5-Chlorothiophene-2-carboxylic Acid [(S)-2-[2 ... - ACS Publications

Article pubs.acs.org/jmc

5‑Chlorothiophene-2-carboxylic Acid [(S)‑2-[2-Methyl-3-(2oxopyrrolidin-1-yl)benzenesulfonylamino]-3-(4-methylpiperazin-1yl)-3-oxopropyl]amide (SAR107375), a Selective and Potent Orally Active Dual Thrombin and Factor Xa Inhibitor Jerome Meneyrol,† Markus Follmann,#,∥ Gilbert Lassalle,*,† Volkmar Wehner,∥ Guillaume Barre,† Tristan Rousseaux,† Jean-Michel Altenburger,‡ Frederic Petit,‡ Zsolt Bocskei,∞,§ Herman Schreuder,∥ Nathalie Alet,† Jean-Pascal Herault,† Laurence Millet,† Frederique Dol,† Peter Florian,∥ Paul Schaeffer,† Freddy Sadoun,⊥ Sylvie Klieber,⊥ Christophe Briot,⊥ Françoise Bono,† and Jean-Marc Herbert† †

Sanofi-Aventis R&D, 195 Route d’Espagne, 31036 Toulouse Cedex, France Sanofi-Aventis R&D, 1 Av. Pierre Brossolette, 91385 Chilly-Mazarin Cedex, France § Sanofi-Aventis R&D, 16 Rue d’Ankara, 67080 Strasbourg Cedex, France ∥ Sanofi-Aventis R&D, Industriepark Höchst, 65926 Frankfurt am Main, Germany ⊥ Sanofi-Aventis R&D, 371, Rue du Professeur Joseph Blayac, 34184 Montpellier Cedex 04, France ‡

S Supporting Information *

ABSTRACT: Compound 15 (SAR107375), a novel potent dual thrombin and factor Xa inhibitor resulted from a rational optimization process. Starting from compound 14, with low factor Xa and modest anti-thrombin inhibitory activities (IC50’s of 3.5 and 0.39 μM, respectively), both activities were considerably improved, notably through the incorporation of a neutral chlorothiophene P1 fragment and tuning of P2 and P3−P4 fragments. Final optimization of metabolic stability with microsomes led to the identification of 15, which displays strong activity in vitro vs factor Xa and thrombin (with Ki’s of 1 and 8 nM, respectively). In addition 15 presents good selectivity versus related serine proteases (roughly 300-fold), including trypsin (1000-fold), and is very active (0.39 μM) in the thrombin generation time (TGT) coagulation assay in human platelet rich plasma (PRP). Potent in vivo activity in a rat model of venous thrombosis following iv and, more importantly, po administration was also observed (ED50 of 0.07 and 2.8 mg/kg, respectively). Bleeding liability was reduced in the rat wire coil model, more relevant to arterial thrombosis, with 15 (blood loss increase of 2-fold relative to the ED80 value) compared to rivaroxaban 2 and dabigatran etexilate 1a.

■

INTRODUCTION Thrombin (factor IIa) and factor Xa1 occupy central positions in the blood coagulation cascade and play prominent roles in various thromboembolic complications. Their importance is reflected in the wide clinical use of heparin and its derivatives and through the arrival on the market of a new generation of approved oral anticoagulants as exemplified by the selective thrombin inhibitor dabigatran etexilate2 1a (Boehringer Ingelheim) and by the selective factor Xa inhibitor rivaroxaban3a−c 2 (Bayer). Recently, two novel factor Xa inhibitors have been approved: edoxaban3d 3 (Daiichi-Sankyo), in Japan for once-daily venous thromboembolism (VTE) prevention, and apixaban3e,f 4 (Bristol-Myers Squibb/Pfizer), in Europe for preventing VTE and to reduce the risk of stroke and blood clots in patients with atrial fibrillation (Figure 1). Hexadecasaccharide 5 (SR123781)4 is a synthetic heparin mimetic that inhibits both factor Xa and thrombin via © 2013 American Chemical Society

antithrombin III (Figure 2) that was advanced to phase I clinical trials. This dual inhibitor demonstrated superior antithrombotic properties in three rat models of thrombosis following iv administration compared to selective factor Xa pentasaccharide fondaparinux (SR90107/Org31540). In addition, novel anti-Xa pentasaccharides coupled to active site thrombin inhibitors to obtain dual inhibitory effects have also been reported.5 Various efforts to obtain orally active small molecules with dual anti-Xa and anti-thrombin properties have been described in the literature (Figure 2). Historically, a first report6 from Boehringer-Ingelheim described benzimidazole derivatives bearing a benzamidine fragment with 6 (BIBM-1015) as a potent dual inhibitor, but the low selectivity of the molecule Received: May 22, 2013 Published: October 31, 2013 9441

dx.doi.org/10.1021/jm4005835 | J. Med. Chem. 2013, 56, 9441−9456

Journal of Medicinal Chemistry

Article

Figure 1. Reference selective thrombin andfactor Xa inhibitors.

Figure 2. Molecules with reported dual anti-Xa and anti-IIa activities.

Scheme 1. Summary of Design Strategy

9442



Article

Aventis colleagues and Bayer researchers14 that had developed other series alerted us to the unique binding properties of chlorinated heteroaryls that enable a Cl−π interaction with Tyr-228 at the bottom of the S1 pocket of factor Xa. Consequently (Table 1), Cl-substituted aryl/heteroaryls were incorporated. Among these, the 2-chlorothienyl-4-

versus trypsin and the presence of this benzamidine precluded further development. Rescaffolding toward a quinoxalinone7 7 did not significantly improve these two aspects. Further optimization led to compound 8a (BIBT-1011), an oral double prodrug (n-propyl ester and benzoylamidine) of tanogitran (BIBT-986)8 8b which was studied in phase I trials. However, the bioavailability was considered too low in humans and development of 8a was discontinued. Several other teams were involved in searching for oral dual inhibitors with good bioavailability without requiring prodrug approaches. For example, excellent in vitro and in vivo anticoagulant efficacy of the dual inhibitor oxazolopyridine 9 was demonstrated by a Merck team,9 but unfortunately the compound exhibited very low levels of oral bioavailability in dogs. Dual active dipeptide mimetics10 were discovered based on substrate analogy, with compound 10 as a prototype, but bioavailability in rats was low (300-fold selectivity); close to 30 μM for plasmin and factor IXa; and above 100 μM for the other enzymes. It is roughly 1000-fold less potent on the digestive enzyme trypsin, which possesses a wide open active site and is known to accommodate many inhibitors. The pharmacokinetic properties of 15 were assessed in rats and dogs following both intravenous (iv) and oral (po) administration. As summarized in Table 7, the compound displayed a moderate clearance in dogs and extensive clearance in male Sprague−Dawley rats (1.7−5.8 L h−1 kg−1) with a large volume of distribution when given iv. When given po, a moderate half-life in dogs (2.4 h) and a long one in rats (6.2 h)

Figure 5. Active site region of the crystal structure of 13 in complex with human thrombin. Compound 13 is shown as a stick model with magenta carbons, red oxygens, deep blue nitrogens, yellow sulfurs, and green fluors. Thrombin is shown in an accessible surface representation with cyan carbons and the same colors as for 13 for the noncarbon atoms. The specificity pockets S1−S4 are indicated as well.

restricted because of the presence of Tyr60A of the insertion loop. Looking again with this information at the crystal structure of the thrombin−13 complex,13a we see that the size of the large S2 substituents, like the 4-difluoromethylenepiperidine in 13, needs to be reduced to remain active on factor Xa, where the S2 pocket is restricted due because of Tyr99. The smaller S3 pocket in factor Xa due to the presence of Phe174 explains why 6 with a larger S3-acetamido group is inactive on factor Xa, but our lead 15, with just a methyl at the S3 position, is active. So the S4 residue also needs to be adapted to the smaller S4 pocket in factor Xa. The result of these modifications can be seen in Figure 6e−h where the crystal structures of the same compounds, 57 and 58, in both thrombin and factor Xa are compared. Main hydrogen bonds between the protein and the inhibitors involve the carbonyl next to the S1 moiety with the main chain NH of Gln192, the nitrogen next to this carbonyl and the carbonyl O of Gly219, and finally one of the sulfonyl oxygens and the main chain NH of Gly219. The general mode of binding of 57 is very similar in both thrombin and factor Xa. This is particularly true in S1, where the superposition of the chlorothiophene groups is excellent. The largest difference is observed in the S4 pocket where the inhibitor has a unique conformation in thrombin but is disordered and assumes two different conformations in factor Xa. One of the conformations is identical to the conformation in thrombin (Figure 6e), while in the other conformation, the P2 substituent is 180° rotated around the exocyclic amid bond (Figure 6f). This conformation can be rationalized by the fact that in thrombin conformation the P2 residue is hindered by the presence of the Tyr99 in factor Xa and that in thrombin, the terminal piperidone moiety in the alternative (up in Figure 6e) conformation would be hindered Tyr60A of the insertion loop. Because of the disordered binding mode in factor Xa, the orientation of the piperidone moiety of 57 in factor Xa is not 9448



