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Conformationally Constrained ortho-Anilino Diaryl Ureas. Discovery of 1-(2-(1'-Neopentylspiro[indoline-3,4'piperidine]-1-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea; a Potent, Selective and Bioavailable P2Y Antagonist 1

Jennifer X Qiao, Tammy C Wang, Réjean Ruel, Carl Thibeault, Alexandre L’Heureux, William A Schumacher, Steven A. Spronk, Sheldon Hiebert, Gilles Bouthillier, John Lloyd, Zulan Pi, Dora M Schnur, Lynn M. Abell, Ji Hua, Laura A Price, Eddie Liu, Qimin Wu, Thomas E Steinbacher, Jeffrey S Bostwick, Ming Chang, Joanna Zheng, Qi Gao, Baoqing Ma, Patricia A McDonnell, Christine S Huang, Robert Rehfuss, Ruth R. Wexler, and Patrick Y. S. Lam J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 28 Oct 2013 Downloaded from http://pubs.acs.org on November 3, 2013

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

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Conformationally Constrained ortho-Anilino Diaryl Ureas. Discovery of 1-(2-(1'-Neopentylspiro[indoline3,4'-piperidine]-1-yl)phenyl)-3-(4(trifluoromethoxy)phenyl)urea; a Potent, Selective and Bioavailable P2Y1 Antagonist Jennifer X. Qiao,* Tammy C. Wang, Réjean Ruel, Carl Thibeault, Alexandre L’Heureux, William A. Schumacher, Steven A. Spronk, Sheldon Hiebert, Gilles Bouthillier, John Lloyd, Zulan Pi, Dora M. Schnur, Lynn M. Abell, Ji Hua, Laura A. Price, Eddie Liu, Qimin Wu, Thomas E. Steinbacher, Jeffrey S. Bostwick, Ming Chang, Joanna Zheng, Qi Gao, Baoqing Ma, Patricia A. McDonnell, Christine S. Huang, Robert Rehfuss, Ruth R. Wexler, and Patrick Y. S. Lam Research and Development, Bristol-Myers Squibb Company, 311 Pennington-Rocky Hill Road, Pennington, NJ 08534 RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to) ABSTRACT Preclinical antithrombotic efficacy and bleeding models have demonstrated that P2Y1 antagonists are efficacious as antiplatelet agents and may offer a safety advantage over P2Y12 antagonists in terms of reduced bleeding liabilities. In this paper, we describe the structural modification of the tert-butyl ACS Paragon Plus Environment

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phenoxy portion of lead compound 1 and subsequent discovery of a novel series of conformationally constrained ortho-anilino diaryl ureas. In particular, spiropiperidine indoline substituted diaryl ureas are described as potent, orally bioavailable small molecule P2Y1 antagonists with improved activity in functional assays and improved oral bioavailability in rats. Homology modeling and rat PK/PD studies on the benchmark compound 3l will also be presented. Compound 3l was our first P2Y1 antagonist to demonstrate a robust oral antithrombotic effect with mild bleeding liability in the rat thrombosis and haemostasis models.

KEYWORDS Antiplatelet, P2Y1 antagonists, conformationally constrained ortho-anilino diaryl ureas, spiropiperidine indolines, homology modeling of P2Y1, pharmacokinetic, rat models of thrombosis and bleeding

INTRODUCTION Adenosine 5′-diphosphate (ADP) acts as a key activator of platelets in the process of haemostasis and thrombosis. Platelet activation is pivotal in thrombus formation under high shear stress such as in arterial circulation. Recent data also suggest that platelet activation may be important in mediating thrombus formation under lower shear stress, such as in venous circulation. ADP activates platelets by simultaneously interacting with two platelet G protein-coupled receptors (GPCRs), P2Y1 and P2Y12, to produce two intracellular signals that synergistically induce complete platelet activation. Both P2Y1 and P2Y12 must be present and functional for the complete effect of ADP-induced platelet aggregation. The P2Y1 receptor, coupled to G-protein q (Gq), mobilizes intracellular Ca2+. This transitory increase of intracellular free Ca2+ leads to a change of the platelet shape and initiates a weak, transient platelet aggregation in response to ADP. The P2Y12 receptor, on the other hand, coupled to G-protein i (Gi), completes and amplifies platelet aggregation in response to ADP and generates a stable platelet ACS Paragon Plus Environment


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aggregate.1 The blockade of either receptor, therefore, significantly decreases both in vitro and ex vivo ADP-induced platelet aggregation and thrombosis formation.2 As a result, an antagonist that specifically binds to either of the two receptors could theoretically halt ADP-induced platelet aggregation. The P2Y12 receptor is a well-established and clinically validated target for antithrombotic therapy because of its central role in the formation and stabilization of a thrombus and its relatively limited tissue distribution in platelets compared to P2Y1.3 The irreversible P2Y12 antagonist clopidogrel (Figure 1A), a thienopyridine analog acting as a prodrug,4 is in world-wide clinical use for the treatment of peripheral artery disease and acute coronary syndrome as well as for the secondary prevention of vascular ischemic events, stroke, myocardial infarction, and vascular death. Combination therapy of clopidogrel with aspirin is the current standard of care for most patients with non-ST-elevated acute coronary syndromes (NSTE-ACS), percutaneous coronary intervention (PCI) and stent replacement. Besides clopidogrel, additional P2Y12 antagonists were recently approved in the United States including prasugrel5 and ticagrelor6 (Figure 1A), with others in development. In 1999, preclinical studies using P2Y1-deficient (P2Y1−/−) mice and selective adenine nucleotidederived small molecule P2Y1 antagonists introduced P2Y1 antagonism as a new antiplatelet mechanism.7a−g In that year, the Leon laboratory reported that P2Y1−/− mice showed resistance to thromboembolism induced by infusion of ADP or a mixture of collagen and adrenaline with no spontaneous bleeding tendency;7a the lab of Fabre independently confirmed the phenotyping of the P2Y1−/− mice in a separate model of thromboembolism induced by a mixture of collagen and ADP.7b P2Y1−/− mice were also shown to be resistant to thrombin-dependent tissue factor-induced thromboembolism by injection of thromboplastin7c and displayed significantly less localized arterial thrombosis generated by ferric chloride- or laser-induced injury of mouse mesenteric arteries.7d These results were subsequently extended to include the inhibition of both venous and arterial thrombosis in P2Y1−/− rats. Similar results were observed in mice or rats treated with P2Y1 antagonists, the bisphosphate nucleotide derivatives MRS21797c−f and MRS25007g (Figure 1B). The latter, the more

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potent and selective P2Y1 antagonist of the two, strongly inhibited both systematic thromboembolism after intravenous injection of collagen and epinephrine and thrombus formation in laser-injured mesenteric arteries in vivo. Experimental data also showed that additive effects of inhibition of thrombosis formation were observed when clopidogrel was combined with either P2Y1-deficiency7d or a P2Y1 antagonist.7g F F A

O

H COOCH3 N S

H3COCO

Cl

NH

clopidogrel

N

N S

HO(H2C)2O

prasugrel

NHCH3

N N

N N

MRS2179

N

N N

S(CH2)2CH3

F HO

B

O HO P O O OH O HO P O OH

N

O HO P O OH O HO P O OH

OH

ticagrelor

NHCH3

N N

N N R

MRS2500 R = I MRS2279 R = Cl

Figure 1. A. Three P2Y12 antagonists in clinical use: thienopyridines clopidogrel and prasugrel and reversible ticagrelor. B. Three representative nucleotide-derived P2Y1 antagonists from the MRS series.

