Patent Application
20120190832
The present invention relates to novel amidines and guanidines, to the production thereof, and to the use thereof as competitive inhibitors of trypsin-like serine proteases, particularly thrombin and the complement proteases C1s and C1r.
The invention also relates to pharmaceutical compositions containing said compounds as active ingredients, and also to the use of said compounds as thrombin inhibitors, anticoagulants, complement inhibitors, or anti-inflammatory agents. A characteristic of the novel compounds is their ability to link a serin protease inhibitor having an amidine or guanidine function to an alkyl group having two or more hydroxyl functions and derived from sugar derivatives. Thus a number of sugar building blocks or building blocks derived from sugars can be linked. This principle of coupling with sugar derivatives provides orally active compounds.
Preferred sugar derivatives include all types of reductive sugars which reductively react with a terminal amine function of the inhibitor.
Reductive sugars are sugars which are capable of reducing Cu(II) ions in solution to Cu(I) oxide.
Reductive sugars include:
Examples of other preferred sugar derivatives are sugar acids which react with a terminal amine function of the inhibitor via the acyl function.
Thrombin is a member of the group of serine proteases and plays a central role as terminal enzyme in the blood coagulation cascade. Both the intrinsic and the extrinsic coagulation cascades cause, via a number of intensification stages, the production of thrombin from prothrombin. Thrombin-catalyzed cleavage of fibrinogen to fibrin then triggers blood coagulation and aggregation of the thrombocytes, which in turn increase the formation of thrombin by binding platelet factor 3 and coagulation factor XIII as well as via a whole series of highly active mediators.
The formation and action of thrombin are central events in the genesis of both white arterial thrombi and red venous thrombi and are therefore potentially effective points of attack for pharmacological agents. Thrombin inhibitors are, unlike heparin, capably of completely inhibiting, simultaneously, the action of free thrombin and thrombin bound to thrombocytes, irrespective of co-factors. They can prevent, in the acute phase, thrombo-embolic events following percutane transluminal coronary angioplasty (PTCA) and cell lysis and serve as anticoagulants in extracorporeal recirculation (heartlung apparatus, haemodialysis). They can also serve in a general way for the prophylaxis of thrombosis, for example, after surgical operations.
Inhibitors of thrombin are suitable for the therapy and prophylaxis of
A number of thrombin inhibitors of the D-Phe-Pro-Arg type is known for which good thrombin inhibition in vitro has been described: WO 9702284-A, WO 9429336-A1, WO 9857932-A1, WO 9929664-A1, U.S. Pat. No. 5,939,392-A, WO 200035869-A1, WO 200042059-A1, DE 4421052-A1, DE 4443390-A1, DE 19506610-A1, WO 9625426-A1, DE 19504504-A1, DE 19632772-A1, DE 19632773-A1, WO 9937611-A1, WO 9937668-A1, WO 9523609-A1, U.S. Pat. No. 5,705,487-1, WO 9749404-A1, EP-669317-A1, WO 9705108-A1, EP 0672658. However, some of this compounds exhibit low oral activity.
In WO 9965934 and Bioorg. Med. Chem. Lett., 9(14), 2013-2018, 1999, benzamidine derivatives of the NAPAP type are described which are coupled through a long spacer to pentasaccharides and thus show a dual antithrombotic principle of action. However, no oral activity of these compounds is described.
Activation of the complement system ultimately leads, through a cascade of ca 30 proteins, inter alia, to lysis of cells. Simultaneously, molecules are liberated which, like C5a, can lead to an inflammatory reaction. Under physiological conditions, the complement system provides a defence mechanism against foreign bodies, such as viruses, fungi, bacteria, or cancer cells. Activation by various routes takes place initially via proteases. By activation, these proteases are made capable of activating other molecules of the complement system, which may in turn be inactive proteases. Under physiological conditions, this system, like blood coagulation, is under the control of regulatory proteins, which counteract exuberant activation of the complement system. In such cases it is not advantageous to take measures to inhibit the complement system.
In some cases the complement system overreacts, however, and thus contributes to the pathologic physiology of diseases. In such cases, therapeutic action on the complement system causing inhibition or modulation of the exuberant reaction is desirable. Inhibition of the complement system is possible at various levels in the complement system by inhibition of various effectors. The literature provides examples of the inhibition of serine proteases at the C1 level with the aid of the C1 esterase inhibitor as well as inhibition at the level of C3 or C5 convertases by means of soluble complement receptor CR1 (sCR1), inhibition at the level of C5 by means of antibodies, and inhibition at the level of C5a by means of antibodies or antagonists. The tools used for achieving inhibition in the above examples are proteins. In the present invention, low-molecular substances are described which are used for inhibition of the complement system.
For such inhibition of the complement system some proteases utilizing various activation routes are particularly suitable. Of the class of thrombin-like serine proteases, such proteases are the complement proteases C1r and C1s for the classical route, factor D and factor B for the alternative route, and also MASP I and MASP II for the MBL route. The inhibition of these proteases then leads to a re-establishment of the physiological control of the complement system in the above diseases or pathophysiological states.
Generally speaking, all inflammatory disorders accompanied by the immigration of neutrophilic blood cells must be expected to involve activation of the complement system. Thus it is expected that with all of these disorders an improvement in the pathophysiological state will be achieved by causing inhibition of parts of the complement system.
The activation of complement is associated with the following diseases or pathophysiological states:
Accordingly, treatment of the above mentioned diseases or pathophysiological states with complement inhibitors is desirable, particularly treatment with low-molecular inhibitors.
