preparative HPLC (reverse phase, 115 mL/min, 10-25% CH3CN/H20,0.1% TFA, 70 minute program).
Appropriate fractions were combined and the solvent removed in vacuo. The remaining liquid was frozen and lyophilized to afford 0.46 g (50%) of 5-6 as a fluffy white powder.
Example 43
Preparation of 5-7
EMI97.1
5-7
The compound of Example 42 (0.46 g, 0.8 mmol) was dissolved with stirring in 50% aqueous CH3CN (10 mL) and then cooled in an ice-water bath. To this solution was added concentrated HC1 (10 mL) and the reaction mixture was stirred 2 hours. Additional concentrated HC1 (5 mL) was added and the reaction mixture was stirred 1 hour.
The reaction mixture was adjusted to pH 5 with saturated sodium acetate and the reaction mixture was purified by preparative HPLC (reverse phase, 115 mL/min, 5-15% CH3CN/H2O, 0.1% TFA, 70 minute program). Appropriate fractions were combined and the solvent removed in vacuo. The remaining liquid was frozen and lyophilized to afford 5-7 as a fluffy white powder. Mass spectral analysis gave an ion m/e = 546 (calc 545.66).
Example 44
Preparation of Na-formyl-Na-2, 3, 4, 6-tetra-O-pivalovl--d- galactopvranosyl-d-4-pvridylqlycyl-qlycine ethyl ester 6-1
EMI98.1
6-1
A solution 2,3,4,6-tetra-O-pivaloyl-ss-Dgalactopyranosylamine (1.6 g, 3 mmol), ethyl isocyanoacetate (344 pL, 3.15 mmol), 4pyridinecarboxaldehyde (286 pL, 3 mmol), and formic acid (124 L, 3.3 mmol) dissolved in anhydrous tetrahydrofuran (12 mL) was cooled in a-30 C (dry ice/CH3CN) bath. To this solution was added a 1M solution of zinc chloride in diethyl ether (6 mL, 6 mmol). The brownish solution was stirred at-30 C for 1 hour, then allowed to slowly warm in a 4 C cold room, where the reaction mixture stirred an additional 12 hours.
The reaction mixture was diluted with ethyl acetate, and washed with 1N NaHS04 (20 mL), saturated
NaHCO3 (20 mL), and brine (20 mL). The organic fraction was dried over Na2S04 and concentrated in vacuo to afford 2.22 g (96%) of 6-1 as a yellowish foam. Rf = 0.53
(ethyl acetate). Analytical HPLC (4.6 x 250 mm column,
C-18 reverse phase, 5 Am particles, 300 A pores, 5-75% CH3CN/H20, 0.1% TFA) gave a two peaks corresponding to two diastereomers (20.2 minutes, 19%; 20.4 minutes, 81%).
Example 45
Preparation of D-4-pyridvlqlycyl-qlycine ethyl ester hydrochloride 6-2
EMI99.1
6-2
The compound of Example 44 (1 g, 1.3 mmol) was treated with HCl saturated ethanol (10 mL) for 8 hours.
To the reaction mixture was added water (10 mL) and the reaction mixture was stirred overnight. The organic solvent was r. emoved in vacuo and the aqueous residue washed with several 10 mL portions of pentane. The aqueous fraction was concentrated to dryness and the product reesterified by treatment with HCl saturated ethanol (10 mL) for 1 hour. The reaction mixture was concentrated in vacuo and triturated with diethyl ether.
The white precipitate was collected by filtration and washed well with diethyl ether affording 231 mg (80%) of 6-2..Low resolution mass spectral analysis confirmed the unit mass (MH+ = 237).
Example 46
Preparation of Na-tert-butoxvcarbonyl-D-4- piperidylqlycyl-qlycine ethyl ester 6-3
EMI100.1
6-3
To a solution of the compound of Example 45 (100 mg, 0.32 mmol) dissolved in dioxane (4 mL), was added di (tert-butyl) dicarbonate (92 mg, 0.42 mmol) and triethylamine (117 pL, 0.84 mmol). The reaction mixture was stirred 1 hour. The reaction mixture was concentrated in vacuo and the product purified by flash chromatography [silica (230-400 mesh), 0.75 x 6 in, ethyl acetate (2 column volumes), 9/1 CH2CL2/methanol] affording 30 mg (28%) of product as a white foam.
A hydrogenation apparatus was charged with the compound of Example 46 (140 mg, 0.41 mmol), ethanol (4 mL), water (1 mL), acetic acid (1 mL), and platinum oxide (14 mg). The apparatus was pressurized to 40 psi H2 and the reaction run 2 hours. The catalyst was removed by filtration, the filtrate was concentrated.
Example 47
Preparation of N=-tert-butoxyzarbonvl-D-4-EN- bis (benzyloxycarbonyl) cfuanyloiperidyl1glycyl-glycine ethyl ester 6-4
EMI101.1
6-4
The residue from Example 46 was dissolved in tetrahydrofuran (5 mL). To this solution was added in order, bis (benzyloxycarbonyl)-S-methylisothiourea (223 mg, 0.62 mmol) and N-methylmorpholine (45 L, 0.41 mmol). The reaction mixture was stirred 12 hours at room temperature and then diluted with ethyl acetate (10 mL). This solution was washed with 1N NaHS04 (2 x 3 mL), saturated NaHC03 (2 x 3 mL), and brine (2 x 3 mL). The organic fraction was dried over Na2SO4 and concentrated in vacuo.
Flash chromatography [silica (230-400 mesh), 0.75 x 6 in], eluting with a gradient of 50/50 ethyl acetate/hexane followed by ethyl acetate afforded 110 mg (41%) of 6-4 as a white foam. Rf = 0. 62 (ethyl acetate).
Example 48
Preparation of D-4- [N- bis (benzvloxycarbonvl) quanylpiperidvllalycyl-qlycine ethyl ester hydrochloride 6-5
EMI102.1
6-5
The compound of Example 47 (110 mg, 0.17 mmol) was dissolved in HC1 saturated ethanol (5 mL) and the reaction mixture was stirred 1 hour at ambient temperature. The solution was concentrated in vacuo to afford 105 mg of 6-5 as a white solid which was used for the procedure described in Example 49 without further purification. Low resolution mass spectral analysis confirmed the unit mass (MH+ = 54.4).
Example 49
Preparation of benzylsulfonyl-D-4-N- bis (benzyloxycarbonyl) guanylpiperidvllqlycyl-qlycine ethyl ester 6-6
EMI103.1
6-6
The compound of Example 48 (105 mg, 0.17 mmol) was dissolved in CH3CN (5 mL) and cooled in an ice-water bath. To this solution was added in order, a- toluenesulfonyl chloride (35.2 mg, 0.18 mmol) in a single portion and a solution of N-methylmorpholine (37.4 ssg, 0.34 mmol) dissolved in CH3CN (1 mL) dropwise.
The reaction mixture was stirred 0.5 hour at 0 C, then 12 hours at ambient temperature. The reaction mixture was concentrated in vacuo and the residue redissolved in ethyl acetate (5 mL). This solution was washed with 1N NaHS04 (2 x 1 mL), saturated NaHC03 (2 x 1 mL), and brine (2 x 1 mL). The organic fraction was dried over Na2SO4 and concentrated in vacuo to afford 90 mg (75%) of 6-6 as a white foam. Rf = 0. 83 (ethyl acetate).