Our in-house animal models of antithrombotic efficacy and bleeding with selective P2Y1 antagonists such as t-butylphenoxypyridine urea analog 1, as well as clopidogrel, demonstrated that P2Y1 and P2Y12 inhibition provided equivalent antithrombotic efficacy in terms of blocking aggregation and reducing thrombus weight, while P2Y1 antagonists may offer safety advantages in terms of a reduced bleeding liability.8a-d P2Y1 may therefore represent a promising new target for the development of new antiplatelet therapies. The aforementioned bisphosphate adenine nucleotide analogs, although potent P2Y1 antagonists exhibit poor pharmacokinetic profiles and limited oral bioavailability. To date, the non-nucleotide P2Y1 antagonists reported in the literature have not been sufficiently optimized.9 Therefore, the discovery of potent orally bioavailable small molecule P2Y1 antagonists remains an important goal.



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Researchers at Bristol-Myers Squibb recently disclosed a series of 2-(phenoxypyridine)-3-phenylureas with good in vitro potency, represented by compound 1, which was derived from a HTS hit series. Compound 1 was the first in-house P2Y1 antagonist to demonstrate thrombus weight reduction and a lower bleeding liability than clopidogrel in rat models of thrombosis and bleeding.8d Compound 1 was relatively potent (Ki of 6 nM) in the P2Y1 primary membrane binding assay,10 but had only moderate in vitro antiplatelet activity in the ADP-induced platelet aggregation assay11 in platelet-enriched human plasma (PA IC50 = 2.1 μM against 2.5 μM of ADP). The platelet aggregation assay is thought to be more predictive of in vivo activity than the membrane binding assay and is sensitive to plasma protein binding. In addition, poor aqueous solubility and low bioavailability precluded compound 1 from being further progressed. Subsequent SAR studies including substitutions on all parts of the molecule, including changing the urea linker to other one- to four-atom linear linkers as well as urea mimetic linkers such as aminoheterocycles, and incorporation of polar groups to reduce plasma protein binding, failed to produce a compound with significantly improved antiplatelet activity, making it difficult to advance this series.12

Design Strategy Concurrent to the lead optimization efforts on the t-butylphenoxypyridinyl phenylurea series, we also searched for novel structurally diverse small molecule P2Y1 antagonist chemotypes.13 Preliminary molecular modeling studies indicated that compound 1 and the bisphosphate nucleotide derivatives bound at least in part to different regions of P2Y1, therefore the published SAR and homology models of the MRS series was difficult to apply to our design of novel bioavailable P2Y1 antagonists. Several structural modifications to constrain the t-butylphenoxy pyridine region of compound 1 were explored. In the most successful approach, we rigidified the t-butylphenoxy pyridinyl region and reduced the rotatable bonds by replacing the ether oxygen atom in the t-butylphenoxy group with a nitrogen and conformationally constrained the resulting 2-anilino diaryl urea by tying the nitrogen atom back to the quaternary carbon of the t-butyl group (Figure 2). This led to the formation of N-arylated ACS Paragon Plus Environment

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bicyclic [6,5] and [6,6] ring systems 2a−2j, such as indoline/tetrahydroquinoline analogs 2a−2d. Further modification of the 3,3-dimethyl region in indolinyl analog 2a by the addition of bulkier spirocycloalkyl rings (2k−2n), introduction of a protonatable nitrogen into potent spiro[cyclohexane-1,3'-indoline] analog 2n, and subsequent substitution of that nitrogen led to the discovery of the spiro[indoline-3,4'piperidine] analogs 3a−3l. Nitrogen walk on the indoline ring of compound 3l led to azaindoline analogs 3m−3p. Changing the spirocycle ring size and the position of the nitrogen atom resulted in compounds 3q−3s. Conformationally constrained ortho-anilino diaryl ureas OCF3

O

X N

N

O

O

N H

O N H

OCF3

N H 1

OCF3

N H

X

N H

N

N H

N H

N

X

2c, X = CH 2d, X = N

R 2e, X = CH2CH2, R = H 2f, X = CH2, R = H 2g, X = CHMe, R = H 2h, X = O, R = H 2i X = O, R = Me 2j, X = NMe, R = H

Early lead

O N H

N

N H

R

2a, X = CH 2b, X = N

P2Y1 Ki 6 nM P2Y1 Flipr IC50 29 nM PA IC50 2.0 μM privileged @2.5 μM ADP substructure

OCF3

O

OCF3

N H 2k, n=1

n 2l, n=2

2m, n=3 2n, n=4

2-Aryl indole as privileged substructure OCF3

O X

N H NH

increasing solubility by insertion of a protonatable nitrogen

N H

4, X = CH, P2Y1 Ki = 75 nM 5, X = N, P2Y1 Ki = 227 nM

O

7

N

6 5

N

N H

N

OCF3

N H 3m,N6 3n, N4 3o, N5 3p, N7

4

azaindolines O

N H

N

OCF3

N H

OCF3

O

alternate ring size N

N

N H

N H

R

3q

Spiropiperidine indoline (Spiro[indoline-3,4'-piperidine]) 3a, R = CBz 3b, R = H 3c, R = CH2COOH 3d, R = CH2CH2OH 3e, R = CH2CH2OMe 3f, R = COOMe 3g, R = CONHCH(CH3)2 3h, R = Me 3i, R = CH(CH3)2 3j, R = CH(CH3)CH2CH3 3k, R = CH2Ph 3l, R = CH2C(CH3)3

N

alternate nitrogen position


O N

N

N H

m

OCF3

N H 3r, m=2 3s, m=1


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Figure 2. Design of conformationally constrained ortho-anilino diaryl ureas as t-butylphenoxy diaryl urea replacements (Compounds of tables 1−4).

Because the phenoxypyridinyl group can be viewed as a privileged substructure for GPCR targets, we also investigated other privileged structures, such as 2-aryl indoles14 as phenoxypyridinyl replacements. The initial 2-phenylindole analog 4 and 2-pyridinyl indole analog 5 gave 75 nM and 227 nM binding affinity towards P2Y1, but neither was active in the ADP-induced platelet aggregation assay. Moreover, as observed in the t-butylphenoxy series, further modification of these two analogs did not yield compounds with significantly improved binding affinity and these compounds did not show in vitro antiplatelet activity. Herein, we describe the structural modification of the tert-butylphenoxy portion of compound 1 and the discovery of a novel series of conformationally constrained ortho-anilino diaryl ureas. In particular this work resulted in a series of potent and orally bioavailable spiropiperidine indoline substituted diaryl ureas. The SAR of the spiropiperidine indoline ring, including piperidinyl nitrogen substitution leading to 3l (3a−3l), azaindolinyl analogs (3m−3p), and variations of ring size and the position of the nitrogen atom (3q−3s), will also be discussed. Finally, homology modeling and PK/PD studies on the benchmark compound 3l will be presented.

Results and Discussion CHEMISTRY Schemes 1−10 illustrate the synthesis of compounds described herein. Scheme 1 depicts the synthesis of various 3,3-disubstituted indolinyl diaryl ureas as conformationally constrained orthoanilino

systems

(2a,

2b,

and

2l−2o).