PUT and FUT derivatives are amidinophenol esters and amidinonaphthol esters respectively and have been described as complement inhibitors (eg, Immunology (1983), 49(4), 685-91).
Inhibitors are desired which inhibit C1s and/or C1r, but not factor D. Preferably, there should be no inhibition of lysis enzymes such as t-PA and plasmin.
Special preference is given to substances which effectively inhibit thrombin or C1s and C1r.
Reagents: thrombin reagent (List No. 126,594, Boehringer, Mannheim, Germany)
Reagents: human plasma thrombin (No. T 8885, Sigma, Deisenhofen, Germany)
Reagents: human plasma thrombin (No. T-8885, Sigma, Deisenhofen, Germany)
The platelet aggregation is measured by turbitrimetric titration at 37° C. (PAP 4, Biodata Corporation, Horsham, Pa., USA). Before thrombin is added, 215.6 μL of PRP are incubated for 3 minutes with 2.2 μL of test probe and then stirred over a period of 2 minutes at 1000 rpm. At a final concentration of 0.15 NIH units/mL, 2.2 μL of thrombin solution produce the maximum aggregation effect at 37° C./1000 rpm. The inhibited effect of the test probes is determined by comparing the rate (rise) of aggregation of thrombin without test substance with the rate of aggregation of thrombin with test substance at various concentrations.
For measuring potential complement inhibitors use is made, in the manner of diagnostic tests, of a test for measuring the classical route (literature: Complement, A practical Approach; Oxford University Press; 1997; pp 20 et seq). The source of complement used for this purpose is human serum. A test of similar layout is, however, also carried out on various serums of other species in a similar manner. The indicating system used comprises erythrocytes of sheep. The antibody-dependent lysis of these cells and the thus exuded haemoglobin are a measure of the complement activity.
Reagents, Biochemical Products:
Stock Solutions:
Buffer:
Evaluation was based on the absorption values at 540 nm.
Factor D plays a central role in the alternative route of the complement system. By reason of the low plasma concentration of factor D, the enzymatic step of cleavage of factor B by factor D represents the rate-limiting step in the alternative way of achieving complement activation. On account of the limiting role played by this enzyme in the alternative route, factor D is a target for the inhibition of the complement system.
The commercial substrate Z-Lys-S-Bzl * HCl is converted by the enzyme factor D (literature: C. M. Kam et al, J. Biol. Chem. 262 3444-3451, 1987). Detection of the cleaved substrate is effected by reaction with Ellmann's reagent. The resulting product is detected spectrophotometrically. The reaction can be monitored on-line. This makes it possible to take enzyme-kinetic readings.
Chemicals:
Buffer:
Stock Solutions:
Procedure:
Batches:
Readings:
Evaluation:
In this test, the serin protease inhibitor FUT-175; Futhan, Torii; Japan was co-used as effective inhibitor.
Confirmation of the inhibition of complement by the alternative route was obtained using a hemolytic test (literature: Complement, A practical Approach; Oxford University Press; 1997, pp 20 et seq).
The test is carried out on the lines of clinical tests. The test can be modified by additional activation by means of, say, Zymosan or cobra venom factor.
Human serum was either procured from various contractors (eg, Sigma) or obtained from test persons in the polyclinic department of BASF Süd.
Guinea pig's blood was extracted and diluted 2:8 in citrate solution. Several batches were used without apparent differences.
Assessment was made using the OD values.
The test probes are dissolved in isotonic salt solution just prior to administration to Sprague Dawley rats in an awake state. The administration doses are 1 ml/kg for intravenous Bolus injection into the cereal vein and 10 ml/kg for oral administration, which is carried out per pharyngeal tube. Withdrawals of blood are made, if not otherwise stated, one hour after oral administration of 21.5 mg·kg−1 or intravenous administration of 1.0 mg·kg−1 of the test probe or corresponding vehicle (for control). Five minutes before the withdrawal of blood, the animals are narcotized by i.p. administration of 25% strength urethane solution (dosage 1 g·kg−1 i.p.) in physiological saline. The A. carotis is prepared and catheterized, and blood samples (2 mL) are taken in citrate tubules (1.5 parts of citrate plus 8.5 parts of blood). Directly after blood sampling, the ecarin clotting time (ECT) in whole blood is determined. Following preparation of the plasma by centrifugation, the plasma thrombin time and the activated partial thromboplastin time (APTT) are determined with the aid of a coagulometer.
Ecarin clotting time (ECT): 100 μL of citrate blood are incubated for 2 min at 37° C. in a coagulometer (CL 8, ball type, Bender & Hobein, Munich, German Federal Republic). Following the addition of 100 μL of warmed (37° C.) ecarin reagent (Pentapharm), the time taken for a fibrin clot to form is determined.
Activated thromboplastin time (APTT): 50 μL of citrate plasma and 50 μL of PTT reagent (Pathrombin, Behring) are mixed and incubated for 2 min at 37° C. in a coagulometer (CL 8, ball type, Bender & Hobein, Munich, German Federal Republic). Following the addition of 50 μL of warmed (37° C.) calcium chloride, the time taken for a fibrin clot to form is determined.
Thrombin time (TT): 100 μL of citrate-treated plasma are incubated for 2 min at 37° C. in a coagulometer (CL 8, ball type, Bender & Hobein, Munich, German Federal Republic). Following the addition of 100 μL of warmed (37° C.) thrombin reagent (Boehringer Mannheim), the time taken for a fibrin clot to form is determined.