Article

Figure 6. Comparison of the active sites of thrombin and factor Xa. Shown are the active site regions of the crystal structures of thrombin and trypsin complexes. Thrombin carbon atoms are colored cyan, and factor Xa carbons are colored orange. Non-carbon atoms have the same colors as in Figure 5. The left column of pictures shows the thrombin surface with the superimposed factor Xa structure as a stick model. The right columns of pictures show the factor Xa surface with the superimposed thrombin structure as a stick model. (a, b) Thrombin and factor Xa complexes with 57, with the 57 left out for clarity. (c, d) Thrombin in factor Xa complexes with 57, with the inhibitor shown as a stick model. The binding mode of 57 in factor Xa is disordered, and two conformations of the inhibitor have been fitted. (e, f) Superposition of the thrombin and factor Xa complexes with 57. In panel e, the “thrombin conformation” of 57 in factor Xa is shown. In panel f, the conformation of 57 is shown where the P2 substuent is 180° rotated. (g, h) Superposition of the thrombin and factor Xa complexes with 58. 58 as bound to thrombin has green carbons, and the factor Xa bound conformation has white carbons.

10 and 30 mg/kg revealed strong antithrombotic activity at 4 h postadministration (70% and 80% inhibition of thrombus weight, respectively) and pharmacological activity remaining at 6 h for the highest dose. In the wire-coil model in rats, a model described as more relevant to arterial thrombosis because of a dual coagulation and platelet dependency, the compound exhibited a clear dose effect following oral administration with an ED50 of 16 mg/kg (Figure 11). Moreover, the ED80 value reflecting the more relevant antithrombotic dose was calculated

were observed, overall resulting in a moderate (in rats) to good (in dogs) absolute bioavailability of 21−47%. The in vivo activities of 15 in animal models of thrombosis are represented in Figures 8−11. In the venous Wessler rat model, the compound exhibited a strong and dose-dependent reduction of thrombus weight with an ED50 of 0.07 mg/kg following iv administration (Figure 8) and an ED50 of 2.8 mg/ kg following po administration at 30 min (Figure 9). In the same model, time course analysis (Figure 10) at oral doses of 9449



Article

Figure 8. Dose-dependent effects of 15 in the Wessler model after iv administration in rats.

Figure 7. Superposition of the crystal structures of the factor Xa−58 complex (white carbons) with a peptidic linker and the factor Xa−31 complex (yellow carbons), which has an isoxazole linker.

Table 5. In Vitro Activity of 15 vs Factor Xa and Thrombin in Comparison to Rivaroxaban 2 and Dabigatran 1b Ki (FXa) Ki (FIIa) TGT (PPP rat) TGT (PPP human) TGT (PRP human)

15

rivaroxaban 2

dabigatran 1b

1.0 nM 8.1 nM 250 nM 66 nM 390 nM

0.4 nM >1 μM 370 nM (Imax = 60%) 61 nM 590 nM

>1 μM 4.5 nM 780 nM 130 nM 400 nM

Figure 9. Dose-dependent effects of 15 in the Wessler model after po administration in rats at 30 min.

Table 6. In Vitro Selectivity Profile of 15 against a Panel of Serine Proteases serine protease

calcd Ki (μM)

serine protease

% inh at 100 μM

chymotrypsin p-kallikrein t-PA trypsin plasmin factor IXa

2.4 2.9 3.6 7.0 29 34.2

XIa VIIa C1s FXIIa APC tryptase uPA

30 21 20 20 13 6 1

Figure 10. Time-course of 15 in the Wessler model after po administration of 10 and 30 mg/kg in rats.

increase of 2-fold of blood loss using the ED80 value. Such increase was shown to be higher than 2-fold for dabigatran etexilate 1a and above 3-fold for rivaroxaban 2, suggesting a more favorable nonbleeding profile in rats for 15. Overall, the dual inhibitor 15 potently inhibited thrombus weight formation in two in vivo models of thrombosis, in agreement with its bioavailability in rats.

or estimated for each compound (Table 8). ED80 was higher than 30 mg/kg for rivaroxaban 2, and for comparison, calculated ED80 values for 15 and dabigatran etexilate 1a were 41 and 23 mg/kg, respectively. A bleeding ratio could be also calculated for each compound by comparing the ED80 values and their corresponding blood losses at these doses. Compound 15 displayed a favorable ratio with an estimated

Table 7. Pharmacokinetic Parameters for 15 in Rats and Dogs after iv and po Administration species rat dog

admin

Cl (L/h)

AUC (ng·h/mL)

a

5.8

510 360 1800 2600

iv pob iva pob

1.7

Tmax (h) 0.25 0.5

C0a or Cmaxb (ng/mL)

Vdss (L/kg)

T1/2 (h)

1300 154 3500 1530

4.6

6.6 6.2 1.1 2.4

1.5

F (%) 21 47

a

Given iv at 3 mg/kg as a 0.9% NaCl aqueous solution. bGiven po at 10 mg/kg as a 0.6% methylcellulose/Tween 80 (99.5/0.5; v/v) aqueous solution. 9450



Article

may help to demonstrate the expected beneficial effects in patients from combining inhibition of both factor Xa and thrombin in one single molecule.

■

General Chemistry Information. Melting points were determined using open capillary tubes on a Buchi 530 apparatus and are uncorrected. Merck Kieselgel 60 (230−400 μm) was used for flash chromatography, and Kieselgel 60 F254 silica plates (0.2 mm) were used for TLC. The structures of the compounds were confirmed spectroscopically by proton and carbon NMR (CDCl3, DMSO-d6, CD3OD, or pyridine-d5) with TMS as the internal standard, using a Bruker AC-250 instrument or Bruker Avance 400 or 500 spectrometer, and by their mass spectra (MS-ES) which were recorded on a VG Autospec (Fisons Instruments) spectrometer. The purity of the compounds was assessed to be above 95% by LCMS/UV Gilson/ Micromass ZMD with a quadrupole mass spectrometer equipped with a Z-spray and operating in electrospray ionization mode (column is Symmetry C18 (2.1 mm × 50 mm) 3.5 I.Jm noWAT200650 with solvent: A = 0.005% TFA + H2O, pH 3.05, B = CH3CN + 0.05% TFA (flow 0.4 mU min), gradient 100% A (0 min) to 90% (10 min) and 5.0 min at 100% B, injection volume = 2 μL, sol. 0.5 mg/mL in MeOH, ionization API-ES+ or GC/MS with column Agil HP-5MS 250 μm × 30 m × 0.25 μm, flow Hel cst, 1.4 mL/min, oven t = 0 to t = 1 min at 55 °C and then 10 °C/min to 300 °C and then 5 min at 300 °C and equilibrium. Injection: split, 250 °C, ratio 40:1, injection volume 2 μL. Detection: 300 °C, flow H2 35 mL/min, flow air 350 mL/min, flow makeup He 20 mL/min. Spectra are available on demand when not mentioned. Results of elemental analysis are available on most of the final target molecules and were obtained within ±0.4% of the theoretical values. Data are available on demand when not mentioned. Optical rotations were recorded using a Perkin-Elmer 343 polarimeter with a sodium source. Reagent grade chemicals were purchased from Sigma-AldrichFluka. All solvents were analytical grade, and anhydrous reactions were performed in oven-dried glassware under an atmosphere of argon or nitrogen. Chemical names of the molecules have been generated using ACDName, version 12, from Advanced Chemistry Development, Inc. Synthesis. Route a. 1,3-Dibromo-2-ethylbenzene (39b). A threenecked round-bottom flask was purged with argon and then filled with dry THF (1.2 L), 1,3-dibromobenzene (121 g, 0.514 mol), and ethyl iodide (95.4 g, 0.611 mol). The mixture was cooled to −78 °C, and LDA (348.5 mL, 0.697 mol) (2 M in THF/n-heptane/ethylbenzene) was added slowly in such a way that the temperature did not rise above −65 °C. After being stirred for 2.75 h, the mixture was poured onto 1 L of sat. aq NH4Cl solution and stirred vigorously for 20 min. Double extraction with DCM yielded a colorless oil (167 g) which was used in the next step without further purification. 1H NMR (250 MHz, DMSO) δ ppm 7.65 (d, J = 7.9 Hz, 2H), 7.09 (t, J = 7.9 Hz, 1H), 2.95 (q, J = 7.0 Hz, 2H), 1.12 (t, J = 7.0 Hz, 3H). GC/MS (CL+) m/z = 263 (M+) 1-Benzylsulfanyl-3-bromo-2-ethylbenzene21 (40b). Intermediate 1,3-dibromo-2-ethylbenzene 39b (528 mg, 2 mmol) in 13 mL of THF was cooled to −78 °C and then treated with n-BuLi (1.25 mL, 2 mmol) (1.6 M in heptane). After the mixture was stirred for 15 min at −78 °C, sulfur (64 mg, 2 mmol) was added under argon atmosphere and the reaction temperature was kept at −78 °C for another 30 min. Then benzyl bromide (0.238 mL, 2 mmol) in 2 mL of THF was added, and stirring at −78 °C was continued for 90 min. The reaction was quenched by addition of 10 mL of sat. aq NH4Cl solution and 150 mL of H2O. After three extractions with DCM, the combined organic layers were washed with water, dried with MgSO4, evaporated to dryness and the product was purified by chromatography on silica gel. Yield: 488 mg, 79%. 1H NMR (400 MHz, DMSO) δ ppm 1.02−1.05 (t, 3H, J = 7.4 Hz); 2.80−2.85 (q, 2H, J = 7.2 Hz); 4.25 (s, 2H); 7.07− 7.11 (t, 1H, J = 8 Hz); 7.11−7.41 (m, 7H). LCMS (ESI+) m/z = 307 (M+). 4-(3-Benzylsulfanyl-2-ethylphenyl)morpholin-3-one (41e). Intermediate 1-benzylsulfanyl-3-bromo-2-ethylbenzene (1.075 g, 3.5

