Discovery and Development of 1-[(2-Bromophenyl)sulfonyl]-5-methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole Dimesylate Monohydrate (SUVN-502): A Novel, Potent, Selective and Orally Active Serotonin 6 (5-HT6) Receptor Antagonist for Potential Treatment of Alzheimer’s Disease

Article

pubs.acs.org/jmc

Discovery and Development of 1‑[(2-Bromophenyl)sulfonyl]-5-

methoxy-3-[(4-methyl-1-piperazinyl)methyl]‑1H‑indole Dimesylate

Monohydrate (SUVN-502): A Novel, Potent, Selective and Orally

Active Serotonin 6 (5-HT₆) Receptor Antagonist for Potential

Treatment of Alzheimer’s Disease

Ramakrishna Nirogi,* Anil Shinde, Rama Sastry Kambhampati, Abdul Rasheed Mohammed,

Sangram Keshari Saraf, Rajesh kumar Badange, Thrinath Reddy Bandyala, Venugopalarao Bhatta,

Kumar Bojja, Veena Reballi, Ramkumar Subramanian, Vijay Benade, Raghava Choudary Palacharla,

Gopinadh Bhyrapuneni, Pradeep Jayarajan, Vinod Goyal, and Venkat Jasti

Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road-5, Avenue-7, Banjara Hills, Hyderabad 500 034, India

S

* Supporting Information

ABSTRACT: Optimization of a novel series of 3-(piperazi-

nylmethyl) indole derivatives as 5-hydroxytryptamine-6

receptor (5-HT₆R) antagonists resulted in identiﬁcation of 1-

[(2-bromophenyl)sulfonyl]-5-methoxy-3-[(4-methyl-1-

piperazinyl)methyl]-1H-indole dimesylate monohydrate (5al,

SUVN-502) as a clinical candidate for potential treatment of

cognitive disorders. It has high aﬃnity at human 5-HT₆R (K_i=

2.04 nM) and selectivity over 100 target sites which include

receptors, enzymes, peptides, growth factors, ion channels, steroids, immunological factors, second messengers, and

prostaglandins. It has high selectivity over 5-HT_2Areceptor. It is orally bioavailable and brain penetrant with robust preclinical

eﬃcacy. The combination of 5al, donepezil, and memantine (triple combination) produces synergistic eﬀects in extracellular

levels of acetylcholine in the ventral hippocampus. Preclinical eﬃcacy in triple combination and high selectivity over 5-HT_2A

receptors are the diﬀerentiating features which culminated in selection of 5al for further development. The Phase-1 evaluation of

safety and pharmacokinetics has been completed, allowing for the initiation of a Phase-2 proof of concept study.

cholinergic receptors and promote memory function. Although

AChEIs can temporarily delay the progression of cognitive

decline in AD, their eﬀects are modest. ACh being present in

both the central and peripheral nervous systems, AChEIs

produce several undesirable side eﬀects, such as gastrointestinal

disturbances, bradycardia, and excessive salivation, which are

associated with peripheral cholinergic receptors.³The other

issues with this class of drugs are that they are poorly tolerated,

and their eﬃcacy is not sustained and requires constant dose-

titration as the disease progresses. This leads to signiﬁcant

patient noncompliance.^4,5The incidence and severity of these

side eﬀects becomes augmented with an increase in dose, and in

general, they are more pronounced at the initiation of the

treatment or after a dose increase.

INTRODUCTION

■

Alzheimer’s disease (AD) is a progressive and fatal neuro-

degenerative disorder manifested by cognitive and memory

deterioration in addition to progressive impairment of activities

of daily living. The current global population with dementia is

rising rapidly. There is no cure for AD to date, and it worsens as

the disease progresses, eventually leading to death. The disease

advances in four stages with a progressive pattern of cognitive

and functional impairments: predementia, early dementia,

moderate AD, and advanced stage of AD.¹The cause for

most Alzheimer’s cases is still not known, with several

competing hypotheses, such as the cholinergic hypothesis,

amyloid hypothesis, and tau hypothesis, attempting to explain

it.²

The glutamatergic system is also involved in learning and

memory, which is a target for AD treatment.⁶It is known that

glutamate neurons have synaptic connections on cholinergic

neurons in brain areas associated with learning and memory.

Activation of glutamatergic NMDA receptors has been shown

The current list of approved cognitive enhancing drugs for

AD is not long and has historically been focused on

acetylcholinesterase inhibitors (AChEIs) such as donepezil,

rivastigmine, and galantamine and N-methyl-^D-aspartate

(NMDA) receptor antagonists, namely memantine. AChEIs

act by inhibiting the hydrolysis of acetylcholine (ACh) into

acetate and choline by targeting acetylcholinesterase enzyme.

Increasing the ACh levels in the synapse can stimulate

Received: November 11, 2016

A

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Chart 1. Selected 5-HT₆R Antagonist Clinical Candidates

Figure 1. Genesis of ligands.

to stimulate the synaptic release of ACh. Memantine, which

acts on the glutamatergic system by inhibiting the NMDA

receptor under conditions of excess stimulation, is another

approved treatment for Alzheimer’s disease. It may act to

protect glutamate neurons from excessive glutamate stimula-

tion, while increasing the signal-to-noise ratio. Memantine has

been shown to be moderately eﬃcacious in the treatment of

moderate to severe Alzheimer’s disease. The eﬀects of

memantine in the initial stages of AD are unknown, and it

does not alter the cognitive ability or delay the onset of AD.⁷

Hence, given the limitations of the currently approved

therapies, there is an unmet need of alternate therapy for

treating cognitive disorders. The recent clinical trial failures of

the disease modifying therapies in AD have prompted

researchers to shift their focus to symptomatic treatment, and

the 5-HT₆receptor (5-HT₆R) is emerging as a promising

alternative target.

The 5-HT₆R is a member of the GPCR family and is almost

exclusively expressed in the central nervous system, particularly

in areas relevant to cognition, such as the hippocampus and

frontal cortex.⁸Because of this central location, it is believed

that modulators of 5-HT₆R would have limited peripheral side

eﬀects, including the ones which are commonly associated with

AChEIs. The published literature described the signiﬁcant

developments in the ﬁeld of medicinal chemistry and

pharmacology of 5-HT₆R antagonists, with particular emphasis

on their potential application as both cognitive enhancers and

antiobesity drugs.⁹⁻²⁰Antagonism of this receptor by several

investigational compounds has been shown to improve learning

and memory in animal models.^15,16In addition, studies have

shown that blockade of 5-HT₆R function increases cholinergic

and glutamatergic neurotransmission.²¹⁻²³Thus, it is possible

that the cognitive eﬀects of 5-HT₆R antagonists could be the

consequence of interactions between cholinergic and gluta-

matergic systems involved in learning and memory.

Several investigators have looked at the possible advantage of

combining memantine with AChEI in AD.²⁴⁻²⁶Cotreatment of

memantine and AChEIs in triple-transgenic mice resulted in

signiﬁcant memory improvement when compared to either of

the treatments.^27,28Similar to the observation made in

transgenic mice in the preclinical experiments, cotreatment of

memantine and donepezil showed beneﬁcial eﬀects on memory

in AD patients.²⁹This combination of donepezil and

memantine is currently approved for the treatment of moderate

to severe AD. Since blocking 5-HT₆R modulates both

cholinergic and glutamatergic activity, one might expect 5-

HT₆R antagonists, in combination with both donepezil and

memantine, to complement and/or augment the eﬀects of

combined donepezil and memantine.

A 5-HT₆R antagonist Idalopirdine³⁰(K_i= 0.83 nM), from

Lundbeck, showed statistically signiﬁcant improvement as an

add-on therapy to donepezil versus donepezil alone, on

cognitive symptoms of AD patients in a Phase-2 study.

Intepirdine,³¹a 5-HT₆R antagonist (K_i= 0.23 nM), from

Axovant, is currently in Phase-3 clinical trials as an add-on

therapy to donepezil. SAM-531³²(K_i= 1.3 nM), is one of the

ﬁrst reported clinical candidates from Pﬁzer (Chart 1).

Genesis of Ligand. Our eﬀorts toward the preclinical

characterization and clinical development of 5-HT₆R antago-

nists have been ongoing for quite some time.³³⁻³⁷The N-aryl

sulfonyl tryptamine, 1-benzenesulfonyl-5-methoxy-N,N-dime-

thyltryptamine, MS-245 (2, K_i= 2.3 nM, Figure 1),³⁸⁻⁴⁰an

analog of endogenous ligand 5-hydroxytryptamine (5-HT, 1),

was used as a starting point for our research. In our earlier

B

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a

Scheme 1

a

Conditions: (a) aq formaldehyde, acetic acid, methanol, 5−10 °C to r.t., 6 h; (b) (i) KOH, THF, 10 °C to r.t., 4 h; (ii) CH₃SO₃H, ethanol, r.t., 3−

24 h; (iii) methanolic HCl, MeOH, r.t., 3 h; (c) (i) HBr in acetic acid, 80 °C, 12 h; (ii) methanolic HCl, MeOH, r.t., 2 h; (d) (i) CH₃SO₃H, acetone,

reﬂux, 3 h; (ii) methanolic HCl, MeOH, r.t., 3 h; (e) benzoyl or acetyl chloride, Et₃N, DCM, r.t., 2 h; (f) benzyl bromide or 1-bromo-2-ﬂuoroethane,

K₂CO₃, DMF, 100 °C, 6 h.

eﬀorts, the N,N-dimethylamino ethyl side chain at the C3

position of indole in compound 2 was rigidized into

pyrrolidine, which resulted in identiﬁcation of a series of

novel, potent, selective, and brain penetrant 5-HT₆R antago-

nists 3,³³as exempliﬁed by (R,S)-1-(2-bromobenzenesulfonyl)-

3-(1-methylpyrrolidin-3-yl)-5-methoxy-1H-indole (K_b= 0.13

nM). As the latter series of compounds were chiral and

required chiral resolutions, the pyrrolidine ring of 3 was further

expanded into a six-membered ring with the introduction of

another nitrogen atom, which resulted in substituted piperazine

compounds 4³⁴that were potent, selective, and brain penetrant

5-HT₆R antagonists, as exempliﬁed by 1-(4-isopropylbenzene-

sulfonyl)-3-(4-methylpiperazin-1-yl)-1H-indole (K_i= 3.4 nM).

In this chemical scaﬀold, the piperazine ring was directly

attached to the C3 of the indole nucleus. In the current

research, a linker in the form of a methylene spacer was

introduced between the piperazine ring and the indole nucleus

to further expand the chemical diversity within this scaﬀold and

to provide space for more ﬂexibility (to mimic serotonin

structure). The benzenesulfonyl group, having both electron

donating and withdrawing substituents, was evaluated to study

the SAR. In the next set of modiﬁcations, the indole central ring

was replaced with diﬀerent halo, alkyl, and alkoxy substituted

indole nuclei. After zeroing in on the indole central ring with

optimal substituents, the basic 6-membered piperazine was

expanded to homopiperazine and the substituents on the

C

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a

Scheme 2

a

Conditions: (a) 9a/9b/9i, KOH, THF, 10 °C to r.t., 4 h; (b) NaBH₄, MeOH, 3 h; (c) SOCl₂, DMF (cat.), DCM, −5 °C to r.t.; (d) (i) 7a/7d,

Et₃N, DCM, −5 °C - r.t., 6 h; (ii) methanolic HCl, MeOH, r.t.

a

Scheme 3

a

Conditions: (a) TBTU, DIPEA, DMF, r.t., 6 h; (b) LiAlH₄, THF, reﬂux, 4 h; (c) (i) 9b/9c, KOH, THF, 10−20 °C to r.t., 4 h; (ii) methanolic HCl,

MeOH, r.t.

piperazine terminal nitrogen were also explored. Replacement

of N-benzenesulfonamide on the indole with N-benzyl and N-

benzoyl groups was explored. During further iterations, the

central indole ring was replaced with diﬀerent heterocycles,

such as azaindoles and indazole. At the end, the linker length

between the central indole ring and the piperazine was also

explored. Focused eﬀort on optimizing in vitro binding aﬃnity,

selectivity, in vivo eﬃcacy, and safety culminated in the

discovery of 1-[(2-bromophenyl)sulfonyl]-5-methoxy-3-[(4-

methyl-1-piperazinyl)methyl]-1H-indole dimesylate monohy-

drate (SUVN-502, 5al), which forms the main feature of this

paper (Figure 1).