The test probes are dissolved in isotonic salt solution just prior to administration to half-breed dogs. The administration doses are 0.1 ml/kg for intravenous Bolus injection and 1 ml/kg for oral administration, which is carried out per pharyngeal tube. Samples of venous blood (2 mL) are taken in citrate tubules prior to and also 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 300, and 360 min (if required, 420 min, 480 min, and 24 H) after intravenous administration of 1.0 mg/kg or prior to and also 10, 20, 30, 60, 120, 180, 240, 300, 360, 480 min and 24 h after oral dosage of 4.64 mg/kg. Directly after blood sampling, the ecarin clotting time (ECT) in whole blood is determined. Following preparation of the plasma by centrifugation, the plasma thrombin time and the activated partial thromboplastin time (APTT) are determine with the aid of a coagulometer.
In addition, the anti-F-IIa activity (ATU/mL) and the concentration of the substance are determined by their anti-F-IIa activity in the plasma by means of chromogenic (S 2238) thrombin assay, calibration curves with r-hirudin and the test substance being used.
The plasma concentration of the test probe forms the basis of calculation of the pharmacokinetic parameters: time to maximum plasma concentration (T max), maximum plasma concentration; plasma half-life, t0.5; area under curve (AUC); and resorbed portion of the test probe (F).
Ecarin clotting time (ECT): 100 μL citrate-treated blood are incubated for 2 min at 37° C. in a coagulometer (CL 8, ball type, Bender & Hobein, Munich, German Federal Republic). Following the addition of 100 μL of warmed (37° C.) ecarin reagent (Pentapharm), the time taken for a fibrin clot to form is determined.
Activated thromboplastin time (APTT): 50 μL citrate-treated plasma and 50 μL of PTT reagent (Pathrombin, Behring) are mixed and incubated for 2 min at 37° C. in a coagulometer (CL 8, ball type, Bender & Hobein, Munich, German Federal Republic). Following the addition of 50 μL of warmed (37° C.) calcium chloride, the time taken for a fibrin clot to form is determined.
Thrombin time (TT): 100 μL of citrate-treated plasma is incubated for 2 min at 37° C. in a coagulometer (CL 8, ball type, Bender & Hobein, Munich, German Federal Republic). Following the addition of 100 μL of warmed (37° C.) thrombin reagent (Boehringer Mannheim), the time taken for a fibrin clot to form is determined.
The present invention relates to peptide substances and peptidomimetic substances, to the preparation thereof, and to the use thereof as thrombin inhibitors or complement inhibitors. In particular, the substances concerned are those having an amidine group as terminal group on the one hand and a polyhydroxyalkyl or polyhydroxcycloalkyl group—which can comprise several units—as the second terminal group on the other hand.
The invention relates to the use of these novel substances for the production of thrombin inhibitors, complement inhibitors, and, specifically, inhibitors of C1s and C1r.
In particular, the invention relates to the use of chemically stable substances of the general formula I, to their tautomers and pharmacologically compatible salts and prodrugs for the production of medicinal drugs for the treatment and prophylaxis of diseases which can be alleviated or cured by partial or complete inhibition, particularly selective inhibition, of thrombin or C1s and/or C1r.
Formula I has the general structure
A-B-D-E-G-K-L (I),
in which
A stands for H, CH3, H—(RA1)iA
NH—(CH2)nK-QK
Preference is given to the following compounds of formula I
A-B-D-E-G-K-L (I),
in which
A stands for H or H—(RA1)iA
A-B stands for
NH—(CH2)nK-QK
and
L stands for
Preferred thrombin inhibitors are compounds of formula I
A-B-D-E-G-K-L (I),
NH—CH2-QK
Preferred complement inhibitors are compounds of formula I
A-B-D-E-G-K-L (I),
in which
A stands for H or H—(RA1)iA
A-B stands for
NH—CH2-QK
Particularly preferred thrombin inhibitors are compounds of formula I
A-B-D-E-G-K-L (I),
in which
A stands for H or H—(RA1)iA
NH—CH2-QK
and
L stands for
Particularly preferred complement inhibitors are compounds of formula I
A-B-D-E-G-K-L (I),
in which
A stands for H or H—(RA1)iA
A-B stands for
NH—CH2-QK
Preferred building blocks A-B are:
The term “C1-x alkyl” denotes any linear or branched alkyl chain containing from 1 to x carbons.
The term “C3-8 cycloalkyl” denotes carbocyclic saturated radicals containing from 3 to 8 carbons.
The term “aryl” stands for carbocyclic aromatics containing from 6 to 14 carbons, particularly phenyl, 1-naphthyl, and 2-naphthyl.
The term “heteroaryl” stands for five-ring and six-ring aromatics containing at least one hetero-atom N, O, or S, and particularly denotes pyridyl, thienyl, furyl, thiazolyl, and imidazolyl; two of the aromatic rings may be condensed, as in indole, N—(C1-3 alkyl)indole, benzothiophene, benzothiazole, benzimidazole, quinoline, and isoquinoline.
The term “Cx-y alkylaryl” stands for carbocyclic aromatics that are linked to the skeleton through an alkyl group containing x, x+1 . . . y−1, or y carbons.
The compounds of formula I can exist as such or be in the form of their salts with physiologically acceptable acids. Examples of such acids are: hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, maleic acid, fumaric acid, succinic acid, hydroxysuccinic acid, sulfuric acid, glutaric acid, aspartic acid, pyruvic acid, benzoic acid, glucuronic acid, oxalic acid, ascorbic acid, and acetylglycine.