Low resolution mass spectral analysis confirmed the unit mass (MH"= 707. 6)
Example 50
Preparation of benzylsulfonvl-D-4-N- bis (benzyloxycarbonyl) guanylniperidvllqlycyl-glycine 6-7
EMI104.1
6-7
To a solution of the compound of Example 49 (90 mg, 0.13 mmol) dissolved in ethanol (1 mL), was added 1N LiOH (260 pL), and the solution was stirred 3 hours at ambient temperature. The reaction mixture was concentrated in vacuo, and the residue was dissolved in water and then washed twice with diethyl ether. The aqueous fraction was adjusted to pH 3 with 1N NaHS04, and this solution was extracted with ethyl acetate (3 x 5 mL).
The organic fraction was dried over Na2SO4 and then concentrated in vacuo to afford to afford 22 mg (25%) of 6-7 as a white foam. Low resolution mass spectral analysis confirmed the unit mass (MH+ = 680).
Example 51
Preparation of benzylsulfonyl-D-4-[N- bis (benzyloxycarbonyl) quanylpiperidyllqlycyl-glycyl- arqinine (nitro) ethyl aminal 6-8
EMI105.1
6-8
To a solution of the compound of Example 50 (22 mg, 0.032 mmol) dissolved in N, N-dimethylformamide (1 mL), were added in order, arginine (nitro) ethyl aminal hydrochloride (9.4 mg, 0.035 mmol), 1-hydroxy-7- azabenzotriazole (4.3 mg, 0.032 mmol), 0- (7- azabenzotriazol-1-yl)-1, 1, 3,3-tetramethyluronium hexafluorophosphate (0.25 mg, 0.064 mmol), and diisopropylethylamine (22 pL, 0.128 mmol). The solution was stirred 5 hours at ambient temperature and then diluted with ethyl acetate (5 mL).
This solution was washed with 1N NaHS04 (2 x 2 mL), saturated NaHCO3 (2 x 2 mL), and brine (2 x 2 mL). The organic fraction was dried over Na2SO4 and concentrated in vacuo to afford 25 mg (88%) of 6-8 as a white solid. Analytical HPLC (4.6 x 250 mm column, C-18 reverse phase, 5 Sm particles, 300
A pores, 5-75% CH3CN/H2O, 0 1% TFA) gave a single peak (16, 9 minutes). Low resolution mass spectral analysis confirmed the unit mass (MH+ = 893. 5).
Example 52
Preparation of benzylsulfonyl-D-4-fN- guanylpiperidyll glycyl-qlycyl-arginine ethyl aminal 6-9
EMI106.1
6-9
A hydrogenation apparatus was charged with the compound of Example 51 (25 mg, 0.028 mmol), ethanol (8 mL), water (1 mL), acetic acid (1 mL), and palladium on carbon (10 mg). The apparatus was pressurized to 30 psi
H2 and the reaction run 4 hours. The catalyst was removed by filtration and the filtrate concentrated in vacuo to afford 6-9 as a clear oil in quantitative yield. Analytical HPLC (4.6 x 250 mm column, C-18 reverse phase, 5 Am particles, 300 A pores, 5-25%
Ch3CN/H2O, 0.1% TFA) gave two peaks (17.5 minutes, 18 minutes).
Low resolution mass spectral analysis confirmed the half-unit mass [ (M + 2)/2 = 290. 5].
Example 53
Preparation of benzylsulfonyl-D-4-N guanylpiperidyllqlycyl-glycyl-arvininal 6-10
EMI107.1
6-10
A solution of the compound of Example 52 (20 mg, 0.034 mmol) dissolved in CH3CN (1 mL) was treated with 6N HC1 (4 mL) for 2 hours. The volume of reaction mixture was brought to 10 mL with water, and the pH was adjusted to neutral with solid ammonium acetate. The product was purified by preparative HPLC (2.2 x 25 cm, C-18 reverse phase, 10 m particle, 300 A pore, 5-25% CH3CN/H20, 0.1%
TFA). Appropriate fractions were combined and lyophilized to afford 1.5 mg (8%) of 6-10.
Analytical
HPLC (4.6 x 250 mm column, C-18 reverse phase, 5 ym particles, 300 A pores, 5-25% CH3CN/H2O, 0.1% TFA) gave a single peak (14.5 minutes). Low resolution mass spectral analysis confirmed the half-unit mass [ (M + 2)/2 = 276. 5].
Examples 54 to 67 describe synthesis of certain intermediates which may be used in synthesis of compounds of the present invention. See Figure 7 (Examples 54 to 58) and Figure 8 (Examples 59 to 66).
Example 54
Preparation of N-- (t-butoxvcarbonyl)-3- (3-iovridyl)-L- alanine methyl ester
EMI108.1
7-2
To a solution of N- (t-butoxycarbonyl)-3- (3- pyridyl) alanine (5.0 g, 18.8 mmol) in methanol (100 mL), was added thionyl chloride (2M solution in dichloromethane, 66 mL, 132 mmol). The resulting solution was stirred overnight at ambient temperature.
The methanol was removed under reduced pressure to a minimum volume and ethyl acetate (100 mL) was added.
The resulting white precipitate was collected in a fritted funnel. To a solution of the collected precipitate in a mixture of tetrahydrofuran/water (40 mL each), was added di-tert-butyl dicarbonate (4.8 g, 21.99 mmol) and sodium carbonate (1.95 g, 18.4 mmol). After stirring for 12 hours at ambient temperature, the reaction mixture was diluted with ethyl acetate (40 mL) and washed with a solution of saturated sodium bicarbonate (25 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum to give crude product. This product was subjected to flash column chromatography on silica gel (230-400 mesh) using a 8 x 52 cm column and eluting with a 10: 90 mixture of ethyl acetate/hexane, followed by a 60: 40 mixture of ethyl acetate/hexane. 4 g (74%) of the title compound was obtained as an oil.
Thin-layer chromatography (silica gel; ethyl acetate), Rf = 0.68.
Example 55
Preparation of N- (t-butoxycarbonyl)-3- ( [R, Sl-3- piperidyl)-L-alanine methyl ester, acetate salt
EMI109.1
7-3
A solution of the compound of Example 54 (5 g, 17.8 mmol) in ethanol (24 mL), acetic acid (6 mL) and water (6 mL) was hydrogenated over platinum oxide (500 mg) at 45 psi for three hours. The catalyst was filtered off and the filtrate was concentrated under vacuum to an oily residue (6.89 g) which was used in the procedure described in Example 56 without further purification.
Thin-layer chromatography yielded two spots corresponding to two diastereomers with Rf values of 0.16 and 0.26, respectively (silica gel; 4: 1: 1 n-butanol/ acetic acid/water).
Example 56
Preparation of N-(t-butoxycarbonyl)-3-[(R, S)-3 piperidyl-(N-guanidino (bis-benzyloxycarbonyl)) l-L- alanine methyl ester
EMI110.1
7-4
To a solution of the compound of Example 55 (6.89 g, 19.9 mmol) in tetrahydrofuran (80 mL), was added Smethylisothiourea bis-benzyloxycarbonyl (7.13 g, 19.9 mmol), followed by N-methylmorpholine (4.37 mL). The reaction mixture was stirred at ambient temperature for 18 hours. The reaction mixture then was concentrated under vacuum and the resulting residue was dissolved in ethyl acetate (100 mL) and washed with 1N sodium bisulfate and saturated sodium chloride (50 mL each).
