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Discovery and Development of N-[4-(l-Cyclobutylpiperidin-4-yloxy) phenyl]-2-(morpholin-4-yl) acetamide dihydrochloride (SUVNG3031): A Novel, Potent, Selective and Orally Active Histamine H3 Receptor Inverse Agonist with Robust Wake-Promoting Activity Ramakrishna Nirogi, Anil K. Shinde, Abdul Rasheed Mohammed, Rajesh Kumar Badange, Veena Reballi, Thrinath Reddy Bandyala, Sangram Keshari Saraf, Kumar Bojja, sravanthi manchineella, Pramod Kumar Achanta, Kiran Kumar Kandukuri, Ramkumar Subramanian, Vijay Benade, Raghava Choudary Palacharla, Pradeep Jayarajan, Santoshkumar Pandey, and Venkat Jasti J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01280 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 11, 2019

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Journal of Medicinal Chemistry

Discovery and Development of N-[4-(lCyclobutylpiperidin-4-yloxy) phenyl]-2-(morpholin4-yl) acetamide dihydrochloride (SUVN-G3031): A Novel, Potent, Selective and Orally Active Histamine H3 Receptor Inverse Agonist with Robust Wake-Promoting Activity Ramakrishna Nirogi*, Anil Shinde, Abdul Rasheed Mohammed, Rajesh Kumar Badange, Veena Reballi, Thrinath Reddy Bandyala, Sangram Keshari Saraf, Kumar Bojja, Sravanthi Manchineella, Pramod Kumar Achanta, Kiran Kumar Kandukuri, Ramkumar Subramanian, Vijay Benade, Raghava Choudary Palacharla, Pradeep Jayarajan, Santoshkumar Pandey and Venkat Jasti Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road-5, Avenue-7, Banjara Hills, Hyderabad 500 034, India *Corresponding Author Tel.: +91-40-23556038; Fax: +91-40-23541152; e-mail: [email protected]

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 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

Keywords-Histamine-3 inverse agonist, Structure-activity relationship, Orexin-B-SAP lesioned rats, Wake promoting activity

ABSTRACT: A series of chemical optimizations guided by in vitro affinity at histamine H3 receptor (H3R), physico-chemical properties and pharmacokinetics in rats resulted in identification of N-[4-(1-Cyclobutyl-piperidin-4-yloxy) phenyl]-2-(morpholin-4-yl) acetamide dihydrochloride (17v, SUVN-G3031) as a clinical candidate. Compound 17v is a potent (hH3R Ki = 8.73 nM) inverse agonist at H3R with selectivity over other 70 targets Compound 17v has adequate oral exposures and favorable elimination half-lives both in rats and dogs. It demonstrated high receptor occupancy and marked wake promoting effects with decreased REM sleep in orexin-B saporin lesioned rats supporting its potential therapeutic utility in treating human sleep disorders. It had no effect on the locomotor activity at doses several fold higher than its efficacious dose. It is devoid of hERG and phospholipidosis issues. Phase-1 evaluation for safety, tolerability and pharmacokinetics, and long term safety studies in animals have been successfully completed without any concern for further development.

INTRODUCTION Narcolepsy is a life-long neurological sleep disorder largely characterized by excessive daytime sleepiness (EDS), sleep paralysis, cataplexy, and hallucinations and disrupted nighttime sleep. The disease is segmented into two categories i.e., narcolepsy with cataplexy (Type 1) which is characterized by sudden loss of muscle tone and narcolepsy without cataplexy (Type 2).1,2 Narcolepsy has substantial impact on the quality of life including social functioning, family relationships, academic performance as well as burden on the associated healthcare resource utilization and costs.3,4