The novel compounds of formula I are competitive inhibitors of thrombin or the complement system, especially C1s, and also C1r.
The compounds of the invention can be administered in conventional manner orally or parenterally (subcutaneously, intravenously, intramuscularly, intraperitoneally, or rectally). Administration can also be carried out with vapors or sprays applied to the postnasal space.
The dosage depends on the age, condition, and weight of the patient, and also on the method of administration used. Usually the daily dose of the active component per person is between approximately 10 and 2000 mg for oral administration and between approximately 1 and 200 mg for parenteral administration. These doses can take the form of from 2 to 4 single doses per day or be administered once a day as depot.
The compounds can be employed in commonly used galenic solid or liquid administration forms, eg, as tablets, film tablets, capsules, powders, granules, dragees, suppositories, solutions, ointments, creams, or sprays. These are produced in conventional manner. The active substances can be formulated with conventional galenic auxiliaries, such as tablet binders, fillers, preserving agents, tablet bursters, flow regulators, plasticizers, wetters, dispersing agents, emulsifiers, solvents, retarding agents, antioxidants, and/or fuel gases (cf H. Sucker et al.: Pharmazeutische Technologie, Thieme-Verlag, Stuttgart, 1978). The resulting administration forms normally contain the active substance in a concentration of from 0.1 to 99 wt %.
The term “prodrugs” refers to compounds which are converted to the pharmacologically active compounds of the general formula I in vivo (eg, first pass metabolisms).
Where, in the compounds of formula I, RL1 is not hydrogen, the respective substances are prodrugs from which the free amidine or guanidine compounds are formed under in vivo conditions. If ester functions are present in the compounds of formula I, these compounds can act, in vivo, as prodrugs, from which the corresponding carboxylic acids are formed.
Apart from the substances mentioned in the examples, the following compounds are very particularly preferred and can be produced according to said manufacturing instructions:
Abu: 2-aminobutyric acid
AIBN: azobisisobutyronitrile
Ac: acetyl
Acpc: 1-aminocyclopentane-1-carboxylic acid
Achc: 1-aminocyclohexane-1-carboxylic acid
Aib: 2-aminoisobutyric acid
Ala: alanine
b-Ala: beta-alanine (3-aminopropionic acid)
am: amidino
amb: amidinobenzyl
4-amb: 4-amidinobenzyl (p-amidinobenzyl)
Asp: aspartic acid
Aze: azetidine-2-carboxylic acid
Bn: benzyl
Boc: tert-butyloxycarbonyl
Bu: butyl
Cbz: carbobenzoxy
Cha: cyclohexylalanine
Chea: cycloheptylalanine
Cheg: cycloheptylglycine
Chg: cyclohexylglycine
Cpa: cyclopentylalanine
Cpg: cyclopentylglycine
d: doublet
Dab: 2,4-diaminobutyric acid
Dap: 2,3-diaminopropionic acid
DC: thin-layer chromatography
DCC: dicyclohexylcarbodiimide
Dcha: dicyclohexylamine
DCM: dichloromethane
Dhi-1-COOH: 2,3-dihydro-1H-isoindole-1-carboxylic acid
DMF: dimethylformamide
DIPEA: diisopropylethylamine
EDC: N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide
Et: ethyl
Eq: equivalent
Gly: glycine
Glu: glutamic acid
fur: furan
guan: guanidino
ham: hydroxyamidino
HCha: homocyclohexylalanine, 2-amino-4-cyclohexylbutyric acid
His: histidine
HOBT: hydroxylbenzotriazol
HOSucc: hydroxysuccinimide
HPLC: high-performance liquid chromatography
Hyp: hydroxyproline
Ind-2-COOH: indoline-2-carboxylic acid
iPr: isopropyl
Leu: leucine
Lsg: solution
Lys: lysine
m: multiplet
Me: methyl
MPLC: medium-performance liquid chromatography
MTBE: methyl-tert-butyl ether
Nva: norvaline
Ohi-2-COOH: octahydroindole-2-carboxylic acid
Ohii-1-COOH: octahydro-isoindole-1-carboxylic acid
Orn: ornithine
Oxaz: oxazole
p-amb: p-amidinobenzyl
Ph: phenyl
Phe: phenylalanine
Phg: phenylglycine
Pic: pipecolic acid
pico: picolyl
PPA: propylphosphonic anhydride
Pro: proline
Py: pyridine
Pyr: 3,4-dehydroproline
q: quartet
RP-18: reversed phase C18
RT: room temperature
s: singlet
Sar: sarcosine (N-methylglycine)
sb: singlet broad
t: triplet
t: tertiary (tert)
tBu: tert-butyl
tert: tertiary (tert)
TBAB: tetrabutylammonium bromide
TEA: triethylamine
TFA: trifluoroacetic acid
TFAA: trifluoroacetic anhydride
thiaz: thiazole
Thz-2-COOH: 1,3-thiazolidine-2-carboxylic acid
Thz-4-COOH: 1,3-thiazolidine-4-carboxylic acid
thioph: thiophene
1-Tic: 1-tetrahydro-isoquinoline carboxylic acid
3-Tic: 3-tetrahydro-isoquinoline carboxylic acid
TOTU: O-(cyanoethoxycarbonylmethylene)amino-1-N,N,N′,N′-tetramethyluronium tetrafluoroboronate(?)
Z: carbobenzoxy
The compounds of formula I can be represented by schemes I and II.
The building blocks A-B, D, E, G and K are preferably made separately and used in a suitably protected form (cf scheme I, which illustrates the use of orthogonal protective groups (P or P*) compatible with the synthesis method used.
