Novel Glycoprotein VI Antagonists as Antithrombotics: Synthesis

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Novel Glycoprotein VI Antagonists as Anti-thrombotics: Synthesis, Biological Evaluation and Molecular Modeling Studies on 2,3-Disubstituted Tetrahydropyrido(3,4-b)indoles. Shome Sankar Bhunia, Ankita Misra, Imran A. Khan, Stuti Gaur, Manish Jain, Surendra Singh, Aaruni Saxena, Thomas Hohlfield, Madhu P. Dikshit, and Anil K. Saxena J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01360 • Publication Date (Web): 07 Dec 2016 Downloaded from http://pubs.acs.org on December 16, 2016

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Journal of Medicinal Chemistry

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Novel Glycoprotein VI Antagonists as Anti-thrombotics: Synthesis, Biological Evaluation and Molecular Modeling Studies on 2,3-Disubstituted Tetrahydropyrido(3,4-b)indoles. Shome S. Bhunia1,4$, Ankita Misra2$, Imran A Khan1, Stuti Gaur1, Manish Jain2, Surendra Singh2, Aaruni Saxena3, Thomas Hohlfield3, Madhu Dikshit2* and Anil K. Saxena1,4*. 1

Division of Medicinal and Process Chemistry, 2Division of Pharmacology, CSIR-CDRI,

Lucknow 226031, India. 3

Institut für Pharmakologie und Klinische Pharmakologie, Heinrich-Heine-Universität

Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany 4

Academy of Scientific and Innovative Research, New Delhi, India.

$

Both authors contributed equally.

Abstract The development of small molecule inhibitors targeting GPVI has promising therapeutic role as they inhibit arterial thrombosis with limited risk of bleeding. Among the compounds showing in vivo antithrombotic activity, the most active compound 6b (ED50 = 28.36 µmol/kg p.o in mice) showed improved inhibition for collagen (IC50 = 6.7 µM), CRPXL (IC50 = 53.5 µM) and convulxin (CVX) (IC50 = 5.7 µM) mediated platelet aggregation as compared to losartan (LOS) (collagen:IC50 = 10.4 µM, CRP-XL:IC50 = 158 µM, CVX:IC50 = 11 µM) than any of its enantiomers S (6c) (collagen:IC50 = 25.3 µM, CRP-XL:IC50 = 181.4 µM, CVX:IC50 = 9 µM) and R(6d) (collagen:IC50 = 126.3 µM, CRP-XL:IC50 > 500 µM, CVX:IC50 = 86.8 µM). The compound 6b also inhibited platelet P-selectin expression and thus may diminish atherosclerosis. The molecular interactions of both enantiomers 6c and 6d at the GPVI receptor have been explained through docking studies. Keywords: GPVI antagonist, antithrombotic, collagen platelet aggregation, Losartan,

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Docking GPVI, Docking convulxin. INTRODUCTION The sequential activation of platelets on interaction with extracellular matrix proteins in artery wall is the key determinant leading to atherothrombotic complications culminating to coronary and cerebrovascular diseases.1 The GPVI a type I transmembrane glycoprotein of the immunoreceptor family at platelet surface has a decisive role in thrombus formation due to its participation in collagen mediated platelet activation and adhesion.2 The clinical and experimental studies have established significant role of GPVI in the haemostasis. Regulation of its expression level has direct link in the prognosis of acute cardiovascular diseases as enhanced expression of GPVI on circulating platelets is an early marker of myocardial infarction and ischaemic stroke.3, 4 The distinguishing role of GPVI in arterial thrombosis has invoked the development of competitive inhibitors targeting collagen-GPVI interaction having the potential to block GPVI-dependent thrombotic events to prevent cardiovascular complications. The current antiplatelet agents in the management of pathological thrombosis such as aspirin (cyclooxygenase inhibitor),

clopidogrel and prasugrel (ADP receptor

antagonists), cilostazol (phosphodiesterase inhibitor) and abciximab (fibrinogen receptor blocking antibodies) are associated with undesirable side effects like gastrointestinal bleeding, cerebral haemorrhage and neutropenia. The extracellular domain of GPVI involved in binding of fibrous collagen is defined by two immunoglobin like domains D1 (N-terminal) and D2 (C-terminal) that share high level of sequence conservation with other leukocyte Ig-like receptor complex (LRC) such as FcαRI and the leukocyte Ig-like(LILR or LIR) and killer-cell Ig-like (KIR) receptor families.5 Interestingly, triggering of platelet activation and downstream signaling through GPVI is restricted not only to physiological collagen but is extended up to the recognition of diverse ligands such as synthetic triple-helical collagen related peptides (CRP) that has a repeated


