Design, Synthesis, and Biological Evaluation of Novel, Non-Brain

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Design, Synthesis and Biological Evaluation of Novel, Non-Brain Penetrant, Hybrid Cannabinoid CB1R Inverse Agonist/Inducible Nitric Oxide Synthase (iNOS) Inhibitors for the Treatment of Liver Fibrosis Malliga R Iyer, Resat Cinar, Alexis Katz, Michael Gao, Katalin Erdelyi, Tony Jourdan, Nathan J Coffey, Pal Pacher, and George Kunos J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01504 • Publication Date (Web): 13 Jan 2017 Downloaded from http://pubs.acs.org on January 15, 2017

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Journal of Medicinal Chemistry

Design, Synthesis and Biological Evaluation of Novel, Non-Brain Penetrant, Hybrid Cannabinoid CB1R Inverse Agonist/Inducible Nitric Oxide Synthase (iNOS) Inhibitors for the Treatment of Liver Fibrosis Malliga R. Iyer,1* Resat Cinar,1 Alexis Katz, 1 Michael Gao,1 Katalin Erdelyi, 2 Tony Jourdan, 1 Nathan J. Coffey,1 Pal Pacher2 and George Kunos1* 1

Laboratory of Physiologic Studies and 2Laboratory of Cardiovascular Physiology and Tissue

Injury, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 5625 Fishers Lane, Rockville, MD 20852, USA. KEYWORDS: CB1R, CB2R, iNOS, DIO

ABSTRACT: We report the design, synthesis and structure-activity relationships of novel dualtarget compounds with antagonist/inverse agonist activity at cannabinoid receptor type 1 (CB1R) and inhibitory effect on inducible nitric oxide synthase (iNOS). A series of 3,4-diarylpyrazoline carboximidamides were synthesized and evaluated in CB1 receptor (CB1R) binding assays and iNOS activity assays. The novel compounds, designed to have limited brain penetrance, elicited

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potent in vitro CB1R antagonist activities and iNOS inhibitory activities. Some key compounds displayed high CB1R binding affinities. Compound 7 demonstrated potent in vivo pharmacological activities such as reduction of food intake mediated by the antagonism of the CB1Rs and antifibrotic effect in the animal models of fibrosis mediated by iNOS inhibition and CB1R antagonism.

INTRODUCTION The endocannabinoid system (EC) includes two main G-protein-coupled receptors (CB1R and CB2R), endogenous ligands, endocannabinoids (ECs), that bind to these receptors along with several enzymes responsible for the biosynthesis and the degradation of these ECs (e.g., fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL))1,2,3. The CB1R and CB2R belong to the family of G-protein coupled receptors (GPCRs) acting through the inhibitory Gprotein (Gi/Go). The CB1 receptor is mainly expressed in the central nervous system, where it is one of the most abundant receptors of GPCRs and is responsible for the psychoactive effects of marijuana and synthetic agonists.4 The behavioral effects of exogenous cannabinoids like memory, cognition and motor functions corroborates the central nervous system (CNS) presence of the CB1R.5 It is also expressed in a number of peripheral tissues like liver, testis and ileum. The CB2 receptor is mainly located in cells of the immune system, and involved in the immunomodulatory effects induced by cannabinoids.6 Recent evidences show that these receptors may also exist in the CNS.7 The modulation of the endocannabinoid system through the antagonism of CB1R has long been a viable therapeutic strategy. Indeed, this system has shown beneficial effects in several diseases like addiction8,9, GI disorders10, cognitive and neuroinflammatory disorders.11


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An overactive endocannabinoid/CB1R system has been implicated in obesity12, its metabolic complications and even fibrosis.13,14 Fibrosis characterized by the build-up of excessive fibrous connective tissue in an organ during chronic inflammation or injury repair is a key pathological process often leading to organ dysfunction and failure (e.g liver, lung and kidney). In the liver, fibrosis can develop as a result of hepatitis C Virus (HCV) infection, chronic alcoholism or fatty liver disease, and can progress to cirrhosis and hepatocellular carcinoma. Fibrosis, which can lead to progressive loss of tissue function and eventual organ failure, has been estimated to contribute to ~45% of deaths in the developed world. Hence new therapeutics to attenuate fibrosis are urgently needed. 15 In the past decade, CB1R inverse agonists (even neutral antagonists) have been the subject of much attention for their ability to treat obesity, diabetes and other metabolic syndromes.16 Despite their promising therapeutic effects, the CNS-mediated adverse effects of these brain-penetrant compounds like anxiety and development of suicidal tendencies precluded successful clinical use of the first-in-class CB1 inverse agonist rimonabant (Figure 1).17 Consequently, the development of similar CB1 antagonists like ibipinabant, taranabant and otenabant were halted at various stages.18 Nevertheless, these molecules by way of detailed studies and the CB1 receptor target itself has so many therapeutic benefits, which can be harnessed, that renewed focus has been established on a new class of CB1R antagonists/inverse agonists that are restricted in the peripheral space.19,20,21 Our group has been recently involved in the study and development of peripherally selective antagonists22–24 based on the variations of ibipinabant scaffold25 using different approaches. One approach has been to produce compounds that have polar surface areas (PSA) > 90 which can result in a low permeability into the CNS. Designing these compounds led us to specific moieties that have use in inhibiting inducible nitric