Calcium

hydride-induced

Brunner

reaction

of

cyclobutanecarboxylic acid 2-phenyl-hydrazide led to the formation of spiro(cyclobutane-1,3'-oxindole) 6, whereas 3,3-dimethyl (8) and spiro(cyclopropane-, cyclopentane-, and cyclohexane-1,3'-oxindoles) (9, 12, and 13) were prepared via double alkylation of 1-acetyloxindole or oxindole with the ACS Paragon Plus Environment

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corresponding alkyl iodides or alkyl dibromides using LiHMDS as the base. Reduction of oxindoles 6, 8, 9, 12, and 13 with lithium aluminum hydride afforded indolines 7, 10, 11, 14 and 15, respectively. Heating the neat mixture of indolines and 2-fluoronitrobenzene or 2-fluoro-3-nitropyridine under microwave irradiation gave the nitro analogs 16a, 16b, and 17−20. Reduction followed by urea formation led to the indolinyl analogs 2a, 2b, and 2k−2n. Scheme 1a

O NH NH2

+

H N

a,b,c

O

Cl 6

Ac N

H N

e

O

d

O

R2 R1 2 10 R = R = CH3 11 R1,R2 = -(CH2)2-

R2

H N

H N

e

O

7 H N

R1 8 R1 = R2 = CH3 9 R1,R2 = -(CH2)2-

1

H N

d

O

X 14 X = CH2, 15 X = CH2CH2

X 12 X = CH2, 13 X = CH2CH2 H N

NO2 +

R1

H N

d

2

R

X

X f

X g,h

NO2

N

N

O N H

F R1

7, 10, 11, 14, 15

R2 1

2

16a X =CH, R = R = CH3 16b X =N, R1 = R2 = CH3 17 X =CH,R1,R2 = -(CH2)218 X =CH,R1,R2 = -(CH2)319 X =CH,R1,R2 = -(CH2)420 X =CH,R1,R2 = -(CH2)5-

N H

OCF3

R2 2a, 2k−2n R1 2a X =CH, R1 = R2 = CH3 2b. X =N, R1 = R2 = CH3 2k X =CH,R1,R2 = -(CH2)22l X =CH,R1,R2 = -(CH2)32m X =CH,R1,R2 = -(CH2)42n X =CH, R1,R2 = -(CH2)5-

a

Reagents and conditions: (a) Et3N, CH2Cl2; (b) CaH2, 240 °C; (c) HCl, MeOH, H2O; 78−86% for 3 steps; (d) LiAlH4, THF, 77−92%; (e) MeI or BrCH2−X−CH2CH2Br, LiHMDS, 87−90%; (f) neat, microwave, 175 °C, 1200s; 40−95%; (g) Zn, NH4Cl, MeOH, 67−95%; (h) OCN-Ph-p-OCF3, CH2Cl2, 78−93%.

Scheme 2 shows the synthesis of several bicyclic [6,6] ring systems such as compounds 2c and 2j. Treatment of aniline and 3-methylbut-2-enoyl chloride followed by Friedel-Crafts alkylation on the resulting

amide

with

trimethylaluminum

and

LAH

reduction

afforded

the

4,4-dimethyl

tetrahydroquinoline 21. Nucleophilic aromatic substitution of 21 with 2-fluoronitrobenzene under the same conditions as employed to prepare the indolinyl analogs above (Scheme 1) did not yield any desired product. However, we found that using 2,4,6-lutidine as the base at high temperature (>200 °C) under microwave irradiation, the reaction proceeded to the desired nitro analog 23, which after ACS Paragon Plus Environment


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hydrogenation gave the aniline 22 in 35% yield. On the other hand, Buchwald-Hartwig cross-coupling of 21 with 2-bromonitrobenzene in the presence of Pd2(dba)3 and rac-BINAP followed by zinc reduction afforded the aniline 22 in an improved 73% yield. Surprisingly, an attempt to further improve the yield by replacing the 2-bromonitrobenzene with 2-iodonitrobenzene gave rise to a C−C coupled product 23 in 18% yield and only a 2% yield of the desired nitro product was obtained in the presence of Pd(OAc)2, rac-BINAP and KO-t-Bu in refluxing toluene. Addition of bromo ethylacetate to ophenylenediamine led to tetrahydroquinoxalinone intermediate 24. Reductive amination of 24 with paraformaldehyde and sodium cyanoborohydride gave the N-methylated derivative 25, which was reduced with LAH to afford 1-methyl-1,2,3,4-tetrahydroquinoxaline (26). Nucleophilic aromatic substitution of 2-fluoronitrobenzene with 26 using KO-t-Bu as the base in DMSO afforded the nitro intermediate, and 2j was subsequently obtained using similar transformations leading to 2c. Compounds 2d−2i (Figure 2) were synthesized similarly following the procedures of 2c or 2j. Scheme 2

a OCF3

O NH2

H N

a,b

NH2

c or d

N

e

N

N H

2c

22

21

O2N

N H

23 OCF3

O NH2

H N

f

NH2 24

N H

O

g

H N

O

H N

h

N

N 25

i,j,k

26

H N

N N

N H

N H

2j

a

Reagents and conditions: (a) 3-methylbut-2-enoyl chloride, CHCl3, reflux, 2 h; then AlCl3, toluene, 80 °C, 2.5 h, 80%; (b) LiAlH4, THF, 0 °C, 93%; (c) with 2-fluoronitrobenzene, neat, 2,4,6-lutidine, microwave, 220−250 °C, 3600s, then Pd/C (10%), MeOH, 35%; (d) with 2-bromonitrobenzene, Pd2(dba)3, rac-BINAP, Cs2CO3, toluene, 100 ºC, 16 h; then Zn, NH4Cl, MeOH, 82%; (e) OCN-p-Ph-OCF3, CH2Cl2, 77%; (f) BrCH2COOEt, Et3N, DMF, rt, 16 h then 80 °C, 3 h, 80%; (g) NaBH3CN, (CHO)n, MeOH, HOAc, 98%; (h) LiAlH4,THF, rt, 95%; (i) 2-fluoronitrobenzene, KOt-Bu, DMSO, 80 °C, 16 h, 45%; (j) Pd-C (10%), H2, rt, 2 h, 90%; (k) OCN-p-OCF3-Ph, CH2Cl2, 76%.