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GPO motif (glycine-proline- hydroxyproline) and CVX (CVX), a multimeric, C type lectinlike toxin from the venom of a rattlesnake having higher affinity towards GPVI as compared to collagen and CRP.6-9 Monoclonal antibody binding assays have led to the determination of distinct sites with sharing of some common residues at the functional domain of GPVI for binding with collagen, CRP and CVX.9 A number of contemporary studies have strengthened the concept of blocking GPVI-collagen interactions as an attractive strategy for the treatment of thromboischaemic diseases. The beneficial role of GPVI fusion (GPVI-Fc) proteins has been shown in the reduction of athero progression10, 11 and infarct size with improvement in arterial remodeling12 and regaining of myocardial function13 in murine models. The GPVI receptor as a pharmacological target has a superlative aspect of minimal bleeding time that indicates antiplatelet strategies targeting GPVI/collagen could be an attractive alternative for preventing pathological thrombosis without haemostatic negotiation.14-16 Hence the development of small GPVI antagonists may have high therapeutic value. Losartan (I) (LOS) and its metabolite (EXP3179) (Figure 1) (concentration of both at 500 µM/L has significant effect on collagen (2 µg/ml) mediated aggregation) were the first small molecules identified as GPVI inhibitors independent of angiotensin 1 (AT1) receptor antagonism.17 In this study Grothusen et al. has reported that LOS prevents collagen-I mediated platelet aggregation when stimulated

by selective GPVI-receptor activating

antibody 4C9 in human PRP (platelet-rich plasma) thus indicating that

LOS prevents

collagen mediated platelet aggregation. The first evidence about the direct interaction of LOS with GPVI protein was reported by Ono et al where LOS has a KD = 1.7x10-4 M for human GPVI receptor.18 However in a recent study it has been reported that LOS does not compete with collagen for binding to GPVI instead it inhibits the collagen induced clustering of GPVI on platelets.19 LOS is followed by other small molecule antagonists such as cinanserin (III)



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HN N N N

HO

HN N N N

O

O N

N Cl

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Cl

NH

N

N

S Cinanserin (III)

EXP3179 (II)

Losartan (I)

HO H2N

F

O

H N

N H O

O

O

O O

O H N

N H

O

O OH

O

NH

OH HO

S14161 (IV)

Pep10L(TDWLYFS) (V)

N N N N H

CompA (VI)

H N

NH

HO

NO2

O (H2C)2

N

O (H2C)3

N N N N H CompB (VII)

O (H2C)4

N N N H

CompC (VIII)

Figure 1. Structures of the compounds that prevent collagen mediated platelet aggregation. (IC50 = 35 µM and 40 µM for CRP (1 µg/ml) and collagen (2 µg/ml) mediated platelet aggregation respectively; Figure 1)20 discovered through structure based

repurposing,

S14161 (IV) (IC50=3.79±1.17 µM for collagen (2 µg/ml) mediated platelet aggregation) a Pan-PI3K inhibitor21 and other small molecules such as CompA(VI) (KD=5.2±0.4x10-5 M), CompB (VII) (no chemical shift perturbation in NMR based study) and CompC (VIII) (KD=8.5±0.5x10-4 M) derived through combination of losartan substructure and an active peptide molecule pep10L(YSDTDWLYFSTS) (KD=5.7x10-5 M) (Figure 1). The binding site of LOS has been interpreted at the hydrophobic pocket of the GPVI receptor through NMR


N

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and docking based interaction studies.18 This binding site has been also postulated to be the binding site for the peptide ligands (pep10L)22. Although these compounds prevent GPVI mediated platelet activation still there is need for specific inhibitors that selectively block GPVI mediated platelet activation. We report here the development of a selective, racemic small-molecule antagonist with βcarboline core, initially screened from a series of potential antithrombotic candidates (based on in vivo activity). The design of these molecules was based on the observed antiaggregatory effect of 3S-1, 2, 3, 4-tetrahydro-β-carboline-3-carboxylic acid moiety present in plant A. Chinese G. Don.23 There are ample evidences about β-carboline alkaloids preventing collagen-induced platelet aggregation.24, 25 Further the synthesized molecules reported by us have a tryptophan substructure in the β-carboline core that is reported to be essential for the binding of the peptide antagonist pep10L at the GPVI active site.22 The binding interactions for the most active compound in the series at the GPVI receptor were further determined with the help of docking studies. The insights gained in this study may provide opportunities for the development of small molecular therapeutics targeting GPVI. RESULTS Chemistry Tryptophan was used as a precursor to synthesize the target 2,3-disubstituted pyridoindoles. Unlike our earlier reported26 and successively used method27 where the first step was the PictetSpengler cyclization of tryptophan with formaldehyde followed by the esterification of the 1,2,3,4-tetrahydro-9H-pyrido(3,4-b)indole carboxylic acid thus obtained, the procedure was changed where

in the first step the methyl ester of tryptophan was synthesized using dry

methanol and thionyl chloride. It was then followed by Pictet-Spengler cyclization reaction of this ester to give the hydrochloride of the methyl 1,2,3,4-tetrahydro-9H-pyrido(3,4-b)indole