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The histamine 3 receptor (H3R) is a member of a seven transmembrane-spanning G-protein coupled receptor (GPCR) family5 and it functions both as a presynaptic autoreceptor and a heteroreceptor. As an autoreceptor it regulates the release of histamine in the brain, particularly in the cortex, striatum, hippocampus, amygdala and substantia nigra. H3R as an inhibitory heteroreceptor, modulates the release of key neurotransmitters like acetylcholine, dopamine, norepinephrine, and serotonin. H3R also associated with many important functions including arousal, regulation of sleep-wake cycle, immune modulation, satiety and cognition.6-8 Wakepromoting effect of H3R inverse agonists most likely depends on histaminergic autoreceptormediated modulation of histaminergic neurotransmission. The anti-cataplectic effect seems to involve not only the histaminergic but also other neurotransmitter systems like dopamine and norepinephrine that are controlled by histamine 3 heteroreceptors.9,10 A number of H3R antagonists/inverse agonists demonstrated wake-promoting effects in several animal species. H3R antagonists/inverse agonists did not show wake promoting effects in H3R knockout mice providing evidence for the involvement of the receptor in wake promoting effects.8,11 Previously reported imidazole based H3R antagonist thioperamide increased the wakefulness and decreased NREM and REM sleep in tested rats and cats12,13 but unlike amphetamine and caffeine, its wake promoting effects were not accompanied by behavioral excitation and sleep rebound.8,14,15 Other H3R antagonists like ciproxifan and carboperamide were also reported to increase the wake promoting effects and were reported to decrease NREM sleep in rats.16,17 The European Medicines Agency (EMA) has granted a marketing authorization valid throughout the European Union for pitolisant (Wakix), a H3R antagonist for the treatment of narcolepsy with or without cataplexy.18 Thus, H3R offers a unique platform for the treatment of sleep-wake disorders without having stimulant activity.



In the past,19,20 researchers have optimized the parameters like hERG and phospholipidosis by modulating the physico-chemical properties like lipophilicity (LogP, LogD) and basicity (pKa). A diamine based H3R antagonist, JNJ-5207852was reported to possess high brain residence time and induced phospholipidosis.21 A related wake promoting compound (JNJ-7737782) was reported subsequently with substantially reduced pKa and reduced half-life in rats.22 Letavic et. al. recently demonstrated23 that, improved human pharmacokinetic profiles can be obtained by fine tuning the physicochemical properties of H3R ligands in order to reduce tissue retention time and half-lives. Hudkins et. al. have reported24 the increased wake duration for CEP-32215 in rat EEG model at 3 to 30 mg/kg p.o. indicating its wake promoting properties. Irdabisant, a H3R antagonist/inverse agonist, reported25 to increase wake promoting activity, decreased slow-wave and rapid-eye movement sleep dose dependently in the tested doses of 3–30 mg/kg p.o. In the past, we have published our initial efforts in identifying novel H3R antagonists.26 Starting from a hit with moderate potency at H3R, a series of six membered cyclic benzamide (lactam) derivatives, exemplified by 2-(1-cyclobutyl piperidin-4-yl)-6-(1-cyclobutyl piperidin-4yloxy)-3,4-dihydro-2H-isoquinolin-1-one (Compound I, Figure 1) were designed, synthesized and evaluated. Compound I exhibited potent in vitro affinity (hH3 Ki = 0.8 nM) at H3R. However, it exhibited delayed absorption accompanied by higher volume of distribution (Vdss) and extended half-life in rats. The Compound I had low brain penetration (Cb/Cp ratio of ~0.40) and also had phospholipidosis inducing potential at concentrations above 20 µM (Supporting information). The ADME features for this compound were less than ideal given the role of H3R antagonist’s in sleep/wake cycle, vigilance and attention. This prompted us to work on a chemically diverse scaffold distinct from lactam based Compound I with pharmacokinetic (PK) profiles more consistent with our goal of developing a rapidly absorbing, shorter-acting clinical


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candidate devoid of phospholipidosis potential. The optimization of chemical scaffold was mainly driven by H3R affinity, modulation of physico-chemical properties and PK properties in rats. Opening of the rigid ring in compound I to obtain the flexible linker resulted in compounds 9a–9f (Series A, Figure 1), transposition of the amide functionality to obtain the acetamide functionality resulted in compounds 17a–17ah (Series B), modulation of ether linkage in the western domain resulted in compounds 26a–26f (Series C), substitution on acetamide nitrogen resulted in compounds 27a and 27b (Series D). O N

N O Compound I

O R1

Ring opening Rearranging amine ring

N

2

R O

3

1 4

Western domain

N

Central core

N H

R1

N

H N

O Series A (9a-9f)

R1

2

R N

O

R2

Eastern domain

O N H

R1

2

R N

N R2

R2

Series C (26a-26f)

R2

O

Transpose amide Reduce linker

R2 N

N R2 3 R Series D (27a-27b)

Series B (17a-17ah)

Figure 1. Design strategy Chemistry. The general synthetic strategy used for the preparation of compounds is summarized in Schemes 1−5. The starting materials 1, 2, 5a–5c, 8a–8c (Scheme 1), 12a–12d, 16a–16q (Scheme 2), 18a–18e (Scheme 3) and 24a–24f (Scheme 4) were commercially procured.