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oxide synthase (iNOS) and that could be manipulated to yield beneficial effects against fibrotic cells and tissues. It is known that endocannabinoids acting via CB1Rs promote fibrosis, whereas CB1R antagonist/inverse agonist like rimonabant mitigates fibrosis in animal models with moderate to low efficacy.12,24,26 Inducible nitric oxide synthase (iNOS), an enzyme that catalyzes the generation of pro-inflammatory reactive nitrogen species, is induced in fibrotic tissue, and iNOS inhibitors, with various amidino warheads like N-(3-(aminomethyl)-benzyl) acetamidine (1400W), have antifibrotic activity in animal models or fibrosis. 24,27,28 Hence we reasoned that a hybrid compound inhibiting both CB1R and iNOS might have beneficial effects in mitigating fibrosis and its related complications. Peripheral restriction of hybrid CB1R/iNOS inhibitors is of paramount importance to minimize any neuropsychiatric side effects secondary to blockade of CB1R in the CNS. Peripheral restriction of these compounds also precludes their ability to reduce brain iNOS activity, which has been proposed to aggravate certain neuroinflammatory conditions.29 RESULTS AND DISCUSSION Chemistry In this paper we report in detail, our efforts toward the design, synthesis and characterization of peripherally restricted dual inhibitors of CB1R and iNOS. The compound ibipinabant is a highly selective CB1 receptor inverse agonist developed by Solvay, whose further development was halted due to related CNS adverse issues arising from brain permeability of the compound.25 This compound has a higher topological PSA than rimonabant, but still falls short of the PSA range for CNS non-permeable compound. Inclusion of an L-Valine amide side attachment as in (S)-2-((S)-3-(4-chlorophenyl)-N'-((4-chlorophenyl)sulfonyl)-4-phenyl-4,5-dihydro-1H-pyrazole1-carboximidamido)-3-methylbutanamide (JD5037)30 (Figure 1) instead of the methyl group


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rendered the molecule non-brain permeable and improved the pharmacokinetic parameters while retaining the potent CB1R inverse agonism. This molecule is under preclinical investigation.22,30 Our approach was to replace the methyl group in ibipinabant not only to favor peripheral restriction, but also potentially to utilize any additional beneficial therapeutic effects that could result from this replacement (Figure 2). This novel approach to ligand design has now led us to several compounds, which seem promising as a dual CB1/iNOS inhibitor with potential for treating fibrosis, diabetes and obesity. We recently disclosed our lead compound 4S-(N-1-aminoethylidene)-3-(4-chlorophenyl)-4phenyl-N'-((4-(trifluoromethyl)phenyl)sulfonyl)-4,5-dihydro-1H-pyrazole-1-carboximidamide which is under investigation for treatment of fibrotic conditions.24 Herein we outline the synthesis and detailed SAR for the dual-target compounds that showed potency in in vivo models and that ultimately led to our lead compound. The synthesis of the novel compounds began with coupling of compound 131 and sulfonyl compounds 2, leading to sulfonyl urea products 3 (Scheme 1).25 Sulfonylurea 3 was subjected to PCl5 treatment in refluxing chlorobenzene to yield a sensitive imidoyl chloride 4 which was immediately utilized in situ without purification to build novel compounds of the formula A bearing the revamped pharmacophore (Scheme 2). The compounds synthesized included a chiral center at the 4-position of the 3,4-diarylpyrazoline ring. Thus the initial promising racemic compounds could be processed on to enantiomeric analogs for further investigations.

Biology and Pharmacology The target compounds were evaluated in vitro at the CB1 receptor utilizing radioligand binding assays and GTPγS functional assays. Our approach initially was to install S-methylisothiourea


