J. Med. Chem. 2004, 47, 1969-1986
1969
Discovery, Modeling, and Human Pharmacokinetics of N-(2-Acetyl-4,6-dimethylphenyl)-3-(3,4-dimethylisoxazol-5-ylsulfamoyl)thiophene-2-carboxamide (TBC3711), a Second Generation, ETA Selective, and Orally Bioavailable Endothelin Antagonist1 Chengde Wu,*,† E. Radford Decker,‡ Natalie Blok,† Huong Bui,† Tony J. You,§ Junmei Wang,§ Andree R. Bourgoyne,‡ Vippra Knowles,† Kurt L. Berens,| George W. Holland,⊥ Tommy A. Brock,X and Richard A. F. Dixon4 Encysive Pharmaceuticals Inc., 7000 Fannin, Houston, Texas 77030 Received October 17, 2003
Sitaxsentan (1) (Wu et al. J. Med. Chem. 1997, 40, 1690) is our first endothelin antagonist being evaluated in clinical trials. It has demonstrated biological effects in an acute hemodynamic study in CHF (Givertz et al. Circulation 2000, 101, 2922), an open-label 20-patient pulmonary hypertension trial (Barst et al. Chest 2002, 121, 1860-1868), and a 31-patient trial in essential hypertension (Calhoun et al. AHA Scientific Sessions 2000). In a phase 2b/3 pulmonary arterial hypertension trial, once a day treatment of 100 mg of sitaxsentan statistically significantly improved 6-min walk distance and NYHA class at 12 weeks (Barst et al. Am. J. Respir. Crit. Care Med. 2004, 169, 441). We have since reported on our efforts in generating follow-up compounds (Wu et al. J. Med. Chem. 1999, 42, 4485) and recently communicated that an ortho acyl group on the anilino ring enhanced oral absorption in this category of compounds (Wu et al. J. Med. Chem. 2001, 44, 1211). Here we report an expansion of this work by substituting a variety of electron-withdrawing groups at the ortho position and evaluating their effects on oral bioavailability as well as structure-activity relationships. As a result, TBC3711 (7z) was identified as our second endothelin antagonist to enter the clinic due to its good oral bioavailability (∼100%) in rats, high potency (ETA IC50 ) 0.08 nM), and optimal ETA/ETB selectivity (441 000-fold). Compound 7z has completed phase-I clinical development and was well tolerated with desirable pharmacokinetics in humans (t1/2 ) 6-7 h, oral availability > 80%). Introduction The 21 amino acid peptide endothelin-1 (ET-1) is a potent and long acting vasoconstrictive and mitogenic peptide.9 Its structure features two disulfide bridges and a carboxyl terminal side chain conserved across ET isoforms,10 and ET-1 has the same amino acid sequence across mammalian species. ET-1 has been implicated in a variety of diseases including chronic heart failure, hypertension, atherosclerosis, pulmonary hypertension, and coronary artery disease.11 ET-1 exerts its biological effects via binding to its two G-protein-coupled receptors: ETA, selective for ET-1, ET-2 over ET-3, and the nonselective ETB.9,10 ETA and ETB receptors are expressed on smooth muscle cells and mediate the vasoconstrictive effects of ET-1.9,10 ETB receptors are found also on endothelial cells where they clear ET-1 and mediate vasodilation through the release of NO and thromboxane.12-14 The potentially wide application of endothelin antagonists has spurred intensive medicinal research and a number of chemical entities are being evaluated in * To whom correspondence should be addressed. E-mail: cwu@ encysive.com. † Medicinal chemistry. ‡ Pharmacology. § Computer modeling. | Clinical development. ⊥ Chemical sciences. X Biological sciences. 4 CSO and Sr. VP, Research.
