Conformationally Constrained Analogs of

Org27569 as Allosteric Modulators of CB1 Cannabinoid Receptor

Master’s Thesis Research

By Siddhi Honavar

Advisor: Dr. Ganesh Thakur, PhD

Department of Pharmaceutical Sciences Northeastern University August 2015

1

Acknowledgements

I would like to express my sincere gratitude to my advisor Dr. Ganesh Thakur for the continuous support during my thesis project. I thank him for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis.

Besides my advisor, I would like to extend a special thanks to Dr. Ganesh Chaturbhuj who has not only been an excellent guide and mentor for my thesis project, but he has also been an inspiration for me to work hard. His continuous encouragement has driven me to being a better researcher. I want to thank him especially for being there to help even across the seas, and being patient and resolving my abundant queries.

I would also like to thank Abhijit Kulkarni for training me in my initial days and being a support through the entire research journey.

My sincere thanks also goes to Dr. Sumantha Garai, Dr. Pushkar Kulkarni, and Dr. Gopalkrushna Waghule, who have been very helpful in guiding me in the lab and shared their experiences and knowledge which have helped me, grow as a researcher.

I thank my fellow lab mates Sharvik, Brinda, Ninad and Prisca for being so understanding and kind, and for all the fun we have had.

I would also like to extend thank you to Robert Laprairie and Dr. Eileen Denovan-Wright from Dalhousie University for characterization of compounds and helping with the biological data.

Last but not the least I would like to thank my parents for their belief in me and being supportive throughout my MS, and also my friends and roommates in Boston who have been with me through all good and bad times.

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CONTENTS:

1. Abbreviations………………………………………………………………………………………………...3

2. List of Figures………………………………………………………………………………………..…….….4

3. List of Tables……………………………………………………………………………………….…………..5

4. Abstract.………………………………………………………………………………………………………....6

5. Specific aims……………………………………………………………………………………………………7

6. Background and Significance……………………………………………………………………………8

7. Research Plan………………………………………………………………………………………………..18

8. Chemistry and Schemes………………………………………………………………..………………22

9. Biological Assay Data…………………………………………………………………………………....28

9.1 Data Analysis………………………………………………………………………………..…………33

10. Conclusion……………………………………………………………………………………………..……..34

Experimental Section……………………………………………………………………………..……..35

11. References…………………………………………………………………………………………………….44

3

Abbreviations:

CB - Cannabinoid

GPCR – G-Protein Coupled Receptor

Org – Organon

2-AG – 2-arachidonolyglycerol

MAPK – Mitogen activated protein kinase

JNK – c-Jun N- terminal kinase

FAAH – Fatty acid amide hydrolase

∆9-THC - ∆9- tetrahydrocannabinol

PAM – Positive allosteric modulator

NAM – Negative allosteric modulator

DMF – Dimethylformamide

DCM – Dichloromethane

TEA – Triethylamine

DPPA – Diphenylphosphoryl azide

THF - tetrahydrofuran quant – quantitative 4

List of Figures:

Figure A: The CB1 receptor signaling

Figure B: Structures of cannabinoid antagonists

Figure C: Examples of endocannabinoids

Figure D: Cannabinoid receptor agonists

Figure E: Allosteric and orthosteric ligands

Figure F: Negative allosteric modulators at cannabinoid receptor

Figure G: Structures of Organon compounds

Figure H: Summary of SAR studies of the Org scaffold

Figure I: Constrained analog approach for Org27569

Figure J: Structures of GAT700 and GAT701

Figure K: Core structure of constrained analog series

Graph A: Data for GAT700 and GAT701 for Modulation of CB1 and Gαq dependent PLCβ3 phosphorylation.

Graph B: Data for GAT700 and GAT701 for pPLCβ3

Graph C: Data for cAMP inhibition by GAT700 and GAT701 5

Graph D: Data of GAT700 and GAT701for ERK1/2 phosphorylation

List of Tables:

Table 1: Structures of proposed compounds

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Abstract:

The Cannabinoid receptors have become the focus of research due to their importance as targets for treating a number of disorders. These receptors which are a part of the G-protein coupled receptor (GPCR) superfamily are of two subtypes, CB1 receptors which are present abundantly in the brain and in the peripheral and adipose tissues and CB2 receptors which are predominantly found in the immune cells. The cannabinoid receptors were always known to show its function through the orthosteric ligand binding, but the discovery of allosteric site on the CB1 receptors, has opened up a whole new horizon for research. Three of Organon analogs displayed a paradoxical allosterism at the CB1 receptors, wherein they were negative allosteric modulators

(NAM) of function but positive allosteric modulators (PAM) of binding of orthosteric ligand at the CB1 receptor.

The SAR around these three molecules has not been explored as much, and as all three almost shared the same phamacophoric properties, Org27569, the most potent analog of these, was selected as the lead compound. Org27569 has shown hypophagic effect independent of the presence of the CB1 receptor, and hence pointing towards the possibility of it‟s off target binding which is a significant limitation in its further development as a drug. Exploring the SAR around

Org27569 would give a better insight into the molecule‟s structural requirements for allosteric modulation at CB1 receptor. The conformational restriction approach is adopted as a tool for molecular modification and design of the analogs. This projects aims at synthesizing conformationally constrained analogs of Org27569 as GAT700 and GAT701, to explore the receptor binding and functional selectivity of the allosteric modulators at the CB1 cannabinoid receptor. 7

Specific Aims:

1. Rational design of constrained analogs:

The project aims at synthesizing analogs of Org27569 that have a restricted orientation, which would give us more control over the stereochemistry of the compound, and which might increase its selectivity for CB1 receptor and pacify the off target binding, giving us more control over the receptor function.

2. The structural characterization of these molecules.

3. Biological testing of analogs, to study the effect on CB1 receptor:

The analogs were tested in different functional assays to measure their functional

potencies and efficacies, which would help in expanding the SAR study for this

chemotype.

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1. Background & Significance

1.1 Endocannabinoid system:

The endocannabinoid system encompasses two of the G-protein coupled receptors (GPCRs),

CB1 and CB2, the endogenous cannabinoid receptor ligands, along with the enzymes and proteins that are involved in their synthesis and inactivation1-4. The ones identified so far are derivatives of long chain polyunsaturated fatty acids, the ones that have been researched the most; these include anandamide (N-arachidonoylethanolamine), and 2-arachinonolyglycerol (2-AG).

Experiments have also shown endocannabinoids to act as retrograde synaptic messengers5-7.

8,9 The CB1 receptors are found in both central and peripheral nervous system , but in abundance in the central nervous system, and most prominently at the neuronal terminals where they play a

2,3 pivotal role in modulation of the neurotransmitter release and the CB2 receptors are mainly occurring in the immune cells and are involved in cytokine release and immune cells migration10.