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Scheme 1. Synthesis of 9a–9fa OTs

O OCH3

O Boc

a

+

OCH3

N Boc 2

HO 1

N

3 O Boc

c

OH

N

N H

R2 N

R2

d

O

5a/5b/5c

O

b

O

O Boc

N

6a-c

4 R2 N

O HN 2HCl

N H O

O

e

R2

R1

8a/8b/8c

N

N H O

7a-c

R2 N

R2

9a-f

O N

N

N

O

NH2

NH2

NH2

(5a)

(5b)

(5c)

(8a)

O

O

(8b)

(8c)

aReagents

and conditions: (a) K2CO3, DMF, 6 h, 110-120 ºC, 72%; (b) LiOH.H2O, THF:H2O, rt, 6 h, 85%; (c) PyBop, Et3N, DCM, 12 h, rt, 65-75%; (d) IPA.HCl: IPA, 3 h, rt, 85-95%; (e) NaBH(OAc)3, Et3N, 1, 2-dichloroethane, 15 h, rt, 80-85%.


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Scheme 2. Synthesis of 17a–17k, 17q–17ac and 17aha OH

OH a

b

8a/8b/8c

N H

N R1 11a-11c

10

12a/12b

R1

R1

4

N

NO2

13a; R1 = cyclopentyl, R = H 13b; R1 = cyclobutyl, R = H 13c; R1 = isopropyl, R = H 13d; R1 = cyclobutyl, R = 2-F

11a; R = cyclopentyl, 11b; R1 = cyclobutyl, 11c; R1 = isopropyl

R

R

O d

N

12c/12d

NH2

c

3

13a-13d

1

O

R

2

O 1

N

R1

X

O

Y

N H

14a-14d

17w-17x

e

O R

1

O

N

Cl

N H

15a-15d

f

R1

16a-16q

N

F

O N N H 16j

N H 16d

N H 16c O

O N N H 16k

R2

NO2

F N

R

16a

N H

R2 N

17a-17k, 17q-17ac, 17ah

F N H 16b

O

2HCl

16a-q N H

R

O

R

N H 16e

N H 16f

N H 16g

F

N H 16i

N H 16h

O O S

O

N H

N H

N H

16l

16m

16n

HO HO

N H 16o

O

O N H 16p

N H 16q

aReagents

NO2

F 12a

OH

N

F

12b

O

N

N

HO

HO 12c

12d

and conditions: (a) NaBH(OAc)3, Et3N, 1, 2-dichloroethane, 24 h, rt, 90-95%; (b) NaH, THF, rt, 5 h, 75-80%; (c) Fe, NH4Cl, Ethanol:H2O, reflux, 4 h, 84-88%; (d) PyBop, Et3N, DCM, rt, 15 h, 50-55%; (e) Chloroacetyl chloride, K2CO3, THF, 10 ºC to  5 ºC, 2 h, 95-98%; (f) i) Methanol, reflux, 4 h, 88-93%; ii) IPA.HCl: IPA, rt, 3 h, quantitative.



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Scheme 3. Synthesis of 17l–17p and 17ad–17aga OTs 2

HO

R 3

1 4

a

+

N Boc

NO2

18a-18e

R

O Boc

b

N

N

Boc

NO2

NH2

19a-19e

2

R

O

20a-20e

18a-18e, where R is; 18a=3-CH3, 18b=3-CF3, 18c=3-F, 18d=3-OCH3, 18e=H R

O

c Boc

O

N

d Cl

N H

16c/16d/16e/16n

Boc

e

N H

R2 N

R2

22a-22j

R R2 N

O

HN

O

N

21a-21e

O

R

O

N H

R2

R

2

O

f R1

8b

N

O

3

1 4

N H

R2 N

R2

17l-17p & 17ad-17ag

23a-23j

aReagents

and conditions: (a) K2CO3, DMF, 110-120 ºC, 6 h, 80-85%; (b) Fe, NH4Cl, Ethanol:H2O, reflux, 4 h, 85-88%; (c) Chloroacetyl chloride, K2CO3, THF, 10 °C to  5 ºC, 2 h, 90-95%; (d) K2CO3, DMF, 75-80 ºC, 4 h, 75-80%; (e) IPA.HCl: IPA, rt, 3 h, then aq. NaOH, 8590%; (f) NaBH(OAc)3, Et3N, 1, 2-dichloroethane, 24 h, rt, 75-80%. Scheme 4. Synthesis of 26a–26fa R1