the clinic. The first oral dual ETA/ETB antagonist bosentan has been approved for pulmonary arterial hypertension. Although ETB receptor disruption has been shown to cause hypertension,15 both selective ETA antagonist and dual antagonists are useful. Our laboratories have been involved in researching selective ETA antagonists, and compound 12 (sitaxsentan, Thelin) is our first such compound identified for clinical development. Compound 1 is an orally active ETA selective endothelin antagonist that attenuates pulmonary vascular hypertension and cardiac hypertrophy16 and prevents matrix metalloproteinase activation late post MI in rats.17 It confers hemodynamic changes including reductions in pulmonary artery pressures and pulmonary vascular resistance in patients with congestive heart failure3 and pulmonary arterial hypertension.4 When administered orally at 2 mg/kg to humans (N ) 4), sitaxsentan has an AUC0-∞ of 33.02 ( 7.08 h‚µg/mL with Cmax of 16.43 ( 5.86 µg/mL. The elimination halflife is 6.46 ( 1.52 h with a Tmax of 1.75 ( 0.96 h.18 In a phase 2b/3 STRIDE trial in 178 pulmonary arterial hypertension patients, once a day treatment of 100 mg and 300 mg of sitaxsentan statistically significantly improved 6-min walk distance and NYHA class at 12 weeks.6,19 Both doses resulted in significant improvements in hemodynamics in these NYHA class II-IV patients.20 The 300 mg dose of sitanxsentan also showed statistically significant improvement in change in percent of predicted peak VO2 at 12 weeks.6,19
10.1021/jm030528p CCC: $27.50 © 2004 American Chemical Society Published on Web 03/16/2004
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Journal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Chart 1. Maximal Plasma Concentration of Selected ET Antagonists in Rats (po 50 mg/kg)
Efforts continued in our laboratories to generate a second clinical candidate that is more potent and more ETA selective than 1 while maintaining the oral pharmacokinetic profile of 1. We have reported progress7 toward these goals by replacing the benzodioxolylacetyl group in 1 with a substituted mesitylcarboxamide, keeping the rest of the molecule constant. Within this scaffold the three methyl groups collectively are important for increasing potency of ∼10-fold over 1. An additional 3-substitutent improves selectivity for ETA receptors on average 10-fold, exerting minimal effect on potency. Accordingly, the strategy was to manipulate the physicochemical properties of this series of compounds by varying the functionality at the 3-position to achieve optimal oral pharmacokinectics. The maximal plasma concentrations in rats following oral administration of 50 mg/kg of selected compounds (1, 2a-k, and 3a,b) are listed in Chart 1. The parent, unsubstituted mesityl amide 2a had a reasonable Cmax of 35.9 µg/mL but to increase its aqueous solubility and lower its hemolytic activity, a polar group was required in the amide moiety of the molecule. Therefore, a range of groups with varying degree of polarity were screened at the 3-postion only to give ∼10-fold reduction in Cmax value: Benzyl alcohol 2b had a Cmax value of 2.4 µg/mL while less polar benzyl acetate 2c, methyl ether 2d, and cyclopropylmethyl ether 2e afforded Cmax of 3.8, 4.0, and 6.1 µg/mL, respectively. The basic dimethylamino compound 2f was also poor with Cmax of 3.2 µg/mL whereas the sulfonamides 2g and 2h provided similarly unsatisfactory values of 2.8 and 2.9 µg/mL, respectively. The exceptions were cyanide 2i (17.3 µg/mL), benzyl cyanide 2j (42.0 µg/mL), and phenol 2k (48.2 µg/mL), regaining
Wu et al.