Chemistry. The general synthetic strategy used for the

preparation of title compounds 5, 11, 12, and 13 has been

summarized in Scheme 1. Various substituted indoles (6a−o)

were either procured commercially or prepared by reported

Fischer indole synthesis,⁴¹or the Leimgruber−Batcho

method.⁴²The key intermediates (8a−s) were synthesized by

the well-known Mannich reaction.⁴³Substituted piperazines

and homopiperazines (7a−d) were reacted with aqueous

formaldehyde to obtain the Mannich adducts, which were in

situ reacted with indoles (6a−o) to obtain compounds (8a−s).

The key intermediates (8a−s) were then reacted with

commercially procured aryl sulfonyl, benzoyl, or benzyl

chlorides (9a−y) to aﬀord compounds 5 (5a−ax and 5bb−

bn). The 5-methoxy indole compounds 5al, 5am, and 5an were

treated with HBr in acetic acid to obtain the corresponding O-

desmethyl compounds 5ay, 5az, and 5ba, respectively.

Deprotection of compounds (10a−e) with either methanolic

HCl or CH₃SO₃H gave the targeted compounds (11a−e).

Compound 11b on reaction with acetyl chloride or benzoyl

chloride aﬀorded compounds 12a and 12b, respectively. The

reaction of 11b with benzyl bromide gave compound 13a,

D

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a

Scheme 4

a

Conditions: (a) 7a/7c, CH₃CO₂H, NaBH(OAc)₃, 1,2-DCE, r.t., 6 h; (b) (i) 9a/9b, KOH, THF, 10 °C to r.t., 4 h; (ii) methanolic HCl, MeOH, r.t.

whereas reaction of 11c with 2-ﬂuoroethyl bromide yielded

compound 13b.

electron withdrawing triﬂuoromethoxy (5m) or a cyano group

(5n) showed decreased potency. The dihalo substituted

compounds, viz., 5o (R³= 2′,4′-diﬂuoro), 5p (R³= 3′,4′-

diﬂuoro), 5q (R³= 2′,6′-diﬂuoro), 5r (R³= 2′,4′-dichloro), 5s

(R³= 2′,6′-dichloro), and 5t (R³= 2′-chloro-4′-ﬂuoro), were

found to be 10−60-fold less potent as compared to monohalo

substituted 5d (R³= 4′-Br), which was the most potent

compound among monohalo substituted derivatives. The 2′,5′-

dimethoxy (5u) and 2′,5′-dimethyl (5v) substituted com-

pounds also showed weaker binding aﬃnity. From the above

results, it can be concluded that the disubstituted aryl

sulfonamide ring is poorly tolerated. Interestingly, the

introduction of a bulky naphthyl group in place of a substituted

phenyl group resulted in a potent compound (5w).

Alternately, some of compound 5 was synthesized as given in

Scheme 2. Commercially procured indole-3-carboxaldehydes

14a and 14b were reacted with sulfonyl chlorides 9a and 9b to

obtain compounds 15a and 15b, respectively, whereas indole-3-

carboxaldehyde 14c on reaction with sulfonyl chlorides 9b and

9i gave compounds 15c and 15d, respectively. Reduction of

compounds 15a−d with NaBH₄followed by treatment with

thionyl chloride gave intermediates 17a−d. Intermediates 17a

and 17b on reaction with 7a yielded compounds 5a and 5ae,

while compounds 17c and 17d on reaction with 7d gave 5bf

and 5bg, respectively.

Compounds 21a and 21b, with an increase in spacer length,

were synthesized as shown in Scheme 3. Compound 18 was

treated with 7a under acid−amine coupling conditions to

obtain carboxamide intermediate 19, which on reduction with

LiAlH₄in THF gave intermediate 20. The intermediate 20 on

further treatment with sulfonyl chlorides 9b or 9c gave

compounds 21a and 21b respectively.

Compounds 5bo, 5bp, and 11f were synthesized as given in

Scheme 4. Intermediates 22a−c were commercially procured.

The reaction of 22a with 7c under reductive amination

conditions gave compound 23a whereas reaction of 22b and

22c with 7a gave intermediates 23b and 23c, respectively. The

reaction of 23a with sulfonyl chloride 9b followed by treatment

with methanolic HCl gave compound 11f, while the reaction of

23b and 23c with sulfonyl chlorides 9a or 9b yielded

compounds 5bo and 5bp, respectively.

With this initial understanding, focus was shifted to the

indole central core while retaining some of the best

substitutions on the aryl sulfonamide ring. Compound 5x

with chloro substitution at the C4 position of the indole (R¹=

4-Cl, R³= 4′-ⁱPr) maintained a similar binding aﬃnity as that of

compound 5i (R¹= H, R³= 4′-ⁱPr). Compounds 5y (R¹= 4-Cl,

R³= 4′-F) and 5z (R¹= 4-Cl, R³= 2′-Br) exhibited a 2-fold

decrease in binding aﬃnity compared to compounds 5h (R¹=

H, R³= 4′-F) and 5b (R¹= H, R³= 2′-Br), respectively. Chloro

substitution at the ﬁfth or sixth position of indole resulted in

compounds 5aa (R¹= 5-Cl, R³= 4′-F), 5ab (R¹= 6-Cl, R³=

H), and 5ac (R¹= 6-Cl, R³= 4′-Br) while compounds 5ad, 5ae,

5af, 5ag, and 5ah had ﬂuoro or bromo substitution at the ﬁfth

position (R¹= 5-F/Br) and a bromo/ⁱPr group at the 4′-

position (R³= F/Br/ⁱPr). Most of these halo substituted

analogs were 2−30-fold less potent than their unsubstituted

counterparts. Among 5-halo substitutions, the 5-ﬂuoro

substituted analogs 5ad, 5ae, and 5af showed better potency,

indicating that the ﬂuoro substitution is optimal compared to

chloro or bromo substitution. Next, we examined the eﬀect of

electron donating groups such as alkoxy substitution on the

indole nucleus. The methoxy substitution at the fourth position

of indole (R¹= 4-OCH₃) resulted in compounds 5ai−5ak,

which are 2−3-fold less potent than their unsubstituted indole

counterparts. Alkoxy substitutions at the ﬁfth position of indole

resulted in compounds 5al−5av with 5-HT₆R binding values

ranging between 1 and 100 nM. Among all the 5-OCH₃

substituted indole derivatives, compounds 5al (R³= 2′-Br),

5am (R³= 4′-ⁱPr), 5an (R³= 4′-F), and 5ao (R³= H) are the

most potent. The other compounds, viz., 5ap, 5aq, 5ar, and 5as

showed modest to good potency. Compound 5at, bearing a

bulky electron withdrawing substitution (R³= -OCF₃),

displayed ∼9-fold lower binding aﬃnity compared to

compound 5ao (R³= H). The compounds with bulky alkoxy

groups at the ﬁfth position, as in compounds 5au (R¹= 5-OⁱPr)

and 5av (R¹= 5-OCH₂CF₃), showed reduced aﬃnity,

RESULTS AND DISCUSSION

■

The in vitro 5-HT₆R binding assay of all ﬁnal compounds was

carried out by radioligand binding assay (Table 1). The SAR

was initiated with the synthesis of N₁-benzenesulfonyl-3-

(piperazinylmethyl) indole (5a, R³= H), which showed potent

in vitro binding aﬃnity toward 5-HT₆R. Several 5a analogs were

synthesized by introducing diverse substitutions, such as halo,

alkyl, alkoxy, and cyano groups on the aryl sulfonamide ring of

5a. Insertion of diﬀerent halogen groups at the ortho, meta, and

para positions of the aryl sulfonamide ring resulted in

compounds 5b−5h. Compounds 5b, 5d, 5f, and 5h with

halogen substitution at the ortho or para position showed

potent in vitro aﬃnity compared to compounds 5c, 5e, and 5g,

having halogen at the meta position.

The alkyl substituted compounds 5i (R³= 4-ⁱPr) and 5j (R³

= 4-CH₃) were found to be equipotent with that of parent

compound 5a (R³= H), indicating that the insertion of alkyl

substitution is well tolerated. Compounds with an electron

donating methoxy group at the third or fourth position showed

moderate aﬃnity (5k and 5l), while compounds with an

E

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Table 1. In Vitro Binding Data at 5-HT₆R for Compounds 5, 8a, 11, 12, 13, and 21

a

Compd. No.

R¹

R²

CH₃

R³

X

m

n

K_i(nM)

5a

H

SO₂

1

6.0 0.3

10.2 1.1

19.0 2.3

2.13 0.1

16.3 1.9

7.8 0.4

58 6.4

5b

H

CH₃

2′-Br

3′-Br

4′-Br

3′-Cl

2′-F

3′-F

5c

H

5d

H

5e

H

5f

H

5g

H

5h

H

4′-F

8.76 0.8

6.0 0.2

9.6 0.9

29.4 3.9

25.1 3.5

150 18

90.5 11.6

30 3.7

5i

H

4′-ⁱPr

5j

H

4′-CH₃

5k

H

3′-OCH₃

4′-OCH₃

4′-OCF₃

4′-CN

2′,4′- difluoro

3′,4′- difluoro

2′,6′- difluoro

2′,4′- dichloro

2′,6′- dichloro

2′-chloro-4′-fluoro

2′,5′-dimethoxy

2′,5′-dimethyl

5l

H

5m

5n

H

5o

H

5p

H

119 16.1

64 8.9

5q

H

5r

H

28.2 3.4

100 16.1

29.0 4.3

91 13.5

64 8.9

5s

H

5t

H

5u

H

5v

H

5w

5x

H

R³together with benzene ring is 1-naphthyl

11.5 1.4

9.2 0.9

31.2 3.9

23.5 3.3

17.5 1.9

35.7 4.1

59.7 7.8

4.0 0.3

12.0 1.8

9.2 0.9

17.0 1.8

27.6 2.9

11.6 1.6

14.3 1.8

18.4 1.9

2.0 0.2

1.63 0.2

5.0 0.6

4.3 0.6

10.3 0.9

7.8 0.9

15.5 1.9

18.3 2.1

37.8 3.7

36.3 4.1

96.3 8.9

36.1 4.7

239 39.4

18.7 1.9

50 5.1

4-Cl

5-Cl

6-Cl

5-F

5-Br

4′-ⁱPr

4′-F

2′-Br

4′-F

5y

5z

5aa

5ab

5ac

5ad

5ae

5af

5ag

5ah

5ai

5aj

5ak

5al

5am

5an

5ao

5ap

5aq

5ar

5as

5at

5au

5av

5aw

5ax

5ay

5az

5ba

5bb

H

4′-Br

4′-ⁱPr

2′-Br

4′-Br

4′-F

4′-ⁱPr

4-OCH₃

5-OCH₃

5-OⁱPr

4′-ⁱPr

2′-Br

4′-F

2′-Br

4′-ⁱPr

4′-F

H

4′-Br

R³together with benzene ring is 1-naphthyl

4′-CH₃

4′-OCH₃

4′-OCF₃

2′-Br

5-OCH₂CF₃

6-OCH₃

5-OH

4′-Br

4′-ⁱPr

4′-F

2′-Br

5-OH

4′-ⁱPr

4′-F

5-OH

34.9 4.7

200 37.1

5-NO₂

2′-Br

F

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Table 1. continued

a

Compd. No.