Scheme I describes the linear structure of the molecule I achieved by elimination of protective groups from P-K-L* (L* denotes CONH2, CSNH2, CN, C(═NH)NH—COOR*; R* denotes a protective group or polymeric carrier with spacer (solid phase synthesis)), coupling of the amine H-K-L* to the N-protected amino acid P-G-OH to form P-G-K-L*, cleavage of the N-terminal protective group to form H-G-K-L*, coupling to the N-protected amino acid P-E-OH to produce P-E-G-K-L*, re-cleavage of the N-terminal protective group to form H-E-G-K-L* and optionally re-coupling to the N-protected building block β-D-U (U=leaving group) to form β-D-E-G-K-L*, if the end product exhibits a building block D.
If L* is an amide, thioamide or nitrile function at this synthesis stage, it will be converted to the corresponding amidine or hydroxyamidine function, depending on the end product desired. Amidine syntheses for the benzamidine, picolylamidine, thienylamidine, furylamidine, and thiazolylamidine compounds of the structure type I starting from the corresponding carboxylic acid amides, nitriles, carboxythioamides, and hydroxyamidines have been described in a number of patent applications (cf, for example, WO 95/35309, WO 96/178860, WO 96/24609, WO 96/25426, WO 98/06741, and WO 98/09950.
After splitting-off the protective group P to form H-(D)-E-G-K-L* (L* denotes C(═NH)NH, C(═NOH)NH, or (═NH)NH—COOR*; R* denotes a protective group or a polymeric carrier with spacer (solid-phase synthesis), coupling is effected to the optionally protected (P)-A-B-U building block (U=leaving group) or by hydroalkylation with (P)-A-B′-U (U=aldehyde, ketone) to produce (P)-A-B-(D)-E-G-K-L*.
Any protective groups still present are then eliminated. If L* denotes a C(═NH)NH spacer polymer support, these compounds are eliminated from the polymeric support in the final stage, and the active substance is thus liberated.
Scheme II describes an alternative route for the preparation of the compounds I by convergent synthesis. The appropriately protected building blocks P-D-E-OH and H-G-K-L* are linked to each other, the resulting intermediate product P-D-E-G-K-L* is converted to P-D-E-G-K-L* (L* denotes C(═NH)NH, C(═NOH)NH, or (═NH)NH—COOR*; R* denotes a protective group or a polymeric support with spacer (solid-phase synthesis), the N-terminal protective group is eliminated, and the resulting product H-D-E-G-K-L* is converted to the end product according to scheme I.
The N-terminal protective groups used are Boc, Cbz, or Fmoc, and C-terminal protective groups are methyl, tert-butyl and benzyl esters. Amidine protective groups for the solid-phase synthesis are preferably Boc, Cbz, and derived groups. If the intermediate products contain olefinic double bonds, then protective groups that are eliminated by hydrogenolysis are unsuitable.
The necessary coupling reactions and the conventional reactions for the provision and removal of protective groups are carried out under standardized conditions used in peptide chemistry (cf M. Bodanszky, A. Bodanszky, “The Practice of Peptide Synthesis”, 2nd Edition, Springer Verlag Heidelberg, 1994).
Boc protective groups are eliminated by means of dioxane/HCl or TFA/DCM, Cbz protective groups by hydrogenolysis or with HF, and Fmoc protective groups with piperidine. Saponification of ester functions is carried out with LiOH in an alcoholic solvent or in dioxane/water. tert-Butyl esters are cleaved with TFA or dioxane/HCl.
The reactions were monitored by DC, in which the following mobile solvents were usually employed:
If column separations are mentioned, these separations were carried out over silica gel, for which the aforementioned mobile solvents were used.
Reversed phase HPLC separations were carried out with acetonitrile/water and HOAc buffer.
The starting compounds can be produced by the following methods:
The compounds used as building blocks A-B are for the most part commercially available sugar derivatives. If these compounds have several functional groups, protective groups are introduced at the required sites. If desired, functional groups are converted to reactive groups or leaving groups (eg, carboxylic acids to active esters, mixed anhydrides, etc.), in order to make it possible to effect appropriate chemical linking to the other building blocks. The aldehyde or keto function of sugar derivatives can be directly used for hydroalkylation with the terminal nitrogen of building block D or E.
The synthesis of building blocks D is carried out as follows:
The building blocks D—4-aminocyclohexanoic acid, 4-aminobenzoic acid, 4-aminomethylbenzoic acid, 4-aminomethylphenylacetic acid, and 4-aminophenylacetic acid—are commercially available.
The synthesis of the building blocks E was carried out as follows:
The compounds used as building blocks E—glycine, (D)- or (L)-alanine, (D)- or (L)-valine, (D)-phenylalanine, (D)-cyclohexylalanine, (D)-cycloheptylglycine, D-diphenylalanine, etc. are commercially available as free amino acids or as Boc-protected compounds or as the corresponding methyl esters.
Preparation of cycloheptylglycine and cyclopentylglycine was carried out by reaction of cycloheptanone or cyclopentanone respectively with ethyl isocyanide acetate according to known instructions (H.-J. Prätorius, J. Flossdorf, M. Kula, Chem. Ber. 1985, 108, 3079, or U. Schöllkopf and R. Meyer, Liebigs Ann. Chem. 1977, 1174). Preparation of (D)-dicyclohexylalanine was carried out by hydrogenation after T. J. Tucker et al, J. Med. Chem. 1997, 40., 3687-3693.