Figure 11. Dose-dependent effects of 15 in the wire-coil model after po administration in rats at 30 min.

Table 8. Bleeding Ratio Determined from the Wire Coil Model in Rats wire coil model

ED80 a (mg/kg)

x-fold blood loss for ED80 a

dabigatran etexilate 1a rivaroxaban 2 15

23 >30 41

>2 >3 2

EXPERIMENTAL SECTION

a

Effective dose corresponding to 80% inhibition of the protein content.

■

CONCLUSIONS The positive dose-dependent anticoagulant responses observed after oral administration during phase I clinical development of the discontinued pure anti-thrombin agent 13 (unpublished data), as well as the successful clinical studies of both dabigatran etexilate 1b, as a potent oral anti-thrombin, and rivaroxaban 2, as a selective anti-f.Xa drug, prompted us to try to identify a back-up for our clinical candidate 13 with an improved profile. Encouraged by the antithrombotic performance of the dual-acting inhibitors of thrombin and factor Xa, we sought to combine activities against factor Xa and thrombin to obtain benefit in terms of antithrombotic efficacy and reduce bleeding liability.12,17 Indeed, optimized molecule 15 (Ki’s of 1 and 8 nM for factor Xa and thrombin, respectively) is as active as enoxaparin (data not shown) when administered iv in a venous thrombosis (Wessler) rat model. After oral administration, potent and sustained antithrombotic effects were observed in this model, suggesting potential once-daily dosing in humans. In the wirecoil rat model, a coagulation and platelet dependent model highlighting the roles of both factor Xa and thrombin, compound 15 is also very active after oral administration with a limited effect on bleeding, suggesting an impressive therapeutic window in this acute model. It remains difficult to determine a priori the appropriate balance required between anti-factor Xa and anti-thrombin activities to observe, in humans, anti-thrombotic effects with minimized bleeding risk. Following pharmacological, physicochemical, PK, and ADMET characterization, 15 was selected for preclinical development18 with a potential advantage in terms of bleeding time compared to dabigatran etexilate 1a and rivaroxaban 2. Full detailed in vitro anticoagulant and in vivo antithrombotic profiling of 15 in comparison with these reference drugs will be reported elsewhere. Despite important efforts in various research laboratories, it remained very challenging to identify dual inhibitors of these two key enzymes of the coagulation cascade with sufficient oral bioavailability in animals. The discovery of development candidate 15 with 21−47% bioavailability in rats and dogs 9451



Article

mmol), morpholin-3-one (389 mg, 3.85 mmol), CuI (67 mg, 0.35 mmol), N,N′-dimethylethylenediamine (75 μL, 0.7 mmol), and K2CO3 (1.064 g, 7.7 mmol) were suspended in toluene (30 mL) under argon and heated to 110 °C for 20 h. After cooling to room temperature, the reaction mixture was quenched by addition of 100 mL of sat. aq NH4Cl, 150 mL of concentrated NH3 in water, and 100 mL of water and extracted three times with ethyl acetate. The organic layers were combined and washed with water and sat. aq NaCl solution, dried with MgSO4, filtered, and evaporated to dryness. The resulting oil crystallizes upon standing and was triturated with nheptane/MTBE (19: 1). Yield: 974 mg, 85%. LCMS/UV (ESI+) m/z = 328 (M + H+). 2-Ethyl-3-(3-oxomorpholin-4-yl)benzenesulfonyl Chloride (42e). Intermediate 4-(3-benzylsulfanyl-2-ethylphenyl)morpholin-3-one 41e (197 mg, 0.6 mmol) was dissolved in 4 mL of DCM and treated with water (44 μL, 2.4 mmol), AcOH (138 μL, 2.4 mmol), and SO2Cl2 (193 μL, 2.4 mmol) at 0 °C. After being stirred for 5 min at 0 °C and 90 min at room temperature, the mixture was cooled back to 0 °C and quenched by addition of 10 mL of water. The aqueous solution was extracted with DCM (three times), and combined organic layers were washed with cold water. Drying over MgSO4 and evaporation to dryness yielded 209 mg of crude intermediate which was used without further purification in the next step. Route b. 2-(3-Bromo-2-methylphenyl)pyridine (43a). A solution of i-PrMgCl (27.49 mL, 2 M in THF, 55 mmol) was added dropwise to commercially available 1,3-dibromo-2-methylbenzene 39a (12.5 g, 50 mmol) at room temperature under argon and then heated at 65 °C for 1.5 h. The mixture was added via a syringe to a suspension of dry ZnCl2 (6.83 g, 50 mmol) in dry THF (20 mL) and cooled to 0 °C under argon. The resulting suspension was stirred at room temperature for 30 min. Then 2-bromopyridine (4.78 mL, 50 mmol) and Pd(dppf)Cl2 (2.03 g) were added and the mixture was refluxed for 2 h. The reaction mixture was then quenched by the addition of a 5% solution of citric acid (200 mL) and extracted with ethyl acetate (2 × 250 mL). The combined organic layers were consecutively washed with a 5% solution of citric acid (150 mL) and brine (150 mL), dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude material was chromatographed on silica gel. Yield: 7.7 g, 78%. 1H NMR (250 MHz, DMSO) δ ppm 8.69 (ddd, J = 4.91, 0.8, 1.0 Hz, 1H), 7.92 (td, J = 7.8 Hz, 1.8 Hz, 1H), 7.69 (dd, J = 8.1, 1.4 Hz, 1H), 7.41 (m, 2H), 7.25 (t, J = 7.9 Hz, 1H), 2.32 (s, 3H). LCMS/UV (ESI+) m/z = 248 (M+). 2-(3-Benzylsulfanyl-2-methylphenyl)pyridine (44a). A mixture of intermediate 2-(3-bromo-2-methylphenyl)pyridine 43a (15.0 g, 60.53 mmol), n-tributylstannylsulfanylmethylbenzene19 (27.5 g, 66.6 mmol), KF (5.3 g, 90.8 mmol), and xantphos (1.05 g, 1.82 mmol) in dry NMP (30 mL) was stirred for 20 min under argon at room temperature. Then Pd2(dba)3 (1.66 g, 1.82 mmol) was added and the mixture was heated for 10 h at 100 °C. After cooling to room temperature, the reaction mixture was treated with a 5% solution of KF (100 mL) for 15 min under stirring and was then diluted with ethyl acetate (200 mL) and filtered over Celite 545. The organic layers were consecutively washed with water (2 × 200 mL) and brine (100 mL), dried over Na2SO4, and evaporated under reduced pressure. The crude mixture was chromatographed on silica gel. Elution with ethyl acetate/ cyclohexane from 0% to 20% of ethyl acetate gave 7.7 g of expected intermediate 44a as a colorless oil. Yield = 78%. 1H NMR (250 MHz, DMSO) δ ppm 8.66 (ddd, J = 4.9, 1.8, 1.0 Hz, 1H), 7.88 (td, J = 7.4, 1.9 Hz, 1H), 7.49−7.14 (m, 10H), 4.27 (s, 2H), 2.21 (s, 3H). LCMS/ UV (ESI+) m/z = 292 (M + H+). 2-Methyl-3-pyridin-2-ylbenzenesulfonyl Chloride (42g). Intermediate 2-(3-benzylsulfanyl-2-methylphenyl)pyridine 44a (2.33 g, 8 mmol) was dissolved in 50 mL of DCM and treated with water (32 mmol), AcOH (32 mmol), and SO2Cl2 (2.4 mmol) at 0 °C. After being stirred for 5 min at 0 °C and 90 min at room temperature, the mixture was cooled back to 0 °C and the reaction was quenched by addition of 10 mL of water. The aqueous solution was extracted with DCM (3×), and combined organic layers were washed with cold water. The crude material was diluted with ethyl acetate (100 mL) and