R¹

R²

CH₃

R³

X

m

n

K_i(nM)

5bc

5bd

5be

5bf

5-SCH₃

2-CH₃

2-Cl, 5-OCH₃

5-OCH₃

H

2′-Br

4′-F

SO₂

CH₂

CO

1

2

1

2

1

194 35.1

55.6 5.9

29.6 3.7

5.7 0.9

2.0 0.1

7.79 0.8

6.7 0.8

1.7 0.1

20 1.1

8.8 0.9

312 28

326 44

482 49

7.5 1.1

4.5 0.9

1.3 0.1

5.7 0.2

6.9 0.8

365 46

385 51

133 15

348 36

398 59

289 26

CH₃

C₂H₅

CH₃

H

4′-ⁱPr

2′-Br

4′-ⁱPr

4′-F

5bg

5bh

5bi

2′-Br

4′-ⁱPr

4′-F

5bj

5bk

5bl

H

5bm

5bn

8a

2′-Br

5-F

H

H at N₁position of indole

11a

11b

11c

11d

11e

12a

12b

13a

13b

21a

21b

H

SO₂

H

2′-Br

4′-ⁱPr

4′-F

2′-Br

3′-Br

5-OCH₃

H

COPh

COCH₃

CH₂Ph

CH₂CH₂F

CH₃

H

5-OCH₃

5-Br

a

Displacement of [³H]-LSD binding to cloned human 5-HT₆receptors stably expressed in HEK293 cells. K_ivalues were determined in duplicate,

and the average values as K_iSEM are reported here. The binding assays were carried out at Novascreen Biosciences Corporation, USA, as per their

standard protocol. Compounds 5a, 5h, 5ad, 5ai, 5aj, 5ay, 5az, 5ba, 5bg, 5bh, 11b, 11d, 21a, and 21b are dihydrochloride salts. Compound 12a is

hydrochloride salt. Compounds 5am, 5an, and 11e are dimesylate salts while 5al is a dimesylate monohydrate salt. Compound 11c is a dimesylate

dihydrate salt. Remaining compounds are free bases.

Table 2. In Vitro Binding Data at 5-HT₆R for Compounds 11f, 5bo, and 5bp

a

Compd. No.

R¹

R²

R³

X

Y

Z

K_i(nM)

95

11f

H

2′-Br

H

N

CH

N

CH

N

9

5bo

CH₃

CH

277 23

348 32

5bp

5-OCH₃

CH₃

2′-Br

CH

a

Displacement of [³H]-LSD binding to cloned human 5-HT₆receptors stably expressed in HEK293 cells. K_ivalues were determined in duplicate,

and the average values as K_iSEM are reported here. The binding assays were carried out at Novascreen Biosciences Corporation, USA, as per their

standard protocol. Compound 11f is dihydrochloride salt; 5bo and 5bp are free bases.

indicating the importance of lower alkoxy groups for

maintaining the potent binding aﬃnity. Methoxy substitution

at the sixth position of indole (R¹= 6-OCH₃) was also

explored. It was found that these compounds (5aw and 5ax)

are less potent as compared to compounds with methoxy

substitutions at the fourth or ﬁfth position. Compounds 5ay,

5az, and 5ba, the 5-hydroxy derivatives, are found to be less

potent as compared to their corresponding 5-methoxy

derivatives 5al, 5am, and 5an, respectively. Replacement of

the methoxy substitution at the ﬁfth position of compound 5al

with −NO₂and −SCH₃led to compounds 5bb and 5bc,

respectively, with a signiﬁcant reduction in in vitro aﬃnity.

Insertion of a methyl group at the second position of indole (R¹

= 2-CH₃) gave compound 5bd, with reduced binding aﬃnity as

compared to its unsubstituted counterpart 5h. Compound 5be,

with 2,5-disubstituted indole (R¹= 2-Cl, 5-OCH₃), has shown

lower binding aﬃnity compared to its des-chloro analog 5am.

Among all the alkoxy substituted compounds, compounds with

a methoxy group at the fourth and ﬁfth position of indole have

displayed potent aﬃnity, whereas the compounds with a

methoxy group at the sixth position displayed lower aﬃnity.

Given the potent aﬃnity of 5-methoxy indole derivatives, 5al,

5am, and 5an, the homopiperazine derivatives 5bf, 5bg, and

5bh, respectively, were synthesized to explore the impact of

ring size on aﬃnity. All the three analogs maintained

equipotent aﬃnity, indicating that the ring expansion is also

well tolerated. Compounds 5bi, 5bj, 5bk, and 5bl, which are N-

ethylpiperazine analogues of 5al, 5am, 5an, and 5ao, also

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showed potent binding aﬃnity. Compounds 11a, 11b, 11c,

11d, and 11e, that are N-des-methylpiperazine analogs of 5a,

5b, 5al, 5am, and 5an, respectively, maintained potent binding

aﬃnity, indicating that the terminal secondary amine is well

tolerated. But, the compound with bulky substitution at R²,

such as N-benzylpiperazine derivative (13a) was found to be

less potent. Replacement of N-alkyl substitution of the

piperazine at R²with electron withdrawing groups such as

benzoyl (12a), acetyl (12b), and 2-ﬂuoroethyl (13b) resulted

in reduced binding aﬃnity, indicating that the basic nature of

the terminal nitrogen of the piperazine ring is critical for

aﬃnity.⁴⁴

Replacement of the N₁-phenylsulfonyl group of indole with

hydrogen (8a), substituted benzyl (5bm), or benzoyl (5bn)

gave less potent compounds, conﬁrming the critical role of the

benzenesulfonamide group for aﬃnity. Replacing the indole

central core with 4-azaindole (11f), 7-azaindole (5bo), or

indazole (5bp) resulted in moderately potent compounds

(Table 2), indicating that the indole central core is optimal for

aﬃnity at 5-HT₆R. The linker between the indole central core

and piperazine was increased to two carbons (m = 2) to

investigate the inﬂuence of chain length. Both the synthesized

compounds 21a and 21b are less potent, indicating that one

carbon linker between the indole central core and piperazine is

optimal.

Supporting Information). Compounds 5al and 5an have shown

high selectivity (>1000-fold) over 5-HT_2Areceptor with respect

to their aﬃnity at 5-HT₆R, whereas 5a, 5h, 5ad, 5ai, 5am, and

5bg have shown potent to moderate aﬃnity toward 5-HT_2A

receptor. None of these compounds (5a, 5h, 5ad, 5ai, 5al,

5am, 5an, and 5bg) have any signiﬁcant aﬃnity at 5-HT_1A, 5-

HT_2C, 5-HT_4B, and 5-HT₇receptors.

Compounds 5a, 5h, 5ad, 5ai, 5al, 5am, 5an, and 5bg were

evaluated for cytochrome P450 inhibition potential (CYP 2D6

and CYP 3A4) in pooled human liver microsomes using

speciﬁc CYP isoform marker activities (Table 4). All the tested

a

Table 4. Human CYP450 Inhibition Data

IC₅₀(μM)

Compd. No.

CYP 3A4

CYP 2D6

5a

>10

1.82

1.4

>10

8.8

5h

5ad

5ai

5al

5am

5an

5bg

7.7

7.1

9.7

1.5

13.23

33.49

12.59

1.9

1.85

1.71

2.2

a

The cytochrome P450 inhibition potential was determined in HLM

Several in vitro potent compounds which represent diversity,

such as 5a, 5h, 5ad, 5ai, 5al, 5am, 5an, and 5bg, were further

evaluated for their functional activity at 5-HT₆R using a

reporter gene based assay to ascertain their ability to modulate

5-HT₆R function.^45,46In an in vitro binding assay, a stable CHO

cell line expressing a recombinant human 5-HT₆R and pCRE-

Luc reporter system is used which refers to a nonradioactive-

based approach to determine the functional activity of

compounds. All the above selected compounds have shown

antagonist activity at 5-HT₆R by inhibiting the 5-HT stimulated

cAMP accumulation, as can be seen from the K_band I_maxvalues,

which are in line with values determined by radioligand binding

assay (Table 3).

using isoform speciﬁc marker activities.

compounds moderately inhibited midazolam (probe substrate

for CYP 3A4) hydroxylation, with the exception of 5a, which

showed IC₅₀> 10 μM, whereas, barring 5bg (IC₅₀= 1.9 μM),

all other tested compounds had weak inhibition potential

toward CYP2D6.

Pharmacokinetic proﬁling of compounds 5a, 5h, 5ad, 5ai,

5al, 5am, 5an, and 5bg was carried out in Wistar rats (Table 5).

Compounds 5h, 5ai, 5am, 5an, and 5bg were found to have

lower exposures and higher volume of distribution with poor

oral bioavailability (<20%). Compounds 5a, 5ad, and 5al were

rapidly absorbed into systemic circulation, and showed

moderate clearance, low volume of distribution, high oral

exposures, and adequate oral bioavailability. As the oral

exposures of compounds 5a, 5ad, and 5al were higher as

compared to compounds 5h, 5ai, 5am, 5an, and 5bg, they were

further evaluated. The pharmacokinetic proﬁle of a reference

compound in rats is generated under similar experimental

conditions (Supporting Information).

Table 3. Functional Activity Data at 5-HT₆R

Compd. No.

5a

5h

5ad

5ai

5al

5am

5an

5bg

a

K_b(nM)

7.2

90

12.3

98

14

99

9.8

98

2.6

0.7

8.4

90

5.7

95

I_max(%)

100

a

K_bvalues were determined using a nonradioactive cell based assay

and are the mean of duplicate experiments.

For a compound that acts at the receptor located in the

central nervous system (CNS), suﬃcient drug delivery into the

brain is a prerequisite criterion for drug to show activity.

Compounds 5a, 5ad, and 5al, that displayed adequate oral

exposure in rat pharmacokinetic studies, were evaluated in a

brain penetration study (Table 5). After oral administration at

10 mg/kg p.o. dose in Wistar rats, all the tested compounds

have shown good brain penetration with C_b/C_pratio ∼1.

Given the high oral exposures, good brain penetration

coupled with potent in vitro aﬃnity (both binding and

functional) and preliminary selectivity over serotonin subtype

receptors, compounds 5a, 5ad, and 5al were further evaluated

in the preliminary in vivo eﬃcacy model, time induced object

recognition task (ORT) at 1, 3, and 10 mg/kg p.o. dose (Figure

2). Compound 5al had reversed the time-induced amnesia at all

the tested doses while 5a and 5ad reversed the time-induced

amnesia at higher doses only. With ORT eﬃcacy at all the

tested doses coupled with high selectivity over 5-HT_2A

The 5-HT₆R antagonists which are in clinical development

have shown variable levels of binding aﬃnity toward 5-HT_2A

receptor.^13,47,48Although it has been hypothesized that this

property may have beneﬁcial eﬀects in AD patients,¹³there is

literature precedence suggesting that 5-HT_2Areceptor antago-

nism may have counterproductive eﬀects on cognition in AD

patients. Literature evidence also reports on species diﬀerence

in the aﬃnity of 5-HT_2Aligands (humans vs preclinical

species).⁴⁹Serotonin stimulates soluble amyloid precursor

protein release through activation of the 5-HT_2Areceptor, and

its abundance decreases in AD patient’s brain.^50,51Hence, 5-

HT_2Aantagonism may not be a desired feature for the

treatment of AD. Compounds 5a, 5h, 5ad, 5ai, 5al, 5am, 5an,

and 5bg were proﬁled for their selectivity in our in-house

selectivity panel of closely related serotonin subtypes such as 5-

HT_1A, 5-HT_2A, 5-HT_2C, 5-HT_4B, and 5-HT₇(refer to the

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a

Table 5. Pharmacokinetic Proﬁle of Compounds 5a, 5h, 5ad, 5ai, 5al, 5am, 5an, and 5bg in Wistar Rats

Compd. No.

5a

5h

5ad

5ai

5al

5am

5an

5bg

Dose (mg/kg) p.o.

C_max(ng/mL)

AUC_t(ng·h/mL)

F (%)

10

22

1225 379

2221 1019

21.39

125.4 18.5

221 55

17.5

1895 1117

7743 5166

91.4

45.6 16.2

138 52

10.7

272.3 127.9

784 326

24.9

56.4 21.7

151 76

4.9

55 34

249 123

13.5

5

157 46

4.0

Dose (mg/kg) i.v.

t_1/2(h)

10

0.89 0.06

1.2 0.3

1.50 0.65

6.3 0.9

1.93 0.84

3.2 1.3

19.5 2.8

0.95

2.12 0.22

6.1 1.1

0.95 0.17

4.41 1.16

53 4.5

1.48

1.12 0.15

5.3 1.3

2.52 0.88

19.2 2.9

92 16

−

5.70 0.8

49.5 6.5

101.1 10.4

−

Vz (L/kg)

CL (mL/min/kg)

14.9

0.82

2

75

6

65

5

54

7

C_brain/C_plasma

−

a

Fasted male Wistar rats. Vehicle used is water for injection for both oral and i.v. routes. Dosing volumes: 10 mL/kg for p.o. and 2 mL/kg for i.v.; −

means not tested.