11 Figure A: The CB1 receptor signaling

9

Similar to many GPCRs, multiplicity of signal transduction is demonstrated by the CB1 receptors. Though it shows preferential coupling with Gi/o type proteins, there is possible

12 13 4 interaction with Gs and Gq type proteins in some conditions . CB1 receptor activation is responsible for the drop of the cAMP levels in cells due to its inhibitory action on adenylyl- cyclase. Along with this CB1 receptor also modulates the mitogen-activated protein kinases activation 14, which comprises of extracellular signal regulated kinase-1 and -2, p38 MAPK, p42/p44 MAPK, and c-Jun N-terminal kinase (JNK) 14. Moreover they can inhibit N- and P/Q

2+ + type voltage-gated Ca channels and activates the A-type, leading to inward rectification of K

15 channels . CB1 receptors have a complicated signaling network which brings up the presence of receptor functions that are controlled by modulatory mechanisms.

1.2 Therapeutic potential of the cannabinoid receptors:

The wide distribution of the cannabinoid receptors in different tissues accounts for the psychotropic and peripheral effects of THC. CB1 receptors are predominant in the neuronal circuit (CNS) and are also present in the periphery, whereas CB2 receptors are restricted to the immune cells. CB1 receptors are involved in neuromodulatory actions which cause the regulation of cardiovascular, garstrointestinal16 and cardiovascular functions17 along with regulation of pain

18 perception . CB1 receptors are also involved in regulation of the hormones in males and females

1,19 and in turn controlling the reproductive functions . Vast evidences show that the CB1 receptor is an important target for the treatment of a number of disorders like obesity20,21, pain22, inflammation23, osteoporosis24, cancer25, gastrointestinal disorders, psychosis, and schizophrenia3.

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1.3 Cannabinoid receptor ligands:

CB1 and CB2 receptors, which have been identified, are primarily located in the central nervous system and on the immune cells respectively. The discovery of these receptors, and the importance of their role in the body as neuromodulators and immunomodulators have driven the researchers to find CB1 and CB2 receptor ligands which activate their effects as well as inhibit it, as both functions can be utilized for therapeutic purposes26,27. The cannabinergic receptor ligands were classified into four classes which includes: the classical cannabinoids, non-classical cannabinoids, aminoalkylindoles and eicosanoids15.

1.3.1 Antagonists at cannabinoid receptors:

AM251, AM281, SR141726A and LY320135 are CB1 selective, and produce inverse cannabimimetic effects. These effects are opposite to those produced by agonists, for example, hyperalgesia, stimulation of intestinal motility and hypophagia7,28.

Figure B: Structures of representative cannabinoid antagonists

SR141716A which was identified in 1994 by Maruani J. et al. more popularly known as

Rimonabant is an inverse agonist of CB1 receptor in addition to acting as an antagonist, which means it elicits a response exactly opposite to that of the agonist but binds just like an agonist. 11

1.3.2 Endogenous ligands:

Endogenous ligands for cannabinoid receptors are known as endocannabinoids, which are synthesized in the body on demand and eliminated from the sites through tissue uptake and metabolized by the enzymes. The two major classes of the endocannabinoids are the acylethanolamides and the acylesters, out of which anandamide is from the acylethanolamide family29,30. These eicosanoids are usually both neuromodulators31 and immunomodulators.

Anandamide is a partial agonist and 2-arahidonoyl glycerol is a full agonist, these two endogenous ligands have gathered wide interest. These neurotransmitters are synthesized as per the need of the body, rather than being stored in the vesciles. Anandmide is broken down by fatty-acid amide hydrolase 1 (FAAH) to arachidonic acid and ethanolamine which are inactive32.

In addition to these, virodhamine and noladin ether had been reported to be endogenous CB1 agonists, but their functions have not been completely verified30.

Figure C: Examples of endocannabinoids

The regulation of the endogenous ligands is run by the body in a controlled manner by the monitored synthesis and enzymatic degradation to inactive metabolites. Molecules such as

Lipoxin A4 mimic the endogenous partial agonist anandamide.

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1.3.4 Agonists at cannabinoid receptors:

The cannabinoid receptor agonists are usually less selective for just one of the subtypes.

Cannabinoid receptor agonists include classical cannabinoids like (-)-∆9-tetrahydrocannabinol

(∆9-THC) and HU-210 and non-classical cannabinoids like R-(+)-WIN55212, CP55940 and R-

(+)-WIN55212. The cannabinoids receptor agonists‟ usually have marginal selectivity and show high affinities at both CB1 and CB2 receptors. CP55940 is a full agonist at both CB1 and CB2 receptors and is very widely used in pharmacological bioassays.

Figure D: Cannabionoid receptor agonists

1.4 Cannabinoid receptor binding sites:

Traditionally the GPCR‟s have been known to possess the orthosteric binding site, which is the primary site that recognizes an endogenous agonist and via which receptors elicit their action.

The ligands that bind to these sites are termed as orthosteric ligands. In 2002 Christopoulos and

Kenakin pointed out the presence of allosteric binding sites on GPCR‟s for endogenous and/or synthetic ligands33. All of the ligands described in the earlier discussion are orthosteric ligands.

Over the last decade several GPCRs have shown to possess allosteric sites, along with the 13 elucidation of the endogenous allosteric compounds. Owing to these findings, corresponding allosteric modulators have been developed.

Allosteric binding sites are different from the orthosteric sites and hence the ligands that are recognized by these sites have different structural requirements. Allosteric modulators, as they are known, have the ability to influence the biological activity of an agonist or inverse agonist at the orthosteric binding site27. Any conformational change in the receptor occurring due to the binding of the allosteric modulator could be communicated to the orthosteric binding site, resulting in modulation of the affinity and/or function of the orthosteric ligand34.

Figure E: Allosteric and Orthosteric ligands35.

1.4.1 Positive Allosteric Modulators (PAM):

When the binding of a allosteric ligand leads the enhancement of the orthosteric agonists effect by either increasing the ligands function or binding affinity at the orthosteric site, such a ligand is called a PAM. RTI-371 is an example of PAM at CB1 receptor which causes enhanced 14 endonnabinoid receptor neutotransmission and hence has a possible therapeutic application in

Parkinson‟s disease36.

1.4.2 Negative Allosteric Modulators (NAM):

The allosteric modulators that negatively modulate the effect on the orthosteric ligands binding and/or signaling when bound to the allosteric site are referred to as negative allosteric modulators. NAMs are inactive in the absence of a bound orthosteric ligand. PSNCBAM-137 and

38 Pepcans negatively modulate the function of the CB1 receptors and they are being researched for their application in treatment of various neurodegenerative diseases and obesity.

Figure F: Allosteric modulators at cannabinoid receptor.

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1.5 Advantage of allosteric modulator approach39,40:

Unlike the orthosteric ligands, the allosteric ligands do not compete for binding with the endogenous ligands, and hence the normal physiological signaling patterns remain preserved.