The initial synthesis of the N-isobutyl and N-neopentyl spiropiperidine indoline diaryl ureas 3j and 3l is shown in Scheme 3. Spiro[indoline-3,4'-piperidine] (27) was prepared according to modified literature procedures by dialkylation of 2-fluorobenzeneacetonitrile with bis(2-chloroethyl)carbamic acid 1,1-dimethylethyl ester followed by deprotection of the Boc group and then reductive spirocyclization.15 The more basic nitrogen in 27 was selectively protected with benzyl 2,5ACS Paragon Plus Environment

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dioxopyrrolidin-1-yl carbonate to give 28. Heating the neat mixture of 28 and 2-fluoronitrobenzene under microwave irradiation gave the corresponding nitro intermediate in 70% yield, which underwent reduction to give the aniline 29 followed by urea formation to afford the urea 3a. Removal of the Cbz group in 3a and then reductive amination with either dimethyl- or trimethylacetaldehyde provided compounds 3j and 3l, respectively. Single crystal X-ray analysis confirmed the structure of the HCl salt of 3l.16 In the crystal structure, the piperidinyl nitrogen is protonated and the Cl anion forms three hydrogen bonds with the protonated piperidinyl nitrogen and the two urea NH, and connects individual molecules into a one-dimensional chain. The urea group adopts a cis orientation (i.e., “W” shaped) in the solid state. Scheme 3

a

HCl

H N

H N

Cl a,b,c,d F Cl

O h

N

N Cbz 28

N H 27

CN

N H

N H

N Cbz 3a

i

NO2

N H

N

N R

N 29 Cbz

F

O

OCF3

NH2

N

f,g

e

HN

OCF3

N H

k

j

3b R = H 3j R = isobutyl 3l R = neopentyl

a

Reagents and conditions: (a) (Boc)2O, Et3N, CH2Cl2, 90%; (b) 2-fluorophenyl-acetonitrile, NaH, THF, 65%; (c) 4 M HCl/dioxane, reflux, 75%; (d) LiAlH(OEt)3 (freshly prepared by EtOH+LAH in ethylene glycol/dimethyl ether), reflux, 2 days, 77%; (e) benzyl 2,5-dioxopyrrolidin-1-yl carbonate, THF, rt, 62%; (f) 200 °C, microwave, 20 min, 70%; (g) Zn, NH4Cl, MeOH; (h) OCN-Ph-p-OCF3, THF, reflux, 67% for two steps; (i) H2, 10% Pd/C, MeOH, HOAc, 96%; (j) (CH3)2CHO, NaB(OAc)3H, ClCH2CH2Cl, HOAc, 72%; (k) t-BuCHO, CH(OMe)3, HOAc, NaBH(OAc)3, NMP, 58%.

A larger scale synthesis of compound 3l was carried out as depicted in Scheme 4. Fischer indole synthesis followed by in situ reduction of the indolenine intermediate was applied to construct the spiropiperidine indoline core. Treatment of 1-neopentylpiperidine-4-carbaldehyde (30) with phenyl hydrazine while heating at 50 °C in HOAc followed by addition of BF3.Et2O and heating at 80 °C yielded the indolenine intermediate, which underwent in situ reduction with NaBH4 in MeOH to afford the desired indoline 31. The spiroindoline-3,3'-piperidine analog 3r (Figure 2) was prepared via a ACS Paragon Plus Environment


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similar sequence starting from the Fischer indole synthesis of benzyl 3-formylpiperidine-1-carboxylate and phenylhydrazine followed by in situ reduction of the resulting imine. Scheme 4

a

OH CO2Et

HN

c

a, b

NH2 d-f

+

N

N

N H

H N

CHO

N 31

30 O g

NO2

N

N 32

h,i

N H

N

OCF3

N H

N 3l

a

Reagents and conditions: (a) (CH3)3CCOCl, CH2Cl2, Et3N, 94%; (b) LiAlH4, THF, 91%; (c) (COCl)2, DMSO, CH2Cl2, 95%; (d) HOAc, 50 °C, 2 h; (e) BF3−Et2O (3 equiv), 80 °C, 1.5 h; (f) NaBH4, MeOH, rt, 38% over 3 steps; (g) 2bromonitrobenzene, Pd2(dba)3, rac-BINAP, Cs2CO3, toluene, 100 °C, 80%; (h) H2, Pd-C (10%), MeOH/EtOAc (2:3), 88%; (i) NCO-Ph-p-OCF3, THF, 99%.

Schemes 5−8 illustrate the synthesis of azaindolinyl analogs 3m−3p. The synthetic route for the spiro[6-azaindoline 3,4'-piperidine]diphenyl urea analog 3m is illustrated in Scheme 5. Nucleophilic aromatic substitution of the anion derived from nitrile 33 and KHMDS as the base with 3-fluoro-4chloropyridine selectively displaced the chlorine atom at the 4-position of the pyridine ring to provide the fluoride 34. Intramolecular cyclization of 34 to form the Boc-protected spiropiperidine 6-azaindoline 35 was achieved using lithium tri-tert-butoxy aluminum hydride in 1,4-dioxane heating at 130 °C under microwave irradiation. Urea 36 was prepared using the same sequence as that employed in 3l. Transforming the Boc group to the pivaloyl group followed by reduction afforded the desired 6-aza indolinyl analog 3m. Scheme 5

a


Page 13 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


N

Cl F

CN

a NC

O c, d, e

b F

N

H N

N

N Boc

NBoc

N

N

N R

35

OCF3

N H

N Boc

34

33

N H

36. R = Boc f, g 3m. R = neopentyl

a

Reagents and conditions: (a) KHMDS, toluene, 0−23 °C, 64%; (b) lithium tri-tert-butoxy aluminum hydride, 1,4-dioxane, microwave, 130 °C, 18%; (c) 2-bromonitrobenzene, Pd2(dba)3, rac-BINAP, Cs2CO3, toluene, 100 °C, 80%; (d) H2, Pd-C (10%), MeOH/EtOAc (2:3), 96%; (e) OCN-Ph-p-OCF3, THF, 74%; (f) TFA, CH2Cl2, 68%; (g) Piv-Cl, DIPEA, CH2Cl2, 63%; (h) LiAlH4, THF, 57%.

6-Chloro-4-azaindolinyl analog 37 was prepared in an analogous fashion to 35 starting from 2,3difluoro-4-chloropyridine (Scheme 6). The chlorine in 37 was then reduced at the aniline stage (38 to 39) and the neopentyl group was introduced at the end of the synthesis via reductive amination with polymer supported cyanoborohyride to afford spiro[4-azaindoline 3,4'-piperidine] diphenyl urea 3n. Scheme 6

a

Cl

F

Cl

NC N

F

NH2

N N

Boc

NC

N

Boc

N

NH2

N

e

Boc

O f,g,h

N H

N

OCF3

N H

N N

Boc 38

N

37

N N

c, d

b N

33

Cl

H N

Cl

a

+ N

F

Boc 39

N

3n

a

Reagents and conditions: (a) KHMDS, toluene, 0−23 °C, 96%; (b) lithium tri-tert-butoxy aluminum hydride, dioxane, 100 °C, 21%; (c) 2-bromonitrobenzene, Pd2(dba)3, rac-BINAP, Cs2CO3, toluene, 100 °C, 58%; (d) H2, Pd-C (10%), MeOH/EtOAc (2:3), 64%; (e) H2, Pd-C (10%), Degussa (wet), MeOH/EtOAc (2:3), 42 psi, 29%; (f) OCN-Ph-p-OCF3, THF, 31%; (g) TFA, CH2Cl2; (h) HCO-tBu, PSN(CH3)3+BH3CN-, CH3COOH, CH2Cl2, 15% for 2 steps.