close to or surpassing the level of the parent compound 2a (35.9 µg/mL), but still comparing unfavorably with the Cmax value of 1 (122.8 µg/mL). It was concluded at this point that although the mesityl amide series offered improved potency and selectivity for ETA receptors over 1, they probably would not match the oral properties seen in 1. A different scaffold of aryl amide would need to be designed that could maintain the high ETA potency, ETA receptor selectivity of the mesityl amide series, with a polar group in the amide arm of the molecule, yet match the oral characteristics of 1. One such amide scaffold, 2-acyl4,6-dimethylphenyl amide, offered possibility to meet all these criteria. We have recently reported8 two leads in this series, the methyl ketone 3a and the isopropyl ketone 3b. Both compounds were highly potent (ETA IC50 ) 0.04 and 0.12 nM for 3a and 3b, respectively), highly selective for ETA receptors (442 000- and 144 000fold), with oral Cmax values of 57 and 59 µg/mL, respectively. The methyl ketone 3a has oral bioavailability of 25% in rats, 42% in cats, and 70% in dogs.8 In addition, due to the electron-withdrawing effect of the ortho acyl group, the linker amide became carboxylic acid like and therefore served as the required polar group. In this paper, we report expanded structureactivity/rat oral pharmacokinetics relationships around 3a and the identification of our second endothelin antagonist 7z which has concluded phase I clinical trials. Synthetic Chemistry. The synthesis of most of the target compounds 7 consisted of (1) the stockpiling of common advanced methoxymethyl-protected sulfonamide acid chlorides 4,7 5,7 or 6 (Scheme 1), (2) the generation of prerequisite anilines (Schemes 2-5), and (3) the coupling of an aniline with 4, 5, or 6 and a subsequent cleavage of the methoxymethyl group to reveal the sulfonamide (Scheme 1). The coupling of electronically deficient anilines with these acid chlorides was accomplished using either 2 equiv of an aniline or, when the aniline was precious, 1 equiv of the aniline plus 1 equiv of 4-dimethylaminobenzonitrile to scavenge in situ generated hydrogen chloride. Heating with methanolic hydrochloric acid then liberated the sulfonamide group, the protection of the sulfonamide group being necessitated by its interference with acid chloride formation and the coupling. The 5-methylthiophene acid chloride 6 was obtained by saponification of ester 10 followed by treatment with oxalyl chloride. The bis(methoxymethyl) compound 10 was in turn generated by a sequence of lithiating thiophenecarboxylic acid 8,21 immediate quenching of the resultant trianion with iodomethane, and a double alkylation of 922 with methoxymethyl bromide. The synthesis of o-acylanilines is depicted in Scheme 2. Boron trichloride mediated Friedel-Crafts acylation reactions23,24 between anilines 11 or 14 with alkyl cyanides were uneventful to furnish required o-acyl anilines 12a-d, 15a, and 15b, respectively. However, a similar attempt using cyclopropylnitrile gave the ringopened 4′-chlorobutyronylaniline 13 as the sole product. Fortunately, the cyclopropyl ring could be regenerated under strong basic conditions25 to yield target compound 7m from 17. Target compound sulfone 7p was accessed via a SN2 displacement of the chloride in 16 with sodium
An Orally Bioavailable Endothelin Antagonist
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 8 1971
Scheme 1. Outline of General Synthesisa
Scheme 2. Synthesis of 2-Acylanilines and 7m, 7pa
a Reagents: (a) aniline/THF with or without 4-dimethylaminobenzonitirle; (b) MeOH/concentrated HCl (2:1), 70 °C 2 h; (c) nBuLi/THF/-78 °C/MeI; (d) Hunig base/THF/bromomethyl methyl ether; (e) 1N NaOH; (f) oxalyl chloride.
salt of methanesulfinic acid. The precursor compounds 16 and 17 were generated using the two-step protocol shown in Scheme 1 with acid chloride 4 and anilines 12d, 13, respectively. Scheme 3 delineates the synthesis of ortho-sulfonylanilines 24a-c. A cuprous iodide-mediated coupling26 of 5-iodo-m-xylene with methanesulfinic acid, sodium salt afforded aryl sulfone 19, a common intermediate for all three sulfonylanilines. Thus, lithiation of 19 followed by quenching with iodomethane (two iterations) or iodoethane provided isopropyl sulfone 21, or n-propyl sulfone 22, respectively. Nitration of 19, 21, or 22 generated only the desired regioisomers 23a-c, which upon reduction with zinc were converted to the orthosulfonylanilines 24a-c, respectively. The synthesis of o-oxazolylaniline 30 required introduction of the oxazole ring first, followed by amination of the benzene ring, whereas a reversal of this sequence was necessitated to access p-oxazolylaniline 38 (Scheme 4). The installation of oxazolyl group consisted three steps: (1) coupling of acid chlorides derived from thionyl choride treatment of benzoic acids 25 or 32 with ethanolamine; (2) ring forming condensation of the resultant hydroxyethylamides 26 or 34, with thionyl chloride27 or PPA, respectively; and (3) aromatization of oxazolines 27 or 36 to the corresponding oxazoles 28 or 37, respectively, using nickel dioxide.28 The amination of benzenes was accomplished via standard nitration of
a Reagents: (a) BCl /RCN/1,2-dichloroethane, HCl/H O/MeOH; 3 2 (b) MeSO2Na/DMF/rt; (c) KOH/MeOH.