The allosteric binding sites are more evolved than the endogenous ligand binding sites, and hence pose a great scope for development of higher receptor subtype selective drugs. In the absence of an endogenous ligand on the receptor, the allosteric ligand does not show any modulation in receptor signaling. By utilizing these advantages it could be possible to produce molecules that are much safer than the competitive agonists or antagonist drugs.

1.6 Allosteric modulators at cannabinoid receptors:

In 2005 first evidence of the existence of allosteric binding site on a cannabinoid receptor was demonstrated by Price et al by the investigation of three pharmacological molecules Org27569,

3 Org 29647, Org 27759 at the cannabinoid CB1 receptor against the CB1 receptor agonist [ H]CP

55 [1R,3R,4R)-3-[2-hydroxy-4-91,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan- ol], and inverse agonist [3H]SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4- dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride]40.

The Org compounds decrease the specific binding of the CB1 receptor inverse agonist, but have a positive modulation on the CB1 agonist binding affinity. Various assays such as, equilibrium

35 binding assay, dissociation kinetic assay, mouse vas deferens and the [ S]GTPγS binding assay, and the human CB1 receptor reporter gene assay were carried out. These assays were performed to confirm the effect of the Org compounds on the agonist and inverse agonist ligand binding and function, for validation of the presence of the allosteric site on the CB1 receptor and prove the

Org compounds as allosteric modulators40. The study carried out revealed interesting aspects 16

about these compounds and the CB1 cannabinoid receptor. The Org compounds demonstrated themselves to be negative allosteric modulators of CB1 receptor function but positive allosteric

40 modulators of CB1 agonist binding .

Figure G: Structures of Organon compounds.

Following the discovery of the allosteric ligand on the cannabinoid receptor, researchers pursued this field to see if there were multiple allosteric sites, and other compounds also behaved as allosteric modulators. In 2007, Horswill et al characterized PSNCBAM-1 was proved to be an allosteric antagonist at CB1 receptor and also was elucidated to possess acute hypophagia and weight loss in male SD rats37. Further the dopamine transporter selective inhibitor RTI-371 was

36 established to be a positive allosteric modulator of CB1 receptor by Novarro et al in 2009 . Post

41 this, Lipoxin A4 an endogenous anti-inflammatory ligand was identified to allosterically

42 enhance the arachidonoylethanolamide i.e. anandamide signaling at CB1 receptor by Pamplona et al in 2012. Also 2012 saw the recognition of certain Pepcans as negative allosteric

38 modulators at CB1 Receptors by Bauer et al. Recently in 2015 there was identification of the 17 fenofibrate as a member of the class of allosteric modulator and ligand at the cannabinoid receptor by Priestley et al43.

2. Research Plan:

The focus of this project revolves around Organon molecules and the striking results of the study conducted by Price et al. was that although these compounds were negative modulators of function at CB1 cannabinoid receptor; they positively modulated the agonist binding. Moreover, they behaved as insurmountable antagonists of receptor function. The advantages of allosteric modulators is that they effect affinity not the efficacy, which mean that they can intensify or calm the effects of the endogenous ligand without altering the inherent patterns of physiological signaling40. In 2012 Ahn et al studied the pharmacology of Org27569 in detail, where they initially hypothesized that the modulation in the agonist and inverse agonist binding affinities could be due to the transition of CB1 receptor conformations between active and inactive forms.

One of the important outcomes was that ligand-biased signaling was enhanced by Org27569 via conformational change of the CB1 cannabinoid receptor leading to the internalization and downstream activation of ERK signaling. The first evidence of the signaling-pathway-selective allosteric modulation via CB1 was hence provided. This opened new avenues in the cannabinoid- based drug discovery research, as distinct therapeutic properties can be explored due to the allosteric modulator‟s receptor-selectivity and signaling-pathway selectivity40,44,45.

Indole-2-carboxamides were noted to be less explored and hence their investigation was taken up keeping Org27569 as the lead molecule3. Chemical modification of the N-phenylethyl-1H- indole-2-carboxamide scaffold gave some analogs that were potent CB1 PAMs. The first SAR study revealed that (1) the carboxamide moiety at 2nd position and (2) piperidin-yl, nitro and 18 dimethyl amino groups at the para position were required for stimulatory effect3. During the pharmacological assays, more analogs of the indole-2-carboxamides were tested to investigate their influence on the β-arrestin-mediated downstream activation of the extracellular signal- regulated kinase (ERK) signaling, where it was seen that the β-arrestin-1 was involved in the induced ERK1/2 phosphorylation, and this biased agonism can be utilized to regulate the

45 function of the CB1 receptor selectively .

Figure I: Summary of SAR studies of the Org scaffold.

Elongation of the alkyl chain on the C-3 position could be tolerated up to n-pentyl. The C-3 substituents determine the allosteric modulator effect, because it is hypothesized that a 19 hydrophobic pocket is present close to the C-3 position, and hence cyclic structures do not work at that position40. Added assays prove that the n-pentyl chain is good for allosteric modulation of the orthosteric ligand binding, but n-hexyl enhances the affinity of the allosteric modulator to the

46 CB1 receptor . Removal or shortening the ethylene linker between the amide and the phenyl ring leads to complete eradication of the allostery of the ligand4,46. Additionally phenyl or piperazine linkers are also not tolerated, thus proving that flexible alkyl linkers are required for the CB1 activity9,46. Hydrophilic substitution on the amino group weakened the affinity of the ligand binding to the allosteric site and also leads to loss of the allosteric modulation of the orthosteric site46. The indole ring is more influential on the binding ability to allosteric sites than on the modulatory effects on the orthosteric site4. Except the methylamino, dimethylamino, and diethylamino no other group was tolerated on replacement of the piperidinyl group. The amino groups showed higher binding affinity for the allosteric site and increased cooperativity to orthosteric site as well. The substituents on the phenyl ring A of indole play a key role in maintaining the binding affinity to allosteric site as well as the cooperativity with the orthosteric site46.

All of the compounds were tested based on two critical parameters: The Equilibrium dissociation constant (KB) which represents the ligands‟ binding affinity to the allosteric site; and the binding cooperativity factor (α) that indicates the allosteric interaction between the allosteric and orthosteric ligands when the ligand is occupied by both. The allosteric ternary complex model is used for the evaluation of both the parameters. If the cooperativity factor α is >1 then the ligand is said to indicate the positive cooperativity and if α <1 then there negative cooperativity at the orthosteric ligand binding. The allosteric modulators that emerged to be promising were then 20 further examined for their effects on the agonist-induced G-protein coupling activity and the β- arrestin mediated ERK1/2 phosphorylation.