After drying over anhydrous sodium sulfate, the solvents were removed under vacuum; the crude title compound was subjected to'flash column chromatography on silica gel (230-400 mesh) using a 8x52 cm column and eluting with 1: 9 ethyl acetate/hexanes (two column volumes) followed by 1 : 1 ethyl acetate/hexanes. 2.75 g the title compound was obtained as a mixture of two diastereomers. Thinlayer chromatography gave two spots with Rf values of 0.57 and 0.62, respectively (silica gel; 1: 1 ethyl acetate/hexanes).
Example 57
Preparation of N- (t-butoxycarbonyl)-3- [ (R S)-3 piperidyl- (N-guanidino (bis-benzyloxycarbonyl)) l-Lalaninol
EMI111.1
7-5
To a stirred solution of the compound of Example 56 (2.23 g, 3.7 mmol) in absolute ethanol (8 mL) and anhydrous tetrahydrofuran (4 mL), was added calcium chloride (844 mg, 7.6 mmol) and sodium borohydride (575 mg, 15.2 mmol). After stirring 12 hours at ambient temperature, the reaction mixture was concentrated under vacuum and the resulting residue was partitioned between ethyl acetate and 1N sodium bisulfate (10 mL each). The two layers were separated; the organic layer was washed twice more with 1N sodium bisulfate, dried over anhydrous sodium sulfate and concentrated under vacuum to give a residue.
Flash column chromatography of the residue on silica gel (230-400 mesh) using a 5. 5x45 cm column and eluting with ethyl acetate gave 1.3 g of the title compound as a white foam. Thin layer chromatography yielded two spots corresponding to two diastereomers with Rf values of 0.18 and 0.27, respectively (silica gel; 1: 1 ethyl acetate/hexanes).
Example 58
Preparation of 3-[(R, S)-3-piperidyl-(N-guanidino (bis benzyloxycarbonyl)) 1-L-alaninol, hydrochloride salt
EMI112.1
7-6
The compound of Example 57 (290 mg, 0.57 mmol) was treated with 2.5N anhydrous hydrochloric acid in ethyl acetate (2.0 mL) at ambient temperature for one hour.
The solvent was removed under vacuum to a sticky-white solid (260 mg).
This solid is used without further isolation to prepare a compound of the present invention having a 3 piperidinyl- (N-guanidino)-alaninal at P1. 1H NMR spectrum taken in CD30D showed no t-butoxycarbonyl protons at 1.4 ppm.
Example 59
Preparation semicarbazid-4-yl diphenylmethane, trifluoroacetate salt
EMI112.2
Step 1 :
A solution of carbonyldiimidazole (16.2 g, 0.10 mole) in 225 mL of dimethylformamide was prepared at room temperature and allowed to stir under nitrogen. A solution of t-butyl carbazate (13.2 g, 0.100 mole) in 225 mL dimethylformamide was then added dropwise over a 30 minute period. Next, diphenylmethylamine (18.3 g, 0.10 mole) was added over a 30 minute period. The reaction mixture was allowed to stir at room temperature under nitrogen for one hour. Water (10 mL) was added and this mixture was concentrated to about 150 mL under vacuum. This solution was poured into 500 mL water and then extracted with 400 mL of ethyl acetate.
The ethyl acetate phase was extracted two times each with 75 mL 1N
HC1, water, saturated sodium bicarbonate and brine, and then was dried with anhydrous magnesium sulfate. The mixture was filtered and the solution was concentrated to give 29.5 g (85% yield) of 1-t-butoxycarbonylsemicarbazid-4-yl diphenylmethane as a white foam. This material may be purified by recrystallization from ethyl acetate/hexane, but was pure enough to use directly in step'2 : mp 142-143 C. lH NMR (CDC13) delta 1.45 (s, 9H), 6.10 (dd, 2H), 6.42 (s, 1H), 6.67 (bs, 1H), 7.21-7.31 (m, 10H). Analysis calculated for ClgH23N303 : C, 66.84;
H, 6.79; N, 12.31.
Found: C, 66.46; H, 6.75; N; 12.90.
Sten 2:
A solution of 3.43 g (10 mmol) of 1-tbutoxycarbonyl-semicarbazid-4-yl diphenylmethane in 12.5 mL of dichloromethane was treated with 12.5 mL of trifluoroacetic acid at 0 C. The reaction mixture was allowed to stir for 30 minutes at this temperature. The reaction mixture was then added dropwise to 75 mL of diethyl ether to give a precipitate. The resulting precipitate was filtered off and washed with diethyl ether to give 2.7 g (80% yield) of the title compound; mp 182-184 C.
Example 60
Preparation of 3-thioamidobenzyl-N-acetylaminomalonic acid diethyl ester
EMI114.1
8-4
To a stirred solution of a-bromo-meta-tolunitrile (45.0 g, 0.24 mole), diethyl acetamidomalonate (48.0 g, 0.22 mole) and potassium iodide (3.0 g, 0.018 mole) in dioxane (500 mL), was added 2.5M sodium ethoxide in ethanol (100 mL) dropwise under an argon atmosphere.
After the addition was complete, the solution was refluxed for 6 hours. The reaction mixture was allowed to stand overnight at room temperature, then diluted with brine (250 mL) and water (250 mL), and extracted with ethyl acetate four times (1.0 L total). The combined extracts were washed with water (100 mL), 10% citric acid (100 mL), water (100 mL) and brine (2 x 50 mL) then dried over anhydrous magnesium sulfate and filtered; the solvent was removed under vacuum. The crude residue was recrystallized from ethyl acetate and diethyl ether in two crops to yield 43.51 g (60%) of the 3-cyanobenzyl-N-acetylaminomalonic acid diethyl ester as yellow crystals.
HZS (g) was bubbled into a rapidly stirring solution of 3-cyanobenzyl-N-acetylaminomalonic acid diethyl ester (44.3 g, 0.13 mmol) in pyridine (300 mL) and triethylamine (100 mL) for 40 minutes. The. eaction mixture was stirred at room temperature for 16 hours, then poured into 3.0 L of water. A yellow precipitate formed immediately. The solution was allowed to stand at 4 C for 4 hours, then was filtered. The crude title compound was recrystallized from ethyl acetate and hexanes to yield 48.1 g (98% yield) of the title compound as yellow crystals, m. p. 183-186 C. 1H NMR <RTI
Example 61
Preparation of 3-amidino-D, L-phenylalanine, dihydrochloride salt
EMI116.1
8-5
The compound of Example 60 (48.1 g, 0.13 mmol) was dissolved in acetone (800 mL). Iodomethane (18.3 mL, 0.19 mole, 1.5 equivalents) was added, and the solution was refluxed for 30 minutes. The solution was cooled to room temperature, and the intermediate thioimidate was filtered, dried and dissolved in methanol (500 mL).