R1

a

R1

O Cl

N H

NH2 24a-24f

O

b 16d/16n

25a-25f

N H

R2 N

R2

26a-26f

R1 = O

O

N

N

R

R

N

N

N

N N

a

b

c

d

e

f

and conditions: (a) Chloroacetyl chloride, K2CO3, THF, 10 ºC to  5 °C, 2 h, 7075%; (b) Methanol, reflux, 6 h, 85-90%. aReagents


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Scheme 5. Synthesis of 27a–27b a O N

O N H

O N

O

a

N

17v aReagents

O N R3

O N

27a-27b

and conditions: (a) CH3I/C2H5I, NaH, DMF, 65-70 ºC, 4 h, 65%.

The tosyl group of compound 2 was displaced with methyl 4-hydroxybenzoate (1), to obtain ester compound 3 which on hydrolysis with LiOH afforded acid compound 4. The acid compound 4 was reacted with various amines, 5a–5c, in presence of PyBop to obtain Bocprotected carboxamide derivatives 6a–6c, which upon Boc deprotection with isopropanolic HCl in isopropanol gave compounds 7a–7c respectively. Reductive amination of compounds 7a with 8a, 8b and 8c gave compounds 9a, 9e and 9f, whereas reaction of compound 7b with 8b yielded compound 9b. The reductive amination of 7c with 8b and 8c provided compounds 9c and 9d respectively (Scheme 1). Reductive amination of compound 10 with carbonyl compounds 8a−8c afforded N−alkylated compounds 11a−11c respectively. Nucleophilic substitution of 12a with 11a−11c in presence of NaH afforded the nitro compounds 13a−13c respectively, while 12b on reaction with 11b gave intermediate 13d. Reduction of nitro group in compounds 13a−13d with activated iron yielded compounds 14a−14d, which on further reaction with chloroacetyl chloride and potassium carbonate afforded compounds 15a−15d respectively. PyBop mediated acid-amine coupling of 14b with 12c and 12d yielded compounds 17w and 17x respectively. Amination of 15a with 16c, 16e and 16n gave target compounds 17i, 17j and 17ac while reaction of 15b with 16a−16h gave 17a−17h respectively. Reaction of 15b with 16i−16q gave 17q−17aa respectively whereas



compound 15c on reacting with 16e and 16n gave compounds 17k and 17ab respectively. Reacting compound 15d with 16n afforded compound 17ah (Scheme 2). Alternate synthetic scheme was followed for getting the target compounds ‘17’ having substitution on central aromatic ring. Variously substituted aromatic nitro compounds (18a–18e) were reacted with compound 2 to obtain compounds 19a–19e which on nitro group reduction followed by chloroacetyl chloride reaction yielded compounds 21a–21e. Amination of compounds 21a with 16c, 16d, 16e and 16n gave compounds 22a, 22b, 22c and 22g respectively while amination of compound 21b with 16c, 16e and 16n gave compounds 22d, 22e and 22h respectively. Amination of compounds 21c, 21d, and 21e with 16n yielded compounds 22f, 22i and 22j respectively. The compounds 22a–22j on Boc deprotection yielded 23a–23j respectively. Reductive amination of 23a, 23b, 23c,and 23g with 8b resulted in target compound 17l, 17m, 17n and 17ae while 23d, 23e, 23f, 23h and 23i on reductive amination with 8b gave 17o, 17p, 17ad, 17af and 17ag respectively (Scheme 3). Substituted aromatic amines (24a–24f) on reaction with chloroacetyl chloride yielded compounds 25a–25f. Amination of 25a–25b with 16d gave target compounds 26a–26b whereas 25c–25f on amination with 16n yielded target compounds 26c–f respectively (Scheme 4). Alkylation of amidic ‘NH’ of compound 17v delivered compounds 27a and 27b (Scheme 5). The compound 28 was synthesized according to Scheme 2 starting with 1-fluoro-2nitrobenzene whereas the compound 29 was synthesized according to Scheme 3 starting from 3nitrophenol. The scope of structure activity relationship (SAR) using Compound I (LogP = 4.0, hH3 Ki = 0.80 nM) was limited because of the cyclic amide nature of its central core (3,4-dihydro-2Hisoquinolin-1-one). Hence the rigid benzamide series was modified and rearranged to a flexible