The discovery of the presence of the allosteric site on the CB1 cannabinoid receptor has given a new angle to the cannabinoid-based drug discovery. The approach of targeting allosteric sites mainly gives the advantage of being able to maintain the spatial and temporal signaling profile of the endogenous ligand along with improving selectivity across the receptor. Indole-2- carboxamides have demonstrated striking pharmacological activity of being allosteric enhancers of binding and affinity and allosteric inhibitors of agonist signaling efficacy. This scaffold has not been investigated much and has a great therapeutic potential as allosteric enhancers of the

CB1 receptor can cause modulation of binding without producing the CNS side effects that are usually the characteristics of direct receptor agonists. The long term goal of the development of these analogs is to target the treatment of addiction, anxiety, neurodegeneration, depression, obesity, cancer and inflammation. Recent studies have also brought to light the function of

PAMs to be protective against β-amyloid-induced neurotoxicity, and hence the indole-2- carboxamides can be explored as neuroprotective agents as well. Overall this scaffold has great scope for further SAR studies which could lead to interesting pharmacological findings for the physiologically vital CB1 receptor.

In 2012 Gamage et al conducted the testing of Org27569 in whole animals which brought to

(+/+) light an interesting finding that the Org27569 reduces food consumption in CB1 as well as

(-/-) CB1 mice, which means that Org compound elicits the hypophagic effect independent of the

47 CB1 . The study indicates that the Org27569 has off target effects and this aspect has questioned the use of Org compounds in future pharmacotherapies. Hence there is a need to develop analogs 21

of this compound which are more selective for CB1 receptors and have such biological activity in-vitro which is translational into the whole animal.

2.1 Constrained analogs approach:

When a molecule has freely rotatable bonds they tend to have numerous possible conformations, and the conformation which perfectly fits into the ligand is unclear, and also it leaves a loop hole for certain conformations to bind off target. In order to get an analog that could mimic the possible “bioactive conformation” the molecules mobility is reduced and the molecule‟s geometry is locked48. Conformational constrains moreover can lead to differentiation of ligands

Figure J: Constrained analog approach for Org27569

affinity to the target, thus improving the selectivity. One of the most commonly used approaches to synthesize conformationaly rigid analogs is the „ring chain transformation‟ approach, which involves connection of the alkyl substituents to give the corresponding cyclic analogs. This is the 22 exact approach that has been used here to synthesize the constrained analogs of Organon as represented in Fig. J. Cyclopropanation of the ethylene linker between the amide and the phenyl ring yielded stereospecificaly cis and trans constrained analogs depending on the reaction conditions. The application of this approach in this project would give us an idea about which conformation is best suited to fit the CB1 allosteric site of the receptor and to derivate if this modification in the structure impacts the selectivity or modulatory activity of the molecule.

3. Chemistry and Schemes:

Constrained analogs of Org27569:

GAT 700 and GAT 701 are the constrained analogs of Org27569 which are synthesized.

Figure K: Structures of GAT700 and GAT701

The challenge was to design and implement synthetic routes which would give specific stereoisomers of the compound and to characterize these isomers based on their Nuclear

Magnetic Resonance spectra and Mass spectroscopy.

J-coupling values as calculated from the NMR to validate the stereochemistry of the compounds

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3.1 Retrosynthesis and Chemistry:

3.1.1 Retrosynthetic Scheme:

The Retrosynthetic scheme involves breaking the final molecule into two fragments, in order to carry out a convergent synthesis. The molecule can be broken down into two fragments: A) The chloro-indole acid and B) The cyclopropanated amine. The Indole acid can be synthesized using the Fisher indole synthesis and the amine can be obtained via the Horner-Wadsworth-Emmons reaction, and further with the Curtius rearrangement. The stereoisomers can be synthesized specifically via these routes.

Scheme A: Retrosynthetic breakdown

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3.2.2 Chemistry:

A. For trans amine:

Scheme I: Synthesis scheme for trans-amine.

The synthesis of 9 which is the trans-cyclopropanated amine hydrochloride is depicted in

Scheme I. The compound 3 was obtained by coupling of aldehyde with piperidine in presence of

K2CO3 and KI in DMF, it was then subjected to Horner-Wadsworth-Emmons reaction in presence of sodium hydride in THF. This gave the trans ester 5 whose ethylene chain was the cyclopropanated using diazomethane in ether and palladium acetate as a catalyst in DCM to give

6 which was then hydrolyzed to the corresponding acid by simple reflux with KOH and methanol. The product 7 was then subjected to Curtius rearrangement to furnish 8 the NHBoc of the Acid. The amine hydrochloride was obtained by treating 8 with HCl/Ether.

25

Scheme II: Synthesis scheme for 5-chloro-3-ethyl-1H-indole-2-carboxylic acid.

For the Left fragment of the molecule Fischer indole synthesis was employed. First 11 were obtained by coupling of 10 with iodopropane in presence of Na/MeOH. Jaap-Klingemann reaction of 11 with 4-choloroaniline in cold conditions gave a mixture of hydrazone and azo compounds as crude (12) which was further cyclized to give the indole (13) in presence of sulphuric acid under overnight reflux, which was then hydrolyzed to the corresponding acid (14) ready to be coupled with the amine 9.

B. For cis-amine:

The major challenge was faced to get the cis cyclopropanated ester because trans seemed to be the most preferred conformation in most of the coupling reactions.

First synthetic route tried is as described in Scheme III, wherein all steps except the first two steps are the same to obtain the amine. The aldehyde is converted to a styrene (16) by potassium 26 tert-butoxide in the presence of methyl triphenylphosphonium bromide in THF. The next step was the most crucial as this was aimed to give the cis-cyclopropanated ester, the styrene was treated with Vit B12 in TFE in the µW for 30mins.

Scheme III: First synthetic route for cis-amine.

The Vit B12 mediated reaction did not give a good yield and hence it became necessary to devise a better synthetic route. The next route tried for this step as described in scheme V, was by subjecting the aldehyde 3 to Horner-Wadsworth-Emmons reaction, where the phosphonate ester used is stereoselective to direct the reaction to give 60:40 ratio of the cis: trans isomer. The cis isomer 17 can be easily isolated by flash chromatography and all the other steps to get the amine

21 from 17 remained the same. 27

Scheme V: Synthesis of cis-amine.

Coupling of the respective amine (9, 21) with the acid (14) was brought about in the presence of

EDCI, HOBt and DIPEA where NMP was used as a solvent at room temperature as shown on

Scheme VI.

Scheme VI: Final coupling 28

The formation of the desired product of the specific stereochemistry was confirmed by NMR of the two compounds.

3.3 Biological Data

A. Modulation of CB1 and Gαq dependent PLCβ3 phosphorylation:

The Phospholipase C-β3 is an enzyme that uses internal release of calcium to activate the many extracellular signals. It is known that agonist stimulation of CB1 receptor cause release of calcium via the PLC/IP3 pathway, which can be activated via the Gαq. This assay gives the information about the effect of the compound on the phosphorylation of PLCβ3.

Graph A: Data for GAT700 and GAT701 for Modulation of CB1 and Gαq dependent PLCβ3 phosphorylation.