Ammonium acetate (14.8 g, 0.19 mole, 1.5 equivalents) was added. The reaction mixture was refluxed for 1 hour, then cooled to room temperature, and poured into ether (1.2 L). The solution was allowed to stand at 4 C for 72 hours. The crude 3-amidinobenzyl-Nacetylaminomalonic acid diethyl ester was filtered, washed with ether, air dried, and then refluxed in concentrated HC1 (250 mL) for 3 hours. The reaction mixture was concentrated under vacuum, diluted with water (0.5 L), and concentrated under vacuum again.
These steps were repeated. The crude title compound was purified by cation-exchange (Sephadex SP-C25) using a gradient of 0-1. ON HC1 as eluent to yield 10.8 g (30% yield) of the title compound as an off-white solid. 1H
NMR (D20) : delta 3.14-3.29 (2H, m), 4.17 (dd, J=7.4, 6.2 Hz, 1H), 7.42-7.69 (4H, m). Analysis calculated for
CloHl3N302 2HCl 1. 9H2O : C, 38.20 ; H, 6.03 ; N, 13.36. Found: C, 38.51; H, 5.64; N, 12.89.
Example 62
Preparation of N-a-Boc-N-omeqa-4-methoxy-2, 3, 6 trimethylbenzenesulfonyl-3-amidino-D,L-phenylalanine
EMI117.1
8-6
3-amidino-D, L-phenylalanine (the compound of
Example 61,) (4.00 g, 13 mmol) was dissolved in 50% aqueous dioxane (20 mL). Sodium bicarbonate. (3.38 g, 40 mmol) was added, followed by di-t-butyl dicarbonate (2.93 g, 13 mmol) in dioxane (4 mL). The reaction mixture was stirred for 18 hours at room temperature.
The solution was cooled in an ice bath, and 4. ON sodium hydroxide was. added dropwise until the solution was pH 12.4-methoxy-2,3,6-trimethylbenzenesulfonyl chloride (8.01 g, 32 mmol) in dioxane (10 mL) was added dropwise.
4. ON sodium hydroxide was added as needed to keep the pH at 12. The ice bath was removed. After 1 hour, 1. ON
HC1 was added to bring the solution to pH 7-8. The solution was diluted with an additional 50 mL of water and then was washed with ethyl acetate two times (20 mL each). The aqueous layer was acidified to pH 1.0 with 1.0 N HC1 and extracted with ethyl acetate three times (100 mL total). The combined organic layers were washed with water (20 mL) and brine twice (10 mL each). The organic layer was dried over anhydrous magnesium sulfate and the solvent was removed under vacuum. The residue was dissolved in a minimum amount of dichloromethane, then added dropwise to ether (25 mL).
Solid impurities were removed by filtering and the solvent removed from the filtrate under vacuum to give 4.90 g (68% crude yield) of the title compound as an off-white foam. A 30 mg sample of the title compound was further purified by preparative thin-layer chromatograph developing with 1% acetic acid/5% isopropanol/dichloromethane to give 9 mg of the title compound in a purer form. Rf = 0.16 (1% acetic acid/5% isopropanol/dichloromethane). 1H NMR (CD30D): delta-1. 32 (s, 9H), 2.14 (s, 3H), 2.63 (s, 3H), 2.71 (s, 3H), 2.93 (dd, J=13.7,9.3 Hz, 1H), 3.22 (dd,
J=13.7,4.3 Hz, 1H), 3.85 (s, 3H), 4.34-4.37 (m, 1H), 6.72 (s, 1H), 7.35-7.47 (2H, m), 7.69-7.75 (m, 2H).
Example 63
Preparation of N-a-Boc-N-omega-4-methoxy-2, 3, 6- trimethylbenzenesulfonyl-3-amidino-D, L-phenylalanine-N methyl-0-methyl-carboxamide
EMI119.1
8-7
To a stirred solution of compound of Example 62 (1.00 g, 1.92 mmol), O, N-dimethyl hydroxylamine hydrochloride (375 mg, 3.85 mmol), hydroxybenzotriazole hydrate (294 mg, 1.92 mmol) and 4-methylmorpholine (1.06 mL, 9.62 mmol) in tetrahydrofuran (4 mL), cooled in an ice bath, was added EDC (406 mg, 2.12 mmol). The ice bath was removed, and the reaction mixture was stirred for 2 hours at room temperature. The reaction mixture was diluted with ethyl acetate (75 mL), washed with water, 10% citric acid, water, saturated sodium bicarbonate, and brine.
The organic layer was dried over anhydrous magnesium sulfate and the solvent was removed under vacuum. 750 mg (69% yield) of the title compound was isolated. 1H NMR (CDC13) : delta 1.33 (s, 9H), 2.14 (s, 3H), 2.66 (s, 3H), 2.75 (s, 3H), 2.80-2.88 (m, 1H), 3.06-3.20 (m, 4H), 3.70 (s, 3H), 3.84 (s, 3H), 4.98-5.06 (m, 1H), 5.21 (d, J=8.7 Hz, 1H), 6.48 (bs, 1H), 6.58 (s, 1H), 7.30-7.34 (m, 2H) 7.60-7.68 (m, 2H), 8.11 (bs, 1H). Analysis calculated for C27H38N40'7S'0. 5H20 :
C, 56.73; H, 6.88; N, 9.80. Found: C, 56.97; H, 6.66; N, 9.43.
Example 64
Preparation of N-a-Boc-N-omega-4-methoxy-2, 3, 6- trimethylbenzenesulfonyl-D, L-3-amidinophenylalaninal
EMI120.1
8-8
To a stirred solution of LiAlH4 (2.00 mL of a 1.0 M solution in tetrahydrofuran, 1.24 mmol) in tetrahydrofuran (8 mL), cooled in a dry ice/acetone bath, the compound of Example 63 (0.75 g, 1.9 mmol in tetrahydrofuran (5 mL)) was added dropwise. The cooling bath was removed and the reaction mixture was allowed to warm to-5 C. The reaction mixture was re-cooled in the dry ice acetone bath and quenched with 3.0 mL of a 1: 2.7 wt./wt. solution of potassium bisulfate in water. The reaction mixture was allowed to warm to room temperature, stirred for 3 hours, filtered and concentrated under vacuum.
The residue was dissolved in ethyl acetate (20 mL), and washed with 10% citric acid (2 mL), water (2 mL), saturated sodium bicarbonate (2 mL) and brine (2 mL). The organic layer was dried over anhydrous magnesium sulfate and the solvent was removed under vacuum to yield 580 mg (86%) of the title compound. 1H NMR (CDC13) :
delta 1.31 (s, 9H), 2.07 (s, 3H), 2.57 (s, 3H), 2.67 (s, 3H), 2.90-3.17 (2H, m), 3.77 (s, 3H), 4.33-4.40 (1H, m), 5.02-5.08 (1H, m), 6.48 (1H, s), 7.23-7.31 (2H, m), 7.50-7.62 (2H, m), 7.94, (1H, bs), 8.05 (1H, bs), 9.55 (1H, s). Analysis calculated for C25H33N306S . SH2O : C, 58. 58 ; H, 6. 69 ; N, 8. 20.
Found: C, 58.57; H, 6.72; N, 7.98.