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open chain benzamide series (Series A, Figure 1). This scaffold was synthetically accessible and sufficiently versatile to support lead optimization. Compounds 9a–9f were synthesized and evaluated in in vitro radioligand binding technique using recombinant histamine H3R,27 the results of which are shown in Table 1. In vitro affinity of 10000

N

H

aThe

affinity of the test compounds were determined using in-vitro radioligand binding assay using recombinant human H3R membrane and [3H]-(R)-α-methylhistamine radioligand. Values are mean of two independent experiments. Compounds 17c, 17e, 17h, 17j, 17k, 17l, 17n, 17o, 17s, 17v, 17u, 17y, 17aa, and 17ab are dihydrochloride salts while 9c, 17f and 17w are L-(+)tartrate salts. Remaining compounds are free bases.



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Table 2. hH3R radioligand binding dataa R1

O N H

R2 N

R1 R

R2 N

O

2

N R3 Series D (27a-27b)

Series C (26a-26f)

R2

R2 N

Comp. Series R1 No

R2

R3

l

m

n

hH3R Ki (nM)

-

-

-

-

1455

-

-

-

-

915

R

26a

C

O

N

26b

C

O

N

26c

C

26d

C

26e

C

26f

C

N R

N

N

N

O

-

-

-

-

114.5

N

O

-

-

-

-

3572

N

O

-

-

-

-

107.6

N

O

-

-

-

-

532.1

N

O

CH3

-

-

-

4.89

N

O

C2H5

-

-

-

10.8

R

N R

N N

N

O

27a

D

N

O

27b

D

N

O

28

N

O

HN N

O


>10000

Page 17 of 51 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

29

N

H N

N O

O

212.1

Pitolisant

27.6

Imetit

0.30

aThe

affinity of the test compounds were determined using in-vitro radioligand binding assay using recombinant human H3R membrane and [3H]-(R)-α-methylhistamine radioligand and are mean of two independent experiments. Pitolisant is HCl salt. Remaining compounds are free bases. To assess the impact of moving from benzamide to acetamide series on the pharmacokinetic parameters, select compounds 17c, 17e and 17l were evaluated in rat PK study. Unfortunately, all these compounds exhibited the same shortcomings as observed with compounds 9a and 9b, such as delayed absorption with higher Vdss and extended half-life (Table 4). However, to our delight, this modification resulted in improved brain exposures for compound 17e (Cb/Cp= 36.6) and 17l (Cb/Cp = 1.20). At this point, in vitro phospholipidosis inducing potential for the select potent compounds (17c, 17e and 17f) was assessed by fluorometric assay as the phospholipidosis phenomenon has been reported to be one of the reason for attrition of the earlier reported H3R ligands.20 Compounds 17c and 17e had shown strong phospholipidosis inducing potential at 100 µM while compound 17f had shown strong phospholipidosis inducing potential at 50 µM (supporting information). The cationic amphiphilic compounds that possess relatively high pKa (basicity) and high LogP (lipophilicity) are known to induce phospholipidosis. Ploemen et al. reported28 use of simple physicochemical properties ClogP and pKa to identify compounds with phospholipidosis inducing potential. The discovery of this acetamide core as a novel, potent H3R chemotype with improved brain penetration properties provided us additional scope for further optimization to modulate the physicochemical properties without affecting the in vitro potency.



In light of literature precedence and the results obtained above, the efforts were made in replacing piperidine ring in compound 17e with various heterocyclic/heteroaryl rings in order to reduce the lipophilicity (by lowering LogP) and modulate the pKa. Theoretically, the introduction of hetero atom increases the polarity thereby decreases LogP and modulates the pKa of the compound. Increase in polarity of the compound may also reduce Vdss and terminal halflife thereby decreases the risk of phospholipidosis potential. This understanding led to the synthesis of compounds 17q–17ac and 23j. Compounds 17q and 17r were prepared by replacing piperidine with alkyl substituted homopiperazines. Both compounds exhibited H3R affinity comparable to its parent analog 17e. Similar results were obtained for compounds 17s and 17t where the piperidine is replaced by piperazine with terminal nitrogen substituted with carbamate group as in compound 17s and acyl group as in compound 17t suggesting that placement of larger groups on the terminal piperazine nitrogen is also tolerated. Compounds 17u, 17v, 17w and 17x with substitutions like thiomorpholine 1,1-dioxide, morpholine, 4-pyridylmethyl and 3pyridylmethyl respectively were well tolerated though the in vitro affinity of these derivatives was 3-4 fold less as compared to compound 17e. Compounds 17y, 17z and 17aa with introduction of heteroatom onto the ring were also tolerated with potent in vitro affinity at H3R. In order to further expand the scope of SAR, the cyclobutyl group on the piperidine of compound 17v was replaced with isopropyl, cyclopentyl and hydrogen to obtain compounds 17ab, 17ac and 23j respectively. Compound 17ab showed similar in vitro affinity as that of 17v while 17ac is 34 fold less potent than 17v whereas compound 23j found to be inactive suggesting alkyl substitution on nitrogen is essential. Few more analogues 17ad–17ah with ortho/meta fluoro, methyl, trifluormethyl and methoxy substituted phenyl ring were prepared. Compounds 17ad and 17ae with fluoro and methyl substituent respectively at ortho position were fivefold less