1)

2)

29

PLCβ3 phosphorylation was measured in HEK293A cells expressing CB1-GFP2 were treated with 1 – 10,000 nM CP55,940 ± 1 μM GAT700, GAT701, and Org27569(Graph A.1) or 0.01 –

10 µM GAT700, GAT701, or Org27569 ± 500 nM CP55940 compound for 10 min (Graph A.2) and PLCβ3 phosphorylation was measured via In-cellTM western. Concentration-response curves were fit using the non-linear regression model (4 parameters). Data are represented as the mean ±

SEM. N = 4.

B. Modulation of β-arrestin1 recruitment

β-arrestins are adapter proteins that complex with GPCRs on agonist binding and phosphorylation of receptors. They trigger the ERK1/2 activation and hence facilitate GPCR stimulated MAPK activation. Org27569 potently inhibits β-arrestin recruitment. β-arrestin1 is critical for Org27569 induced ERK1.2, MEK1/2 and c-Src phosphorylation and hence proving that Org27569 provides CB1 with downstream signaling activity.

Graph B: Data for GAT700 and GAT701 for β-arrestin recruitment

1)

2)

HEK 293A cells expressing 30

2 hCB1-GFP and β-arrestin1-Rluc were treated with 1 – 10µM CP55,940 ± 1 μM GAT700,

GAT701, or Org27569 compound (Graph B.1) or 0.01 – 10 µM GAT700, GAT701, or

Org27569 ± 500 nM CP55940 for 30 min and BRETeff was measured (Graph B.2).

Concentration-response curves were fit using the non-linear regression model (4 parameters).

Data are represented as the mean ± SEM. N = 4.

C. Modulation of CB1 and G protein dependent cAMP inhibition

Org27569 inhibits Gαi-mediated agonist–induced inhibition of cAMP production and hence, it acts as an allosteric agonist by increasing the cAMP production. This assay allows us to evaluate the effect of GAT compounds on the cAMP inhibition.

Graph C: Data for cAMP inhibition by GAT700 and GAT701

2 cAMP inhibition was measured in HEK-CRE cells expressing CB1-GFP via relative light emission 1 h after treatment with 10 µM forskolin and 0.01 – 10 µM CP55940 ± 1 µM GAT700,

GAT701, or Org27569 (Graph C). cAMP levels were measured as firefly luciferase activity driven by the cAMP response element reporter (in HEK-CRE cells). Concentration-response 31 curves were fit using the non-linear regression model (4 parameters). Data are represented % inhibition relative to the Emax of CP55,940 alone (mean ± SEM). N = 4.

D. Modulation of CB1 and Gαi/o dependent ERK phosphorylation

Extracellular signal-related kinase (ERK1/2) forms an important component of Mitogen activated protein kinase (MAPK) signal transduction pathways. Its significance lies in the fact that it is the common endpoint of different signaling cascades despite internalization from different α subunits. Org27569 is found to have a downstream ERK phosphorylation and hence is said to be Gi protein dependent, this assay will help to understand if GAT compounds show similar downstream signaling pattern.

Graph D: Data of GAT700 and GAT701for ERK1/2 phosphorylation

1)

2)

32

HEK293A cells expressing hCB1 were treated with 1 – 10,000 nM CP55,940 ± 1 μM GAT700,

GAT701, or Org27569 or 500 nM ± 1–10,000 nM GAT700, GAT701, or Org27569 ± 500nM

CP55,940 compound for 10 min and ERK phosphorylation was measured via In-cellTM western.

Concentration-response curves were fit using the non-linear regression model (4 parameters).

Data are represented as the mean ± SEM. N = 4.

Data Analysis:

• Gαq dependent PLCβ3 phosphorylation :

Treatment with GAT700 and GAT701 increased the agonist-dependent PLCβ3

phosphorylation as opposed to Org27569 which reduces the phosphorylation (Graph A).

GAT700 and GAT700 had no effect on the basal PLCβ3 phosphorylation in the absence

of orthosteric ligand (Graph A.2). There is a rise in the phosphorylation when GAT700

and GAT701 are administered with the agonist, and this rise is more prominent for

GAT701 than GAT700, this is in contrast to the effect of Org27569 which shows reduced

PLCβ3 phosphorylation. Hence it can be concluded that GAT700 and GAT701, enhance

Gαq dependent PLCβ3 phosphorylation.

• β-arrestin recruitment assay :

The BRETeff measurement showed that GAT 700 and GAT701 improved the agonist-

induced β-arrestin recruitment evidently (Graph B). Both the compounds have no effect

on the basal signal in the absence of the orthosteric agonist (Graph B.2) and GAT701 has

a more pronounced effect than GAT700. Conclusively GAT700 and GAT701, enhances

orthosteric agonist- induced β-arrestin recruitment and acts as a PAM as against the effect

of Org27569 which decreases agonist-induced β-arrestin recruitment. 33

• Modulation of CB1 and G protein dependent cAMP inhibition:

GAT700 and GAT701 shifted the dose response curve up relative to the orthosteric

agonist alone (Graph C). Hence these compounds are seen to elevate the effects of the

agonist to inhibit cAMP and act as an efficacious PAM, whereas Org27569 causes a

downward shift of the curve indicating its function as a NAM. GAT700 and GAT701

enhance orthosteric agonist- induced Gαi cAMP inhibition, and GAT701 is has a more

evident inhibitory effect.

• CB1 and Gαi mediated ERK1/2 production:

Treatment with GAT700 and GAT701 caused an increase in orthosteric agonist- induced

Gαi mediated ERK1/2 phosphorylation (Graph D). These compounds did not affect the

basal ERK1/2 phosphorylation in absence of the orthosteric agonist, but they enhanced

the phosphorylation in the presence of orthosteric agonist hence behaving as a PAM, in

comparison to Org27569 which caused a decrease in the phosphorylation. GAT701

showed more prominent increase in phosphorylation (Graph D.2.). Hence we can say

that, both that compounds eventually lead to inhibition of the ERK production.