oxazolylbenzene 28 and reduction of the nitro group of 29 or 37 with tin chloride.29 The oxidation of a benzylic methyl group in nitromesitylene (31) proceeded to give both isomers 32 and 33, the separation of which was strategically postponed until when the formation of the oxazoline ring was completed. Some additional anilines with an ortho cyano, amido, or phenyl substituent were prepared as demonstrated in Scheme 5. Cuprous cyanide treatment30 of bromoanilines 40a, 40b, provided the desired cyanoanilines 41a and 41b, respectively, whereas a Suzuki reaction31 of 40a with phenylboronic acid furnished aminobiphenyl 42. The bromoanilines 40a and 40b were readily accessible via bromination of anilines 39a and 39b. A standard sequence of nitration, EDCI/HOBtmediated coupling, methylation, and tin chloride reduction afforded neopentylamide 47, while a CDI-mediated coupling reaction of anthranilic acid 48 and dimethylamine furnished dimethyl anthranilic amide 49. For the synthesis of the final target compounds using cyanoanilines 41a and 41b, the acidic conditions employed to unmask the sulfonamide group proved harsh enough to hydrolyze the cyano group to a primary amide. An interesting steric effect on the nitrile hydrolysis was observed. If the cyano group was flanked with only one amino group (41a), it was completely converted to the corresponding primary amide (7d); however, the more hindered cyano group with an
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Journal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Scheme 3. Synthesis of 2-Sulfonylanilinesa
Wu et al.
Scheme 4. Synthesis of Oxazolylanilinesa
a Reagents: (a) MeSO Na/CuI/DMF/100 °C; (b) nBuLi/THF; (c) 2 MeI; (d) EtI; (e) KNO3/H2SO4/0 °C; (f) Zn/NH4Cl/MeOH/H2O.
additional ortho methyl (41b) partially survived the MOM deprotection to give a 1:1 mixture of nitrile 7s and amide 43. The derivatization of methyl ketone 3a is presented in Scheme 6. The keto group was amenable to standard ketone chemistry such as reduction to alcohol 7v and oxime formation to give 7w, methyloxime 7x. The methylation of the 5-position of thiophene in 3a required a lithiation/immediate quenching with iodomethane step22 at the very beginning of the synthesis (Scheme 1, part 3). Similarly, a chloro to methyl switch at the 4-position of the isoxazole ring in 3a also necessitated the use of dimethylisoxazole instead of chloromethylisoxazole at the outset of the synthetic scheme. Standard coupling of acid chlorides 6 and 5 with the aminoacetophenone 50 followed by MOM group cleavage furnished 3a derivatives 7y and 7z. The synthetic methods, yields of last steps, melting points, and the molecular formulas of all target compounds (7a-z) are listed in Table 5 Results and Discussion Sitaxsentan (1) binds competitively to human ETA and ETB receptors with an IC50 of 1.4 nM and 9.8 µM, respectively.2 Our goals have been to generate second generation endothelin antagonists with significantly increased ETA potency and ETA/ETB selectivity versus 1. The inhibition of endothelin binding to ETA and ETB receptors was measured using 125I-labeled ET-1 competition assays. Selectivity for ETA over ETB was expressed as the ratio of ETB IC50 value over that of ETA. ETA binding potency and ETA/ETB selectivity are presented in Tables 1-4. Selected compounds from Tables 1-4 with high ETA potency and selectivity were administered orally at 50 mg/kg to rats. Area under the curve (AUC), number of rats, maximal plasma concentration (Cmax), and plasma
a Reagents: (a) SOCl /ethanolamine; (b) SOCl ; (c) NiO /PhH; 2 2 2 (d) KNO3/H2SO4; (e) SnCl2/HCl;(f) CrO3/HOAc/H2SO4; (g) PPA.