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potent than their parent compound 17v while compounds 17af and 17ag with ortho –CF3 and – OCH3 substituents respectively were ~8 fold less potent. Compound 17ah with fluoro substitution at meta position was also ~8 fold less potent than the parent analog 17v. It is evident from the results obtained as shown in Table 3, the introduction of the polar rings as in compound 17v and 17ab resulted in reduction of LogP (by a unit) and in pKa (~half log unit) as against their counterparts 17e and 17k respectively. Table 3. Measured physicochemical properties of select compounds LogP

LogD (at pH 7.4)

pKa1 pKa2

17c

3.2

1.2

9.1 and 7.4

17e

3.7

1.3

9.2 and 7.5

17f

4.3

1.7

9.2 and 7.6

17k

3.4

1.6

9.4 and 7.2

17v

2.2

0.4

8.5 and 4.9

17y

2.1

0.6

8.9 and 6.4

17ab

1.8

0.5

8.9 and 5.0

Comp. No

and

The detailed rat pharmacokinetic profile for compounds 17v, 17w, 17y and 17ab is given in Table

4.

As

envisaged,

the

PK

profile

of

compound

17y

having

polar

2-

hydroxymethylpyrrolidine substituent showed shorter half-life (LogP = 2.1; i.v.t1/2 = 1.88 h) as compared to pyrrolidine compound 17c (LogP = 3.2; i.v.t1/2 = 42.87 h). Also noteworthy is the reduction in Vdss for compound 17y (9.0 L/Kg) as against compound 17c (34.78 L/Kg) that complements its shorter half-life. Similarly, pyridine substituted compound 17w also showed shorter half-life (i.v.t1/2 = 1.12 h). However, both of compounds 17w and 17y showed poor brain exposures. The morpholine substituted analogues 17v and 17ab have shown favorable rat PK



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profile as compared to their piperidine analogue 17e and 17k respectively. Unlike compound 17e, compounds 17v and 17ab showed rapid absorption, reduced half-life, reduced Vdss and excellent bioavailability. We tentatively attributed this observation to a reduced Vdss which is likely driven by the reduction in LogP and pKa of 17v and 17ab compared to 17e and 17k respectively (Table 3). The brain to plasma (Cb/Cp) ratio for compound 17v was ~1 while that for compound 17ab was relatively less at 0.33. The in vitro phospholipidosis inducing liability was also assessed for both of compounds 17v and 17ab by fluorometric assay. Gratifyingly and in the expected lines, both of the compounds did not show any phospholipidosis inducing potential or any cytotoxicity up to the highest tested concentration of 300 µM (supporting information). In rodent and non-rodent long term in vivo safety studies done on compound 17v, phospholipidosis was not observed up to the tested doses and thus complementing the in vitro observation. In summary, these findings showed that the design strategy of modulating physicochemical properties (lowering LogP and moderate reduction of pKa) by introducing polar substituents was a viable approach for the identification of compound devoid of phospholipidosis without impacting the in vitro affinity and potency. Compounds 26a–26f were prepared (Series C) in order to understand the western domain SAR of the “N-cyclobutyl-4-hydroxypiperidine” motif in driving potency across this series. Compounds with alternate cyclic amines like morpholin-4-yl (26a) or morpholin-4-ylmethyl (26b),

pyrrolidin-1-ylmethyl

(26c),

2-methyl-pyrrolidin-1-ylmethyl

(26d),

2-(2-methyl-

pyrrolidin-1-yl)-ethyl (26e) and 4-cyclobutyl-piperazin-1-yl (26f) were made. The in vitro affinity of all these compounds was >100 nM indicating the importance of N-cyclobutyl-4hydroxypiperidine for H3R affinity.