34

Conclusion:

In the recent years significant research has been done to identify the allosteric site and synthesize compounds that act as allosteric modulators, because of it various advantages over orthosteric ligands, such as selectivity amongst the receptor subtypes and the ability to retain the physiological signaling of the endogenous ligands. Org27569 is one such compound which was identified to be an allosteric modulator at the CB1 receptor. Org27569 was distinct because it was a NAM of function but a PAM of binding. Owing to the interesting effects of Org27569 it was studied and detailed, and one of the studies brought to light that the compound has an off target activity and shows hypophagic effects irrespective of the presence of CB1 receptors. Exploration of the SAR of indole-2-carboxamides elucidated the key structural elements of the structure which are responsible for the allosteric modulation. A structural modification which would help in elimination of the off-target activity and increase the ligands affinity towards CB1 receptor was required. Locking of a molecule in a particular geometry leads to decrease in the freely rotatable bonds, and this makes it more specific for binding, this approach was used here with introduction of a cyclopropyl group, which furnished GAT700 and GAT701 as constrained analogs of Org27569. GAT700 and GAT70 were synthesized with an aim to constrain the geometry of the compound and identify the effect of this structural rigidity on the allosteric modulatory effects, as compared to the lead molecule Org27569. Although the lead molecule is a

NAM of function, both GAT700 and GAT701 are seen to enhance the agonist activity and hence act as efficacious positive allosteric modulators at CB1 receptor. A molecular switch is observed when the function of the constrained analogs is compared to that of Org27569 in bioassays. A further study of these molecules will allow more intensive synthesis of allosteric modulators of

CB1 receptors which are pharmacologically as well as physiologically significant. 35

Experimental Section:

Chemistry: All commercial chemicals and solvents were purchased from Sigma-Aldrich Inc,

Alfa Aesar and unless otherwise specified they were used without further purification. The progress of the reaction was monitored by thin layer chromatography (TLC) using commercially prepared silica gel 60 F254 glass-backed plates. All compounds were visualized under ultraviolet

(UV) light. NMR spectra and other 2D spectra were recorded in DMSO-d6, unless otherwise stated, on a Varian 500 MHz. Chemical shifts are recorded in parts per million (δ) relative to internal tetramethylsilane (TMS). Multiplicities reported in hertz (Hz). LC-MS analysis was performed using a Waters Alliance reverse-phase HPLC (electrospray ionization).

4-(piperidin-1-yl)benzaldehyde (3):

To a DMF solution of 4-fluorobenzaldehyde, 1 (10.0 g, 80.57 mmol) and piperidine, 2 (13g,

152.6 mmol) in 250 ml round bottom flask, added powdered K2CO3 (33.4 g, 241.7 mmol) and KI

(1.0 g, 6.0 mmol). Heated the reaction mixture at 80 C for 19 h. DMF was evaporated under reduced pressure and residue quenched with 100 ml ice-water mixture. Precipitated solid was filtered, dried under vacuum and recrystallized form water-methanol mixture to yield pure 3 as a

1 pale yellow plated (13.71g; yield 93%). H NMR (500 MHz, CDCl3): δ 9.7 (s, 1H), 7.72 (d, J =

10 Hz, 2H), 6.89 (d, J = 10 Hz, 2H), 3.39-3.42 (m, 4H), 1.66-1.69 (m, 6H). MS m/z – 190.26

[M+H]+.

Ethyl (E)-3-(4-(piperidin-1-yl)phenyl)acrylate (5):

Sodium hydride (1.2 g 60% suspension, 31.70 mmol) was suspended in THF, and then ethyl 2-

(diethoxyphophanyl)acetate 4 (7.69 g, 34.34 mmol) was added gradually to the reaction at room temperature and this was allowed to stir for 20 mins, when rapid hydrogen gas evolution was 36 seen. Then a THF solution of 4-(piperidin-1-yl)benzaldehyde 3 (5 g, 26.41 mmol) was added to the reaction mixture and was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure, the residual reaction mixture was quenched with water and crude was extracted with DCM, and dried under reduced pressure. The crude was purified by flash column chromatography to get pure white solid 5 (0%-30%, EtOAc: hex, 5.5 g, 80%) . 1H NMR (500

MHz, CDCl3): δ 7.62 (d, J = 15.5 Hz, 1H), 7.41 (d, J =9 Hz, 2H), 6.87 (d, J =8.5 Hz, 2H), 6.26

(d, J =16 Hz, 1H), 4.24 (q, J = 7 Hz, 2H), 3.27 (m, J = 5.5 Hz, 4H), 1.66-1.70 (m, 4H), 1.60-1.64

(m, 2H), 1.32 (t, 3 H). MS m/z – 260.35 [M+H]+.

Trans-ethyl-2-(4-(piperidin-1-yl)phenyl)cyclopropane-1-carboxylate (6):

Solution of 5 (4.5 g,17.35 mmol) in DCM was taken and palladium (II) acetate (0.05g,

0.24mmol) was added to it and stirred at -10 C. Etheral solution of diazomethane was added drop wise to the reaction while stirring. The reaction was monitored by TLC in 30% EtOAc:hex until complete conversion of starting to the product was observed. Reaction was then filtered off through celite, and solvent was evaporated to get pure white solid (4.5g, 95%). 1H NMR (500

MHz, CDCl3): δ 7.0 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 9 Hz, 2H), 4.16 (q, J =7 Hz, 2H), 3.10-3.12

(m, 4H), 2.42-2.46 (m, 1H), 1.79-1.82 (m, 1H), 1.62-1.71 (m, 4H), 1.54-1.58(m, 2H), 1.51-1.53

(m, 1H), 1.25-1.28 (t, J = 7 Hz, 3H), 1.22-1.24 (m, 1H). MS m/z – 274.38 [M+H]+.

Trans-2-(4-(piperidin-1-yl)phenyl)cyclopropane-1-carboxylic acid (7):

The compound 6 (4.5 g, 16.4 mmol) was dissolved in methanol and to this KOH (6.0g,

10.69mmol) and about 5-7 ml of water was added. The reaction was refluxed for 10-12 h at

85 C. The complete conversion to acid was observed on TLC. After cooling the reaction to room temperature, the solvent was evaporated and residue was dissolved in water and the pH of the 37 reaction was adjusted to 5 with glacial acetic acid, extracted with DCM, washed with brine and dried over Na2SO4 and evaporated to get a yellowish solid which was then purified by flash column chromatography using EtOAc:hex (10%-50%, EtOAc:hex 3.0g, 75%). 1H NMR (500

MHz, CDCl3): δ 7.10 (d, J = 8 Hz, 2H), 6.87 (d, J = 8 Hz, 2H), 3.12 (t, J = 5 Hz, 4H), 2.53 (m,

1H), 1.78-1.81 (m, 1H), 1.61-1.63 (m, 4H), 1.58-1.60 (m, 1H), 1.51-1.55 (m, 2H), 1.32-1.35 (m,

1H). MS m/z – 246.32 [M+H]+.

Trans-tert-butyl-2-(4-(piperidin-1-yl)phenyl)cyclopropyl)carbamate (8):

Compound 7 (2.0g,8.15mmol), diphenylphophorylazide (2.46g, 8.96mmol) and TEA (123g,

12.20mmol) are all taken in dry tert-butanol and the reaction was refluxed at 85 C for 12 h. The tert-butanol was evaporated under reduced pressure and reaction was quenched with water and extraction of crude by ethyl acetate was done, it was washed with brine and dried over Na2SO4 and evaporated to get a sticky liquid. The crude was then purified by flash chromatography (0%-

20%, EtOAc: hex) to get the product as an off white solid (1.5 g, 58%). 1H NMR (500 MHz,

CDCl3): δ 7.0 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 8.5 Hz, 2H), 4.82 (bs, 1H), 3.06-3.09 (m, J = 5.5,

4H), 2.60-2.70 (m, 1H) 1.93-1.97 (m, 1H), 1.67-1.71 (m, 4H), 1.58 -1.61 (m, 1H), 1.52-1.57 (m,

2H), 1.32-1.36 (m, 1H). MS m/z – 317.45 [M+H]+.