Scheme 7 illustrates the synthetic route to 5-azaindoline diphenyl urea 3o. The chloromethyl dihydropyridine 40, prepared according to a literature method in three steps17 was used as an electrophile to react with 3-bromo-2-chloropyridin-4-amine using LiHMDS as the base. The secondary amine 41 was protected with a trifluoromethylacetyl group followed by an intramolecular Heck reaction which afforded spiro[indoline-3,4'-tetrahydropyridine] intermediate 42 in 49% yield. Buchwald-Hartwig cross coupling followed by reduction of the nitro group and urea formation provided 43. The double ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 52

bond in 43 was reduced with triethylsilane in the presence of trifluoroacetic acid, generating the spiropiperidine 44. Reductive amination resulted in N-neopentyl analog 45. High pressure hydrogenation of 45 afforded the spiro[5-azaindoline 3,4'-piperidine] diphenyl urea 3o. Scheme 7

a

Cl

O

N

a,b,c

N H

N Cl

N Boc

N H

HN

NO2 42

N H

N

N Boc

OCF3

N H

k H

N H

Br

O

O N

g,h,i

N

41 N Boc

Cl 43

H N

Cl

OCF3 j

e,f

d

N Boc 40

O N

Br

Br Cl

N Boc

Cl

N

NH2

O

N H

N N R

N

OCF3

N H

l

45 (R = Cl) 3o (R = H)

44

a

Reagents and conditions: (a) tetramethylpiperidine, n-BuLi, THF, -78 °C, 30 min, then methylphenyl sulfoxide, -78 to 23 °C; (b) KO-tBu, tBuOH, 80 °C, 2 h, 31% for 2 steps; (c) SOCl2, 2,6-lutidine, toluene, 40 °C, 30 min, 85%; (d) LiHMDS, THF, 47%; (e) TFAA, Et3N, CH2Cl2, 79%; (f) Pd(OAc)2, Et3N. tBu4NBr, DMA, 90 °C, 49%; (g) Pd2(dba)3, rac-BINAP Cs2CO3, toluene, 100 °C, 80%; (h) H2, Pd-C (10%), EtOAc, 60 °C, 98%; (i) OCN-Ph-p-OCF3, THF, 99%; (j) Et3SiH, TFA, CH2Cl2, 50%; (k) PSN+(CH3)3B+H3CN, CH3COOH, CH2Cl2, 70%; (l) H2, Pd-C (10%), Degussa (wet), EtOH, 60 psi, 80%.

The spiro[7-azaindoline 3,4′-piperidine] diphenyl urea 3p was prepared via dialkylation of 2(trimethylsilyl)ethoxymethyl

(SEM)-protected

1,3-dihydro-2H-pyrrolo[2,3-b]pyridin-2-one

(47)

(Scheme 8). It is worth mentioning that the Fischer indole synthesis was an efficient method for the construction of compound 3l and substituted indolinyl analogs; however, reaction of 2pyridinylhydrazine with N-Boc-4-piperidinylaldehyde failed to give any desired cyclization product under several reaction conditions. The dialkylation of 47 was carried out using cesium carbonate in DMF to afford the SEM-protected spiro[7-azaindoline 3,4′-piperidine] 48. Removal of the SEM group was achieved in a two-step one-pot process (48 to 50). Acylation of 50 followed by reduction afforded the N-neopentyl spiro[7-azaindoline 3,4′-piperidine] 51. Scheme 8a


Page 15 of 52

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N

H N

N

a,b

SEM N O Br 46

f

N

d

Br

SEM N O

e

N 48 Boc

47

g,h,i,j

N

N

N H

N

N

R N O

N H 49 R = CH2OH 50 R = H

O

H N

51

N

c

N

SEM N O

OCF3

N H

N 3p

a

Reagents and conditions: (a) NaH, SEM-Cl, DMF, 100%; (b) Br2, PyH+Br-, dioxane; (c) Zn, NH4Cl; 77% for 3 steps; (d) (CH2CH2Cl)2NBoc, Cs2CO3, DMF, 80 °C, 30%; (e) 4 N HCl in dioxane, rt, 1h, then ethylene glycol, 70 °C, 15 h; (f) trimethylacetyl chloride, Hunig’s base, CH2Cl2, rt, 4 h, 75% for 2 steps; (g) LiAlH4, THF, rt, 25 min; (h) 2Bromonitrobenzene, Pd2dba3, rac-BINAP, Cs2CO3, toluene, 100 °C, 16 h, 36% for 3 steps; (i) Zn, NH4Cl, MeOH/EtOAc (1:1), rt, 16 h; (j) OCN-Ph-p-OCF3, THF, rt, 16 h, 9% for 2 steps.

2',3'-Dihydro-spiro[piperidine-4,4'(1'H)-quinoline] diphenyl urea 3q was prepared as illustrated in Scheme 9. The Boc protecting group in 1-oxospiro[indane-3,4'-piperidine] analog 52 was switched to the trifluoroacetyl group. Ring expansion of the indene-3-one 53 via Schmidt rearrangement with hydrogen azide, generated in situ from sodium azide and conc. sulfuric acid, gave the spiro[piperidine4,4'(1'H)-quinolin]-2'(3'H)-one (54) in 54% yield after separation of the two regioisomers via flash column chromatography. The trifluoromethylacyl group was then changed to the pivaloyl group and treatment of 55 with LAH gave 56, which was converted to urea 3q as above. The spiro[indoline-3,3'-pyrrolidine] diphenyl urea 3s was prepared as shown in Scheme 10. Treatment of tetrahydro-β-carboline 57 with tert-butylhypochloritein the presence of triethylamine afforded the chloroindolenine intermediate 58, which was poured directly into a refluxing mixture of NaOH in MeOH to give the complete rearranged product, β-spiro[pyrrolidinoindolenine] 59. Reduction of 59 with LAH afforded the spiropyrrolidine indoline 60. Scheme 9a



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

O

O

H N

a-b

Page 16 of 52 O

O

H N

c

d-e

N

N Boc

O

52

N CF3

O

53

54

g-i

N H

N

N

N CF3

O

O

CF3

55

OCF3

O

H N f

O

NH

N H

N N 56

3q

a

Reagents and conditions: (a) TFA, CH2Cl2, 0 °C to rt, 30 min; (b) (CF3CO)2O, Et3N, DMAP (cat.), CH2Cl2, rt, 2 h, 92% for two steps; (c) NaN3, conc. H2SO4, H2O, CHCl3, 0 °C to rt, 3 h, then conc. H2SO4, rt to 50 °C, 54%; (d) KOH, H2O, MeOH, rt, 16 h; (e) t-BuCOCl, Et3N, CH2Cl2, rt, 4 h, 79% for 2 steps; (f) LiAlH4, THF, rt, 16 h, 81%; (g) 2-bromonitrobenzene, Pd2(dba)3, rac-BINAP, Cs2CO3, toluene, 100 °C, 16 h, 90%; (h) Zn, NH4Cl, EtOH, rt, 16 h, 63%; (i) OCN-Ph-p-OCF3, THF, rt, 3 days, 42%.

Scheme 10

a H N N

c

N

N

58

59

O

H N

d,e,f N

60

OCH3

b Cl

57

N

N

a

N H

N

OCF3

N H

N 3s

a

Reagents and conditions: (a) tBu-OCl, Et3N, CH2Cl2, rt; (b) NaOH, MeOH, reflux, 93% for 2 steps; (c) LiAlH4, Et2O, 88%; (d) 2-bromonitrobenzene, Pd2(dba)3, rac-BINAP, Cs2CO3, toluene, 75%; (e) H2, Pd/C, (f) OCN-Ph-p-OCF3, THF, rt, 66% for 2 steps.