The said amino acids were provided by well-known methods with an N-terminal or C-terminal protective group depending on requirements.
Synthesis of the building blocks G was carried out as follows:
The compounds used as building blocks G—(L)-proline, (L)-pipecolinic acid, (L)-4,4-difluoroproline, (L)-3-methylproline, (L)-5-methylproline, (L)-3,4-dehydroproline, (L)-octahydroindole-2-carboxylic acid, (L)-thiazolidine-4-carboxylic acid, and (L)-azetidine carboxylic acid—are commercially available as free amino acids or as Boc-protected compounds or as corresponding methyl esters.
(L)-Methyl thiazolidine-2-carboxylate was prepared after R. L. Johnson, E. E. Smissman, J. Med. Chem. 21, 165 (1978).
Synthesis of the building blocks K was carried out as follows:
Preparation of this building block was carried out as described in WO 95/35309.
Preparation of this building block was carried out as described in WO 96/25426 or WO 96/24609.
Preparation of this building block was carried out as described in WO 95/23609.
Preparation of this building block was carried out starting from 2-formyl-4-cyanothiophen in a manner similar to that described for 2-formyl-5-cyanothiophen (WO 95/23609).
Preparation was carried out according to G. Videnov, D. Kaier, C. Kempter and G. Jung, Angew. Chemie (1996) 108, 1604, where the N-Boc-protected compound described in said reference was deprotected with ethereal hydrochloric acid in dichloromethane.
Preparation of this building block was carried out as described in WO 96/17860.
Preparation of this building block was carried out as described in WO 96/17860.
Preparation of this building block was carried out as described in WO 99/37668.
Preparation of this building block was carried out as described in WO 99/37668.
Preparation of this building block was carried out as described in WO 99/37668.
Preparation of this building block was carried out as described in WO 99/37668.
1H-NMR (DMSO-d6, in ppm): 8.16 (s, 1H, NH), 7.86 (t, broad, 1H, NH), 7.71 and 7.59 (2×s, broad, each 1H, NH2), 4.42 (d, 2H, CH2), 1.41 (s, 9H, tert-butyl)
1H-NMR (DMSO-d6, in ppm): 8.98 (s, broad, 2H, NH2), 8.95 (s, 1 h, Ar—H), 4.50 (s, 2H, CH2)
Synthesis of this compound was carried out starting from 5-aminomethyl-3-cyanothiophene by reaction with (Boc)2O to form 5-tert-butyl-oxycarbonylaminomethyl-3-cyanothiophene, conversion of the nitrile function to the corresponding thioamide by the addition of hydrogen sulfide, methylation of the thioamide function with iodomethane, reaction with ammonium acetate to produce the corresponding amidine followed by protective group elimination with hydrochloric acid in isopropanol to give 5-aminomethyl-3-amidinothiophene bishydrochloride.
Building blocks for solid-phase synthesis:
3-Amidino-5-aminomethylthiophene bishydrochloride (1.3 g, 5.7 mmol) was placed in DMF (15 mL), and N,N-diisopropylethylamine (0.884 g, 6.84 mmol) was added. Following stirring for 5 min at room temperature there were added acetyldimedone (1.25 g, 6.84 mmol) and trimethoxymethane (3.02 g, 28.49 mmol). Stirring was continued for 2.5 h at room temperature, after which the DMF was removed in high vacuum and the residue was stirred with DCM (5 mL) and petroleum ether (20 mL). The solvent was decanted from the pale yellow product and the solid matter was dried in vacuo at 40° C. Yield: 1.84 g (5.2 mmol, 91%).
1H-NMR (400 MHz, [D6]DMSO, 25° C., TMS): delta=0.97 (s, 6H); 2.30 (s, 4H); 2.60 (s, 4H); 4.96 (d, J=7 Hz, 2H); 7.63 (s, 1H); 8.60 (s, 1H); 9.07 (sbr, 2H); 9.37 (sbr, 1H).
The synthesis of the H-G-K-CN building block is exemplarily described in WO 95/35309 for prolyl-4-cyanobenzylamide, in WO 98/06740 for 3,4-dehydroprolyl-4-cyanobenzylamide and in WO 98/06741 for 3,4-dehydroprolyl-5-(2-cyano)thienylmethylamide. The preparation of 3,4-dehydroprolyl-5-(3-cyano)thienylmethylamide is similarly carried out by coupling Boc-3,4-dehydroproline to 5-aminomethyl-3-cyanothiophen hydrochloride followed by protective group elimination.
The synthesis of 3,4-dehydroprolyl[2(4-cyano)thiazolmethyl]amide hydrochloride was carried out by coupling Boc-3,4-dehydroproline to 2-aminomethyl-4-cyanothiazole hydrochloride followed by protective group elimination.
The synthesis of the building block H-E-G-K-C(═NOH)NH2 is exemplarily described for H-(D)-Cha-Pyr-NH—CH2-2-(4-ham)thiaz
The synthesis of the H-E-G-K-C(═NH)NH2 building block is exemplarily described for H-(D)-Cha-Pyr-NH—CH2-2(4-am)thiaz.
The preparation of the building block H-E-G-K-C(═NH)NH2H-(D)-Chg-Aze-NH 4-amb is described in WO 94/29336 Example 55. H-(D)-Chg-Pyr-NH—CH25-(3-am)-thioph was synthesized in a similar manner to that used for H-(D)-Cha-Pyr-NH—CH2-2-(4-am)-thiaz, the formation of amidine being effected using the corresponding nitrile precursor Boc-(D)-Chg-Pyr-NH—CH2-5-(3-CN)-thioph as described in WO 9806741 Example 1 via intermediate stages Boc-(D)-Chg-Pyr-NH—CH2-5-(3CSNH2)-thioph and Boc-(D)-Chg-Pyr-NH—CH2-5-(3—C(═NH)S—CH3)-thioph.