Figure 2. Eﬀect of compounds 5al, 5a, and 5ad in time induced object recognition task. *p < 0.05, **p < 0.01 vs familiar object (Paired t-test), n =

7−12/group, dosing (p.o.); 60 min prior to test. Vehicle- W.F.I 1 mL/kg, p.o.

receptor, compound 5al was selected for detailed preclinical

ADME, eﬃcacy (animal models of cognition and neuro-

chemistry) and detailed safety proﬁling. Part of the structural

similarity of compound 5al with 2, which is essentially a

sulfonamide mimic of the endogenous ligand 1, also has favored

the selection of 5al. A reference 5-HT₆R antagonist compound

showed activity at 1 and 3 mg/kg s.c. dose (Supporting

Information).

To identify any other oﬀ-target activities, compound 5al was

evaluated at Novascreen in their commercial selectivity panel of

over 100 target sites including receptors (64), enzymes (5),

peptides (18), growth factors (5), ion channels (6), steroids,

immunological factors, second messengers, and prostaglandins

(Supporting Information). Compound 5al at 1 μM showed

<50% inhibition at all the tested receptors, barring dopamine

D₃, and adrenergic alpha 2A and 2C. In an independent

experiment, 5al showed a K_ivalue of 616 nM at dopamine D₃

and 2570 nM adrenergic alpha 2A receptor. At alpha 2C

receptor, the functional aﬃnity (K_b) value was 619 nM. The

lead candidate, 5al, has shown no signiﬁcant binding toward the

hERG potassium channel when tested in an astemizole binding

assay, and no changes in the QTc intervals were noticed in

human Phase-1 clinical trials.

found to be 99.0, 98.6, and 98.0% in rat, dog, and human,

respectively.

The pharmacokinetic study of 5al was carried out in

nonrodent species, Beagle dogs (at 10 mg/kg p.o. and 3 mg/

kg i.v. dose). It was well absorbed into systemic circulation with

high oral exposures (AUC_0‑t1356 1067 ng.h/mL), favorable

terminal half-life 3.5 h, and high intravenous clearance (46.4

10.9 mL/min/kg). The volume of distribution was 7.5 L/kg,

indicating that 5al was widely distributed in tissues. Absolute

oral bioavailability was found to be 35%.

The CYP3A4 inhibition for compound 5al was assessed with

testosterone, another probe substrate. It inhibited the

testosterone hydroxylation with an IC₅₀value of 2.64 μM.

Compound 5al was also evaluated for the inhibitory potential

against other CYP isoforms, CYP1A2, 2A6, 2B6, 2C8, 2C9,

2C19, and 2E1, in pooled human liver microsomes using

speciﬁc CYP isoform marker activities. It had moderately

inhibited mephenytoin hydroxylation (2C19) with IC₅₀values

of 5.7 μM and did not inhibit the other CYP450 enzymes

signiﬁcantly (IC₅₀> 10 μM). Also the CYP induction potential

of 5al was evaluated in human plateable hepatocytes using both

mRNA expression and enzyme activities as end points. The

results indicated that 5al did not induce CYP1A2, CYP2B6, and

CYP3A4 at tested concentrations of 0.01, 0.1, and 1.0 μM.

Compound 5al was evaluated in additional animal models of

cognition and neurochemistry. It reversed the scopolamine

induced emotional memory deﬁcits in a contextual fear

conditioning task at 1, 3, and 10 mg/kg p.o. dose (Figure 3).

5-HT₆R antagonists are implicated in the modulation of

cholinergic function and play a vital role in the treatment of

The permeability of 5al was evaluated in the Caco-2

intestinal epithelial cell line, where it showed high permeability

(P_app= 19.6 × 10⁻⁶cm/s). The eﬄux ratio [Papp (B - A)/Papp

(A - B)] was less than 2, indicating minimal interaction with

eﬄux transporters such as P-gp. The extent of protein binding

for 5al was determined in rat, dog, and human plasma using

equilibrium dialysis. The plasma protein binding of 5al was

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Treatment with donepezil (1 mg/kg s.c.) and memantine (1

mg/kg s.c.) combination produced an increase in hippocampal

acetylcholine levels, which reached to a maximum of 1203

106% of basal levels. Compound 5al, in combination with

donepezil and memantine, produced a mean maximum increase

of 1383 194% and 2136 288% after 1 and 3 mg/kg p.o.,

respectively. An increase in acetylcholine produced by the

combination of 5al (1 and 3 mg/kg p.o.), donepezil, and

memantine was signiﬁcantly higher than the increase produced

by a donepezil and memantine combination. This increase of

acetylcholine in the cotreatment group was devoid of any

cholinergic side eﬀects. In a separate study involving

pharmacokinetic evaluation, a nonsigniﬁcant diﬀerence in the

plasma exposures of donepezil, memantine, or compound 5al

was observed when administered either alone or in triple

combination, thus ruling out the possibility of a pharmacoki-

netic interaction mediated eﬀect (data on ﬁle). The

aforementioned results from the preclinical study provide the

support for the potential therapeutic utility of 5-HT₆R

antagonist 5al in combination with donepezil and memantine

for the treatment of cognitive disorders.

Compound 5al is nonmutagenic in all four strains of

Salmonella typhimurium, viz., TA1537, TA1535, TA98, and

TA100, and Escherichia coli WP2 uvrA both in the absence and

presence of metabolic activation. It is nonclastogenic in in vitro

chromosomal aberration tests in human peripheral blood

lymphocytes.

To support the increasing demand of 5al for clinical studies,

a suitable four stage scale up process of 5al was developed at

the ∼75 kg level⁵³starting from commercially available building

blocks. Compound 5al, a dimesylate monohydrate salt, is

crystalline in nature with high water solubility (∼1200 mg/

mL), and it falls under the BCS Class I category.

Figure 3. Eﬀect of 5al on scopolamine induced memory deﬁcits in

contextual fear conditioning. Data represents mean SEM of duration

of freezing. N = 9/group. **p < 0.01, *p < 0.05 vs scopolamine

treatment.

learning and memory disorders.⁵²Thus, the modulation of

acetylcholine in the ventral hippocampus by 5al was evaluated

in brain microdialysis studies at 1 and 3 mg/kg p.o. Basal

acetylcholine levels in all treatment groups were non-

signiﬁcantly diﬀerent and per oral administration of vehicle

produced no change in hippocampal acetylcholine levels.

Treatment with 5al produced a signiﬁcant increase in

acetylcholine levels in hippocampus at 3 mg/kg p.o., whereas

no signiﬁcant eﬀect was observed at subtherapeutic dose of 1

mg/kg p.o. (Figure 4).

Having seen the increase in acetylcholine levels upon

standalone treatment, the eﬀects of therapeutic (3 mg/kg

p.o.) and subtherapeutic (1 mg/kg p.o.) doses of 5al were

evaluated in combination with donepezil (1 mg/kg s.c.) and

memantine (1 mg/kg s.c.) for modulation of acetylcholine in

the ventral hippocampus of male Wistar rats (Figure 5). This

study design is based on the premise that this combination may

result in improved therapeutic eﬃcacy and also oﬀer several

therapeutic beneﬁts, for instance, reduction in the doses of

therapeutic agents, which may evade the side eﬀects associated

with dosing of higher amounts. If the hypothesis is correct,

these scenarios may oﬀer a novel mechanism with improved

drug eﬃcacy and tolerability compared to the existing approved

therapeutic options.

In Phase-1 human clinical trial studies,⁵⁴compound 5al

showed a favorable safety and pharmacokinetic proﬁle in a

single ascending dose in healthy male and female subjects and

multiple ascending doses for 14 days in healthy elderly male

subjects. Gender and food did not have a signiﬁcant eﬀect on

the pharmacokinetics of compound 5al. It was well tolerated in

humans with adequate plasma exposures for eﬃcacy and

favorable half-life suitable for once a day treatment. A Phase-2

proof of concept study (ClinicalTrials.gov Identiﬁer:

NCT02580305) is ongoing for evaluating the safety and

eﬃcacy of 5al in subjects with moderate AD, receiving stable

Figure 4. Eﬀect of per oral administration of 5al on acetylcholine modulation in the ventral hippocampus of male Wistar rats. Arrow indicates the

point of treatment. Data expressed as mean SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs vehicle (Bonferroni post test).

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Figure 5. Eﬀect of 5al, donepezil, and memantine combination on acetylcholine modulation in the ventral hippocampus of male Wistar rats. Data

expressed as Mean SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs donepezil and memantine combination (Bonferroni post test).

doses of donepezil and memantine. To the best of our

knowledge, this is the ﬁrst clinical trial wherein a triple

combination [5-HT₆R antagonist (5al) + Donepezil +

Memantine] is being used for evaluating the eﬃcacy of a 5-

HT₆R antagonist.

the Purpose of Control and Supervision of Experiments on

Animals (CPCSEA), India.

EXPERIMENTAL SECTION

■

Chemistry. All the reagents and chemicals used were of “reagent

grade”. All the reactions monitoring was done by thin layer

chromatography (TLC). The TLC was performed on Merck silica

gel 60 F254 plates, and spots visualization was accomplished with UV

light (254 nm) and/or iodometry. Puriﬁcation of crude compounds

was achieved by column chromatography using 60−120 mesh silica gel

under ﬂash chromatography conditions. All the yields given here refer

to isolated pure products. Infrared spectra were recorded on KBr disk

and in solid state using a PerkinElmer model 1600 FT-IR

spectrophotometer (PerkinElmer, Norwalk, CT, USA). Electrospray

ionization mass spectra were recorded on an API 2000 triple

quadrupole instrument (MDS-SCIEX, Concord, Ontario, Canada).

¹H NMR and ¹³C NMR spectra were obtained on a Bruker NMR

spectrometer (Fallanden, Switzerland) at 400 and 100 MHz,

respectively. Deuterated reagents were used as solvents and were

commercially procured. Tetramethylsilane (TMS) was used as an

internal standard. Chemical shift values were expressed in parts per

million (δ), and coupling constants were expressed in hertz. The purity

of the ﬁnal compounds (>95%) was established using an Agilent 1100

high-performance liquid chromatography (HPLC) system. The

chromatographic conditions used are as follows: a YMC-pack-ODS-

AM column, 250 mm × 4.6 mm, S-5 μm, with detection at 220 nm,

using a UV−visible detector; ﬂow rate = 1 mL/min; oven temperature

30 °C, gradient elution over a run time of 35 min using buﬀer

acetonitrile (buﬀer: 0.05% Et₃N in water, adjusted to pH 2.5 with

CF₃COOH). Melting points were determined using an Electro

thermal (model IA 9300 series) open capillary apparatus and are

uncorrected. DSC experiments were carried out on DSCQ100,

Water’s model Q100-1001. Elemental analyses were carried out in

an “Elementar” GmbH, Vario micro cube instrument, and the

“analyses indicated by the symbols of the elements were within

0.4% of the theoretical values”.

In summary, the focused SAR around the 3-(piperazinyl-

methyl) indole scaﬀold led to the ﬁnding that the central indole

core is optimal for aﬃnity at 5-HT₆R. Replacing it with 4-

azaindole, 7-azaindole, or indazole resulted in moderately

potent compounds. Smaller halo and alkoxy substitutions were

tolerated, preferably at the C5 position of indole. 3-

(Piperazinylmethyl) indole is optimal for aﬃnity. Further

chain elongation, i.e. 3-(piperazinylethyl)indole, is not

tolerated. N-Aryl sulfonamide is critical for aﬃnity; replacing

it with benzyl and benzoyl gave less potent compounds. Halo

and lower alkoxy are preferred substitutions on the N-aryl

sulfonamide ring. The in vitro aﬃnity, selectivity, physicochem-

ical properties, in vivo eﬃcacy, and safety evaluations resulted in

the identiﬁcation of 5al, as a developmental candidate. 5al is a

crystalline dimesylate monohydrate salt with very high water

solubility and permeability. It weakly inhibited recombinant

cytochrome P450 3A4 while it does not inhibit CYP 2D6.

Compound 5al is an orally bioavailable and brain penetrant 5-

HT₆R antagonist with robust eﬃcacy in cognition models such

as ORT and the contextual fear conditioning model. The triple

combination of 5al, donepezil, and memantine produced

additive augmentation in extracellular levels of acetylcholine

in the ventral hippocampus without any cholinergic side eﬀects,

thus providing a neurochemical basis for the pro-cognitive

activity observed in various behavioral models. The preclinical

eﬃcacy in triple combination coupled with selectivity over the

5-HT_2Areceptor is the diﬀerentiating feature of this compound.