Trans-2-(4-(piperidin-1-yl)phenyl)cyclopropan-1-amine HCl salt (9):

HCl in ether (2 M solution, 10 ml) was added to the compound 8 (1.5 g, 3.63 mmol), and it was stirred at room temperature for 10 h. As the product was unstable the reaction was not quenched.

The HCl and solvent was evaporated under reduced pressure and crude product was stored under argon as a sticky orange liquid (768mg, 75%). Crude reaction mixture was taken ahead for the 38 next step without any further purification. NMR could not be recorded due to products instability.

Ethyl 2-acetylpentanoate (11):

Sodium (0.01 mmol) was carefully dissolved in ethanol under argon environment while stirring at room temperature, iodopropane (1g, 4.7mmol) was added drop-wise and it was refluxed at

75 C for 3 h. The solvent was evaporated under reduced pressure and residue was dissolved in water and extracted with ethyl acetate. It was washed with brine and dried over Na2SO4 and evaporated under reduced pressure. The crude was purified by flash chromatography (0%-20%

1 EtOAc: hex) to get the pure white solid (920mg, 70%). H NMR (500 MHz, CDCl3): δ 4.19 (q,

J = 5 Hz, 2H), 3.41 (t, J = 7.5 Hz, H), 2.22 (s, 3H), 1.76-1.89 (m, 2H), 1.30-1.34 (m, 2H), 1.27 (t,

J = 5 Hz, 3H), 0.931 (t, J = 5 Hz, 3H). MS m/z – 173.22 [M+H]+.

Ethyl 5-chloro-3-ethyl-1H-indole-2-carboxylate (12 & 13):

An ethanolic solution of 11 (500 mg, 2.33 mmol) was taken in round bottom flask in ethanol and sodium acetate (80 mg, 0.96 mmol) was added, reaction was stirred at room temperature for 1 h.

In another round bottom flask 4-chloro aniline (298 mg, 2.33 mmol) was taken in ethanol and cooled to 0 C and 5-7 ml of 27% HCl was added to it, after which a solution of sodium nitrate

(196.1 mg, 2.87 mmol) in water was added gradually at 0 C. the reaction was stirred at this temperature for 1 h. The contents of Flask are then added into Flask A at 0 C, and then the pH was adjusted to 5 by addition of sodium acetate. The reaction was then left to stir for 3 h, from

0 C to room temperature. The reaction was quenched with water and extracted with DCM to get the crude 12, which was washed with brine and dried over Na2SO4 and evaporated under reduced pressure. The crude 12 is a mixture (650 mg, 90%) and is taken ahead to the next step without 39 any further purification. Crude 12 was treated with about 10 ml 10% sulphuric acid in ethanol, and refluxed overnight at 80 C. The solvent was evaporated and residue was quenched with water, and extracted with ethyl acetate, dried over Na2SO4 and evaporated under reduced pressure. The crude was purified to get desired product by flash chromatography (5%-30%

1 EtOAc: hex) to get pure 13 (500mg, 82%). H NMR (500 MHz, CDCl3): δ 8.7 (s, 1H), 7.65 (s, J

= 5 Hz, 1H), 7.30 (d, J = 5 Hz, 1H), 7.50 (dd, J = 5 Hz, J = 2 Hz, 1H), 4.43 (q, J = 5 Hz, 2H),

3.07 (q, J = 10 Hz, 2H), 1.42 (t, J = 5 Hz, 3H), 1.26 (t, J = 5 Hz, 3H). MS m/z – 252.71 [M+H]+.

5-chloro-3-ethyl-1H-indole-2-carboxylic acid (14):

The indole ester 13 (250mg, 0.85mmol) was dissolved in methanol, KOH (66mg, 1.12mmol) was added with 2ml of water and the reaction was refluxed at 85 C for 3-5 h. Complete conversion to a pure product was observed on TLC. The reaction was quenched with 10% HCl and water till no more precipitate was formed and then product was extracted with DCM to get a

1 pure white solid (175mg, 79%). H NMR (500 MHz, CDCl3): δ 13.06 (s, 1H), 11.57 (s, 1H), 7.71

(s, J = 5 Hz, 1H), 7.39 (d, J = 10 Hz, 1H), 7.23 (dd, J = 9 Hz, 2 Hz, 1H), 3.02 (q, J = 7.5 Hz,

2H), 1.16 (t, J = 7.5 Hz, 3H). MS m/z – 224.66 [M+H]+.

Ethyl (Z)-3-(4-(piperidin-1-yl)phenyl)acrylate (18):

Potassium tert-butoxide (0.781 g, 6.97 mmol) was suspended in THF, and then ethyl-2-(bis(o- tolyloxy)phosphoryl)acetate (2.2 g, 5.81 mmol) was added gradually to the reaction at room temperature and this was allowed to stir for 20 mins, when rapid hydrogen gas evolution was observed. THF solution of 4-(piperidin-1-yl)benzaldehyde 3 (1.16g, 6.16mmol) was added to it anhydrously, the reaction was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure, the residual reaction mixture was quenched with water and crude was 40

extracted with DCM, which was washed with brine, dried over Na2SO4 and evaporated under reduced pressure. The crude was purified by flash column chromatography to get pure yellowish

1 crystalline solid 18 (0%-10% EtOAc:hex, 800mg, cis: 60%). H NMR (500 MHz, CDCl3): δ

7.71 (7.71, J = 9.5 Hz, 2H, 6.85 (d, J = 9 Hz, 2H), 6.77 (d, J = 13 Hz, 1H), 5.72 (d, J = 13 Hz,

1H), 4.19 (q, J = 7.5 Hz, 2H), 3.26 (t, J = 5.5 Hz, 4H), 1.67-1.69 (m, 4H), 1.59-1.64 (m, 2H),

1.29 (t, J = 7.5 Hz, 3H). MS m/z – 260.35 [M+H]+.

Cis-ethyl-2-(4-(piperidin-1-yl)phenyl)cyclopropane-1-carboxylate (17):

Solution of 18 (650 mg,17.35 mmol) in DCM was taken and palladium (II) acetate (7.5 mg,

0.033 mmol) was added to it and stirred at -10 C. Etheral solution of diazomethane was added drop wise to the reaction while stirring. The reaction was monitored by TLC in 30% EtOAc:hex until complete conversion of starting to the product was observed. Reaction was then filtered off through celite, and solvent was evaporated to get a pure white solid 17 (550 mg, 89.5%). 1H

NMR (500 MHz, CDCl3): δ 7.13 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 8.5 Hz, 2H), 3.88 (q, J = 7 Hz,

2H), 3.10 (t, J = 5.5 Hz, 4H), 2.45-2.53 (m, 1H), 2.00 (m, 1H), 1.53-1.58 (m, 2H), 1.24-1.30 (m,

2H), 1.00 (t, J = 7.5 Hz, 2H). MS m/z – 274.38 [M+H]+.