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moiety, a potent iNOS inhibitor32 to the parent scaffold to obtain compound 5a. This compound however had a lower CB1R binding affinity (60 nM). We then switched the R group to methyl (Q moiety to an acetamdine), as this group is prevalent in selective iNOS inhibitors. When the putative iNOS-inhibiting component was changed to Me instead of SMe, the binding affinity for CB1R improved markedly for compound 6a. The results are reported in Table 1. This prompted us to change the R’ groups to glean the effect of steric and electronic effects on the CB1R binding affinity in the SMe and Me series. Interestingly, the 4-position at the R’ substituent contributed greatly to the binding CB1R affinities. Electron withdrawing, bulky groups enhanced the CB1R affinities, whereas the affinities dropped considerably in presence of smaller substituents like H, F or electron donating substituents like OMe, Me. Compound 5c with a strong iNOS inhibitory pendant showed a CB1R binding affinity of 10 nM, whereas the 4-Cl substituent 5a had a much lower binding affinity of 60 nM. The binding affinities for CB1R improved markedly for compounds 6a-d in the comparable 5a-5d series (Table 1). Varying the 4-R’ substituents while keeping R= Me retained the inverse agonism functionality in the compounds tested. The SMe amidine compounds in the 5a-5d series also retained CB1R inverse agonism albeit with low binding affinity. To expand the scope of substituents tolerated, we next turned our attention to keeping the R’ substituent constant (4-Cl) and the R groups in the formula A were varied to incorporate additional easily accessible amidino-type moieties keeping in mind the physicochemical parameters and the possible synergism for a dual target. This led to interesting CB1R binding affinities and clear SAR from further functional studies (Table 2). With the binding results in hand, it was clear that replacement of the methyl group in ibipinabant with amidine substituted moiety, the CB1R high affinity was retained in many of the


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compounds tested. The in vitro functional assays revealed that like SMe and Me substituents at the R position, cyclopropyl and t-butyl group retained the CB1R inverse agonism functionality. Replacing the alkyl R-groups with Ar-alkyl (benzene ring) switched the function to neutral antagonism for compounds 12-20 (Table 2) (Figure 3). This was interesting particularly, in the light of ibipinabant, as replacement of methyl group with bulky or aromatic amino groups could potentially lead to neutral brain-penetrant CB1R antagonists. Selected compounds were then tested for their iNOS inhibitory activity (Figure 4).

We initially picked compound 5c for further in vivo studies due its good binding affinity for CB1R, dual-target synergism and functional potency of 65nM in GTPγS binding assay. However solubility issues and bioavailability prevented further investigations on compound 5c and its enantiomers (not shown). Having recently published our findings with compound 6b24, here we present data on compound 6a. We turned our attention to compound 6a and subjected it to chiral prep-HPLC giving pure enantiomers 7 and 7’ (Scheme 3). Compound 7 binds stereoselectively to the CB1R in mouse brain as shown in Figure 5 and its potency as an inverse agonist is similar to ibipinabant and rimonabant. The X-ray structure of the eutomer, compound 7 revealed the compound to be a 4S-enantiomer (Figure 6). In line with in vitro results, the in vivo CB1R antagonism of 7 was further confirmed by the dose-dependent reversal of Arachidonyl-2'chloroethylamide (ACEA) induced reduction in upper GI motility (Figure 7). To assess the brain penetrance of 7, tissue distribution studies were performed and it revealed a very low brain penetrance ( 1000

6h

Me

H

114

2041

> 1000

6i

Me

OMe

182

70

> 1000

6j

Me

-(t-butyl)

48

30

> 1000


57


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6k

Me

(isopropyl)

10

90

> 1000

6l

Me

OCF3

138

76

> 1000

a

Binding affinity was determined from binding displacement isotherms using [3H]-CP55940 as the labeled ligand and plasma membranes from CB1R-expressing or CB2R-expressing CHO-K1 cells, respectively. Ki values were calculated using the Cheng-Prusoff equation. Data represent mean ± SEM from 3 independent experiments performed in triplicates.


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Table 2: In vitro CB1R binding results of the dual-target 4-chlorophenyl compoundsa

Compd #

Ki CB1 GTPγS (nM) IC50 (nM)

Ki CB2 (nM)

7

182

> 1000

9

13

241

> 1000

10

29

>1000

> 1000

11

29

N.D.

836

15

226

> 1000

13

26

117

1000

14

7.9

116

173

15

10

18

158

16

27

N.D.

> 1000

R

8

12

adamantyl


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17

34

603

> 1000

18

7.7

22

106

19

56

292

250

20

19

72

107

21

86

33

161

22

26

33

> 1000

23

72

125

> 1000

24

60

101

> 1000

25

67

534

> 1000

26

166

395

> 1000

27

127

213

> 1000

28

287

441

> 1000

29

190

2642

> 1000

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a

Binding affinity was determined from binding displacement isotherms using [3H]-CP55940 as the labeled ligand and plasma membranes from CB R-expressing or CB2R-expressing CHO-K1 cells, respectively. K values were calculated using the Cheng-Prusoff equation. Data represent mean ± SEM from 3 independent experiments performed in triplicates. 1

i


60

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Table 3. Profile for Compound 7 Target Profile

Compound 7

MW

514.4

CLogPa

6.01

PSAa

100.48

CB1R (Ki nM)

7.9

Tmax mouse (PO)

1h

T1/2 mouse (PO)

2h

Tmax rat (PO)

45 min

T1/2 mouse (PO)

2h

Bioavailability (F:%) rat

55

AMES test

negative

Plasma/brain

~ 0.03

hERG

>100 µM

hCB1R/hCB2R selectivity

~57

a

Theoretical values.


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TOC graphic:


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Design, Synthesis, and Biological Evaluation of Novel, Non-Brain

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