Compound 5al is nonmutagenic and nonclastogenic. The

clinical portions of the single and multiple ascending dose

studies assessing safety and pharmacokinetics have been

completed, allowing for the initiation of the Phase-2 proof of

concept study. The complete biological characterization of 5al

covering the detailed rationale for the use of a triple

combination in a Phase-2 POC trial will be reported in a

forthcoming biology publication.

General Procedures. Formaldehyde solution was added to a

mixture of substituted piperazine/homopiperazine (1.15 mmol), acetic

acid (0.8 mmol), and water (1 mmol) at 10 °C to 15 °C and stirred at

25 °C to 30 °C for 2 h to obtain a thick syrupy mass which was added

to a solution of substituted/unsubtituted indoles (6, 1 mmol) in

methanol at 5 °C to 10 °C and stirred for 3 h to obtain the

intermediates 8. These intermediates 8 (1 mmol) were treated with

potassium hydroxide (3 mmol) and substituted aryl sulfonyl, benzoyl,

or benzyl chlorides (9, 1.3 mmol) in THF, and stirred for 3 h, to

obtain the target compounds 5 or boc protected intermediates 10. The

boc protected intermediates 10 (1 mmol) were treated with

methanolic HCl or methanesulfonic acid (10 mmol) to obtain target

Animal Care. All animal care and experiments were carried

out according to the protocols approved by the Institutional

Animal Ethics Committee (IAEC) of Suven Life Sciences Ltd.

constituted according to the directions of the Committee for

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compounds 11 as hydrochloride salts or mesylate salts. The

compounds 12 were synthesized by the addition of acid chloride

(1.5 mmol) to a solution of compounds 11 (1 mmol) and Et₃N (3

mmol) in dichloromethane at 0 °C to 5 °C and further stirring at 25

°C to 30 °C for 2 h. The compounds 11 (1 mmol) were reacted with

either 2-ﬂuoroethyl bromide or benzyl bromide in the presence of

potassium carbonate (3 mmol) in N,N-dimethylformamide and stirred

at 100 °C for 6 h to obtain target compounds 13. The demethylation

of the methoxy group was performed by reacting the compounds 5al,

5am, and 5an (1 mmol) with HBr in acetic acid (10 vol.) at 80 °C for

12 h to obtain target compounds 5ay, 5az, and 5ba, respectively

(Scheme 1).

Powdered KOH (51.42 mmol) was added to a mixture of 14 (17.14

mmol) in THF (50 mL) stirred for 1 h followed by addition of a

solution of 9 (20.55 mmol) in THF (20 mL) at 25 °C to 30 °C and

then stirred overnight to obtain target compounds 15. Sodium

borohydride (19.6 mmol) was added in portions to a stirred mixture of

15 (9.8 mmol) under stirring at 25 °C to 30 °C (exothermic) and then

maintained for 3 h to obtain compounds 16. Thionyl chloride (10.44

mmol) was added to a stirred solution of 16 (6.96 mmol) in

dichloromethane (50 mL) and N,N-dimethylformamide (catalytic,

0.05 mL) at 0 °C to 5 °C and then stirred for 6 h at 25 °C to 30 °C to

obtain compounds 17. Compound 7 (4.76 mmol) was added to a

solution of 17 (3.97 mmol) and Et₃N (11.9 mmol) in dichloro-

methane (30 mL) at 0 °C to 5 °C, and then stirred at 25 °C to 30 °C

for 10 h, to aﬀord pure free base. The latter compound was dissolved

in methanol (10 mL), treated with methanolic HCl (7.91 mmol), and

stirred at 25 °C to 30 °C for 1 h to obtain the target compounds 5

(Scheme 2).

1-Methylpiperazine (1.1 mmol) followed by DIPEA (2.5 mmol)

was added to a solution of compound 18 (1 mmol) and TBTU (1.2

mmol) in N,N-dimethylformamide (3 mL) at 25 °C to 30 °C and

stirred for 2 h to obtain the target compound 19. LiAlH₄(2.5 mmol)

powder was added in portions to a solution of 19 (1 mmol) in THF at

0 °C to 5 °C and reﬂuxed for 4 h to obtain target compound 20. A

solution of arylsulfonyl chloride (1.2 mmol) in THF (3 mL) was

added to a suspension of compound 20 (1 mmol) and potassium

hydroxide (3 mmol) in THF (10 mL), and the mass was stirred at 25

°C to 30 °C overnight to obtain target compound 21 (Scheme 3).

Sodium triacetoxyborohydride (1.5 mmol) was added to a mixture

of 22 (1 mmol), 7 (1.2 mmol), and acetic acid (1 mmol) in EDC (10

mL) and stirred for 6−12 h to obtain compounds 23. The compounds

23 were treated with KOH and substituted/unsubstituted aryl sulfonyl

chlorides in THF followed by treatment with methanolic HCl (7.91

mmol) and stirring at 25 °C to 30 °C for 1 h to obtain the target

compounds 5 (Scheme 4).

General Procedure for the Preparation of Dihydrochloride

Salts. Methanolic HCl (20% w/v solution, 3 mmol) was added to a

solution of compound 5 (1 mmol) in methanol (5 mL) and stirred at

25 °C to 30 °C and maintained for 1 h. The resulting mixture was

concentrated to obtain the title compound as a hydrochloride salt, or

methanolic HCl (20% w/v solution, 10 mmol) was added to a solution

of compounds 10 (1 mmol) in methanol (5 mL), stirred at 25 °C to

30 °C for 3 h. The resulting mixture was concentrated to obtain the

title compound as a hydrochloride salt.

analysis calculated (%) for C₂₀H₂₅Cl₂N₃O₂S: C 54.30, H 5.70, N 9.50.

Found: C 54.45, H 5.68, N 9.53.

1-[(4-Fluorophenyl)sulfonyl]-3-[(4-methyl-1-piperazinyl)methyl]-

1H-indole Dihydrochloride (5h). IR (KBr, cm⁻¹): 3433, 2986, 2918,

1

1590, 1372, 1179, 979, 674, 567. H NMR (D₂O, δ ppm): 7.92−7.96

(4H, m), 7.62−7.64 (2H, d, J = 7.8 Hz), 7.30−7.41 (2H, m), 7.14−

7.18 (2H, t, J = 8.6 Hz), 4.40 (2H, s), 3.42−3.48 (8H, bs), 2.87 (3H,

s); ¹³C NMR (D₂O, δ ppm): 167.2, 164.7, 134.2, 131.8, 130.0, 129.9,

129.8, 129.3, 125.9, 124.5, 119.5, 117.0, 116.8, 113.4, 110.0, 50.3, 50.2,

48.2, 42.7; ESI mass (m/z): 388.2 [M + H]⁺. HPLC = 98.5%;

Elemental analysis calculated (%) for C₂₀H₂₄Cl₂FN₃O₂S: C 52.18, H

5.25, N 9.13. Found: C 52.33, H 5.26, N 9.16.

5-Fluoro-1-[(4-isopropylphenyl)sulfonyl]-3-[(4-methyl-1-

piperazinyl)methyl]-1H-indole Dihydrochloride (5ad). IR (KBr,

cm⁻¹): 3420, 2963, 2873, 1595, 1375, 1174, 662, 594; ¹H NMR

(D₂O, δ ppm): 7.97 (1H, s), 7.62−7.64 (2H, d, J = 8.1 Hz), 7.53−7.56

(1H, m), 7.26−7.28 (1H, d, J = 8.1 Hz), 7.02−7.04 (2H, d, J = 8.2

Hz), 6.60−6.64 (1H, m), 4.32 (2H, s), 3.41 (8H, bs), 2.87 (3H, s),

2.44−2.50 (1H, m), 0.71−0.73 (6H, d, J = 6.74 Hz); ESI mass (m/z):

430.3 [M + H]⁺; HPLC = 98.4%; Elemental analysis calculated (%)

for C₂₃H₃₀Cl₂FN₃O₂S: C 54.98, H 6.02, N 8.36. Found: C 54.79, H

6.04, N 8.39.

1-[(4-Isopropylphenyl)sulfonyl]-4-methoxy-3-[(4-methyl-1-

piperazinyl)methyl]-1H-indole Dihydrochloride (5ai). IR (KBr,

1

cm⁻¹): 3427, 2962, 2922, 2848, 1496, 1369, 12962, 1180, 1094; H

NMR (D₂O, δ ppm): 7.81 (1H, s), 7.69−7.71 (2H, d, J = 8.1 Hz),

7.44−7.46 (1H, d, J = 8.3 Hz), 7.22−7.26 (1H, m), 7.14−7.16 (2H, d,

J = 8.1 Hz), 6.70−6.72 (1H, d, J = 7.8 Hz), 4.55 (2H, s), 3.83 (3H, s),

3.52 (8H, bs), 2.88 (3H, s), 2.65−2.70 (1H, m), 0.91−0.92 (6H, d, J =

6.6 Hz); ESI mass (m/z): 442.5 [M + H]⁺; HPLC = 97.7%; Elemental

analysis calculated (%) for C₂₄H₃₃Cl₂N₃O₃S: C 56.03, H 6.46, N 8.17.

Found: C 56.22, H 6.48, N 8.20.

1-[(2-Bromophenyl)sulfonyl]-5-methoxy-3-[(4-methyl-1-

piperazinyl)methyl]-1H-indole Dimesylate Monohydrate (5al).

Powdered potassium hydroxide (9.25 g, 231.3 mmol) was added to

a solution of 8h (20 g, 77.1 mmol) in THF (80 mL) at 15 °C to 20 °C

under stirring and maintained for 60 min at 15 °C to 20 °C. Then a

solution of 9b (25.6 g, 100.2 mmol) in THF (50 mL) was added to

the above mixture at 15 °C to 25 °C and stirred for 3 h at 25 °C to 35

°C. After completion of the reaction, the reaction mass was poured

into chilled water (400 mL) under stirring at 10 °C to 15 °C, the pH

of the resulting mass was adjusted to ∼9.5−10 with an aqueous

solution of potassium hydroxide, and the product was extracted with

ethyl acetate (100 mL × 3). The combined organic layer was washed

with brine solution (50 mL × 2), dried over anhydrous sodium sulfate,

ﬁltered, and concentrated to obtain a residual mass.

Puriﬁcation. Methanol (35 mL) and isopropanol (70 mL) were

charged to the above residual mass. The reaction mass was heated to

reﬂux to obtain a clear solution. The mass was allowed to cool to 15

°C to 20 °C and maintained under stirring for 2 h at 15 °C to 20 °C.

The solids were ﬁltered, the cake on the ﬁlter was washed with 30 mL

of isopropanol (15 °C to 20 °C), and solvents were removed via

vacuum. The wet solid material was dried in vacuo to obtain the

compound 1-(2-bromobenzenesulfonyl)-5-methoxy-3-[(4-methyl pi-

perazin-1-yl) methyl]-1H-indole as free base (30.2 g, 82% yield). IR

(KBr, cm⁻¹): 2931, 2786, 1607, 1474, 1369, 1222, 1178, 1032, 737,

1

General Procedure for the Preparation of Dimesylate Salts.

Methanesulfonic acid (2.2 mmol) was added to a mixture of 5 (1

mmol) in ethanol (5 mL), stirred at 25 °C to 30 °C for 24 h, and then

the solids were ﬁltered and dried under reduced pressure to obtain

dimesylate salt, or methanesulfonic acid (3.5 mmol) was added to a

mixture of 10 (1 mmol) in acetone (5 mL) under reﬂux and

maintained for 3 h; precipitated solids were ﬁltered and dried under

reduced pressure to obtain dimesylate salt.