half-life (t1/2) are reported in Table 6. Those compounds from Table 6 with a decent oral pharmacokinetics profile were subjected to a full rat pharmacokinetic study to measure their oral bioavailability. Their in vivo effects were then evaluated in an acute hypoxia-induced pulmonary hypertension model in rats.16 Oral bioavailability and the oral dose to effect 50% inhibition of mean pulmonary arterial pressure (MPAP) are shown in Table 7. The first aryl system studied (Table 1) was 2,4-xylenyl with a different 6-position substituent, mostly electronwithdrawing groups. Comparing to 1, a methyl group (2a) at 6-position caused a 10-fold increase of ETA binding affinity with minimal effect on receptor subtype selectivity. A methyl to chloro switch (2a to 7a) had a modest effect on potency (0.15 to 0.09 nM), whereas selectivity was significantly enhanced (7000 to 166 000fold). A phenyl group (7b) reduced both potency (1.47 nM) and selectivity (1900-fold). Further erosion of ETA potency and selectivity was caused by a carboxyl group (7c) with IC50 of 2.62 nM and selectivity of only 600fold. The primary amide 7d exhibited similar activity (0.14 nM) and selectivity profiles to 2a, whereas the N,N-dimethylamide 7e was more potent (0.07 nM) and
An Orally Bioavailable Endothelin Antagonist
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 8 1973
Scheme 5. Synthesis of 2-Cyano-, Amido-, and Phenylanilinesa
a Reagents: (a) NBS/DCM; (b) CuCN/DMF/heat; (c) PhB(OH) /Pd(PPh ) /Na CO /EtOH; (d) HNO /H SO ; (e) EDCI/HOBt/neopentyl3 3 4 2 3 3 2 4 amine; (f) NaH/MeI; (g) SnCl2/HCl; (h) CDI/THF/dimethylamine.
more selective for ETA receptors (12 300-fold) than 2a. Replacing one of the two methyl groups on amide nitrogen in 7e with neopentyl resulted in a 10-fold loss of potency (0.07 to 0.68 nM). The amide groups seemed to have a size dependent effect on selectivity going from 8100 to 12 300 and to 15 900-fold for unsubstituted- 7d, dimethyl- 7e, and N-methyl-N-neopentylamide 7f, respectively. Sulfonyl groups (7g-i) at the 6-position generally increased ETA potency by 2-5-fold comparing with 2a without much size effect. On the other hand, the small methyl sulfone 7g improved selectivity significantly to 325 000-fold whereas a longer propyl (7h) and a branched isopropyl (7i) sulfone had little effect on selectivity. An aromatic 2-oxazolyl group in 7j improved potency ∼18-fold vs a phenyl group in 7b, with much higher receptor subtype selectivity. However, a positional switch of the o-oxazole and the p-methyl group in 7j completely eliminated those enhancements (7k). To summerize for this series, small to mid-sized electron-withdrawing groups such as halogen, amide, sulfonyl, or oxazole are more effective in potency enhancement than phenyl and carboxyl, affording com-
pounds with ETA potency similar to that of methyl ketone 3a. Five analogues in this were selected for oral pharmacokinetics (PK) evaluation (50 mg/kg) in rats (Table 6). The chloro compound 7a had an AUC value of 172.3 h‚µg/mL and a t1/2 of 2.6 h, with an acceptable Cmax of 47.5 µg/mL which was better than the corresponding methyl compound 2a (35.9 µg/mL) and comparable to the methyl ketone 3a (57.6 µg/mL). Both the dimethylamide 7e and methyl sulfone 7g had low values of AUC (27.2 and 27.8 h‚µg/mL), Cmax (10.9 and 10.3 µg/mL), and t1/2 (1.2 and 1.7 h, respectively). A higher homologue of 7g, n-propyl sulfone 7h, gave even more discouraging results: AUC, 5.9 h‚µg/mL; Cmax 2.9 µg/mL; and t1/2, 1.3 h. In contrast, the 2-oxazolyl anilide 7j exhibited the best oral profile of this mini-series with AUC, Cmax, and t1/2 values of 362.5 h‚µg/mL, 66.0 µg/mL, and 5.3 h, respectively. The Cmax of 7j (66.0 µg/mL) was higher than that of the methyl analogue 2a (35.9 µg/mL) and acetophenone 3a (57 µg/mL), but still only half of that of 1 (122.8 µg/mL) disqualifying it as a follow-up clinical candidate. Efforts were then focused on expanding methyl ketone 3a into a series where the aryl side of the ketone
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Journal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Scheme 6. Modification of 3aa
Wu et al. Table 2. Effect of Ortho Keto Groups on ETA Affinity and Selectivity
a Reagents: (a) NaBH ; (b) NH OH‚HCl, NaOH (aq), 60 °C, 3 4 2 h; (c) NH2OMe‚HCl, Na2CO3, EtOH, 60 °C.