washed with a 1% solution of NaHCO3 (50 mL), water (50 mL), and brine (50 mL), dried over Na2SO4, and evaporated under reduced pressure. The crude material was quickly chromatographed on silica gel. Elution with ether/pentane from 0% to 100% of ether gave 0.95 g of intermediate 42g as a colorless oil. Yield = 44%. Route c. 1-Benzylsulfanyl-3-bromo-2-chlorobenzene (40c). Phenylmethanethiol was stirred (14.8 g, 119.36 mmol) in 175 mL of DMF. Cs2CO3 (38.89 g, 119.36 mmol) was added under argon. After 10 min, 1-bromo-2-chloro-3-fluorobenzene 45 (25 g, 119.36 mmol) in 25 mL of DMF was added and stirring was continued overnight at room temperature and then 3 h at 80 °C. After cooling, the mixture was diluted with ethyl acetate/water and washed subsequently with 1 N HCl and brine. The organic layer was dried with MgSO4, filtered, and evaporated. Purification by chromatography on silica gel yielded 22 g (59%) as an amorphous, colorless solid. 1H NMR (250 MHz, DMSO) δ ppm 7.55 (dd, J = 8.2, 1.4 Hz, 1H), 7.47− 7.20 (m, 7H), 4.33 (s, 2H). GC/MS (Cl+) m/z = 213 (M+). 4-(3-Benzylsulfanyl-2-chlorophenyl)morpholin-3-one (41j). Intermediate 1-benzylsulfanyl-3-bromo-2-chlorobenzene 40c was treated with morpholin-3-one according to the procedure described above for 1-(3-benzylsulfanyl-2-methylphenyl)pyrrolidin-2-one 41b. Yield after chromatography on silica gel (n-heptane/ethyl acetate): 7.2 g (68%). 2-Chloro-3-(3-oxomorpholin-4-yl)benzenesulfonyl Chloride (42j). Intermediate 4-(3-benzylsulfanyl-2-chlorophenyl)morpholin-3-one 41j (1 g, 3.05 mmol) was converted to the title sulfonyl chloride using the procedure described above for 2-methyl-3-(pyrrolidin-2-one)benzenesulfonyl chloride 42j. An amount of 0.9 g of the crude material was used without further purification in the next step. (S)-2-tert-Butoxycarbonylamino-3-[(5-chlorothiophene-2carbonyl)amino]propionic Acid Methyl Ester (48). Commercially available (S)-3-amino-2-tert-butoxycarbonylaminopropionic acid 46 (18.09 g, 88.56 mmol) was suspended in 100 mL of DCM with Nhydroxysuccinimide (16.99 g, 147.61 mmol) and N,N′-diisopropylcarbodiimide (11.43 mL, 73.8 mmol). Then 5-chlorothiophene-2carboxylic acid 47 (12 g, 73.8 mmol) and DIPEA (25.51 mL, 147.61 mmol) in 60 mL of DCM were slowly added. The mixture was stirred at room temperature for 2 h, then cooled to 0 °C, and trimethylsilyldiazomethane was slowly added. The mixture was stirred for 2 h at room temperature. HCl solution (0.5 M) was added, and the organic layer was separated, washed with H2O, dried with Na2SO4, filtered, and evaporated to dryness. The resulting solid was triturated 16 h with diisopropyl ether, filtered, and washed with diisopropyl ether and dried under vacuum at 60 °C. An amount of 24.4 g of crude product was obtained and used without further purification. 1H NMR (250 MHz, DMSO) δ ppm 8.64 (t, J = 5.7 Hz, 1H), 7.60 (d, J = 4.3 Hz, 1H), 7.21 (br d, J = 8.0 Hz, 1H), 7.20 (d, J = 3.9 Hz, 1H), 5.48 (d, J = 7.8 Hz, 1H), 4.23 (q, J = 8.0 Hz, 1H), 3.62 (s, 3H), 3.54 (t, J = 6.0 Hz, 1H), 1.38 (s, 9H). LCMS/UV (ESI+) m/z = 363 (M + H+). (S)-2-Amino-3-[(5-chlorothiophene-2-carbonyl)amino]propionic Acid Methyl Ester Hydrochloride (49). An amount of 33 g (90.95 mmol) of intermediate 48 was dissolved in 600 mL of ethyl acetate, and 250 mL of HCl (2 N in diethyl ether) was added. After 20 h of stirring at room temperature, the mixture was filtered and the solid dried under reduced pressure. Yield: 26.64 g, 98%. 1H NMR (250 MHz, DMSO) δ ppm 9.18 (t, J = 5.5 Hz, 1H), 8.73 (br s, 3H), 7.85 (d, J = 4.0 Hz, 1H), 7.20 (d, J = 4.0 Hz, 1H), 4.19 (t, J = 5.1 Hz, 1H), 3.78−3.68 (m, 5H). LCMS/UV (ESI+) m/z = 263 (M + H+). (S)-3-[(5-Chlorothiophene-2-carbonyl)amino]-2-[2-methyl-3-(2oxopyrrolidin-1-yl)benzenesulfonylamino]propionic Acid Methyl Ester (50). Intermediate (S)-2-amino-3-[(5-chlorothiophene-2carbonyl)amino]propionic acid methyl ester hydrochloride 49 (3.46 g, 11.59 mmol) was dissolved in 25 mL of DCM and TEA (8.49 mL, 61.03 mmol). Intermediate 2-methyl-3-(pyrrolidin-2-one)benzenesulfonyl chloride 42c (3.34 g, 12.21 mmol) in 20 mL of DCM was slowly added at 0 °C, and the mixture was stirred at room temperature for 16 h. Then the solution was evaporated to dryness and purified by silica gel chromatography. Yield: 5.69 g, 93%. 1H NMR (250 MHz, DMSO) δ ppm 8.66 (t, J = 6.2 Hz, 1H), 8.61 (br s, 1H), 7.78 (dd, J = 8.2, 1.2 Hz, 1H), 7.52 (d, J = 4.2 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.17 (d, J = 4.2 Hz, 1H), 4.03 (t, J = 9452



Article

Measurement of Factor Xa and Thrombin Inhibition. The compounds were tested for factor Xa/thrombin inhibition using a chromogenic assay. Compounds were assayed from 10 μM to 10 pM in buffer (50 mM Tris, 100 mM NaCl, 0.1% BSA, pH 7.5) with a maximal final DMSO concentration of 0.1% on 25 μL of enzyme (human coagulation factor Xa from Enzyme Research Laboratories hFXa, final concentration 0.003 IU/mL and on human thrombin from CTS Strasbourg with a final concentration of 0.125 IU/mL). The reagents were mixed, centrifuged, and incubated for 10 min at 37 °C in a 96-well microtiter plate. The enzyme reaction was started by adding 50 μL of substrate (for factor Xa, S-2765, Biogenic ref 821413 in a final concentration of 62.5 μM or at different concentrations from 30 to 125 μM for IC50 or Ki determination, respectively; for thrombin, S2238, Biogenic ref 820324 in a final concentration of 83 μM for IC50 determination or from 40 to 165 μM in order to allow Ki calculation). The time course of the reaction was monitored at 405 nm in a microtiter plate reader (Tecan M200) for 20 min. The IC50 was calculated from the mean of duplicates from a dilution series of the compound with the internal software Speed 1.1. The Ki constants were calculated from three independent experiments from a dilution series of the compound against a dilution series of the substrate with the help of Preclinical and Research Biostatistics department and the internal software SAS 2.0. The IC50 evaluation was performed as a screening approach for the chemical optimization, whereas the Ki determination was only performed for the optimized compound 15 in comparison to reference compounds (rivaroxaban 2 and dabigatran etexilate 1a). Determination of Inhibitor Constants for Human Enzymes. The ability of the compounds to inhibit other enzymes like factor VIIa, plasmin, or trypsin in comparison to thrombin or factor Xa inhibition was assessed by determining the concentration of the compound that inhibits enzyme activity by 50%, i.e., the IC50 value, which is related to the inhibition constant Ki. Purified enzymes were used in chromogenic assays. The concentration of inhibitor that causes a 50% decrease in the rate of substrate hydrolysis was determined by linear regression after plotting the relative rates of hydrolysis (compared to the uninhibited control) versus the log of the concentration of the compound of formulas I or Ia. For calculating the inhibition constant Ki, the IC50 value was corrected for competition with substrate using the formula