In vitro biological results The 3,3-dimethylindoline analog 2b was the first conformationally constrained ortho-anilino diaryl urea made according to the aforementioned design strategy. It showed a three-fold drop of P2Y1 binding affinity compared to 1 and had IC50 >10 μM in the ADP-induced platelet aggregation (PA) assay. Surprisingly, changing the central 2-pyridyl ring to the phenyl ring (2a) improved both binding affinity (Ki = 8.7 nM) and in vitro antiplatelet activity (PA IC50 = 8.5 μM at 2.5 μM of ADP). The same SAR trend was observed with 4,4-dimethyl tetrahydroquinoline analogs 2c and 2d. Compound 2c had very ACS Paragon Plus Environment

Page 17 of 52

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similar binding affinity and antiplatelet activity (Ki = 7.3 nM and PA IC50 = 2.2 μM, respectively) as the t-butylphenoxypyridine urea compound 1. Both methyl groups in 2c were required for binding, as removing either one (2g) or both (2f) showed reduced potency. Ring expansion to the [6,7] bicyclic compound 2e resulted in much lower binding affinity. Because of the promising activity shown with 2c, we looked at several structurally related [6,6] bicyclic analogs of 2c hoping to reduce lipophilicity while maintaining activity. While changing from the 4-carbon atom in 2f to an oxygen or N-methyl group (2h, and 2j) did reduce HPLC logP18 (HPLC logP of 2h and 2j is 5.7 and 5.5 respectively, compared with > 6.2 for 2c), these compounds showed reduced binding affinity and significant loss of antiplatelet activity. Adding a gem-dimethyl group at the C−3 position of the benzomorpholine 2h, which provided 2i, did not result in much improvement in potency.

Table 1. Human P2Y1 receptor binding (Ki) and in vitro platelet aggregation (PA) activity of selected N-aryl indolines, tetrahyhydroquinolines and related bicyclic analogsa OCF3

O X M

Compd #

N H

N H

P2Y1 Ki (nM)a

PA IC50 (μM)b

6.0±0.6

2.1±0.3 (n=7)

CH

8.7±1.7

8.5

N

20.3±4.6

>10

CH

7.3±0.6

2.2

N

16.0±2.7

11.1±0.2

X

M

1

2a

N Me

2b

Me

N Me

Me

N

2c Me Me N

2d Me Me



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2e

2f

N

N

Page 18 of 52

CH

830±5

NT

CH

36.5±5.3

>10

CH

29.5±5.5

>10

CH

65±15

>10

CH

39±11

>10

CH

94±15

>10

N

2g Me

2h

N O

2i

N O

N

2j

N Me

a

Ki values are reported as the mean ±SD, n≥2. See reference 10 for details. bPA activity was tested against 2.5 μM of ADP in human platelet-rich plasma, n=1 unless stated. See reference 11 for details. NT: not tested.

The tetrahydroquinoline analog 2c had relatively low metabolic stability (percentage remaining after 10 min liver microsomal incubation: 51% (human), 6% (rat), and 63% (mouse)) presumably because of the extensive oxidation on the saturated carbons of the 4,4-dimethyltetrahydroquinoline ring. Therefore, subsequent SAR studies focused on analogs of the indolinyl 2a. Previous SAR suggested that the hydrophobicity of the gem-dimethyl group in 2a was important for binding affinity and in vitro functional activity. This was further explored with spirocycloalkyl analogs by varying the spirocycle ring size. As shown in Table 2, increasing the carbocyclic ring size of the spirocycle from cyclopropyl 2k to cyclobutyl 2l to cyclopentyl 2m to cyclohexyl 2n led to improved binding potency and platelet aggregation activity. Spirocyclopentyl analog 2m (Ki = 6.6 nM, PA IC50 = 2.4 μM) and spirocyclohexyl analog 2n (Ki = 5.9 nM, PA IC50 = 2.0 μM) were equipotent to the t-butyl phenoxy lead 1 in both the binding and the platelet aggregation assays. Insertion of a 4-nitrogen atom to introduce polarity into the cyclohexyl ring in 2n, resulted in 3b with a three-order-of-magnitude reduction of P2Y1 binding affinity but with improved aqueous solubility (2n: 10

7.6±0.7

10.5±0.9

4.1

6.6±0.3

NT

2.4

5.9±0.4

2.2±0.2

2.0

36.6±9.9

5.9±0.2

>10

N

2m

N

2n

N

3a N

Cbz



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 52

N

4500±450

3b

NT

NT

NH

a

Ki values are reported as the mean ±SD, n≥2. See reference 10 for details. bFLIPR was tested with 0.5 nM of ADP, and IC50 values are reported as the mean±SD, n=2 unless stated, see reference 20 for details. cPA activity was tested against 2.5 μM of ADP in human platelet-rich plasma, n=1 unless stated. See reference 11 for details. NT: not tested.

We continued to investigate the N-1' substitution on the piperidinyl ring in 3b (see Table 3). Polar substitutions such as acid (3c) and alcohol (3d) functional groups were not well tolerated. The methylethyl ether analog 3e showed similar activity to the Cbz analog 3a but with improved aqueous solubility. Neutralization of the basic NH in 3b with small functional groups such as carbamate (3f) and urea (3g) groups improved binding affinity compared with the unsubstituted 3b, with methylcarbamate 3f being the most potent (Ki = 13 nM). Larger liphophilic groups were more potent, a trend visible from the N-methyl substituted 3h to N-isobutyl substituted 3j to the N-neopentyl substituted 3l; although as expected with an increase in logP, reduced solubility was observed. A methylene linker between the piperidinyl nitrogen atom and the aryl group (3k) or the bulky alkyl group (3l) was preferred, with the neopentyl substitution in 3l being optimal. A threshold concentration of 2.5 μM level of agonist ADP was initially used in the platelet aggregation assay due to the relative low potency and high protein binding of the compounds tested. Because the significant potency improvement seen in compounds such as 2n and 3l in the P2Y1 FLIPR functional assay (IC50 = 2.2 and 2.5 nM for 2n and 3l, respectively), the agonist concentration for the PA assay was increased to 10 μM, a concentration believed to be clinically relevant because it is the lowest concentration that produces a maximum aggregation response in rat platelet rich plasma.8c To our delight, compound 3l had a PA IC50 of 0.67 μM against 2.5 μM ADP and a PA IC50 of 4.9 μM against 10 μM ADP. This was the first compound in the program that demonstrated good antiplatelet activity against 10 μM ADP.

Table 3. In vitro data of N-1' substitution of spiropiperidine indolinyl analogs 3b−3l ACS Paragon Plus Environment

Page 21 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O N

N H

OCF3 insert a protonatable nitrogen

N H

O N H

N

2n

N H

OCF3

3b−3l N R

PA IC50 μMb at 2.5 μM ADP NT

PA IC50 μMb at 10 μM ADP NT

Solubility μg/ml19 84 (partial cryst.)

HPLC logP18

3b

H

P2Y1 Ki (nM)a 4500±1900

3c

CH2COOH

13500

NT

NT

5 (amorphous)

4.4

3d

CH2CH2OH

4500±450

NT

NT

123 (partial cryst.)

4.6

3e

CH2CH2OMe

390±80

25.4

NT

66 (amorphous)

5.1

3f

COOMe

13.3±5.5

5.8

54.2

1 (cryst.)

6.0

3g

CONH-isopropyl

200±30

>10

NT

1 (partial cryst.)