Example 65
Preparation of N-a-Boc-N-Q-4-methoxy-2, 3, 6- trimethylbenzenesulfonyl-D, L-3-amidinophenylalaninal- semicarbazonyl-4-N-diphenylmethane
EMI121.1
8-9
The compound of Example 64 (0.58 g, 1.9 mmol), the compound of Example 65 (410 mg, 1.15 mmol) and sodium acetate trihydrate (188 mg, 1.38 mmol) were refluxed in 75% aqueous ethanol (10 mL) for 1 hour. After the reaction mixture was cooled to room temperature, it was diluted with ethyl acetate (50 mL), washed with 1. ON HC1 (5 mL), water (5 mL), saturated sodium bicarbonate (5 mL) and brine (2x5 mL), and dried over anhydrous magnesium sulfate.
The solvent was removed under vacuum to yield 750 mg (89% yield) of the title compound as an off-white foam. Analysis calculated for C39H46N606S-1. 0 H2O : % C, 62.88; % H, 6.49; % N, 11.28. Found: % C, 63.14; % H, 6.35 % N, 11.10. Calculated molecular weight was726.90.
Example 66
Preparation of N-omecra-4-methoxy-2, 3, 6-trimethylbenzene sulfonyl-D, L-3-amidinophenylalaninal-semicarbazonyl-4-N- diphenylmethane, trifluoroacetate salt
EMI122.1
8-10
The compound of Example 65 (750 mg, 1.9 mmol) was treated with 50% trifluoroacetic acid/dichloromethane (3 mL) for 30 minutes at room temperature. The reaction mixture was added dropwise to ether (50 mL). The solution was allowed to stand at 4 C for 18 hours. The product was filtered, and dried under vacuum to yield 600 mg (79% yield) of the title compound as an off-white solid. Analysis calculated for C34H38N 4S-1. 3CF3CO2H : % C, 56.72; % H, 5.11; % N, 10.84.
Found: % C, 56.34; % H, 5.47; % N, 11.49. The salt had a calculated molecular weight of 740.8.
Example 67
Preparation of Compound 9-6
Compound 9-6 of Figure 9 is prepared according to the method of Examples 54 to 58 with the substitution of t-butoxycarbonyl-4- (4-pyridyl) alanine in Example 54.
By following the teachings of the Detailed
Description of the Invention and the Examples and using the appropriate starting materials and reagents, the compounds depicted in Figures 10A to 10D were made.
By following the teachings of the Detailed
Description of the Invention and the Examples and using the appropriate starting materials and reagents, the compounds depicted in Figures 11A to 11G are made.
Example A
In vitro enzyme Assays for specificity determination
The ability of compounds of the present invention to act as a selective inhibitor of factor Xa catalytic activity was assessed by determining the concentration of test-compound which inhibited the activity of this enzyme by 50%, (ICso), and comparing this value to that determined for all or some of the following related serine proteases : recombinant tissue plasminogen activator (rt-PA), plasmin, activated protein C, chymotrypsin, thrombin and trypsin.
The buffer used for all assays was HBSA (10 mM
HEPES, pH 7.5,150 mM sodium chloride, 0.1% bovine serum albumin).
The assay for ICSO determinations was conducted by combining in appropriate wells of a Corning microtiter plate, 50 microliters of HBSA, 50 microliters of the test compound at a specified concentration (covering a broad concentration range) diluted in HBSA (or HBSA alone for Vo (uninhibited velocity) measurement), and 50 microliters of the enzyme diluted in HBSA. Following a 30 minute incubation at ambient temperature, 50 microliters of the substrate at the concentrations specified below were added to the wells, yielding a final total volume of 200 microliters. The initial velocity of chromogenic substrate hydrolysis was measured by the change in absorbance at 405 nm using a
Thermo Maux3 Kinetic Microplate Reader over a 5 minute period in which less than 5% of the added substrate was utilized.
The concentration of added inhibitor which caused a 50% decrease in the initial rate of hydrolysis was defined as the ICso value.
Thrombin (fIIa) Assay
Enzyme activity was determined using the chromogenic substrate, Pefachrome t-PA (CH3SO2-D- hexahydrotyrosine-glycyl-L-Arginine-p-nitroaniline, obtained from Pentapharm Ltd.). The substrate was reconstituted in deionized water prior to use. Purified human a-thrombin was obtained from Enzyme Research
Laboratories, Inc. The buffer used for all assays was
HBSA (10 mM HEPES, pH 7.5,150 mM sodium chloride, 0.1% bovine serum albumin).
ICso determinations were conducted where HBSA (50 HL), a-thrombin (50 l) (the final enzyme concentration is 0.5 nM) and inhibitor (50 y1) (covering a broad concentration range), were combined in appropriate wells and incubated for 30 minutes at room temperature prior to the addition of substrate
Pefachrome-t-PA (50 1) (the final substrate concentration is 250 SM, about 5 times Km). The initial velocity of Pefachrome t-PA hydrolysis was measured by the change in absorbance at 405nm using a Thermo Maux@
Kinetic Microplate Reader over a 5 minute period in which less than 5% of the added substrate was utilized.
The concentration of added inhibitor which caused a 50% decrease in the initial rate of hydrolysis was defined as the ICsa value.
Factor Xa
Factor Xa catalytic activity was determined using the chromogenic substrate S-2765 (N-benzyloxycarbonyl-Darginine-L-glycine-L-arginine-p-nitroaniline), obtained from DiaPharma Group (Franklin, OH). All substrates were reconstituted in deionized water prior to use.
The final concentration of S-2765 was 250 SM (about 5times Km). Purified human Factor X was obtained from
Enzyme Research Laboratories, Inc. (South Bend, IN) and
Factor Xa (FXa) was activated and prepared from it as described [Bock, P. E., Craig, P. A., Olson, S. T., and
Singh, P. Arch. Biochem. Biophys. 273: 375-388 (1989)].
The. enzyme was diluted into HBSA prior to assay in which the final concentration was 0.25 nM.
Recombinant tissue plasminogen activator (rt-PA) Assay
rt-PA catalytic activity was determined using the substrate, Pefachrome t-PA (CH3SO2-D-hexahydrotyrosine- glycyl-L-arginine-p-nitroaniline, obtained from
Pentapharm Ltd.). The substrate was made up in deionized water followed by dilution in HBSA prior to the assay in which the final concentration was 500 micromolar (about 3-times Km). Human rt-PA (Activasee) was obtained from Genentech Inc. The enzyme was reconstituted in deionized water and diluted into HBSA prior to the assay in which the final concentration was 1.0 nM.
Plasmin Assay
Plasmin catalytic activity was determined using the chromogenic substrate, S-2366 [L-pyroglutamyl-L-prolyl
L-arginine-p-nitroaniline hydrochloride], which was obtained from DiaPharma group. The substrate was made up in deionized water followed by dilution in HBSA prior to the assay in which the final concentration was 300 micromolar (about 2.5-times Km). Purified human plasmin was obtained from Enzyme Research Laboratories, Inc.
The enzyme was diluted into HBSA prior to assay in which the final concentration was 1.0 nM.
Activated Protein C (aPC) Assay
aPC catalytic activity was determined using the chromogenic substrate, Pefachrome PC (delta carbobenzloxy-D-lysine-L-prolyl-L-arginine-p- nitroaniline dihydrochloride), obtained from Pentapharm
Ltd.). The substrate, was made up in deionized water followed by dilution in HBSA prior to the assay in which the final concentration was 400 micromolar (about 3times Km). Purified human aPC was obtained from Hematologic Technologies, Inc. The enzyme was diluted into HBSA prior to assay in which the final concentration was 1.0 nM.