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CO2CH3

COOH a

NH2

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N H

N H

1

2

CO2CH3

b,c

NH2.HCl

NH

N H

2a=RS 2b=S 2c=R

3

3a=RS 3b=S 3c=R

e

d CONH2

CO2CH3

NH

N H

N O S O R

N H

5

4

4a: (SR), R = Methyl 4b: (S), R = Methyl 4c: (R), R = Methyl 4d: (SR), R = Quinolin-8-yl 4e: (S), R = Quinolin-8-yl 4f: (R), R = Quinolin-8-yl 4g: (SR), R = 2,5-Dichlorophenyl 4h: (S), R = 2,5-Dichlorophenyl 4i: (R), R = 2,5-Dichlorophenyl 4j: (SR), R = 5-Dimethylaminonapthyl 4k: (SR), R = 2,4,6-Trimethylphenyl 4l: (SR), R = 2-Trifluromethylphenyl

CO2CH3

CO2H

e

N O S O R

N H

5a=RS 5b=S 5c=R

d

7

7a: (SR), R = 2-Napthyl 7b: (SR), R = Phenyl

CONH2 N H 6

6a: (SR), R = 2,5-Dichlorophenyl 6b: (SR), R = 4-Methoxyphenyl 6c: (S), R = 4-Methoxyphenyl 6d: (R), R = 4-Methoxyphenyl 6e: (SR), R = 4-Flurophenyl 6f: (SR), R = 2-Napthyl 6g: (SR), R = 3-Nitrophenyl 6h: (SR), R = Quinolin-8-yl 6i: (SR), R = 2,4,6-Trimethylphenyl

COR1

f

N O S O R

N H

N O S O R

8

8a: (SR), R = 2-Napthyl 8b: (SR), R = Phenyl

N H 9

N O S O R

9a: (SR), R = 2-Napthyl, R1 = Propylamino 9b: (SR), R = 2-Napthyl, R1 = Isopropylamino 9c: (SR), R = Phenyl, R1 = Propylamino 9d: (SR), R = Phenyl, R1 = Isopropylamino

Figure 2.(a) SOCl2, dry methanol, -10°C to 30°C; (b)HCHO, MeOH,H2O; (c) NaHCO3 saturated solution; (d) R-SO2-Cl, dry acetone, TEA; (e) MeOH,NH3; (f) 2N NaOH, H2O, 1,4dioxane (g) COCl2, dry THF (h) TEA, R-NH2, dry THF.


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carboxylate hydrochloride salt which on treatment with sodium bicarbonate (NaHCO3) yielded the free base (3). The free base (3) thus obtained was converted to respective sulphonamides (4a-4l) by treatment with individual sulphonyl chlorides using a combination of DMF as solvent with triethylamine (TEA) at room temperature. The 1,2,3,4-tetrahydro-9H-pyrido(3,4-b)indole carboxamide (5) was synthesized by dissolving the ester (3) in methanolic ammonia under sealed conditions and kept overnight. The reaction of the 1,2,3,4-tetrahydro-9H-pyrido(3,4-b)indole carboxamide

(5), with different sulphonyl

chlorides yielded the corresponding sulphonamides (6a-6i) under similar reaction conditions as described above for the conversion of esters to their sulphonamides. The amine substitutions at position 3 of the tetrahydropyridoindole were further explored by synthesizing compounds 9a9d. The compounds 9a-9d were synthesized by de-esterification of the starting compounds 7a and 7b to yield their corresponding acids (8a, 8b) followed by the conversion of the acids to acid chlorides using oxalyl chloride. The reaction mixture containing the acid chlorides were concentrated under high vacuum conditions and without further isolation were treated with individual amines in presence of dry THF and TEA to yield 9a-9d. Structure activity relationship: In order to investigate the structural requirements involving the pyridoindole core we have synthesized 26 derivatives with a substitution of diverse functionalities to rationalize the antithrom-botic activity of these compounds in terms of structural modifications. The antithrombotic activity was examined in the mouse antithrombotic model employed primarily for the screening of antiplatelet agents. Through this screening method we have identified some promising antiplatelet agents (ED50 = 28 – 91 µmol/kg). Among these synthesized compounds the compound substituted by a methylsulphonyl and methyl carboxylate at 2 and 3 positions of 1,2,3,4- tetrahydro-pyridoindole (4a) showed good activity (ED50 = 54.09 µmol/kg). Since it