Page 21 of 51 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-alkylation on compound 17v resulted in compounds 27a and 27b (Series D). This modification though tolerated in terms of in vitro affinity, however the rat half-life (~6 h) was 3 times more for 27a than the half-life of 17v. At the end, the position of the ether linkage with respect to acetamide group was explored. Changing of the position from para to ortho or meta was not well tolerated and compounds 28 (ortho substitution) and 29 (meta substitution) turned out to be inactive at H3R. The radioligand binding affinity of Pitolisant at H3R was found to be 27.6 nM when tested in our in-house radioligand binding assay. In comparison, under the similar tested conditions 17v has a Ki of 8.73 nM. The reported Ki value10 for the Pitolisant for human recombinant H3R is 2.7 nM. In our in house study we have used [3H]-R--methyl histamine while in the reported study the radioligand used was [125I]-Iodoproxyfan. We assume that the probable disparity in the Ki value of Pitolisant might be due to the difference in radioligand employed and their pharmacological properties. In the same study, the reference compound imetit shown a Ki of 0.3 nM which is in agreement with literature reports. Table 4. Pharmacokinetic properties of compound set in rats

Comp. No

AUC AUC0-24hr Tmax (ng*h/mL) (ng*h/mL) (h) p.o. i.v.

Compound I

1429

9a

205

9b

1743

Vdss (L/Kg)

CL t1/2 (mL/min/kg) (h) i.v.

i.v.

2

F (%)

Cbrain/Cplasma at 1 h (total)

27

0.41

6

13

174

3.33

135.25

156

13.18

48

NT

347

255

1.33

45

51

13.81

46

0.07

17c

556

757

3.33

34.78

13.57

42.87

17

NT

17e

1387

1855

3.33

20

30

11.18

37

36.6

23.49



Page 22 of 51

17l

1349

3345

2.17

27.03

13.21

26.31

20

1.20

17v

626

263

0.42

5

63

1.45

79

0.93

17w

545

145

0.25

7

113

1.12

125

0.16

17y

63

212

0.58

9

67

1.88

10

0

17ab

850

340

0.42

6

48

2.04

85

0.33

aFasted

male Wistar rats, Vehicle used: water for injection for both p. o. and i.v. routes. Dosing Volumes: 10 mL/kg for p.o. and 2 mL/kg for i.v.; NT: Not tested

Having zeroed in on compound 17v, the most optimized one among the tested lot, the PK of compound 17v was evaluated in Beagle dogs (at 3 mg/kg p.o. and 1 mg/kg i.v., dose). Compound 17v showed rapid absorption (Tmax between 0.25 – 1.00 h) with high oral exposure (AUC0-t = 1120 ± 227 ng*h/mL) and with a favorable terminal i.v. half-life of 1.7 ± 0.2 h. Intravenous clearance (36 ± 3 mL/min/kg) and volume of distribution (5.2 ± 0.4 L/kg) were high. The observed absolute oral bioavailability (% F) was 83 ± 14%. Given the good in vitro affinity and acceptable pharmacokinetic properties in both rats and dogs, compound 17v was selected for further profiling. Rat in-vitro radioligand binding H3R Ki values for compound 17v was 9.8 nM which is close to hH3R Ki of 8.73 nM suggesting no interspecies variation in binding affinity. Compound 17v on evaluation for its selectivity towards other receptors, showed

minimal binding against over seventy target sites including

neurotransmitter related receptors, enzymes, brain/gut peptides, growth factors/hormones, ion channels, steroids, immunological factors, second messengers and prostaglandins (Caliper life sciences, data on file). Compound 17v is characterized as dihydrochloride salt which was picked up from over 10 different salts based on its better physico-chemical properties. The dihydrochloride salt is nonhygroscopic and crystalline solid (observed under cross polarized light and crystalline compounds showed birefringence) with high water solubility (730 mg/mL).