Chymotrypsin Assay
Chymotrypsin catalytic activity was determined using the chromogenic substrate, S-2586 (methoxysuccinyl-L-arginine-L-prolyl-L-tyrosyl-p-nitroanilide), which was obtained from DiaPharma Group. The substrate was made up in deionized water followed by dilution in
HBSA prior to the assay in which the final concentration was 100 micromolar (about 9-times Km). Purified (3Xcrystallized; CDI) bovine pancreatic alpha-chymotrypsin was obtained from Worthington Biochemical Corp. The enzyme was reconstituted in deionized water and diluted into HBSA prior to assay in which the final concentration was 0.5 nM.
Trypsin Assay.
Trypsin catalytic activity was determined using the chromogenic substrate, S-2222 (benzoyl-L-isoleucine-Lglutamic acid- (gamma-methyl ester]-L-arginine-pnitroanilide), which was obtained from DiaPharma Group.
The substrate was made up in deionized water followed by dilution in HBSA prior to the assay in which the final concentration was 250 micromolar (about 4-times Km).
Puri-fied (3X-crystallized ; TRL3) bovine pancreatic trypsin was obtained from Worthington Biochemical Corp.
The enzyme was reconstituted in deionized water and diluted into HBSA prior to assay in which the final concentration was 0.5 nM.
Table I lists the determined ICso values for certain of the enzymes listed above for compounds of the present invention and demonstrate the high degree of specificity for the inhibition of alpha-thrombin compared to these related serine proteases.
EMI128.1
<tb>
Compound <SEP> No. <SEP> Xa <SEP> Ila <SEP> Trypsin <SEP> Ila/Xa <SEP> Trypsin/Xa
<tb> <SEP> A3 <SEP> 1. <SEP> 23 <SEP> > 2500 <SEP> 120 <SEP> > 2032 <SEP> 97. <SEP> 6
<tb> <SEP> A4 <SEP> 0. <SEP> 917 <SEP> > 2500 <SEP> 31 <SEP> > 2726 <SEP> 33. <SEP> 8
<tb> <SEP> A6 <SEP> 1. <SEP> 08 <SEP> 2750 <SEP> 124 <SEP> 2546. <SEP> 3 <SEP> 114. <SEP> 8
<tb> <SEP> A7 <SEP> 1.37 <SEP> > 250 <SEP> 14.5 <SEP> > 182.5 <SEP> 10. <SEP> 6
<tb> <SEP> A8 <SEP> 2. <SEP> 15 <SEP> > 2500 <SEP> 241 <SEP> > 1163 <SEP> 112. <SEP> 1
<tb> <SEP> A9 <SEP> 4. <SEP> 25 <SEP> 352 <SEP> 32. <SEP> 5 <SEP> 82. <SEP> 8 <SEP> 7. <SEP> 6
<tb> <SEP> A5 <SEP> 0. <SEP> 825 <SEP> > 2500 <SEP> 169 <SEP> > 3030 <SEP> 204. <SEP> 8
<tb> <SEP> A10 <SEP> 0.935 <SEP> > 2500 <SEP> 147 <SEP> > 2674 <SEP> 157. <SEP> 2
<tb> <SEP> A12 <SEP> 2. <SEP> 23 <SEP> > 2500 <SEP> 145 <SEP> > 1121 <SEP> 65.
<SEP> 0
<tb> <SEP> A13 <SEP> 4. <SEP> 26 <SEP> > 2500 <SEP> 211 <SEP> > 586. <SEP> 6 <SEP> 49. <SEP> 5
<tb> <SEP> A14 <SEP> 94. <SEP> 8 <SEP> 2500 <SEP> 158 <SEP> > 26. <SEP> 4 <SEP> 1. <SEP> 7
<tb> <SEP> A15 <SEP> 87. <SEP> 6 <SEP> > 2500 <SEP> 160 <SEP> > 28. <SEP> 5 <SEP> 1. <SEP> 8
<tb> <SEP> A16 <SEP> 5. <SEP> 3 <SEP> > 2500 <SEP> 447 <SEP> > 471. <SEP> 7 <SEP> 84. <SEP> 3
<tb> <SEP> A17 <SEP> 135 <SEP> > 2500 <SEP> 412 <SEP> > 18. <SEP> 5 <SEP> 3. <SEP> 05
<tb> <SEP> A18 <SEP> 2. <SEP> 11 <SEP> 2260 <SEP> 240 <SEP> 1071 <SEP> 113.
<tb>
<SEP> A19 <SEP> 2. <SEP> 63 <SEP> > 2500 <SEP> 537 <SEP> > 950. <SEP> 6 <SEP> 204. <SEP> 2
<tb> <SEP> A11 <SEP> 1. <SEP> 07 <SEP> > 2500 <SEP> 25. <SEP> 3 <SEP> > 2337 <SEP> 23. <SEP> 6
<tb> <SEP> A1 <SEP> 2. <SEP> 59 <SEP> 546 <SEP> 77. <SEP> 3 <SEP> 210. <SEP> 8 <SEP> 29. <SEP> 8
<tb> <SEP> A2 <SEP> 2. <SEP> 95 <SEP> 2190 <SEP> 273 <SEP> 742. <SEP> 4 <SEP> 92. <SEP> 5
<tb>
Claims
We claim: 1. A compound of the formula
EMI129.1
wherein
(a) X is selected from the group consisting of -S (0) 2-,-N (R')-S (0) 2-,- (C=O)-,-OC (=O)-,-NH-C (=O)-, and a direct link, wherein R'is hydrogen, alkyl of 1 to about 4 carbon atoms, aryl of about 6 to about 14 carbon atoms or aralkyl of about 7 to 16 carbon atoms;
(b) R1 is selected from the group consisting of:
(1) alkyl of 1 to about 12 carbon atoms which is optionally substituted with Y1,
(2) alkyl of 1 to about 3 carbon atoms substituted with cycloalkyl of about 5 to about 8 carbon atoms which is optionally mono-, di-, or tri-substituted on the ring with Yl, Y2 and/or Y3,
(3) cycloalkyl of 3 to about 15 carbon atoms, which is optionally mono-, di-, or tri-substituted on the ring with Yl, Y2 and/or Y3,
(4) heterocycloalkyl of 4 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen, and S (O) i, wherein i is 0, 1 or 2, which is optionally mono-, di-, or tri-substituted on the ring with Y1,
Y2 and/or Y3,
(5) heterocyclo of 4 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen, and S (O) i, including
EMI130.1
wherein
EMI130.2
is a 5 to 7 member heterocycle of 3 to 6 ring carbon atoms, where V is-CH2-,-O-,-S (=O)-,
S (0) 2- or-S-, which is optionally mono-, di-, or trisubstituted on the ring carbons with Y1, Y2 and/or Y3,
(6) alkenyl of about 2 to about 6 carbon atoms which is optionally substituted with cycloalkyl of about 5 to about 8 carbon atoms, which is optionally mono-, di-, or tri-substituted on the ring carbons with Y1,
Y2 and/or Y3 #
(7) aryl of about 6 to about 14 carbon atoms which is optionally mono-, di-or tri-substituted with Y1, Y2, and/or Y3, t
(8) heteroaryl of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-, di-, or tri-substituted with Y1, Y2, and/or Y3,
(9) aralkyl of about 7 to about 15 carbon atoms which is optionally substituted on the alkyl chain with hydroxy or halogen and mono-, di-, or trisubstituted in the aryl ring with Y1, Y2, and/or Y3,
(10) heteroaralkyl of 5 to 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur,
and which is optionally substituted on the alkyl chain with hydroxy or halogen. and optionally mono-, di-or tri-substituted on the ring with Y1, Y2, and/or Y3,