1-(Phenylsulfonyl)-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

Dihydrochloride (5a). IR (KBr, cm⁻¹): 3432, 2948, 1366, 1171, 981,

596; ¹H NMR (D₂O, δ ppm): 7.94 (1H, s), 7.83−7.88 (3H, s), 7.57−

7.59 (1H, d, 7.82 Hz), 7.50−7.52 (1H, m), 7.37−7.41 (2H, m), 7.31−

7.35 (1H, m), 7.27−7.29 (1H, m), 4.48 (2H, s), 3.51 (8H, bs), 2.88

(3H, s); ESI mass (m/z): 370.3 [M + H]⁺; HPLC = 99.1%, Elemental

597; H NMR (CDCl₃, δ ppm): 8.03−8.05 (1H, dd, J = 1.5, 7.8 Hz),

7.64−7.66 (2H, m), 7.53−7.55 (1H, d, J = 9.0 Hz), 7.45−7.47 (1H, t, J

= 7.5 Hz), 7.36−7.40 (1H, dt, J = 1.5, 7.6 Hz), 7.19−7.20 (1H, d, J =

2.4 Hz), 6.83−6.86 (1H, dd, J = 2.4, 8.9 Hz), 3.62 (2H, s), 3.83 (3H,

s), 2.32−2.50 (8H, bs), 2.29 (3H, s); ¹³C NMR (CDCl₃, δ ppm):

156.1, 138.0, 135.9, 134.4, 131.5, 131.1, 129.5, 127.6, 127.0, 120.4,

117.8, 113.6, 113.2, 103.2, 55.6, 55.1, 53.3, 53.0, 45.9; ESI mass (m/z):

478.1, 480.1 [M + H]⁺; Melting range (°C): 128.1−129.2; HPLC

purity: 99.3%.

Methanesulfonic acid (14.12 g, 147.1 mmol) was added slowly to a

stirred suspension of 1-(2-bromobenzenesulfonyl)-5-methoxy-3-[(4-

methyl piperazin-1-yl)methyl]-1H-indole (32.02 g, 66.88 mmol) in

absolute ethanol (190 mL) under stirring at 25 °C to 35 °C, and the

mass was further stirred for 24 h. The mass was ﬁltered under nitrogen

L

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atmosphere, and solvents were removed via vacuum. The cake was

washed thoroughly with 50 mL of ethyl alcohol followed by washing

with n-hexane (30 mL). The wet cake was dried in vacuo to obtain

44.5 g of 1-(2-bromobenzenesulfonyl)-5-methoxy-3-[(4-methyl piper-

azin-1-yl)methyl]-1H-indole dimesylate. HPLC purity: 99.6%.

A carbon slurry prepared by mixing 6.7 g of carbon powder with 20

mL of aqueous ethanol (95% ethanol and 5% water) was added to a

suspension of aqueous ethanol (450 mL, 95% ethanol, and 5% water)

and 1-(2-bromobenzenesulfonyl)-5-methoxy-3-[(4-methyl piperazin-

1-yl)methyl]-1H-indole dimesylate (44.5 g, 66.35 mmol). The reaction

mass was heated at 75 °C to 80 °C for 45 min, and then the mass was

ﬁltered hot and washed with 50 mL of hot aqueous ethanol (95%

ethanol and 5% water) at 75 °C to 80 °C. The clear ﬁltrate was

allowed to cool to 25 °C to 30 °C, and then further cooled to 10 °C to

15 °C and maintained under stirring for 3 h. The crystalline material,

thus obtained, was washed with 20 mL of chilled aqueous ethanol

(95% ethanol and 5% water), and the material was dried in vacuo to

obtain the title product 5al (37.7 g, 94.5% yield). Melting range (°C):

218.0−220.0; Salt content (Dimesylate): 27.56%; IR spectra (KBr,

cm⁻¹): 3148, 3012, 1611, 1590, 1471, 1446, 1439, 1382, 1220, 1194,

1180, 1045, 775, 596; ¹H NMR (D₂O, δ ppm): 8.14−8.16 (1H, d, J =

7.9 Hz), 8.04 (1H, s), 7.54−7.56 (1H, d, J = 7.7 Hz), 7.44−7.47 (1H,

t, J = 7.6 Hz), 7.32−7.38 (2H, m), 7.10−7.11 (1H, d, J = 1.9 Hz),

6.75−6.78 (1H, dd, J = 2.0, 9.1 Hz), 4.46 (2H, s), 3.70 (3H, s), 3.52

(8H, bs), 2.89 (3H, s), 2.65 (6H, s); ¹³C NMR (D₂O, δ ppm): 156.2,

135.9, 135.2, 132.1, 131.8, 130.1, 128.5, 128.2, 119.6, 114.0, 113.7,

108.2, 102.5, 55.8, 50.3, 48.1, 42.7, 38.4; ESI mass (m/z): 478, 480 [M

+ H]⁺; Elemental analysis calculated (%) for C₂₃H₃₄BrN₃O₁₀S₃: C

40.12, H 4.98, N 6.10. Found: C 40.24, H 4.99, N 6.12; HPLC purity:

99.7%.

119.5, 114.2, 113.6, 107.6, 104.3, 50.3, 50.2, 48.7, 48.0, 42.7; ESI mass

(m/z): 464.2, 466.1 [M + H]⁺.

1-(4-Isopropylbenzenesulfonyl)-5-methoxy-3-(4-methyl-[1,4]-

diazepan-1-ylmethyl)-1H-indole Dihydrochloride (5bg). Compound

7d (0.54 g, 4.76 mmol) was added to a solution of 17d (1.5 g, 3.97

mmol) and Et₃N (1.2 g, 11.9 mmol) in dichloromethane (30 mL) at 0

°C to 5 °C, and the mixture was stirred at 25 °C to 30 °C for 10 h.

The reaction mixture was then poured into water (25 mL) and

extracted using dichloromethane (25 mL × 2). The organic extracts

were combined, dried over anhydrous sodium sulfate, and

concentrated in vacuo to obtain a residual mass that was chromato-

graphed (silica gel, 5:95, methanol:ethyl acetate) to aﬀord 1-(4-

isopropylbenzenesulfonyl)-5-methoxy-3-(4-methyl-[1,4]diazepan-1-yl-

methyl)-1H-indole (1.2 g, 66% yield). This compound was dissolved

in methanol (10 mL), treated with methanolic HCl (7.91 mmol, 20%

w/v solution), and stirred at 25 °C to 30 °C for 1 h. The reaction mass

was concentrated in vacuo to obtain the target compound 1.32 g, 95%

yield). IR (KBr, cm⁻¹): 3407, 2959, 2469, 1612, 1594, 1369, 1174,

1030, 662; ¹H NMR (D₂O, δ ppm): 7.95 (1H, s), 7.61−7.64 (3H, m),

7.06−7.06 (1H, d, J = 1.8 Hz), 6.99−7.01 (2H, d, J = 8.3 Hz), 6.71−

6.74 (1H, dd, J = 1.8, 9.1 Hz), 4.46 (2H, s), 3.65−3.68 (4H, bs), 3.63

(3H, s), 3.39−3.40 (4H, bs), 2.86 (3H, s), 2.51−2.54 (1H, m), 2.14

(2H, m), 0.76−0.78 (6H, d, J = 6.8 Hz); ¹³C NMR (D₂O, δ ppm):

156.9, 156.3, 133.1, 130.5, 130.5, 129.1, 127.6, 126.9, 114.4, 114.3,

110.6, 102.4, 55.8, 55.2, 52.8, 51.1, 50.5, 48.3, 44.0, 33.5, 22.4, 20.3;

ESI mass (m/z): 456.4 [M + H]⁺. HPLC = 99.5%; DSC: 175.63−

184.62 °C; Elemental analysis calculated (%) for C₂₅H₃₅Cl₂N₃O₃S: C

56.81, H 6.67, N 7.95. Found: C 56.67, H 6.65, N 7.97.

1-(2-Bromobenzyl)-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole

(5bm). IR (KBr, cm⁻¹): 2932, 2793, 1461, 1330, 1162, 741; ¹H NMR

(CDCl₃, δ ppm): 7.75−7.82 (1H, m), 7.58−7.62 (1H, m), 7.07−7.25

(6H, m), 6.54−6.60 (1H, m), 5.38 (2H, s), 3.75 (2H, s), 2.38−2.70

(8H, bs), 2.30 (3H, s); ¹³C NMR (DMSO-d₆, δ ppm): 137.3, 136.6,

133.0, 129.7, 128.6, 128.6, 128.4, 128.3, 122.2, 121.8, 119.9, 119.3,

111.6, 110.2, 55.2, 53.3, 52.8, 49.5, 46.1; ESI mass (m/z): 398.3, 400.1

[M + H]⁺; HPLC = 98.1%.

(2-Bromophenyl)-[5-ﬂuoro-3-(4-methylpiperazin-1-ylmethyl)-

indol-1-yl]methanone (5bn). IR (KBr, cm⁻¹): 2936, 2795, 1693,

1449, 922, 785; ¹H NMR (CDCl₃, δ ppm): 7.68−7.70 (1H, d, J = 7.8

Hz), 7.40−7.49 (4H, m), 7.23 (1H, m), 7.11 (1H, m), 6.86 (1H, m),

3.53 (2H, s), 2.42−2.51 (8H, bs), 2.27 (3H, s); ESI mass (m/z):

430.3, 432.1 [M + H]⁺; HPLC = 98.5%.

1-[(4-Isopropylphenyl)sulfonyl]-5-methoxy-3-[(4-methyl-1-

piperazinyl)methyl]-1H-indole Dimesylate (5am). IR (KBr, cm⁻¹):

1

3459, 3010, 2935, 1594, 1373, 1193, 782; H NMR (D₂O, δ ppm):

7.94 (1H, s), 7.58−7.60 (2H, d, J = 7.5 Hz), 7.45−7.47 (1H, d, J = 8.8

Hz), 7.03 (1H, s), 6.88−6.90 (2H, d, J = 7.6 Hz), 6.40−6.42 (1H, d, J

= 8.4 Hz), 4.37 (2H, s), 3.46−3.51 (8H, bs), 2.90 (3H, s), 2.56 (6H,

s), 2.32−2.35 (1H, m), 0.60−0.62 (6H, d, J = 6.2 Hz); ¹³C NMR

(D₂O, δ ppm): 156.4, 156.3, 133.6, 130.6, 130.1, 128.9, 127.5, 126.9,

114.1, 114.0, 110.6, 102.4, 55.6, 50.5, 48.2, 42.7, 38.3, 33.4, 23.6, 22.4;

ESI mass (m/z): 442.2 [M + H]⁺; DSC: 175.32−178.05 °C;

Elemental analysis calculated (%) for C₂₆H₃₉N₃O₉S₃: C 49.27, H

6.20, N 6.63. Found: C 49.17, H 6.18, N 6.61.

1-Benzenesulfonyl-3-(4-methylpiperazin-1-ylmethyl)-1H-pyrrolo-

[2,3-b]pyridine (5bo). Powdered potassium hydroxide (0.29 g, 5.21

mmol) was added to a solution of 23b (0.4 g, 1.73 mmol) at 25 °C to

30 °C in THF (10 mL) under N₂atmosphere and stirred for 1 h

followed by the addition of 9a (0.6 g, 2.08 mmol). The reaction

mixture stirred for 12 h, poured into water (25 mL), and extracted

with ethyl acetate (25 mL × 3). The organic extracts were combined,

dried over sodium sulfate, and concentrated in vacuo to obtain a

residual mass that was chromatographed (silica gel, 5:95, methanol:

1-[(4-Fluorophenyl)sulfonyl]-5-methoxy-3-[(4-methyl-1-

1

piperazinyl)methyl]-1H-indole Dimesylate (5an). H NMR (D₂O, δ

ppm): 7.84−7.87 (3H, m), 7.70−7.73 (1H, d, J = 9.1 Hz), 7.07−7.12

(3H, m), 6.91−6.93 (1H, dd, J = 2.3, 9.1 Hz), 4.42 (2H, s), 3.74 (3H,

s), 3.47−3.51 (8H, bs), 2.90 (3H, s), 2.67 (6H, s); ¹³C NMR (D₂O, δ

ppm): 167.1, 164.6, 156.3, 131.9, 130.4, 129.9, 129.8, 129.0, 116.9,

116.7, 114.4, 114.3, 110.2, 102.4, 55.8, 50.3, 48.1, 42.7, 38.4; ESI mass

(m/z): 418.2 [M + H]⁺; DSC: 148.4−150.5 °C; Elemental analysis

calculated (%) for C₂₁H₂₄FN₃O₃S: C 60.41, H 5.79, N 10.06 Found: C

60.28, H 5.77, N 10.09.