H-(D)-Cha-Pyr-NH—CH2-2-(4-am)-thiaz dihydrochloride (2.0 g, 4.19 mmol) was dissolved in methanol (30 mL), and to the solution there were added D-(−)-arabinose (0.63 g, 4.19 mmol) and molecular sieve (4 Angstrom). The mixture was stirred over a period of 1 h at room temperature and sodium cyanoborohydride was then added portionwise, during which operation slight generation of gas occurred. Following stirring overnight at room temperature, the molecular sieve was filtered off in vacuo, the filtrate concentrated in vacuo and the residue stirred in acetone. The crude product filtered off in vacuo was purified by means of MPLC(RP-18 column, acetonitrile/watter/glacial acetic acid) and then lyophilized to give 840 mg of (D)-Arabino-(D)-Cha-Pyr-NH—CH2-2-(4-am)thiaz×CH2COOH as a white solid (yield 34%).
ESI-MS: M+H+: 539
This compound was synthesized in a manner similar to that described in Example 1 but starting from L-(+)-arabinose.
ESI-MS: M+H+: 539
This compound was synthesized in a manner similar to that described in Example 1 but starting from D-(+)-erythrose.
ESI-MS: M+H+: 509
This compound was synthesized in a manner similar to that described in Example 1 but starting from L-(+)-erythrose.
ESI-MS: M+H+: 509
This compound was synthesized in a manner similar to that described in Example 1 but starting from D-(+)-glycerinaldehyde.
ESI-MS: M+H+: 479
This compound was synthesized in a manner similar to that described in Example 1 but starting from L-(+)-glycerinaldehyde.
ESI-MS: M+H+: 479
This compound was synthesized in a manner similar to that described in Example 1 but starting from L-rhamnose.
L-rhamnnose (0.82 g, 5 mmol) was dissolved in water (20 mL) at room temperature and H-(D)-Cha-Pyr-NH—CH2-2(4-am)thiaz dihydrochloride (2.4 g, 5 mmol) was stirred in. The clear solution became viscous after 20 min. Sodium cyanoborohydride was added portionwise in an equimolar amount over a period of 4 h to give a white precipitate, which dissolved on the addition of ethanol (2 mL). 5 mL of 1M HCl set the pH to 3 and solid was precipitated 3 times with 300 mL of acetone each time. The solid was removed by centrifugation and dissolved in water (100 mL). Following lyophilization there were obtained 2.6 g of (L)Rhamno-(D)-Cha-Pyr-NH—CH2-2-(4-am)-thiaz xHCl as a white powder.
This compound was synthesized in a manner similar to that described in Example 7 but starting from D-melibiose.
D-melibiose (1.8 g, 5 mmol) was dissolved in water (20 mL) at room temperature and H-(D)-Cha-Pyr-NH—CH2-2-(4-am)-thiaz dihydrochloride (2.4 g, 5 mmol) was stirred in. The clear pale yellow solution became viscous after 20 min. An equimolar amount of sodium cyanoborohydride was added portionwise over a period of 4 h. There was obtained a white solid precipitate, to which 2 mL of ethanol were added to give a clear solution. The pH was set to pH 5 with 5 mL of 1M HCl and precipitation was effected 3 times with 300 mL of acetone each time. Following centrifugation, the sediment obtained was taken up in 100 mL of water and the solution lyophilized. Yield: 3.2 g of (D)-Melibio-(D)-Cha-PyrNH—CH2-2-(4-am)-thiaz×HCl.
This compound was synthesized in a manner similar to that described in Example 7 but starting from D-glucose.
D-glucose (1.0 g, 5.6 mmol) was dissolved in 20 mL of water at room temperature and H-(D)-Chg-Pyr-NH—CH2-5-(3-am)-thioph dihydrochloride (3.0 g, 6.5 mmol) was stirred in. The clear solution became viscous after 10 min. An equimolar amount of sodium cyanoborohydride was added portionwise over a period of 4 h to give a white precipitate. After cooling in an ice bath with 3×5 mL of H2O the mixture were shaken and the sediment was taken up in 20 mL of H2O and the pH set to pH 5.0 with ca 5 mL of 0.1 M NaOH. 1st precipitation using 300 mL of acetone. 2nd precipitation: the sediment was taken up in 30 mL of H2O and 300 mL of acetone were added. The sediment was dissolved in H2O and neutralized with 2 mL of 1M HCl; the solution was then lyophilized. Yield: 1.52 g (D)-Gluco-(D)-Chg-Pyr-NH—CH2-5-(3-am)-thioph×HCl als weiβes Pulver.
This compound was synthesized in a manner similar to that described in Example 7 but starting from maltohexaose.
Maltohexaose (2 g, 2 mmol) was dissolved in water (20 mL) at room temperature and H-(D)-Chg-Pyr-NH—CH2-5-(3-am)-thioph dihydrochloride (0.92 g, 2 mmol) was stirred in. The clear solution became viscous after 10 min; an equimolar amount of sodium cyanoborohydride was added portionwise over a period of 4 h; after cooling in an ice bath, precipitation was effected with 8 volumes of ethanol. The sediment was reprecipitated with 300 mL of ethanol. The sediment was dissolved in water and the solution lyophilized.