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has a asymmetric centre at 3-position its two enantiomers S(4b) and R(4c) were evaluated where the racemic 4a (50% protection; ED50 = 54.09 µmol/kg) showed better activity than the corresponding 4b (20% protection) and 4c (20% protection) enantiomers. However, the compounds having aryl/heteroaryl sulphonyl substituent at 2- position showed improved activity viz the compound with a quinonylsulphonyl, 4d (ED50=41.94 µmol/kg) and with a 2, 5-dichloro phenylsulphonyl substitution 4g (ED50=36.1 µmol/kg). Thus aromatic hydrophobic substituents at this position showed positive contribution towards activity. Similar to the compound 4a, the enantiomers of the compounds 4d (4e and 4f) and 4g (4h and 4i) also exhibited lower activity than their corresponding racemates. The compound 4j with 5-dimethylaminonapthylsulphonyl substitution showed promising antithrombotic activity while the compounds 4k with 2,4,6trimethylphenylsulphonyl substitution showed lower activity than 4j. The compound 4l with the 2- trifluoromethyl phenylsulphonyl substitution showed improvement in activity. To explore further, the compound 4g having a methyl ester at the 3-position of the 1,2,3,4tetrahydropyridoindole

moiety was modified to an amide functionality that resulted in

compound 6a (ED50 = 68.34 µmol/kg). The other amide analogs such as compound 6b with a 4methoxyphenylsulphonyl (ED50 = 28.36 µmol/kg), 6e (ED50 = 40.79 µmol/kg) with a 4-fluro phenylsulphonyl, and 6f with a 2-Napthylsulphonyl moiety showed better antithrombotic activity compared to 6a. The compound 6g with a 3-nitrophenylsulphonyl substitution at the N2 atom showed a moderate decrease in antiplatelet activity (ED50=80.68 µmol/kg). No significant loss in activity was observed for the amide 6h as compared to ester compound 4d while decrease in activity was observed on amidation of the compound 4k to 6i. As the compound 6f with a napthylsulphonyl and carboxamido groups at 2 and 3 positions respectively showed better activity hence further modifications were performed at the 3- position where the carboxamide group was modified with propylamide (9a) and isopropylamide groups (9b). However both these compounds 9a (ED50 = 66.54 µmol/kg) and 9b (ED50 = 91.19 µmol/kg)


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showed a decrease in activity, hence the 2-napthylsulphonyl moiety was replaced with a 2phenylsulphonyl group that resulted in improved activity. The compound 9c (ED50 = 42.64 µmol/kg) substituted by 3-propylamido group displayed better activity than the compound 9d (ED50 = 63.62 µmol/kg) having a isopropylamido group. As 6b turned to be the most potent compound in the series, further investigations on its isomers 6c and 6d were performed but none of them was found to be equipotent compared to 6b. Biology Collagen-epinephrine induced pulmonary thromboembolism in mice: The compounds were screened for their antithrombotic efficacy in a mice model of collagen-epinephrine induced pulmonary thromboembolism. The compounds 4g and 6b exhibited Table 1. The In vivo antithrombotic activity of the pyridoindole compounds. Sl.no Comp

1

4a

Antithrombotic activity (% protection at 30 µmol/kg) 50

ED50 (µmol/kg)

Sl.no

Comp

ED50 (µmol/kg)

6b

Antithrombotic activity (% protection at 30 µmol/kg) 70

54.09

14

2

4b

20

-

15

6c

40

-

3

4c

20

-

16

6d

30

-

4

4d

35

41.94

17

6e

60

40.79

5

4e

20

-

18

6f

70

-

6

4f

20

-

19

6g

45

80.68

7

4g

60

36.10

20

6h

30

-

8

4h

25

-

21

6i

30

-

9

4i

30

-

22

9a

40

66.54

10

4j

40

-

23

9b

40

91.19

11

4k

20

-

24

9c

60

42.64

12

4l

55.5

-

25

9d

50

63.62

13

6a

40

68.34

26

Aspirin

38±3

-


28.36


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Figure3.Effect of Compound 6b on collagen-epinephrine induced pulmonary thromboembolism in mice in (a) time dependent study (b) dose dependent study. Results are expressed as Mean±SEM (n=5, 10 animals/group/experiment). (c) Effect of compound 6b on bleeding time (Aspirin: 170 µmol/kg, Clopidogel: 70 µmol/kg, Losartan: 100 µmol/kg, 6b: 30 µmol/kg). Effect of compound 6b on coagulation cascade in human plasma (in vitro) at various concentrations (d) Thrombin Time, (e) Prothrombin Time, (f) activated partial thromboplastin time. (n=3) Results are expressed as Mean + SEM (g) Effect of compound


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6b on human platelet adhesion (in vitro) on collagen coated surface in presence and absence of Mg2+(n=3) Data shown as Mean + SEM. ***p

Novel Glycoprotein VI Antagonists as Antithrombotics: Synthesis

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