1-[(2-Bromophenyl)sulfonyl]-5-hydroxy-3-[(4-methyl-1-

piperazinyl)methyl]-1H-indole Dihydrochloride (5ay). HBr (10 mL,

33 wt % in acetic acid) was added to 5al (1 g, 2.09 mmol), and the

resulting mixture was heated to 80 °C and maintained for 12 h. After

completion of reaction, the reaction mass was cooled to 25 °C to 30

°C, poured into water (25 mL), basiﬁed with ammonia solution (pH ∼

10), and extracted with chloroform (25 mL × 3). The organic extracts

were combined, dried over sodium sulfate, and concentrated in vacuo

to obtain a residual mass that was puriﬁed by ﬂash column

chromatography using methanol−chloroform (5:95) to obtain the

target compound (0.82 g, 85% yield). It was converted to

dihydrochloride salt by following the general procedure given above.

¹H NMR (D₂O, δ ppm): 8.24−8.26 (1H, d, J = 7.9 Hz), 8.07 (1H, s),

1

ethyl acetate) to obtain the title compound (0.45 g, 71% yield). H

NMR (CDCl₃, δ ppm): 8.41−8.42 (1H, d, J = 4.5 Hz), 8.18−8.20

(2H, d, J = 7.8 Hz), 8.01−8.03 (1H, d, J = 7.8 Hz), 7.61 (1H, s), 7.55−

7.58 (1H, t, J = 7.4 Hz), 7.46−7.49 (2H, t, J = 7.6 Hz), 7.15−7.18 (1H,

m), 3.6 (3H, s), 2.44−2.47 (8H, bs), 2.27 (3H, s); ESI mass (m/z):

371.3 [M + H]⁺; HPLC = 98.1%.

5-Methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole (8h).

Twelve milliliters of demineralized water was added to 7a (14.97 g,

0.15 mol) at 10 °C to 15 °C followed by the addition of acetic acid

(6.19 g, 0.103 mol). The mass was stirred for 15 min at 10 °C to 15

°C, and aqueous formaldehyde solution (16.3 mL, 30% w/v, 0.16 mol)

was added at 10 °C to 15 °C. The resultant thick reaction mass was

stirred for another 2 h at 25 °C to 30 °C to obtain the Mannich

adduct. The adduct, thus obtained, was added slowly to a stirred

solution of 5-methoxyindole (20 g, 0.13 mol) in methanol (125 mL) at

5 °C to 10 °C in 15−20 min and stirred for 3 h at 25 °C to 30 °C.

After completion of the reaction (TLC), the reaction mass was poured

slowly into chilled water (100 mL), pH was adjusted to ∼10−12 using

lye solution, and the product was extracted with ethyl acetate (100 mL

7.66−7.67 (1H, d, J = 7.8 Hz), 7.53−7.57 (1H, m), 7.47−7.51 (1H,

m), 7.38−7.41 (1H, d, J = 8.9 Hz), 7.07 (1H, s), 6.78−6.80 (1H, d, J =

8.8 Hz), 4.45 (2H, s), 3.50−3.54 (8H, bs), 2.92 (3H, s); ¹³C NMR

(D₂O, δ ppm): 152.6, 135.7, 135.1, 132.2, 131.6, 130.2, 128.0, 127.9,

M

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× 3). The combined organic layers were washed with brine (50 mL),

dried over anhydrous sodium sulfate, and concentrated in vacuo to

obtain the crude compound. The crude compound was taken in

toluene (60 mL) and heated to 90 °C to obtain a clear solution. The

resulting solution was cooled to 10 °C to 15 °C, maintained for 3 h,

and the precipitated solids were ﬁltered, washed with chilled toluene

(10 mL × 2), and dried in vacuo to obtain crystalline compound 8h

(22.05 g, 62% yield). IR (KBr, cm⁻¹): 3125, 2951, 1875, 1622, 1585,

was chromatographed (silica gel, 5:95, methanol:ethyl acetate) to

aﬀord the title compound as a syrupy mass (0.42 g, 77% yield). IR

(KBr, cm⁻¹): 2934, 2812, 1611, 1475, 1371, 1178, 730, 597; ¹H NMR

(DMSO-d₆, δ ppm): 8.05−8.06 (1H, d, J = 7.1 Hz), 7.84−7.86 (1H, d,

J = 7.4 Hz), 7.70 (1H, s), 7.60−7.66 (2H, m), 7.48−7.50 (1H, d, J =

9.0 Hz), 7.25 (1H, d, J = 1.7 Hz), 6.87−6.89 (1H, dd, J = 1.8, 8.9 Hz),

4.43−4.57 (2H, m), 3.75 (3H, s), 3.61 (2H, s), 2.62−2.63 (2H, m),

2.49−2.52 (8H, bs); ESI mass (m/z): 510.1, 511.8 [M + H]⁺; HPLC =

98.5%.

1

1492, 1351, 1288, 1215, 1059, 930, 654; H NMR (CDCl₃, δ ppm):

8.91 (1H, s), 7.18−7.20 (2H, m), 7.01 (1H, d, J = 2.1 Hz), 6.83−6.86

(1H, dd, J = 2.7, 8.8 Hz), 3.86 (3H, s), 3.71 (2H, s), 2.5 (8H, bs), 2.30

(3H, s); ¹³C NMR (CDCl₃, δ ppm): 153.7, 131.4, 128.4, 124.8, 111.8,

111.7, 111.3, 101.3, 55.8, 55.1, 53.3, 52.7, 45.8; ESI mass (m/z): 260.3

[M + H]⁺; Melting range (°C): 139−140.6; HPLC purity: 99.7%.

1-[(2-Bromophenyl)sulfonyl]-5-methoxy-3-[(4-t-butyloxycarbon-

yl-1-piperazinyl)methyl]-1H-indole (10c). Compounds 10 were

synthesized according to the general procedure given for the synthesis

of compounds 5 with some noncritical variations. IR (KBr, cm⁻¹):

2967, 2857, 1688, 1609, 1475, 1370, 1226, 1175, 1037, 1000, 889, 757,

596; ¹H NMR (CDCl₃, δ ppm): 8.06−8.08 (1H, d, J = 7.8 Hz), 7.64−

7.66 (2H, m), 7.52−7.55 (1H, d, J = 8.9 Hz), 7.43−7.47 (1H, t, J = 7.5

Hz), 7.36−7.40 (1H, t, J = 7.1 Hz), 7.19 (1H, d, J = 2.0 Hz), 6.83−

6.86 (1H, dd, J = 2.1, 8.3 Hz), 3.82 (3H, s), 3.63 (2H, s), 3.42 (4H,

bs), 2.42 (4H, bs), 1.45 (9H, s); ¹³C NMR (CDCl₃, δ ppm): 156.1,

154.6, 137.9, 135.9, 134.5, 131.4, 131.2, 129.5, 127.7, 127.1, 120.3,

117.3, 113.7, 113.2, 103.2, 79.4, 55.5, 53.3, 52.7, 28.3; HPLC: 99.4%;

ESI mass (m/z): 564.3, 566.3 [M + H]⁺.

1-(4-Isopropylbenzenesulfonyl)-5-methoxy-1H-indole-3-carbox-

aldehyde (15d). Powdered KOH (2.87 g, 51.42 mmol) was added to a

mixture of 14c (3 g, 17.14 mmol) in THF (50 mL) at 25 °C to 30 °C

and maintained for 1 h. Then a solution of 9i (4.5 g, 20.55 mmol) in

THF (20 mL) was added dropwise to the above mixture under stirring

at 25 °C to 30 °C, stirred overnight, poured into water (100 mL), and

extracted with ethyl acetate (100 mL × 2). The organic extracts were

combined, dried over sodium sulfate, and concentrated in vacuo to

obtain a residual mass. This mass was chromatographed (silica gel,

20:80, ethyl acetate:n-hexane) to aﬀord the title compound (3.8 g, 62%

1

yield). IR (KBr, cm⁻¹): 2936, 2829, 1675, 1373, 1181, 974, 593; H

NMR (CDCl₃, δ ppm): 10.06−10.10 (1H, s), 8.18 (1H, s), 7.83−7.87

(3H, m), 7.71−7.72 (1H. d, J = 2.6 Hz), 7.33−7.35 (2H, d, J = 8.4

Hz), 7.00−7.03 (1H, dd, J = 2.6, 9.1 Hz), 3.85 (3H, s), 2.89−2.96

(1H, m), 1.19−1.23 (6H, d, J = 6.9 Hz); ESI mass (m/z): 358.2 [M +

H]⁺.

[1-(4-Isopropylbenzenesulfonyl)-5-methoxy-1H-indol-3-yl] Meth-

anol (16d). Sodium borohydride (0.75 g, 19.6 mmol) was added in

portions to a stirred mixture of 15d (3.5 g, 9.8 mmol) under stirring at

25 °C to 30 °C (exothermic), maintained for 3 h, and concentrated to

obtain a residual mass. It was diluted with water (50 mL) and extracted

with ethyl acetate (50 mL × 3), and the combined organic extracts

were dried over anhydrous sodium sulfate and concentrated in vacuo

to obtain the title compound (2.85 g, 81% yield). IR (KBr, cm⁻¹):

3495, 2966, 2873, 1447, 1361, 1170, 1024, 884, 594; ¹H NMR

(CDCl₃, δ ppm): 7.88−7.90 (1H, d, J = 8.9 Hz), 7.76−7.78 (2H, d, J =

6.7 Hz), 7.50 (1H, s), 7.25−7.27 (2H, d, J = 8.3 Hz), 7.05 (1H, d, J =

2.4 Hz), 6.94−6.96 (1H, dd, J = 2.5, 9.0 Hz), 4.79 (2H, bs), 3.83 (3H,

s), 2.85−2.92 (1H, m), 1.18−1.19 (6H, d, J = 6.9 Hz); ESI mass (m/

z): 342.2 [M - 18]⁺.

1-[(2-Bromophenyl)sulfonyl]-5-methoxy-3-[(1-piperazinyl)-meth-

yl]-1H-indole Dimesylate Dihydrate (11c). Compound 10c (1.5 g,

2.65 mmol) was dissolved in acetone (10 mL), and the temperature of

the reaction mass was raised to reﬂux temperature (56 °C).

Methanesulfonic acid (0.89 g, 9.3 mmol) diluted with acetone (5

mL) was added to the above mass at reﬂux temperature and

maintained for 4 h, during which solids precipitated out. Then the

mass was cooled to 25 °C and stirred for 2 h, the solids were ﬁltered,

washed with acetone (5 mL × 2), and dried to obtain the title

compound (1.57 g, 90% yield). IR (KBr, cm⁻¹): 3428, 3009, 1613,

1

1478, 1383, 1207, 1180, 1045, 784, 598; H NMR (D₂O, δ ppm):

8.15−8.17 (1H, d, J = 7.8 Hz), 8.06 (1H, s), 7.54−7.56 (1H, d, J = 7.8

Hz), 7.44−7.48 (1H, d, J = 7.7 Hz), 7.34−7.38 (1H, t, J = 7.6 Hz),

7.32−7.34 (1H, t, J = 9.3 Hz), 7.11 (1H, d, J = 1.7 Hz), 6.75−6.78

(1H, dd, J = 1.9, 8.9 Hz), 4.50 (2H, s), 3.70 (3H, s), 3.53 (4H, bs),

3.48 (4H, bs), 2.64 (6H, s); ¹³C NMR (CDCl₃, δ ppm): 156.1, 135.7,

135.4, 135.3, 132.2, 131.3, 130.0, 128.2, 127.9, 119.5, 114.0, 113.3,

107.6, 102.0, 55.5, 50.4, 47.8, 40.5, 38.3; HPLC: 99.9%, DSC: 198.15−

199.37 °C; ESI mass (m/z): 464.2, 466.2 [M + H]⁺.

1-{4-[1-(2-Bromobenzenesulfonyl)-1H-indol-3-ylmethyl]-pipera-

zin-1-yl} Ethanone (12b). Acetyl chloride (0.14 g, 1.72 mmol) was

added to a solution of 11b (0.5 g, 1.15 mmol) and Et₃N (0.35 g, 3.5

mmol) in dichloromethane (10 mL) at 0 °C to 5 °C under stirring.