This compound was synthesized in a manner similar to that described in Example 7 but starting from cellobiose.
Cellobiose (2 g, 6 mmol) was stirred into water (20 mL) at 50° C. and H-(D)-Chg-Pyr-NH—CH2-5-(3-am)-thioph dihydrochloride (2.8 g, 6 mmol) added. The turbid solution became viscous as an equimolar amount of sodium cyanoborohydride was added portionwise over a period of 4 h. Stirring was continued for approximately one hour at 50° C. Approximately 10 mL of 1M HCl were added to set the pH to 3. Precipitation was then effected twice with 300 mL of acetone. Following cooling in an ice bath, the sediment was taken up in 60 mL of water and reprecipitated with 600 mL of acetone. The sediment was dissolved in water and the solution lyophilized. Yield: 4.4 g (D)-Cello-bio-(D)-Chg-Pyr-NH—CH2-5(3-am)-thioph×HCl.
This compound was synthesized in a manner similar to that described in Example 7 but starting from the sodium salt of D-glucuronic acid.
The sodium salt of D-glucuronic acid×H2O (1.4 g, 6 mmol) was dissolved in water (20 mL) at room temperature and H-(D)-Chg-Pyr-NH—CH2-5-(3-am)thioph dihydrochloride (2.8 g, 6 mmol) was stirred in at room temperature. The clear solution turned pale yellow after 10 min. An equimolar amount of 330 mg of sodium cyanoborohydride was added portionwise over a period of 4 h to give a solid, compact precipitate. 4 mL of 0.1 M NaOH were added and the supernatant was decanted off and the precipitate stirred up in acetone. The sediment was taken up in 40 mL of H2O and 3 mL of 1M HCl were added to give a pH of 4. The compound passed into solution. Precipitation was effected with 400 mL of acetone. The sediment was then dissolved in water and the solution lyophilized. Yield: 3.1 g (D)-Glucuronic-(D)-Chg-Pyr-NH—CH2-5(3-am)-thioph.
This compound was synthesized in a manner similar to that described in Example 7 but starting from D-glucose.
D-glucose (2.5 g, 14 mmol) was dissolved in water (40 mL) at room temperature and H-(D)-Chg-Aze-NH-4-amb (WO 94/29336 Example 55; 6.8 g; 15.4 mmol) was stirred in. An equimolar amount of sodium cyanoborohydride was added portionwise over a period of 4 h and the mixture was then stirred overnight. There was obtained a greasy, viscous emulsion. 50 mL of water were added, after which ethanol was added until the solution became clear.
The pH was adjusted to 4.0 with ca 15 mL of 0.1M HCl. 1st precipitation using 600 mL of acetone. 2nd precipitation: the sediment was taken up in 50 mL of water and 600 mL of acetone were added; the sediment was redissolved in water and the solution lyophilized. Yield: 7.8 g (D)-Gluco-(D)-Chg-Aze-NH-4-amb×HCl.
This compound was synthesized in a manner similar to that described in Example 7 but starting from maltose.
Maltose×H2O (5 g, 14 mmol) was dissolved in 40 mL of water at room temperature and H-Chg-Aze-NH-4-amb (6.8 g; 15.4 mmol) was stirred in. There followed a portionwise addition of an equimolar amount of sodium cyanoborohydride over a period of 4 h. The initially clear, viscous solution slowly changed to a greasy, viscous emulsion. 50 mL of water were added followed by ca 15 mL 0.1 M HCl to give a pH of 4.0. 1st precipitation using 600 mL of acetone. 2nd precipitation: the sediment was taken up in 50 mL of water and 600 mL of acetone were added; the sediment was redissolved in water and the solution lyophilized. Yield: 10.1 g Malto-(D)-Chg-Aze-NH-4-amb×HCl.
This compound was synthesized in a manner similar to that described in Example 1 but starting from L-(+)-erythrose and H-(D)-Cha-Pyr-NH—CH2-2-(4-ham)thiaz.
ESI-MS: M+H+: 525
This compound was synthesized in a manner similar to that described in Example 1 but starting from L-(+)-arabinose and H-(D)-Cha-Pyr-NH—CH2-2-(4-ham)thiaz.
ESI-MS: M+H+: 555
This compound was synthesized in a manner similar to that described in Example 1 but starting from maltose.
H-(D)-Cha-Pyr-NH—CH2-2-(4-ham)-thiaz Maltose×H2O (2.2 g, 6 mmol) was dissolved in 40 mL of water and 60 mL of ethanol at room temperature and H-(D)-Cha-Pyr-NH—CH2-2-(4-ham)-thiaz (2.8 g, 6.6 mmol) was stirred in. The portionwise addition of an equimolar amount of sodium cyanoborohydride over a period of 8 h gave a highly viscous, clear, brownish solution. 1st precipitation using 500 mL, of acetone. The sediment was dissolved in 50 mL of water and set to pH 7.5 with 0.1 M of HCl followed by precipitation with 500 mL of acetone. The sediment was dissolved in 100 mL of water and the solution lyophilized. Yield: 3.6 g Malto-(D)-Cha-Pyr-NH—CH2-2-(4-ham)thiaz.
For the following compounds, the thrombin time was determined according to Example A:
| Number | Date | Country | Kind |
|---|---|---|---|
| 10049937.6 | Oct 2000 | DE | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12850545 | Aug 2010 | US |
| Child | 13277829 | US | |
| Parent | 10398269 | Aug 2003 | US |
| Child | 12850545 | US |