6.9 Hz, 1H), 3.65−3.59 (m, 2H), 3.53 (q, J = 6.4 Hz, 1H), 3.40−3.34 (m, 4H), 2.44 (t, J = 8.2 Hz, 2H), 2.36 (s, 3H), 2.14 (q, J = 7.3 Hz, 2H). LCMS/UV (ESI+) m/z = 500 (M + H+). (S)-3-[(5-Chlorothiophene-2-carbonyl)amino]-2-[2-methyl-3-(2oxopyrrolidin-1-yl)benzenesulfonylamino]propionic Acid (51). Intermediate (S)-3-[(5-chlorothiophene-2-carbonyl)amino]-2-[2-methyl-3-(2-oxopyrrolidin-1-yl)benzenesulfonylamino]propionic acid methyl ester 50 was dissolved in 20 mL of THF, and lithium hydroxide (1 N water solution) (38.16 mL, 38.16 mmol) was added. After 2 h of being stirring at room temperature, the reaction mixture was extracted with diethyl ether and 1 N HCl (38.16 mL, 38.16 mmol) was added to the aqueous layer. The aqueous layer was extracted with n-butanol. The organic layer was dried with Na2SO4, filtered, and evaporated to dryness. An amount of 8.28 g of crude product was obtained and used without further purification. 1H NMR (250 MHz, DMSO) δ ppm 12.82 (br s, 1H), 8.60 (t, J = 5.4 Hz, 1H), 8.33 (d, J = 9.8 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 3.9 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.16 (d, J = 4.1 Hz, 1H), 3.95 (q, J = 6.8 Hz, 1H), 3.59 (t, J = 7.1 Hz, 2H), 3.51 (m, 1H), 3.35 (m, 1H), 2.43 (t, J = 8.3 Hz, 2H), 2.37 (s, 3H), 2.14 (q, J = 7.6 Hz, 2H). LCMS/UV (ESI+) m/z = 486 (M + H+). 5-Chlorothiophene-2-carboxylic Acid [(S)-2-[2-Methyl-3-(2-oxopyrrolidin-1-yl)benzenesulfonylamino]-3-(4-methylpiperazin-1-yl)3-oxopropyl]amide Hydrochloride (15). Intermediate (S)-3-[(5chlorothiophene-2-carbonyl)amino]-2-[2-methyl-3-(2-oxopyrrolidin1-yl)benzenesulfonylamino]propionic acid 51 (802 mg, 1.65 mmol) was dissolved in 10 mL of DCM, and N-methylpiperazine (0.73 mL, 6.60 mmol) was added followed by DIPEA (0.55 mL, 3.3 mmol) and TBTU (636 mg, 1.98 mmol). After 1 h of being stirred at room temperature, the solution was evaporated to dryness and purified by silica gel chromatography. The compound was dissolved in DCM, HCl (2 M in diethyl ether (5 mL) was added, and the solvents were evaporated yielding a white powder. Yield: 651 mg, 70%. 1H NMR (250 MHz, DMSO) δ ppm 10.40 (br s, 1H), 8.90 (br d, J = 34.5 Hz, 1H), 8.43 (d, J = 9.6 Hz, 1H), 7.83 (m, 1H), 7.61 (br s, 1H), 7.49− 7.25 (m, 2H), 7.20 (d, J = 3.8 Hz, 1H), 4.47 (br s, 1H), 4.33−4.00 (m, 2H), 3.61 (br s, 2H), 3.49−3.14 (m, 6H), 3.02−2.59 (m, 5H), 2.44 (t, J = 8.1 Hz, 2H), 2.36 (s, 3H), 2.15 (q, J = 7.6 Hz, 2H). LCMS/UV (ES+): m/z = 568.2, tR = 8.53 min. [α]20D +50.3° (c 3 g/L, MeOH). Mp 205 °C. Anal. Calcd for C24H30ClN5O5S2·HCl, H2O: C, 46.1; H, 5.32; N, 11.03. Found: C, 46.30; H, 5.34; N, 11.25. See additional data in Supporting Information. (2S,5R)-2-[5-(5-Chlorothiophen-2-yl)isoxazol-3-ylmethyl]-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine (54). Commercially available (R)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine 52 (2.0 g, 10.86 mmol) in dry THF (12 mL) under argon was cooled to −75 °C. n-BuLi (8 mL, 13.03 mmol, 1.6 M solution in hexanes) was added slowly, and stirring was continued for 30 min. After that, 3bromomethyl-5-(5-chlorothiophen-2-yl)isoxazole20a,b 53 (3.628 g, 13.03 mmol) in THF (15 mL) was added dropwise under stirring and stirring was continued for 30 min at −78 °C. Then the mixture was warmed to 0 °C and stirred for 1 h before it was quenched with sat. aq NaHCO3 solution. The mixture was extracted with ethyl acetate, and the combined organic phase was dried with MgSO4, filtered, and evaporated to dryness. A crude oil was obtained (4.1 g) and the diastereomeric excess (88% de) was determined by 1H NMR from that crude mixture. The mixture was separated by column chromatography on silica gel (n-heptane/ethyl acetate 6:1). Yield: 3.3 g, 80%. (S)-2-Amino-3-[5-(5-chlorothiophen-2-yl)isoxazol-3-yl]propionic Acid Methyl Ester (56). Intermediate (2S,5R)-2-[5-(5-chlorothiophen2-yl)isoxazol-3-ylmethyl]-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine 54 (3.25 g, 8.51 mmol) in acetonitrile (183 mL) was treated with TFA (90 mL, 2 M in water) and stirred at room temperature overnight. After complete conversion the mixture was neutralized with sat. aq NaHCO3 solution, the majority of the acetonitrile was evaporated and the remainder extracted with ethyl acetate. The combined organic phases were washed with brine, dried with MgSO4, filtered, and evaporated to dryness. The resulting crude oil (2.5 g) was used in the next steps without further purification.

calculated K i = IC50 /{1 + (substrate concentration/K m)} where Km is the Michaelis−Menten constant.22a,b Determination of Thrombin Generation Time (TGT) in Plasma. Continuous monitoring of endogenous thrombin generation in human plasma was performed by a method adapted from those described by Hemker et al.23 and Nieuwenhuys et al.24 Thrombin generation experiments were carried out in defibrinated plasma obtained by mixing an aliquot of platelet-poor plasma with ancrod (50 U/mL), letting a clot form for 10 min at 37 °C and keeping the clotted plasma at 0 °C for 10 min. The fibrin thus formed was discarded before thrombin generation determination. Then 100 μL of 0.25 mM chromogenic substrate, which is sufficiently slowly converted by thrombin and which still shows reasonable specificity for thrombin, and 100 μL of recombinant tissue factor and 100 μL of Ca2+ buffer (0.05 M Tris-HCl, 0.1 M NaCl, 100 mM CaCl2, pH 7.35, and 0.05% ovalbumin) were added to a disposable plastic microcuvette. Then the compound in 100 μL of buffer (0.05 M Tris-HCl, 0.1 M NaCl, pH 7.35, and 0.05% ovalbumin) was added. The reaction was started at zero time by adding defibrinated plasma. The reagents were prewarmed to 37 °C, and the cuvette was thermostatically controlled at that temperature during the measurement. The optical density at 405 nm was recorded at the rate of 10 measurements per minute using a spectrophotometer. From the obtained curve, endogenous thrombin potential (ETP) was calculated using the method described by Nieuwenhuys et al.23 Rat Venous (Wessler) Thrombosis Model. Fasted male CD rats (300−410 g; Charles River, L’Arbresle, France) were anesthetized with pentobarbitone sodium (60 mg/kg ip) and placed on a heated jacket to control body temperature (Harvard, Les Ulis, France). The left jugular vein was cannulated for intravenous injection of drugs. The 9453



Article

Unfortunately the crystals were twinned with twinning vector k,h,−l. For the 31 complex, the twinning fraction was less than 0.1, and here no detwinning was used. For the 57 and 58 complexes, various detwinning protocols were tested and the best results were finally obtained by detwinning using Fcalc’s CNS detwin_perfect protocol.32 Model building and inhibitor fitting was done with Quanta, and parameters for the inhibitors were generated using the program Prodrg.33 Refinement was done with Refmac34 and Buster27 for the 31 complex. The final statistics, given in the Supporting Information, are reasonable, but one should keep in mind that the detwinning procedure used leads to an additional, artificial reduction in R and Rfree.