Page 23 of 51 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


In vitro intestinal bi-directional permeability and P-gp substrate liability of compound 17v was tested (at 10 µM) in presence and absence of P-gp inhibitor Verapamil using Caco-2 cell lines. Compound 17v exhibited similar permeability in basolateral to apical (B - A) direction compared with the apical to basolateral (A - B) direction. The similar permeability in both absorptive and secretory directions in Caco-2 cells with a Papp: 30.8 x 10-6 cm/sec suggests that this compound has high permeability and is not a substrate for the efflux transporter protein. Based on the high solubility and high permeability compound 17v is considered to be a BCS Class I compound. The extent of protein binding was determined in fresh plasma obtained from rat, dog and human using Rapid Equilibrium Dialysis (RED) at a concentration of 1.0 µM. The unbound fraction of compound 17v was found to be 37, 52, and 70% in rat, dog, and human plasma respectively. The unbound fraction in rat brain homogenate, an important property for CNS compounds, was also high (57%) and comparable with that of unbound fraction found in rat plasma. To understand the in vitro metabolism of compound 17v, the metabolic stability was determined in various test systems like liver microsomes (rat, dog, monkey and human) and hepatocytes (rat, dog and human). Moderate (rat, 27%; dog, 13%; monkey, 35%) to low (human, 10 µM) in pooled human liver microsomes using specific CYP isoform marker activities, indicating no interaction potential as perpetrator for compound 17v. Compound 17v was evaluated in [35S]-GTPγS binding assay29 using CHO-K1 cells expressing human H3R membranes at Eurofins Panlabs Taiwan Ltd, Peitou, Taipei, Republic of China (Taiwan). Compound 17v exhibited an IC50 of 20 nM with progressive inhibition of (R)-α-



methylhistamine (0.03 µM) induced agonist activity below the basal levels of agonism which is a characteristic feature of an inverse agonist. Thioperamide, a reported H3R inverse agonist used as positive control exhibited an IC50 of 140 nM with inhibition slightly below the basal, at its maximum tested concentration of 1 µM.

125 % R- -Methyl histamine max

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 24 of 51

Thioperamide

100

Compound 17v

75 50 25 Basal activity

0 -25 -50 -75 10 -9

10 -8

10 -7 10 -6 Concentration [M]

10 -5

Figure 2. Assessment of compound 17v functional activity at human H3R. The positive control thioperamide and compound 17v were tested in duplicates at human H3R using [35S]-GTPγS binding assay.

Compound 17v was further evaluated for its functional efficacy in (R)-α-methylhistamine (RAMH) induced dipsogenia. RAMH is a selective H3 agonist which induces water drinking in the rat when administered either peripherally or centrally. Blocking of this effect by H3R antagonists is used as an in vivo surrogate measure of H3R functional activity.30 Compound 17v, at doses of 0.3, 0.5, 0.7, 1, 3 and 10 mg/kg, p.o., reversed (R)-α-methylhistamine induced dipsogenia (Figure 3) demonstrating it to be a H3R functional antagonist. The efficacy is consistent with in-vitro potency and high CNS penetration of compound 17v. Pitolisant at the


Page 25 of 51

tested doses reversed (R)-α-methylhistamine induced dipsogenia (Figure 3) consistent with the effects of 17v. Vehicle 1 mL/kg, p.o.+ Vehicle 1 mL/kg, s.c. Vehicle 1 mL/kg, p.o.+ R--Methyl histamine 2.5 mg/kg, s.c. Compound 17v 0.3 mg/kg, p.o + R-Methyl histamine 2.5 mg/kg, s.c. Compound 17v 0.5 mg/kg, p.o + R-Methyl histamine 2.5 mg/kg, s.c. Compound 17v 0.7 mg/kg, p.o + R-Methyl histamine 2.5 mg/kg, s.c. Compound 17v 1 mg/kg, p.o + R-Methyl histamine 2.5 mg/kg, s.c. Compound 17v 3 mg/kg, p.o + R-Methyl histamine 2.5 mg/kg, s.c. Compound 17v 10 mg/kg, p.o + R-Methyl histamine 2.5 mg/kg, s.c. Pitolisant 1 mg/kg, p.o + R-Methyl histamine 2.5 mg/kg, s.c. Pitolisant 3 mg/kg, p.o + R-Methyl histamine 2.5 mg/kg, s.c. Pitolisant 10 mg/kg, p.o + R-Methyl histamine 2.5 mg/kg, s.c. 7.5 Water intake (mL)

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


5.0

2.5

0.0

** ****

****

* ****

**** ****

Treatment

Figure 3. Blockade of (R)-α-methylhistamine induced water drinking by compound 17v and Pitolisant in rats. Data represents mean ± SEM of water intake. *p

acetamide Dihydrochloride - ACS Publications - American Chemical

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