(11) aralkenyl of about 8 to about 16 carbon atoms which is optionally mono-, di-, or tri-substituted on the aryl ring with Yi, Y2, and/or Y3,
(12) heteroaralkenyl of 5 to 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-, di-or tri-substituted on the ring with Y1, Y2, and/or Y3,
EMI131.1
EMI131.2
EMI132.1
EMI132.2
(17) fused carbocyclic alkyl of about 5 to about 15 carbon atoms,
(18)
difluoromethyl or perfluoroalkyl of 1 to about 12 carbon atoms,
(19) perfluoroaryl of about 6 to about 14 carbon atoms,
(20) perfluoraralkyl of about 7 to about 15 carbon atoms, and
(21) hydrogen when X is a direct link;
(c) R2 is
EMI132.3
wherein x is 0 or 1, xl is an integer from 0 to 6 and R8 is hydrogen or alkyl of 1 to about 3 carbon atoms;
(d) T is a divalent radical selected from:
(i) a divalent cycloalkyl group of about 3 to about 8 carbon atoms;
(ii) a divalent aryl group of about 6 to about 14 carbon atoms which is optionally substituted with Yl ;
(iii) a divalent heteroaryl group of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen and sulfur and which is optionally substituted with Y1 ;
(iv) a divalent heterocyclo group of 4 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from the group consisting of oxygen, nitrogen and S (O) i wherein i is O, 1 or 2, which is optionally substituted with 1 to 2 substituents independently selected from an oxo group, Y1 and Y2 ;
and
(v) a divalent unsaturated heterocyclo group of about 5 to about 10 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteratoms are selected from the group consisting of oxygen, nitrogen and S (O) i, wherein i is 0,1, or 2, optionally substituted with Y1 ;
(e) J is-C (=E)-D or-NH-C (=E)-D, wherein D is R6 or NR6R, wherein R6 and R, are independently selected from
H, aryl of about 6 to about 10 carbon atoms and lower alkyl of 1 to about 6 carbon atoms, provided that D is not H, and E is 0, S or NR6 ;
(f) R3 is selected from the group consisting of
(1) hydrogen;
(2) alkyl of 1 to about 8 carbon atoms optionally substituted with Y4 ;
(3)alkyi of 1 to about 3 carbon atoms substituted with cycloalkyl of about 5 to about 9 carbon atoms optionally substituted on the ring with Y4 ;
(4) cycloalkyl of 3 to about 15 carbon atoms, which optionally is substituted on the ring with Y4 ;
(5) alkenyl of about 3 to about 6 carbon atoms which is optionally substituted with cycloalkyl of about 5 to about 8 carbon atoms, and which optionally is substituted on a ring carbon with Y4 ;
(6) aryl of about 6 to 14 carbon atoms which is optionally mono or di-substituted with Y1 and/or Y2;
(7) aralkyl of about 7 to about 15 carbon atoms which is optionally mono or di-substituted on the aryl ring with Y1 and/or Y2 ;
and
(8) heteroaralkyl of about 5 to about 14 ring atoms with the ring atoms selected from carbon and heteroatoms, wherein the heteroatoms are selected from oxygen, nitrogen, and sulfur, and which is optionally mono-or di-substituted on the ring with Y1 and/or Y2 ;
(g) R4 is selected from the group consisting of hydrogen and alkyl of 1 to about 7 carbon atoms;
(h) alternatively R3 and R4 taken together are - (CH2) q-, where q is 2,3, or 4, to form a cyclic amino acid residue;
(i) Rs is selected from
EMI135.1
wherein d is an integer from 0 to 5 and W is-N-or
-CH- ;
and
(j) each Y1, Y2, Y3 and Y4 is
(1) independently selected from the group consisting of halogen, cyano, nitro, tetrazolyl optionally substituted with alkyl of 1 to about 6 carbon atoms, guanidino, amidino, methylamino, methylguanidino, -CF3,-CF2CF3,-CH (CF3) 2,-C (OH) (CF3) 2,-OCF3,-OCF2CF3, -OCF2H,-OC (O) NH2,-OC (O) NHZ1,-OC (O) NZ1Z2, -NHC (O) Z1, -NHC (O) NH2,-NHC (O) NHZ1, -NHc (O) NZ1Z2, -C (O) OH,-C (O) OZ1, -C (O) NH2,-C (O) NZlZ2,-P (O) 3H2,-P (0) 3 (z1)2, -S(O)3H, -S (O) mZ1, -Z1, -OZ1, -OH1,
-NH2, -NHZ1, -NZ1Z2,
N-morpholino, and-S (O) m (CF2) qCF3, wherein m is 0,1 or 2, q is an integer from 0 to 5, and Z1 and Z2 are independently selected from the group consisting of alkyl of 1 to about 12 carbon atoms, aryl of about 6 to about 14 carbon atoms, heteroaryl of about 5 to about 14 atoms having 1 to about 9 carbon atoms, aralkyl of about 7 to about 15 carbon atoms, and heteroaralkyl of about 5 to about 14 ring atoms having about 3 to about 9 carbon atoms, or
(2) Y1 and Y2 are selected together to be -0 [C (Z3) (Z4)] rO-, wherein r is an integer from 1 to.
4 and
Z3 and Z4 are independently selected from the group consisting of hydrogen, alkyl or 1 to about 12 carbon atoms, aryl of about 6 to about 14 carbon atoms, heteroaryl of about 5 to about 14 ring atoms having 1 to about 9 carbon atoms, aralkyl of about 7 to about 15 carbon atoms, and heteroaralkyl of about 5 to about 14 ring atoms having about 3 to about 9 carbon atoms, and pharmaceutically acceptable, salts thereof with the proviso that when R1X is benzylsulfonyl or Boc, the sum of x and xl is 1, T is unsubstituted phenyl, J is -C (=NH) NH2 or-NHC (=NH) NH2, R3 is H, R4 is H, and R8 is H
EMI136.1
then Rs is not 2.
A compound according to claim 1 wherein X is selected from the group consisting of-S (O) 2- and -N (R')-S (O) 2-- 3. A compound according to claim 2 wherein X is -S (O) 2-.
4. A compound according to claim 3 wherein R1 is selected from the group consisting of cycloalkyl, aryl and aralkyl.
5.-A compound according to claim 4 wherein R1 is selected from the group consisting of substituted or unsubstituted phenyl, benzyl and naphthyl.
6. A compound according to claim 5 wherein R1 is optionally substituted with Y1.
7. A compound according to claim 6 wherein Y1 is . selected from optionally substituted tetrazolyl, -C (O) OH, and-C (O) Z1.