abdominal vena cava was exposed, and two silk sutures, 1 cm apart, were placed around the vessel to form a snare. Thrombus formation was induced by the intravenous injection of 20 ng/kg rabbit thromboplastin (La Technique Biologique, Paris, France) into the left femoral vein, followed 10 s later by tightening the two snares around the vena cava to induce blood stasis. Stasis was maintained for 15 min, after which time the thrombus was removed and immediately weighed. In the intravenous administration protocol, 15 and reference antithrombotic agents were administered 5 min before thrombus formation. In the oral study, 15 or vehicle was administered as single oral doses 30 min before injection of thromboplastin. Wire-Coil Adapted Model of Thrombosis and Bleeding in Rats. The wire coil model was modified to simultaneously evaluate thrombosis and bleeding as follows: the vena cava was exposed and incised in anesthetized rats and a metallic wire coil was then retrogradely inserted. This device was removed after a period of 90 min, and the protein contents sticking around it were measured using a classical protein assay. The blood loss was quantified by the measurement of the hemoglobin contents and the weighing of sterile gauze pads placed into the abdominal cavity just after the wire coil insertion and removed at the same time 90 min later. Each compound to be evaluated or its vehicle was administered either 30 or 90 min before thromboplastin. Metabolic Stability on Microsomes. Incubation conditions with hepatic microsomal fractions and further experimental conditions used throughout were as follows: microsomal proteins concentration = 1 mg/mL, bovine serum albumin (BSA) concentration = 1 mg/mL; substrate concentration = 5 μM; incubation duration = 20 min; cytochrome P-450 monooxygenases (CYPs) and flavin-containing monooxygenases (FMOs) cofactor = 1 mM NADPH. Enzyme activity was stopped with 1 volume of acetonitrile (ACN). Hepatic microsomal fractions: from Swiss CD1 male mouse (m7), Sprague− Dawley male rat (m21), humans (pool H-19, six donors). Inhibitor: quinidine at a final concentration of 8 μM (20-fold its Ki for CYP2D6) were used for the specific and potent inhibition of enzyme reactions catalyzed by CYP2D6. Ketoconazole at a final concentration of 1.5 μM (100-fold its Ki for CYP3A4) was used for the specific and potent inhibition of enzyme reactions catalyzed by CYP3A4. For each test compound and for each microsomal preparation, three incubations were prepared: absolute reference in buffer (without enzyme material, i.e., microsomes); incubation without NADPH cofactor (with microsomal fractions); incubation with NADPH (with microsomal fractions). For most compounds, biotransformation, as observed in hepatic microsomal fractions in the presence of the NADPH cofactor, consists of oxidative reactions catalyzed by either CYP or FMO. In these conditions, the percentage of total metabolism, which corresponds to oxidative metabolism, was determined as follows: [% total metabolism] ≈ [% oxidative metabolism] = [1(UC peak area − NADPH UC peak area + NADPH)] × 100, where NADPH corresponds to the enzyme cofactor for oxidation reactions catalyzed by either CYP or FMO, and UC represents the unchanged compound. Protein X-ray Crystallography. Thrombin was purchased from Haematologic Technologies Inc. (VT, U.S.) crystallized in the presence of 57 and 58 following the procedure of SkrzypczakJankun.26 The crystals were flash frozen, and data were collected at 100 K in-house. Data were processed with HKL2000.27 The inhibitor was fitted using Coot,28 and the structure was refined with Buster.29 Data processing and refinement statistics are given in the Supporting Information. Purified human factor Xa was purchased from Enzyme Research Lab (South Bend, IN). The Gla-domain was removed, and the Gla-less factor Xa was crystallized in hanging drops as described earlier.25 Factor Xa crystals were soaked with inhibitors as described.20a The crystals were flash frozen, and crystallographic data were collected at 100 K at the European synchrotron radiation facility (ESRF) in Grenoble, France. Data processing and scaling were carried out using the XDS package30 or the CCP4 program Scala31 for the 31 complex. The crystals diffracted to resolutions between 2.27 and 2.59 Å resolution and contained two factor Xa molecules per asymmetric unit.

■

ASSOCIATED CONTENT

S Supporting Information *

Metabolite identification study for compound 29; chiral chromatography, NMR, LCMS/UV spectra related to the final characterization of 15; omit maps for the factor Xa and thrombin crystal structures; and data processing and refinement statistics related to the X-ray structures. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes

PDB accession codes are 4bti, 4btt, and 4btu for the factor Xa complexes with 58, 31, and 57, respectively, and 4lxb for the thrombin complex with 58.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +33.5.34.63.23.90. Fax: +33.5.34.63.29.35. E-mail: gilbert.lassalle@sanofi.com. Present Addresses #

M.F.: Medicinal Chemistry Wuppertal, Global Drug Discovery, Bayer Pharma AG, Aprather Weg 18a, 42113 Wuppertal, Germany. ∞ Z.B.: Sanofi R&D, LGCR-SDI, 13 Quai Jules Guesde, 94403 Vitry-sur-Seine, France. Author Contributions

All medicinal chemists from Frankfurt and Toulouse contributed equally. Biological characterizations were mainly performed in Toulouse. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank Dr. J.-P. Maffrand for constant support and strong scientific challenge, Dr. L. Lafferrere, Dr. Wehland, and colleagues (Sanofi R&D, Chemical Development) for providing large amounts of intermediates and final compounds, Dr. P. Hamley and colleagues (Sanofi R&D, Frankfurt) for library synthesis, Dr. H. Matter (Sanofi R&D, Frankfurt) for early in silico support concerning ADME issues, Dr. S. Maignan (Sanofi R&D, Toulouse) for modeling studies, and Dr. C. Ponthus and colleagues (Sanofi R&D, Toulouse) for their essential support in structural determinations. We thank C. Stehlin-Gaon (Sanofi R&D, Strasbourg) for the crystallization of thrombin, V. Brachvogel for the preparation and crystallization of desGla factor Xa, A. Liesum for help with mounting crystals and data collection, and P. Loenze for help with data processing and refinement (all from Sanofi R&D, Frankfurt). Also special thanks are given to L. Laplanche and colleagues from the DSAR Department (Sanofi R&D, Montpellier) for their support in ADME studies and to B. Misi for technical assistance during the preparation of the manuscript. 9454