The reaction mass was further stirred for 2 h at 25 °C to 30 °C. After

completion of reaction, the reaction mixture was poured into water

(10 mL) and extracted with dichloromethane (25 mL × 2). The

organic extracts were combined, dried over sodium sulfate, and

concentrated in vacuo to obtain a residual mass. The mass was

chromatographed (silica gel, 5:95, methanol:ethyl acetate) to aﬀord

the title compound as syrupy mass (0.4 g, 73% yield). ¹H NMR

(DMSO-d₆, δ ppm): 8.16−8.18 (1H, d, J = 7.8 Hz), 7.85−7.87 (1H, d,

J = 7.9 Hz), 7.77−7.79 (2H, m), 7.57−7.69 (3H, m), 7.26−7.28 (2H,

m), 3.68 (2H, s), 3.37−3.41 (4H, m), 2.35−2.41 (4H, m), 1.96 (3H,

s); ESI mass (m/z): 476.2, 478.3 [M + H]⁺.

3-Chloromethyl-1-(4-isopropylbenzenesulfonyl)-5-methoxy-1H-

indole (17d). Thionyl chloride (1.24 g, 10.44 mmol) was added to a

stirred solution of 16d (2.5 g, 6.96 mmol) in dichloromethane (50

mL) and N,N-dimethylformamide (0.05 mL) at 0 °C to 5 °C and

stirred for 6 h at 25 °C to 30 °C. After completion of the reaction, the

reaction mass was washed with water (25 mL), dried over sodium

sulfate, and concentrated in vacuo to obtain solids. These solids were

triturated using diethyl ether (15 mL × 2) and dried to obtain the title

compound (1.52 g, 58% yield). IR (KBr, cm⁻¹): 3129, 2964, 2837,

1

1595, 1482, 1362, 1238, 1169, 881, 693. H NMR (CDCl₃, δ ppm):

7.86−7.89 (1H, d, J = 9.0 Hz), 7.77−7.79 (2H, d, J = 8.4 Hz), 7.58

(1H, s), 7.27−7.29 (2H, d, J = 8.4 Hz), 7.04−7.05 (1H, d, J = 2.4 Hz),

6.95−6.98 (1H, dd, J = 2.5, 9.0 Hz), 4.71 (2H, s), 3.84 (3H, s), 2.86−

2.93 (1H, m), 1.18−1.20 (6H, d, J = 6.9 Hz).

1-(4-Methylpiperazin-1-yl)-2-(5-bromo-1H-indol-3-yl) Ethanone

(19). A solution of 18 (0.5 g, 1.96 mmol) and TBTU (0.76 g, 2.36

mmol) in DMF (5 mL) was reacted with 7a (0.21 g, 2.16 mmol) and

DIPEA (0.63 g, 4.92 mmol). The reaction mass was stirred at 25 °C to

30 °C for 2 h, and then the mass was poured into water (25 mL) and

the product was extracted with ethyl acetate (25 mL × 3). The organic

layer was separated, dried over Na₂SO₄, and concentrated to obtain

1

the title compound (0.59 g, 89% yield). H NMR (CDCl₃, δ ppm):

8.26 (1H, bs), 7.72 (1H, s), 7.27−7.28 (1H, m), 7.2−7.22 (1H, d, J =

8.5 Hz), 7.08 (1H, s), 3.77 (2H, s), 3.67−3.70 (2H, t, J = 4.5 Hz),

3.48−3.51 (2H, t, J = 4.8 Hz), 2.29 (3H, s); ESI mass (m/z): 336.2,

338.1 [M + H]⁺.

5-Bromo-3-[(4-methylpiperazin-1-yl)ethyl]-1H-indole (20).

LiAlH₄(0.14 g, 3.72 mmol) was added in portions to a stirred

mixture of 19 (0.5 g, 1.48 mmol) in THF (15 mL) at 0 °C to 5 °C,

and then the reaction mass was reﬂuxed for 2 h. After completion of

the reaction, the reaction mixture was cooled to 10 °C, and water (2

1-(2-Bromobenzenesulfonyl)-3-[4-(2-ﬂuoroethyl)piperazin-1-yl-

methyl]-5-methoxy-1H-indole (13b). 1-Bromo-2-ﬂuoroethane (0.21

g, 1.61 mmol) was added to a suspension of 11c (0.5 g, 1.07 mmol)

and K₂CO₃(0.45 g, 3.23 mmol) in DMF (5 mL), and the resulting

mixture was heated to 100 °C and maintained for 6 h. After

completion of reaction, the reaction mass was cooled to 25 °C to 30

°C, poured into water (25 mL), and extracted with ethyl acetate (25

mL × 3). The organic extracts were combined, dried over sodium

sulfate, and concentrated in vacuo to obtain a residual mass. This mass

N

DOI: 10.1021/acs.jmedchem.6b01662

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

mL) was added carefully and then diluted with ethyl acetate (50 mL).

The reaction mass was ﬁltered through Celite, and the organic layer

was separated, dried over Na₂SO₄, and concentrated to obtain a

residual mass. This residual mass was chromatographed (silica gel,

variance (time and treatment), followed by Bonferroni’s post-test.

Statistical signiﬁcance was considered at a p value less than 0.05.

Incorrect probe placement was considered as a criterion to reject the

data from animal.

1

ethyl acetate) to obtain the title compound (0.28 g, 60% yield). H

NMR (CDCl₃, δ ppm): 8.07 (1H, bs), 7.73 (1H, s), 7.20−7.26 (2H,

m), 7.04 (1H, s), 2.89−2.93 (2H, t, J = 8.5 Hz), 2.65−2.69 (2H, t, J =

8.5 Hz), 2.51−2.54 (8H, bs), 2.31 (3H, s); ESI mass (m/z): 322.2,

324.2 [M + H]⁺.

ASSOCIATED CONTENT

■

S

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.6b01662.

1-(2-Bromobenzenesulfonyl)-5-bromo-3-[(4-methylpiperazin-1-

yl)ethyl]-1H-indole Dihydrochloride (21a). A solution of 9b (0.24 g,

0.93 mmol) in THF (5 mL) was added to a stirred mixture of 20 (0.25

g, 0.77 mmol) and powdered KOH (0.13 g, 2.32 mmol) in THF at 25

°C to 30 °C and kept under stirring overnight. Then the reaction mass

was poured into water (10 mL) and extracted with ethyl acetate (15

mL × 3). The organic extracts were combined, dried over Na₂SO₄, and

concentrated to obtain crude compound that was chromatographed

(silica gel, ethyl acetate), and the obtained compound (0.19 g, 45%

yield) was treated with methanolic HCl to obtain the title compound.

¹H NMR (DMSO-d₆, δ ppm): 11.42−11.52 (2H, bs), 8.14−8.15 (1H,

Spectroscopic characterization data of representative

derivatives, in vitro assay protocols, biological assay

protocols, and aqueous solubility protocol (PDF)

Molecular formula strings (CSV)

AUTHOR INFORMATION

■

Corresponding Author

*Tel.: +91-40-23556038; Fax: +91-40-23541152; e-mail:

nvsrk@suven.com.

d, J = 7.2 Hz), 8.00 (1H, s), 7.84−7.89 (2H, m), 7.63−7.70 (2H, m),

7.56−7.58 (1H, d, J = 8.7 Hz), 7.44−7.47 (1H, d, J = 8.7 Hz), 3.42−

3.68 (4H, m), 3.08−3.28 (8H, m), 2.83 (3H, s); ESI mass (m/z):

540.1, 542.2, 544.1 [M + H]⁺.

ORCID

3-(4-Methylpiperazin-1-ylmethyl)-1H-pyrrolo[2,3-b]pyridine

(23b). Sodium triacetoxyborohydride (1.08 g, 5.13 mmol) was added

to a stirred mixture of 22b (0.5 g, 3.42 mmol), 7a (0.41 g, 4.1 mmol),

and acetic acid (0.2 g, 3.42 mmol) in 1,2-dichloroethane (25 mL)

under N₂atmosphere at 25 °C. The reaction mixture was stirred for 6

h, poured into water (25 mL), basiﬁed with lye solution, and extracted

using dichloromethane (25 mL × 3). The organic extracts were

combined, dried over sodium sulfate, and concentrated in vacuo to

obtain a residual mass that was chromatographed (silica gel, 5:95,

methanol:ethyl acetate) to obtain the title compound (0.42 g, 54%

Ramakrishna Nirogi: 0000-0002-2045-0784

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The support from the analytical chemistry team, and the moral

and ﬁnancial support received from Venkateswarlu Jasti, CEO,

Suven Life Sciences Ltd., Hyderabad, India, are greatly

acknowledged.

1

yield). H NMR (CDCl₃, δ ppm): 8.57−8.59 (1H, m), 7.75−7.77

(1H, m), 7.38−7.40 (1H, m), 6.50−6.51 (1H, m), 5.10 (1H, bs), 3.51

(2H, s), 2.43−2.46 (8H, bs), 2.27 (3H, s); ESI mass (m/z): 231.2 [M

+ H]⁺.

ABBREVIATIONS USED

■

In Vivo Brain Microdialysis.⁵⁵Male Wistar rats (240−300 g body

weight) were stereotaxically implanted with a microdialysis guide

cannula in ventral hippocampus (AP: −5.2 mm, ML: +5.0 mm, DV:

−3.8 mm) under isoﬂurane anesthesia. Coordinates were taken

according to the atlas for rat brain with reference points taken from

bregma and vertical from the skull. The rats were allowed to recover

individually for 4 days in a round-bottom Plexiglas bowl with free

access to feed and water. After surgical recovery of 4−5 days, male

Wistar rats were connected to a dual quartz lined two-channel liquid

swivel (Instech, UK) on a counter balance lever arm, which allowed

unrestricted movements of the animal. Sixteen hours before the study,

a pre-equilibrated microdialysis probe (4 mm dialysis membrane) was

inserted into the ventral hippocampus through the guide cannula. On

the day of study, a probe was perfused with artiﬁcial cerebrospinal ﬂuid

(aCSF; NaCl 147 mM, KCl 3 mM, MgCl₂1 mM, CaCl₂·2H₂O 1.3

mM, NaH₂PO₄·2H₂O 0.2 mM and Na₂HPO₄·7H₂O 1 mM, pH 7.2) at

a ﬂow rate of 1.5 μL/min, and a stabilization period of 2 h was

maintained. Five basal samples were collected at 20 min intervals prior

to the treatment of compound 5al (1 or 3 mg/kg p.o.) or vehicle. For

studies involving combination, donepezil (1 mg/kg s.c.) and

memantine (1 mg/kg s.c.) were administered 30 min after

administration of compound 5al. Dialysates were collected for an

additional period of 6 h post-treatment (4 h post-treatment for studies

involving combination) of compound 5al, and dialysates were stored

below −50 °C until quantiﬁcation of acetylcholine by LC-MS/MS.

Concentrations of acetylcholine were converted as percent change

from mean basal acetylcholine concentrations with 100% deﬁned as

the average of ﬁve predose values. The percent changes in

acetylcholine levels were compared with vehicle. In studies involving

combination, percent changes in acetylcholine levels after combination

of compound 5al, donepezil, and memantine were compared with the

donepezil and memantine combination using two-way analysis of

5-HT₆R, 5-hydroxytryptamine 6 receptor; AD, alzheimer’s

disease; AChEI, acetylcholinesterase inhibitors; NMDA, N-

methyl-^D-aspertate; Ach, acetylcholine; AChE, acetylcholines-

terase enzyme; GPCR, G-protein coupled receptor; SAR,

structure−activity relationships; Compd. No, compound

number; Boc, tert-butyloxycarbonyl; HEK293, human embry-

onic kidney; CHO cells, Chinese hamster ovary cells; CNS,

central nervous system; ORT, object recognition task; ADME,

absorption, distribution, metabolism, and excretion; ECG,

electrocardiogram; BCS, biopharmaceutics classiﬁcation sys-

tem; DSC, diﬀerential scanning calorimetry; HLM, human liver

microsomes

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DOI: 10.1021/acs.jmedchem.6b01662

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery and Development of 1-[(2-Bromophenyl)sulfonyl]-5-methoxy-3-[(4-methyl-1-piperazinyl)methyl]-1H-indole Dimesylate Monohydrate (SUVN-502): A Novel, Potent, Selective and Orally Active Serotonin 6 (5-HT6) Receptor Antagonist for Potential Treatment of Alzheimer’s Disease

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