8. A compound according to claim 1 wherein T is 3-piperdinyl or 4-piperdinyl.
9. A compound according to claim 8 wherein J is -C (=E)-D.
10. A compound according to claim 9 wherein D is NR6R7 and E is NR6.
11.A compound according to claim 10 wherein R6 and R7 are hydrogen.
12. A compound according to claim 11 wherein the sum of x and xl is 0,1 or 2.
13..-A compound according to claim 11 wherein Rs is
EMI138.1
14. A compound according to claim 13 wherein the sum of x and xl is 0, 1 or 2 and d is 2.
15. A compound according to claim 15 wherein T is selected from divalent cycloalkyl, divalent heteroaryl, divalent heterocyclo and divalent unsaturated heterocyclo.
16. A compound-according to claim 15 wherein T is selected from divalent cycloalkyl and divalent heterocyclo.
17. A compound according to claim 16 having an argininal mimic at P1.
18. A compound according to claim 16 having an argininal at P1.
19. A compound according to claim 18 selected from the compounds depicted in Figures 10A to 10D.
20.. A compound according to claim 18 selected from the compounds depicted in Figures 11A to 11G.
21. A compound according to claim 18 wherein T is a divalent heterocyclo group of 4 ring atoms.
22. A compound according to claim 18 wherein T is a divalent heterocyclo group of 7 to 10 ring atoms.
23. A compound according to claim 16 wherein T is selected from divalent cycloalkyl groups having 4 or 5 carbon atoms and divalent heterocyclo groups having 4 or 5 ring atoms.
24. A compound according to claim 23 having an argininal mimic at P1.
25. A compound according to claim 23 wherein T is a divalent heterocyclo group of 4 ring atoms.
26. A pharmaceutical composition for treating or decreasing the'incidence of a condition characterized by abnormal thrombus formation in a mammal comprising a pharmaceutical acceptable carrier and a therapeutically effective amount of a compound of claim 1,8,15,19 or 20 effective to decrease abnormal thrombus formation.
27. A method for treating or decreasing the incidence of a condition characterized by abnormal thrombus formation in a mammal which comprises administering to said mammal a therapeutically effective amount of a compound of claim 1,8,15,19 or 20, effective to decrease abnormal thrombus formation.
28. A method of treating a pathologic condition characterized by abnormal thrombosis by preventing or decreasing said abnormal thrombosis, which comprises administering a compound of claim 1,8,15,19 or 20.
29. A method treating a pathologic condition characterized by abnormal thrombosis by preventing or decreasing said abnormal thrombosis, which comprises administering a compound of claim 1 which selectively inhibits factor Xa.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US41490399A | 1999-10-08 | 1999-10-08 | |
| US09/414,903 | 1999-10-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2001027141A1 true WO2001027141A1 (en) | 2001-04-19 |
Family
ID=23643507
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US2000/027615 WO2001027141A1 (en) | 1999-10-08 | 2000-10-06 | Inhibitors of factor xa having an arginine or arginine aldehyde mimic |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2001027141A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7629318B2 (en) | 2002-03-22 | 2009-12-08 | Gpc Biotech Ag | Immunosuppressant compounds, methods and uses related thereto |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5371072A (en) * | 1992-10-16 | 1994-12-06 | Corvas International, Inc. | Asp-Pro-Arg α-keto-amide enzyme inhibitors |
| WO1996040743A2 (en) * | 1995-06-07 | 1996-12-19 | Cor Therapeutics, Inc. | INHIBITORS OF FACTOR Xa |
-
2000
- 2000-10-06 WO PCT/US2000/027615 patent/WO2001027141A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5371072A (en) * | 1992-10-16 | 1994-12-06 | Corvas International, Inc. | Asp-Pro-Arg α-keto-amide enzyme inhibitors |
| WO1996040743A2 (en) * | 1995-06-07 | 1996-12-19 | Cor Therapeutics, Inc. | INHIBITORS OF FACTOR Xa |
Non-Patent Citations (1)
| Title |
|---|
| TAMURA S Y ET AL: "Guanylpiperidine peptidomimetics: potent and selective bis-cation inhibitors of factor Xa", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS,GB,OXFORD, vol. 10, no. 8, April 2000 (2000-04-01), pages 745 - 749, XP004203487, ISSN: 0960-894X * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7629318B2 (en) | 2002-03-22 | 2009-12-08 | Gpc Biotech Ag | Immunosuppressant compounds, methods and uses related thereto |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6025472A (en) | N-substituted glycine derivatives as enzyme inhibitors | |
| US5637599A (en) | Arginine mimic derivatives as enzyme inhibitors | |
| US5932733A (en) | 3-amino-2-oxo-1-piperidineacetic derivatives containing an arginine mimic as enzyme inhibitors | |
| US5656645A (en) | Aromatic heterocyclic derivatives as enzyme inhibitors | |
| US6008351A (en) | Aromatic hetherocyclic derivatives as enzyme inhibitors | |
| US5658930A (en) | Aromatic heterocyclic derivatives as enzyme inhibitors | |
| US5703208A (en) | 3-amino-2-oxo-1-piperidineacetic derivatives as enzyme inhibitors | |
| WO1995035311A9 (en) | 3-amino-2-oxo-1-piperidineacetic derivatives as enzyme inhibitors | |
| WO1996018644A9 (en) | Aromatic heterocyclic derivatives as enzyme inhibitors | |
| WO1995035311A1 (en) | 3-amino-2-oxo-1-piperidineacetic derivatives as enzyme inhibitors | |
| EP1100814A2 (en) | Inhibitors of urokinase and blood vessel formation | |
| NZ502876A (en) | Fused bicyclic lactam selective factor Xa inhibitors for treating thrombosis related diseases | |
| WO1999026920A1 (en) | Substituted 3-amino-2-hydroxyphenylacetamide derivatives as enzyme inhibitors (ii) | |
| US5696231A (en) | N-substituted glycine derivatives as enzyme inhibitors | |
| WO2001079261A1 (en) | Tetrahydro-azepinone derivatives as thrombin inhibitors | |
| NZ500352A (en) | Cyclic diaza selective factor Xa inhibitors | |
| US6506754B1 (en) | Non-covalent thrombin inhibitors | |
| US6777431B2 (en) | Non-convalent thrombin inhibitors | |
| US6541467B1 (en) | Thrombin inhibitors having a lactam at P3 | |
| WO2001027141A1 (en) | Inhibitors of factor xa having an arginine or arginine aldehyde mimic | |
| US5883107A (en) | Arginine mimic derivatives as enzyme inhibitors | |
| US6011047A (en) | Substituted 3-amino-2-hydroxyphenylacetamide derivatives as enzyme inhibitors | |
| US6204384B1 (en) | Substituted 3-amino-2-hydroxyphenylacetamide derivatives as enzyme inhibitors (II) | |
| WO2001079193A2 (en) | Substituted hydrazinyl heteroaromatic inhibitors of thrombin | |
| WO2001079195A9 (en) | Pyridine and pyrazine derivatives as thrombin inhibitors |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AK | Designated states |
Kind code of ref document: A1 Designated state(s): CA JP |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
| 122 | Ep: pct application non-entry in european phase | ||
| NENP | Non-entry into the national phase |
Ref country code: JP |