■

Article

(4) (a) Herbert, J.-M.; Herault, J.-P.; Bernat, A.; Savi, P.; Schaeffer, P.; Driguez, P.-A.; Duchaussoy, P.; Petitou, M. SR123781A, a synthetic heparin mimetic. Thromb. Haemostasis 2001, 85, 852−860. (b) Petitou, M.; Duchaussoy, P.; Driguez, P.-A.; Jaurand, G.; Hérault, J.-P.; Lormeau, J.-C.; van Boeckel, C. A. A.; Herbert, J.-M. First synthetic carbohydrates with the full anticoagulant properties of heparin. Angew. Chem., Int. Ed. 1998, 37 (21), 3009−3014. (c) Petitou, M.; Hérault, J.P.; Bernat, A.; Driguez, P.-A.; Duchaussoy, P.; Lormeau, J.-C.; Herbert, J.-M. Synthesis of thrombin-inhibiting heparin mimetics without side effects. Nature 1999, 398, 417−422. (d) Petitou, M.; Duchaussoy, P.; Driguez, P.-A.; Hérault, J.-P.; Lormeau, J.-C.; Herbert, J.-M. New synthetic heparin mimetics able to inhibit thrombin and factor Xa. Bioorg. Med. Chem. Lett. 1999, 9 (8), 1155−1160. (e) Li, W.; Johnson, D. J. D.; Esmon, C. T.; Huntington, J. A. Structure of the antithrombin−thrombin−heparin ternary complex reveals the antithrombotic mechanism of heparin. Nat. Struct. Mol. Biol. 2004, 11, 857− 862. (5) (a) De Kort, M.; Buijsmain, R. C.; Van Boeckel, A. A. Synthetic heparin derivatives as new anticoagulant drugs. Drug Discovery Today 2005, 10, 769−779. (b) Petitou, M.; Nancy-Porteboi, V.; Dubreucq, G.; Motte, V.; Meuleman, D.; De Kort, M.; Van Boeckel, C. A. A.; Vogel, G. M. T.; Wisse, J. A. J. The long way to a new pentasaccharidebased neutralisable anticoagulant with an unprecedented pharmacological profile. Thromb. Haemostasis 2009, 102, 804−810. (6) Nar, H.; Bauer, M.; Schmid, A.; Stassen, J. M.; Wienen, W.; Priepke, H. W.; Kauffmann, I. K.; Ries, U. J.; Hauel, N. H. Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors. Structure 2001, 9, 29−37. (7) Ries, U. J.; Priepke, H. W.; Hauel, N. H.; Handschuh, S.; Mihm, G.; Stassen, J. M.; Wienen, W.; Nar, H. Heterocyclic thrombin inhibitors. Part 2: Quinoxalinone derivatives as novel, potent antithrombotic agents. Bioorg. Med. Chem. Lett. 2003, 13, 2297−2302. (8) Graefe-Mody, E. U.; Schühly, U.; Rathgen, K.; Stähle, H.; Leitner, J. M.; Jilma, B. Pharmacokinetics and pharmacodynamics of BIBT 986, a novel small molecule dual inhibitor of thrombin and factor Xa. J. Thromb. Haemostasis 2006, 4, 1502−1509. (9) Deng, J. Z.; McMasters, D. R.; Rabbat, P. M.; Williams, P. D.; Coburn, C. A.; Yan, Y.; Kuo, L. C.; Lewis, S. D.; Lucas, B. J.; Krueger, J. A.; Strulovici, B.; Vacca, J. P.; Lyle, T. A.; Burgey, C. S. Development of an oxazolopyridine series of dual thrombin/factor Xa inhibitors via structure-guided lead optimization. Bioorg. Med. Chem. Lett. 2005, 15, 4411−4416. (10) Dönnecke, D.; Schweinitz, A.; Stürzebecher, A.; Steinmetzer, P.; Schuster, M.; Stürzebecher, U.; Nicklisch, S.; Stürzebecher, J.; Steinmetzer, T. From selective substrate analogue factor Xa inhibitors to dual inhibitors of thrombin and factor Xa. Part 3. Bioorg. Med. Chem. Lett. 2007, 17, 3322−3329. (11) Young, R. J.; Brown, D.; Burns-Kurtis, C. L.; Chan, C.; Convery, M. A.; Hubbard, J. A.; Kelly, H. A.; Pateman, A. J.; Patikis, A.; Senger, S.; Shah, G. P.; Toomey, J. R.; Watson, N. S.; Zhou, P. Selective and dual action orally active inhibitors of thrombin and factor Xa. Bioorg. Med. Chem. Lett. 2007, 17, 2927−2930. (12) (a) Maryanoff, B. E.; McComsey, D. F.; Costanzo, M. J.; Yabut, S. C.; Lu, T.; Player, M. R.; Giardino, E. C.; Damiano, B. P. Exploration of potential prodrugs of RWJ-445167, an oxyguanidine based dual inhibitor of thrombin and factor Xa. Chem. Biol. Drug Des. 2006, 68, 29−36. (b) Giardino, E. C.; Haertlein, B. J.; De Garavilla, L.; Costanzo, M. J.; Damiano, B. P.; Andrade-Gordon, P.; Maryanoff, B. E. Cooperative antithrombotic effect from the simultaneous inhibition of thrombin and factor Xa. Blood Coagulation Fibrinolysis 2010, 21, 128− 134. (13) (a) Altenburger, J.-M.; Lassalle, G. Y.; Matrougui, M.; Galtier, D.; Jetha, J.-C.; Bocskei, Z.; Berry, C. N.; Lunven, C.; Lorrain, J.; Herault, J.-P.; Schaeffer, P.; O’Connor, S. E.; Herbert, J.-M. SSR182289A, a selective and potent orally active thrombin inhibitor. Bioorg. Med. Chem. 2004, 12, 1713−1730. (b) Berry, C. N.; Lassalle, G.; Lunven, C.; Altenburger, J.-M.; Guilbert, F.; Lalé, A.; Hérault, J.-P.; Lecoffre, C.; Pfersdorff, C.; Herbert, J.-M.; O’Connor, S. E. SSR182289A, a novel, orally active thrombin inhibitor: in vitro profile

ABBREVIATIONS USED AcOH, acetic acid; ADME, absorption, distribution, metabolism, and excretion; ADMET, absorption, distribution, metabolism, excretion, and toxicity; aq, aqueous; n-BuLi, nbutyllithium; Caco-2, adenocarcinoma of the colon; CYP, cytochrome P450; DAPA, L-2,3-diaminopropionic acid; dba, dibenzylidenacetone; DCM, dichloromethane; DIPEA, diisopropylethylamine; DMF, N,N-dimethylformamide; DMSO, dimethylsulfoxide; dppf, 1,1′-bis(diphenylphosphino)ferrocene; f.Xa, factor Xa; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′tetramethyluronium hexafluorophosphate; HCl, hydrochloric acid; LDA, lithium diisopropylamide; MeOH, methanol; MTBE, tert-butyl methyl ether; NHS, N-hydroxysuccinimide; NEM, N-ethylmorpholine; NMP, N-methylpyrrolidone; PK, pharmacokinetics; RT, room temperature 20−25 °C; sat., saturated; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; TEA, triethylamine; NHS, Nhydroxysuccinimide; THF, tetrahydrofuran; TFA, trifluoroacetic acid; TMS, tetramethylsilane; xantphos, 9,9-dimethyl-4,5bis(diphenylphosphino)xanthene

■

REFERENCES

(1) For reviews, see the following: (a) Lassen, M. R.; Laux, V. Emergence of new oral antithrombotics: a critical appraisal of their clinical potential. Vasc. Health Risk Manage. 2008, 4 (6), 1373−1386. (b) Weitz, J. I. Factor Xa and thrombin as targets for new oral anticoagulants. Thromb. Res. 2011, 127 (2), S5−S12. (c) Straub, A.; Roehrig, S.; Hillisch, A. Oral, direct thrombin and factor Xa inhibitors: the replacement for warfarin, leeches, and pig intestines. Angew. Chem., Int. Ed. 2011, 50, 4574−4590. (2) (a) Hauel, N. H.; Nar, H.; Priepke, H.; Ries, U.; Stassen, J. M.; Wienen, W. Structure-based design of novel potent nonpeptide thrombin inhibitors. J. Med. Chem. 2002, 45, 1757−1766. (b) Ieko, M. Drug evaluation: dabigatran etexilate, a thrombin inhibitor for the prevention of venous thromboembolism and stroke. Curr. Opin. Invest. Drugs 2007, 8 (9), 758−768. (c) Van Ryn, J.; Stangier, J.; Haertter, S.; Liesenfeld, K. H.; Wienen, W.; Feuring, M. Dabigatran etexilatea novel, reversible, oral direct thrombin inhibitor: interpretation of coagulation assays and reversal of anticoagulant activity. Thromb. Haemostasis 2010, 103, 1116−1127. (d) Hankey, G. J.; Eikelboom, J. W. Dabigatran etexilate: a new oral thrombin inhibitor. Circulation 2011, 123, 1436−1450. (3) (a) Perzborn, E.; Roehring, S.; Straub, A.; Kubitza, D.; Misselwitz, F. The discovery and development of rivaroxaban, an oral, direct factor Xa inhibitor. Nat. Rev. Drug Discovery 2011, 10, 61−75. (b) Straub, A.; Roehrig, S.; Hillisch, A. Entering the era of non-basic P1 site groups: discovery of Xarelto (rivaroxaban). Curr. Top. Med. Chem. 2010, 10, 257−269. (c) Misselwitz, F.; Berkowitz, S. D.; Perzborn, E. The discovery and development of rivaroxaban. Ann. N.Y. Acad. Sci. 2011, 1222, 64−75. (d) Furugohri, T.; Isobe, K.; Honda, Y.; KamisatoMatsumoto, C.; Sugiyama, N.; Nagahara, T.; Morishima, Y.; Shibano, T. DU-176b, a potent and orally active factor Xa inhibitor: in vitro and in vivo pharmacological profiles. J. Thromb. Haemostasis 2008, 6, 1542−1549. (e) Pinto, D. J. P.; Orwat, M. J.; Koch, S.; Rossi, K. A.; Alexander, R. S.; Smallwood, A.; Wong, P. C.; Rendina, A. R.; Luettgen, J. M.; Knabb, R. M.; He, K.; Xin, B.; Wexler, R. R.; Lam, P. Y. S. Discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, BMS-562247), a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa. J. Med. Chem. 2007, 50, 5339−5356. (f) Wong, P. C.; Crain, X. B.; Xin, B.; Wexler, R. R.; Lam, P. Y. S.; Pinto, D. J.; Luettgen, J. M.; Knabb, R. M. Apixaban, an oral, direct and highly selective factor Xa inhibitor: in vitro, antithrombotic and antihemostatic studies. J. Thromb. Haemostasis 2008, 6, 820−829. 9455