Cis-2-(4-(piperidin-1-yl)phenyl)cyclopropane-1-carboxylic acid (19):

The compound 18 (720 mg, 2.6 mmol) was dissolved in methanol and to this KOH (205mg,

3.65mmol) and about 5-7 ml of water was added. The reaction was refluxed for 10-12 h at 85 C.

The complete conversion to acid was seen on TLC. After cooling the reaction to room temperature, the solvent was evaporated and residue was dissolved in water and the pH of the reaction was adjusted to 5 with glacial acetic acid, extracted with DCM, washed with brine and dried over Na2SO4 and evaporated to get an off white solid which was then purified by column 41 chromatography (0%-10%, EtOAc:hex) to get a pure off white solid 19 (453mg, 70%). 1H NMR

(500 MHz, CDCl3): δ 7.17 (d, J = 8 Hz, 2H), 6.93 (d, J = 8 Hz, 2H), 3.13 (t, J = 5 Hz, 4H), 2.54

(q, J = 9 Hz, 1H), 1.98-2.04 (m, 1H), 1.67-1.72 (m, 4H), 1.60-1.64 (m, 1H), 1.53-1.57 (m, 2H),

1.31-1.34 (m, 1H). MS m/z – 146.32 [M+H]+.

Cis-tert-butyl-2-(4-(piperidin-1-yl)phenyl)cyclopropyl)carbamate (20) :

Compound 19 (500 mg, 2.04mmol), diphenylphophorylazide (673 mg, 2.44 mmol) and TEA

(312 mg, 3.06 mmol) are all taken in dry tert-butanol and the reaction was refluxed at 85 C for

12 h. The tert-butanol was evaporated under reduced pressure and reaction was quenched with water and extracted with ethyl acetate, and washed with brine and dried over Na2SO4 and evaporated to get a dark sticky liquid. The crude was then purified by flash chromatography

(5%30%, EtOAc: hex) to get the pure product as a sticky liquid 20 ( 325mg, 50.4%). 1H NMR

(500 MHz, CDCl3): δ 7.05 (d, J = 5 Hz, 2H) , 6.89 (d, J = 5 Hz, 2H), 3.14 (t, J = 5 Hz, 4H), 2.84-

2.9 (m, 1H), 2.12-2.17 (m, 1H), 1.69-1.73 ( m, 4H), 1.55-1.59 (m, 2H), 1.35 (s, 9H), 1.19-1.31

(m, 1H), 0.89-0.91 (m, 1H). MS m/z – 317.45 [M+H]+.

Cis-2-(4-(piperidin-1-yl)phenyl)cyclopropan-1-amine HCl salt (21)

To the compound 20 (325 mg) was added HCl in ether (2 M solution, 5-7 ml) and it was stirred at room temperature for 10 h. As the product was unstable the reaction was not quenched. The

HCl and solvent was evaporated under reduced pressure and dry product was stored under argon as a sticky liquid (180mg, 81%). The crude reaction mixture was taken ahead without any further purification. NMR could not be recorded due to products instability.

42

Trans-5-chloro-3-ethyl-N-(2-(4-(piperidin-1-yl)phenyl)cyclopropyl)-1H-indole-2- carboxamide (22)

The acid 14 (200 mg, 0.89 mmol) , amine 9 (211 mg, 0.98 mmol), ethyl-(3- dimethylaminopropyl)carbodiimide hydrochloride (166 mg, 1.068 mmol), hydroxybenzotriazole

(165.5 mg, 1.068 mmol) is taken in NMP as solvent and stirred for 2 min till all contents dissolve, then add DIPEA (541 mg, 4.2 mmol) and let the reaction stir under an argon environment for 2h. The compound might precipitate out either directly in the reaction, or after quenching with water. The precipitate is filtered and washed with water, and ethyl acetate. Due to poor solubility it is hard to carry out chromatographic purification, but most of the pure precipitates out, or can be recrystallized with acetone. The solvent was decanted and then residual was evaporated under reduced pressure to get pure off white solid 22 (173mg, 46%). 1H

NMR (500 MHz, CDCl3): δ 11.27 (s, 1H), 8.27 (s, 2H), 7.66 (s, 1H), 7.40 (d, J = 10 Hz, J = 2.5

Hz, 1H), 7.19 (dd, J = 10 Hz, J = 2.5 Hz, 1H), 7.03 (d, J = 10 Hz, 2H), 6.86 (d, J = 10 Hz, 2H),

3.07 (t, J = 5 Hz, 4H), 2.96-3.02 (m, 2H), 2.91-2.95 (m, 1H), 1.97-2.01 (m, 1H), 1.59-1.64 (m,

4H), 1.49-1.55 (m, 2H), 1.19-1.24 (m, 1H), 1.15 (t, J = 7.5 Hz, 4H). MS m/z – 422.97 [M+H]+.

Cis-5-chloro-3-ethyl-N-(2-(4-(piperidin-1-yl)phenyl)cyclopropyl)-1H-indole-2-carboxamide

(23):

The acid 14 (200 mg, 0.89 mmol) , amine 9 (211 mg, 0.98 mmol), ethyl-(3- dimethylaminopropyl)carbodiimide hydrochloride (166 mg, 1.06 mmol), hydroxybenzotriazole

(165.5 mg, 1.06 mmol) is taken in NMP as solvent and stirred for 2 min till all contents dissolve, then add DIPEA (541 mg, 4.2 mmol) and let the reaction stir under an argon environment for 2h.

The compound might precipitate out either directly in the reaction, or after quenching with water. 43

The precipitate is filtered and washed with water, and ethyl acetate. Due to poor solubility it is hard to carry out chromatographic purification, but most of the pure precipitates out, or can be recrystallized with acetone. The solvent was decanted and then residual was evaporated under

1 reduced pressure to get pure off white solid 23 (150mg, 40%). H NMR (500 MHz, CDCl3): δ

11.21 (s, 1H), 7.57 (t, J = 2.5 Hz, 2H), 7.34 (d, J = 8.5 Hz, 1H), 7.14 (dd, J = 10 Hz, J = 2.5 Hz,

1H), 7.07 (d, J = 9 Hz, 2H), 6.77 (d, J = 5 Hz, 2H), 3.10-3.15 (m, 1H), 3.01 (t, J = 5 Hz, 4H),

2.60-2.73 (m, 2H), 2.19 (q, J = 10 Hz, 1H), 1.53-1.57 (m, 4H), 1.44-1.48 (m, 2H), 1.24-1.29 (m,

1H), 1.12-1.15 (m, 1H), 0.89 (t, J = 7.5 Hz, 3H). MS m/z – 422.97 [M+H]+.

44

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