5.7

3h

Me

690±140

NT

NT

100 (amorphous)

4.6

3i

isopropyl

300±260

4.7±0.8 (n=2)

29.2±0.6 (n=2)

71 (partial cryst.)

4.5

3j

isobutyl

20.7±3.2

2.2±0,2 (n=3)

8.8±2.7 (n=2)

7 (partial cryst.)

5.5

3k

CH2Ph

18.6±1.1

3.3±1.0 (n=3)

18.6

6.3

3l

neopentyl

4.3±2.6

0.67±0.31(n=6)

4.9±1.7 (n=267)

4 (cryst.)

>6.2

Compd# R

4.3

a

Ki values are reported as the mean ±SD, n≥2. See reference 10 for details. bPA activity was tested against either 2.5 μM of ADP or 10 μM of ADP in human platelet-rich plasma, n=1 unless stated. See reference 11 for details. NT: not tested.

A nitrogen walk on the phenyl ring of the indoline ring provided four azaindoline analogs 3m−3p (Table 4). Among them, compounds 3m and 3n were much more potent in binding and more active in the platelet activation assay compared with the other two azaindoline analogs 3o and 3p. In addition to being most potent, the spiropiperine-6-azaindoline analog 3m also showed improved aqueous solubility (25 μg/mL at pH=6.5, albeit with amorphous compound). Modifying the spirocycle ring size of 3l led to the [6,6]spirocycle analog 3q, which had activity comparable to 3l. Changing the position of the piperidinyl nitrogen in 3l as shown in the spiro[indoline-3,3'-piperidine] 3r resulted in much reduced activity compared with 3l, and the [5,5]spirocycle analog 3s, which, despite having similar binding affinity as 3l, was 4-fold less active in PA assay.

Table 4. In vitro data of N-neopentyl substituted analogs 3m−3s ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

O N H

M

N

N

N

N

N 3l

3m

N

N

N

N

N

N

M=

OCF3 N H

N

N

N

Page 22 of 52

N

N 3n

3o

N

N

N

3r

3q

3p

N 3s

Solubility19 P2Y1 FLIPR IC50 PA IC50 (μM) P2Y1 HPLC logP18 a b c Ki (nM) (nM) μg/mLd at 10 μM ADP 4.3±2.6 2.5±0.5 4.9±1.7 (n=267) 4 >6.2 3l 11.5±1.0 10.7±3.8 4.0±2.7 (n=2) 25 >6.0 3m 28.7±7.8 NT 15.1±2.7 (n=2) 5 >6.2 3n 617.2±99.8 346 >40 428 5.1 3o 253±35 503±249 >40 NT NT 3p 4.6±0.9 9.9±0.9 6.3 16 >6.2 3q 12.8±4.0 NT >40 6.2 3r 5.8±0.9 NT 18.5 1 >6.2 3s a b Ki values are reported as the mean±SD, n≥2. See reference 10 for details. FLIPR was tested with 0.5 nM of ADP, and IC50 values are reported as the mean±SD, n=2, see reference 20 for details. cplatelet aggregation (PA) was tested with 10 μM of d ADP in human platelet-rich plasma, n=1 unless stated. See reference 11 for details. solubility data reported are from amorphous material except for compound 3l which was crystalline. NT: note tested. Compd #

Pharmacokinetics Based on potency in the in vitro antiplatelet assay and metabolic stability, compounds 3j, 3l, 3m and 3q were selected for PK studies. Table 5 illustrates the in vitro profile and the rat PK data for these compounds. In rats, compound 3l had the best profile with relatively low clearance (CL) and medium volume of distribution (Vdss,3.1 L/kg), slightly high PO exposure, and 32% oral bioavailability. In dogs using the same vehicle as the rat PK studies (0.1 mg/kg IV and 0.2 mg/kg PO), compound 3l demonstrated low CL (5.3 mL/min/kg) and relatively small Vdss (2.8 L/kg) with an oral half-life of 9.7 h and oral bioavailability of 55%. Compound 3l was highly bound to serum proteins with a free fraction less than 0.2% in all five species tested (human: < 0.1%, rat: < 0.1%, mouse: 0.2%, rabbit: 0.1%, and dog: 0.2%).

Table 5. In vitro potency and rat PKa profile of representative compounds 3j, 3l, 3m, and 3q P2Y1

PA IC50

HLM

d

RLM

d

IV CL

IV Vdss


T1/2

PO

F%

Page 23 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3j

b

c

3l

b

3m 3q

b


(mL/min/kg)

(L/kg)

(h)

AUC total (nM*h)

84

27.1

12.8

7.5

147

26

89

87

9.1

2.8

7.5

587

32

4.0

67

73

17.7

5.6

6.1

167

19

6.3

90

87

14.5

4.1

7

278

33

Ki (nM)

(μM) at 10 μM ADP

20.7

8.8

79

4.3

4.9

11.5 4.6

a. IV bolus and PO administration: dose: 0.5 mg/kg; vehicle: 10% Cremophor/10% N,N-dimethylacetamide (DMAC)/10%EtOH/70%H2O. b. Cassette PK. c. Discrete PK. d. Percentage remaining after 10 min incubation. HLM: human liver microsomes, RLM: rat liver microsomes.

Compound 3l was selective against P2Y12 and P2Y2 with IC50 > 20 μM in the P2Y12 cell binding assay21 and IC50 > 10 μM in the P2Y2 FLIPR functional assay22 and achieved > 600-fold selectivity against P2X1 in the FLIPR functional assay23 (IC50 = 1.6 μM for P2X1). Compound 3l was clean against a broad kinase panel. In the MDS panel, 3l showed much cleaner profile compared to the previous disclosed lead molecules bearing amphiphilic amines,12a,d but did show inhibition against opiate receptor (non selective, IC50 = 2.7 μM), rat Ca channel L-type and Na channel site 2 (78 % and 88% at 10 μM respectively. Despite these potential selectivity issues, compound 3l provided the best overall profile and was selected as a benchmark compound and advanced into in vivo studies in rat models of thrombosis and bleeding. Binding model of Compound 3l The single crystal X-ray structure of the HCl salt of the N-neopentyl spiropiperidine indoline 3l showed that the urea group adopted a “cis” orientation. A cis urea was also preferred by 3l in solution, as determined by 2D NMR experiments including ROESY and NOESY in either DMSO-d6 or 9:1 H2O:DMSO-d6 (see supporting information for experimental details). Efforts to develop a binding model of P2Y1 bound to compound 3l began with an externally developed model of the receptor bound to antagonist MRS2279, a bisphosphate nucleotide derivative (Figure 1B), which was kindly provided by Kenneth Jacobson and Stefano Costanzi.24 The MRS model ACS Paragon Plus Environment