Article

and ex vivo anticoagulant activity. J. Pharmacol. Exp. Ther. 2002, 303, 1189−1198. (c) Lorrain, J.; Millet, L.; Lechaire, I.; Lochot, S.; Ferrari, P.; Visconte, C.; Sainte-Marie, M.; Lunven, C.; Berry, C. N.; Schaeffer, P.; Herbert, J.-M.; O’Connor, S. E. Antithrombotic properties of SSR182289A, a new, orally active thrombin inhibitor. J. Pharmacol. Exp. Ther. 2002, 304, 567−574. (d) Altenburger, J.-M.; Cremer, G.; Lassalle, G.; Matrougui, M. N-(Heterocyclyl)benzene or Pyridine Sulphonamides as Antithrombotic Agents and Anticoagulants. WO200170736 A1, 2001. (14) (a) Maignan, S.; Guilloteau, J.-P.; Choi-Sledeski, Y. M.; Becker, M. R.; Ewing, W. R.; Pauls, H. W.; Spada, A. P.; Mikol, V. Molecular structures of human factor Xa complexed with ketopiperazine inhibitors: preference for a neutral group in the S1 pocket. J. Med. Chem. 2003, 46, 685−690. (b) Choi-Sledeski, Y. M.; Kearney, R.; Poli, G.; Pauls, H.; Gardner, C.; Gong, Y.; Becker, M.; Davis, R.; Spada, A.; Liang, G.; Chu, V.; Brown, K.; Collussi, D.; Leadley, R., Jr.; Rebello, S.; Moxey, P.; Morgan, S.; Bentley, R.; Kasiewski, C.; Maignan, S.; Guilloteau, J.-P.; Mikol, V. Discovery of an orally efficacious inhibitor of coagulation factor Xa which incorporates a neutral P1 ligand. J. Med. Chem. 2003, 46, 681−684. (c) Matter, H.; Nazaré, M.; Güssregen, S.; Will, D. W.; Schreuder, H.; Bauer, A.; Urmann, M.; Ritter, K.; Wagner, M.; Wehner, V. Evidence for C-Cl/C-Br···π Interactions as an Important Contribution to Protein−Ligand Binding Affinity. Angew. Chem., Int. Ed. 2009, 48, 2911−2916. (d) Roehrig, S.; Straub, A.; Pohlmann, J.; Lampe, T.; Pernerstorfer, S.; Schlemmer, K.-H.; Reinemer, P.; Perzborn, E. Discovery of the novel antithrombotic agent 5-chloro-N-({(5S)-2-oxo-3- [4-(3-oxomorpholin-4-yl)phenyl]1,3-oxazolidin-5-yl}methyl)thiophene- 2-carboxamide (BAY 59-7939): an oral, direct factor Xa inhibitor. J. Med. Chem. 2005, 48, 5900−5908. (15) Knochel, P.; Dohle, W.; Gommermann, N.; Kneisel, F. F.; Kopp, F.; Korn, T.; Sapountzis, I.; Vu, V. A. Highly functionalized organomagnesium reagents prepared through halogen−metal exchange. Angew. Chem., Int. Ed. 2003, 42, 4302−4320. (16) Groth, U.; Schöllkopf, U. Asymmetric syntheses via heterocyclic intermediates; XVII1. Enantioselective synthesis of (R)-α-methyl-Sbenzylcysteine methyl ester and (R)-α-methyl-S-t-butylcysteine methyl ester using L-valine as chiral auxiliary reagent. Synthesis 1983, 1, 37−38. (17) (a) Gould, W. R.; McClanahan, T. B.; Welch, K. M.; Baxi, S. M.; Saiya-Cork, K.; Chi, L.; Johnson, T. R.; Leadley, R. J. Inhibitors of blood coagulation factors Xa and IIa synergize to reduce thrombus weight and thrombin generation in vivo and in vitro. J. Thromb. Haemostasis 2006, 4, 834−841. (b) Giardino, E. C.; Haertlein, B. J.; de Garavilla, L.; Costanzo, M. J.; Damiano, B. P.; Andrade-Gordon, P.; Maryanoff, B. E. Cooperative antithrombotic effect from the simultaneous inhibition of thrombin and factor Xa. Blood Coagulation Fibrinolysis 2010, 21, 128−134. (18) (a) Follmann, M.; Wehner, V.; Meneyrol, J.; Altenburger, J.-M.; Petit, F.; Lassalle, G.; Herault, J. P. Preparation of Chlorothiopheneamides as Inhibitors of Coagulation Factors Xa and Thrombin. WO2009103440, 2009. (b) Follmann, M.; Wehner, V.; Altenburger, J.M.; Lassalle, G.; Herault, J.-P.; Preparation of Chlorothiopheneisoxazoles as Inhibitors of Coagulation Factors Xa and Thrombin. WO2009103439, 2009. (19) Gutierrez, C. G.; Leo, R.; Summerhays, L. R. Organotinmediated selective desulfurization: tri-n-butyltin hydride reduction of unsymmetric sulfides. J. Org. Chem. 1984, 49 (26), 5206−5213. (20) (a) Nazaré, M.; Will, D. W.; Matter, H.; Schreuder, H.; Ritter, K.; Urmann, M.; Essrich, M.; Bauer, A.; Wagner, M.; Czech, J.; Lorenz, M.; Laux, V.; Wehner, V. Probing the subpockets of factor Xa reveals two binding modes for inhibitors based on a 2-carboxyindole scaffold: a study combining structure−activity relationship and X-ray crystallography. J. Med. Chem. 2005, 48, 4511−4525. (b) Nazaré, M.; Essrich, M.; Will, D. H.; Matter, H.; Ritter, K.; Urmann, M.; Bauer, A.; Schreuder, H.; Dudda, A.; Czech, J.; Lorenz, M.; Laux, V.; Wehner, V. Factor Xa inhibitors based on a 2-carboxyindole scaffold: SAR of neutral P1 substituents. Bioorg. Med. Chem. Lett. 2004, 14, 4191−4195. (21) Ham, J.; Yang, I.; Kang, H. A facile one-pot synthesis of alkyl aryl sulfides from aryl bromides. J. Org. Chem. 2004, 69, 3236−3239.

(22) (a) Cheng, Y.; Prusoff, W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973, 22, 3099−3108. (b) Segal, I. H. Enzyme Kinetics; John Wiley & Sons: New York, 1975; pp 100−125. (23) Hemker, H. C.; Beguin, S. Thrombin generation in plasma: its assessment via the endogenous thrombin potential. Thromb. Haemostasis 1995, 74, 134−138. (24) Nieuwenhuys, C. M.; Feijge, M. A.; Beguin, S.; Heemskerk, J. W. Monitoring hypocoagulant conditions in rat plasma: factors determining the endogenous thrombin potential of tissue factoractivated plasma. Thromb. Haemostasis 2000, 84, 1045−1051. (25) Matter, H.; Defossa, E.; Heinelt, U.; Blohm, P.-M.; Schneider, D.; Mueller, A.; Herok, S.; Schreuder, H.; Liesum, A.; Brachvogel, V.; Loenze, P.; Walser, A.; Al-Obeidi, F.; Wildgoose, P. Design and quantitative structure−activity relationship of 3-amidinobenzyl-1Hindole-2-carboxamides as potent, nonchiral, and selective inhibitors of blood coagulation factor Xa. J. Med. Chem. 2002, 45, 2749−2769. (26) Skrzypczak-Jankun, E.; Rydel, T. J.; Tulinsky, A.; Fenton, J. W.; Mann, K. G. Human D-Phe-Pro-Arg-CH2-alpha-thrombin crystallization and diffraction data. J. Mol. Biol. 1989, 20 (206(4)), 755−757. (27) Otwinowski, Z.; Minor, W. Processing of X-ray Diffraction Data Collected in Oscillation Mode. In Macromolecular Crystallography. Part A; Carter, C.W., Jr., Sweet, R. M., Eds.; Methods in Enzymology, Vol. 276; Academic Press: New York, 1997; pp 307−326. (28) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. 2004, D60, 2126−2132. (29) Bricogne, G.; Blanc, E.; Brandl, M.; Flensburg, C.; Keller, P.; Paciorek, W.; Roversi, P.; Sharff, A.; Smart, O. S.; Vonrhein, C.; Womack, T. O. BUSTER, version 2.11.2; Global Phasing Ltd.: Cambridge, U.K., 2011. (30) Kabsch, W. Evaluation of single-crystal X-ray diffraction data from a position sensitive detector. J. Appl. Crystallogr. 1988, 21, 916− 924. (31) Collaborative Computational Project, Number 4.. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. 1994, D50, 760−763. (32) Brunger, A. T.; Adams, P. D.; Clore, G. M.; Gros, P.; GrosseKunstleve, R. W.; Jiang, J.-S.; Kuszewski, J.; Nilges, N.; Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L. Crystallography & NMR System (CNS), a new software suite for macromolecular structure determination. Acta Crystallogr. 1998, D54, 905−921. (33) Schüttelkopf, A. W.; van Aalten, D. M. F. PRODRGa tool for high-throughput crystallography of protein−ligand complexes. Acta Crystallogr. 2004, D60, 1355−1363. (34) Pannu, N. J.; Murshudov, G. N.; Dodson, E. J.; Read, R. J. Incorporation of prior phase information strengthen maximumlikelihood structure refinement. Acta Crystallogr. 1998, D54, 1285− 1294.

9456


5-Chlorothiophene-2-carboxylic Acid [(S)-2-[2 ... - ACS Publications

Recommend Documents