Table 1. Effect of Ortho Substituents on ETA Affinity and Selectivity
entry
X
IC50 ETA (nM) (n)
selectivity for ETA
1 2a 7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 3a
CH3 Cl C6H5 CO2H CONH2 CON(CH3)2 CONCH3(neopentyl) SO2CH3 SO2-n-Pr SO2-i-Pr oxazol-2-yl see structure COCH3
1.4 ( 0.5 (5) 0.15 (1) 0.09 (1) 1.47 ( 0 (3) 2.62 (1) 0.14 (1) 0.07 (1) 0.68 (1) 0.03 (1) 0.06 (1) 0.05 (1) 0.08 (1) 1.27 (1) 0.04 ( 0 (5)
6500 7000 165600 1900 600 8100 12300 15900 325000 6100 8800 118900 1900 442000
carbonyl was maintained while different alkyl groups and a phenyl were screened at the other side of the keto group (Table 2). Small alkyl groups such as methyl (3a), ethyl (7l), or cyclopropyl (7m) afforded similarly potent compounds (ETA IC50 ) 0.03-0.06 nM) with comparable ETA/ETB selectivity (214 000-442 000-fold). A branched
entry
R
IC50 ETA (nM) (n)
selectivity for ETA
3a 7l 3b 7m 7n 7o 7p
Me Et i-Pr cyclo-Pr cyclo-Hex Ph CH2SO2CH3
0.04 ( 0 (5) 0.06 (1) 0.11 (1) 0.03 (1) 1.37 (1) 0.39 (1) 0.10 (1)
442000 214000 144000 331400 13000 46000 66500
isopropyl group (3b) and a much more polar methanesulfonylmethyl group (7p) reduced potency by approximately 2-fold vs 3a with slightly diminished but still good selectivity (144 000- and 66 500-fold). Potency was decreased to the level of 1 with a bulkier cyclohexyl group (7n) with selectivity slightly better than 1 (13 000fold). Comparing to 3a, both binding affinity and subtype selectivity were lowered by ∼10-fold (0.39 nM and 46 000-fold, respectively) by a phenyl group (7o). Three compounds (cyclopropyl ketone 7m, phenyl ketone 7o, and sulfonyl ketone 7p) from this small series were selected for rat pharmacokinetic studies (Table 6). Comparing to methyl ketone 3a, cyclopropyl compound 7m had a similar Cmax (53.4 vs 57.6 µg/mL), but a longer t1/2 (5.4 vs 4.0 h) and hence a larger value of AUC (346.6 vs 272.4 h‚µg/mL). Replacing the ketonic methyl group in 3a with a phenyl (7o) lowered Cmax (33.4 vs 57.6 µg/mL) and AUC (141.5 vs 272.4 h‚µg/mL) values, with a similar t1/2 of 3.95 h. Interestingly, the keto sulfone 7p, with a Cmax comparable to phenyl ketone 7o (37.3 vs 33.4 µg/mL), had an extended t1/2 of 12.2 h and an AUC 2.3-fold higher than 3a. Full rat pharmacokinetics was conducted with 7p and its oral bioavailability was determined to be 16% (Table 7) vs 25% for 3a. In an acute hypoxia-induced pulmonary hypertension rat model, 7p was much less efficacious than 1 or 3a with an EC50 of >5 mg/kg vs 2.5, and