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was based on the structure of bovine rhodopsin, which was the only GPCR that had been crystallized at the time. It was evident that refinement of the model was required, primarily because the binding site was too small to fit compound 3l and its analogs, and the two antagonist series have little obvious chemical similarity (for example, the MRS analogs have a high negative charge density, with two phosphate groups, in contrast to the relatively hydrophobic and cationic compound 3l). Several mutants of residues in the binding site in the rhodopsin-based model —Tyr100Ala, Phe131Ala, His132Ala, Asn197Ala, Thr201Leu, Thr221Ala, Phe226Ala, Phe276Ala, and Ser314Ala— were produced to provide experimental data for model refinement.24a The mutants were tested in the aforementioned primary binding assay using [33P] 2-methylthioADP (2MeSADP) as a probe with a wide range of P2Y1 antagonists from several chemotypes, including conformationally constrained ortho-anilino diphenylurea compounds (e.g., 3l) and bisphosphate derivatives. Almost without exception, the only ligand/mutant combinations for which the Ki values differed more than 10-fold from the wild-type were diphenylurea compounds with the Phe131Ala mutant. It should be noted that five of these mutants had been previously reported to be less responsive to 2MeSADP in an inositol phosphate accumulation functional assay.24a The difference in the mutant data may be due to the different assays: these residues may be important for agonism but contribute little to overall binding. Nevertheless, the fact that so many of these residues appear to be uninvolved in binding any of the chemotypes is strong evidence that rhodopsin is not a suitable template for a homology model of P2Y1. A new homology model was constructed from the crystal structure of the CXCR4 receptor,25 which has the greatest residue identity in the transmembrane domain (TM) regions with P2Y1 (24%) among all receptors that had been crystallized to date. The CXCR4 was also recently found by another group to be the best template for a reversible series of P2Y12 antagonists.26 A binding model of compound 3l (Figure 3, cyan) was obtained by docking and refined by molecular dynamics (MD). Consistent with the crystallography and NMR results, the ligand pose contains a cis urea. Consistent with mutagenesis results, most of the mutated residues (Figure 3, green) ACS Paragon Plus Environment

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are relatively far from the ligand binding site and do not interact with the ligand in the model. However, Phe131 (Figure 3, red) forms two crucial aromatic edge-to-face interactions via both the indoline phenyl ring and the adjacent phenyl ring.

Figure 3. A. Compound 3l (cyan carbons) bound in P2Y1. Key complex interactions are shown as orange dashes between 3l and the labeled residues. Residues for which P2Y1 mutants were tested but had no effect on 3l binding are shown as green spheres. B. The binding model with the protein represented by ribbons and surface. The view is rotated slightly from A, but the colors of the residues are the same.

Key SAR data can be explained by the binding model. The fact that at least one hydrogen bond donor is required in the region of the urea may be due to the hydrogen bonding with the side chain of Asp204 on extracellular loop 2 (ECL2) (Figure 3, magenta). The original pose based on docking hinted at these hydrogen bonds, and the dynamics indicated that they were reasonably stable. At least one direct hydrogen bond between the urea and Asp204 was retained for more than 8 ns of simulation, and even after both H-bonds were broken, the Asp204 side chain remained near, suggesting that the interactions could break and reform easily. An alternative explanation for the requirement for the urea “left-hand” NH is that intramolecular interactions between this donor and the indoline N lone pair help stabilize the bioactive conformation of the core. The phenyl ring of the OCF3-phenyl may form a πstacking interaction with Tyr203 on ECL2 (Figure 3, purple), although this interaction was less prevalent in the MD than the hydrogen bonds with Asp204. It should be noted that residues on ECL2 ACS Paragon Plus Environment


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are not always well-placed in GPCR homology models because of variability in this loop, but Tyr203 and Asp204 are directly adjacent to Cys202, which generally can be placed with greater certainty because of the disulfide linkage formed with Cys124 on TM3. Mutagenesis of these two residues would be required to confirm these interactions. Another feature of the SAR explained by the model is the requirement for hydrophobic bulk in the basic piperidinyl series. The only hydrogen bond acceptors in the region of the basic NH are backbone carbonyls of TM7, so binding must result in either the uncompensated desolvation of the cationic amine or the disruption of the α-helical hydrogen bonding. Either of these would probably be so unfavorable that compensation by hydrophobic interactions alone would require a rather large hydrophobic contact area. The model has a deformed peptide bond between Arg310 and Gly311 that allows the carbonyl of Arg310 to accept the hydrogen bond from the ligand, although the formation of this interaction (as opposed to maintenance of the usual peptide bond and the absence of any hydrogen bond with the ligand) is not a crucial part of the model. Directly adjacent to the basic amine is a hydrophobic pocket formed by the side chains of Leu54, Tyr58, and Tyr111 and the TM7 backbone, including Gly311 (Figure 3, yellow). This pocket is filled up almost completely by the neopentyl group of compound 3l, satisfying the requirement for a significant burial of hydrophobic surface. In addition, it is likely that the alpha CH2 in the neopentyl group of 3l forms a cation−π interaction with the phenyl ring of Tyr111 based on the proximity of these two groups.27 Scan of phenyl−urea dihedral angle The urea group of compound 3l in the binding model is considerably out of plane with respect to the core phenyl ring, with a dihedral angle of 123° between the urea’s proximal N−C bond and the phenyl plane. To further ensure that this was a reasonable conformation, a torsional scan of a model of compound 3l was performed, with energies determined as the sum of the quantum mechanical energy (at the RIMP2/cc-pVTZ//B3LYP/6-31+G** level of theory) and the free energy of solvation (calculated


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with Cosmo-RS methodology28) (Figure 4). The scan confirms that the conformer with this dihedral angle has a low energy (0.25 kcal/mol) relative to the global minimum.

Figure 4. A. Torsional scan relating the energy of the compound 3l model system (inset) to the dihedral angle τ. There is a relatively broad well from -180° to -120°. The binding pose of 3l has τ = -123.5° (purple), which falls within this well. B. The conformation of the model system after minimization with τ constrained at -123.5° (purple carbons) overlaid onto the binding pose (cyan carbons). The urea, phenyl, and dihydropyrrole groups align well.

Pharmacology The effect of the benchmark compound 3l on arterial and venous thrombosis and provoked bleeding time was determined in pentobarbital-anesthetized rats using previously described procedures.29 Occlusive thrombosis was induced by a topical application of FeCl2-saturated filter paper to either the carotid artery (50% FeCl2 solution for 10 min) or vena cava (15% FeCl2 solution for 2 min), while bleeding time was measured by needle puncture of mesenteric arteries. Given the good solubility and bioavailablity of compound 3l observed in rat PK studies, the same vehicle was also used for both IV and PO routes in the PD studies and compared to compound 1, which was dosed only IV. Compound 3l inhibited arterial thrombus formation and improved blood flow in a dose-dependent manner when administered by either IV (Figure 5) or PO (Figure 6) routes. Maximum thrombus weight ACS Paragon Plus Environment


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reductions of 66% and 64% were observed in comparison to vehicle at doses of 0.6 mg/kg + 2.25 mg/kg/hr, IV and 10 mg/kg, PO, respectively. At these dose levels, vascular occlusion was prevented in all drug-treated rats and integrated blood flow was maintained at >95% of control levels. 7

proportion of the total number of rats in which the artery occluded

Vehicle 0.02 + 0.075

6/6 4/4 4/6

0.06 + 0.225

1/7 * 0/6 *

0.2 + 0.75 0.6 + 2.25

6

Compound 3l (mg/kg + mg/kg/hr, i.v.)

Carotid Blood Flow (ml/min)

*

0

*

* 100

4

80 Integrated Blood 60 Flow Over 60 min 40 (% Control) 20

3 2 1

-66%

1

=SEM

5

*

Thrombus 4 Weight 3 (mg) 2

* p

Anilino Diaryl Ureas - ACS Publications - American Chemical Society

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