Discovery and Preclinical Characterization of Usmarapride (SUVN-D4010): A Potent, Selective 5-HT4Receptor Partial Agonist for the Treatment of Cognitive Deficits Associated with Alzheimer's Disease

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Drug Annotation

Discovery and Preclinical Characterization of Usmarapride (SUVN-

D4010): A Potent, Selective 5‑HT Receptor Partial Agonist for the

4

Treatment of Cognitive De ﬁ cits Associated with Alzheimer ’s Disease

Ramakrishna Nirogi,* Abdul Rasheed Mohammed, Anil Karbhari Shinde, Shankar Reddy Gagginapally,

Durga Malleshwari Kancharla, Srinivasa Rao Ravella, Narsimha Bogaraju, Vanaja Reddy Middekadi,

Ramkumar Subramanian, Raghava Choudary Palacharla, Vijay Benade, Nageswararao Muddana,

Renny Abraham, Rajesh Babu Medapati, Jagadeesh Babu Thentu, Venkat Reddy Mekala, Surendra Petlu,

Bujji Babu Lingavarapu, Sivasekhar Yarra, Narendra Kagita, Vinod Kumar Goyal, Santosh Kumar Pandey,

and Venkat Jasti

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sı Supporting Information

ABSTRACT: A series of oxadiazole derivatives were synthesized

and evaluated as 5-hydroxytryptamine-4 receptor (5-HT R) partial

4

agonists for the treatment of cognitive deﬁcits associated with

Alzheimer’s disease. Starting from a reported 5-HT R antagonist, a

4

systematic structure−activity relationship was conducted, which

led to the discovery of potent and selective 5-HT R partial agonist

4

1

-isopropyl-3-{5-[1-(3-methoxypropyl) piperidin-4-yl]-[1,3,4]-

oxadiazol-2-yl}-1H-indazole oxalate (Usmarapride, 12l). It showed

balanced physicochemical−pharmacokinetic properties with robust

nonclinical eﬃcacy in cognition models. It also showed disease-

modifying potential, as it increased neuroprotective soluble

amyloid precursor protein alpha levels, and dose-dependent target

engagement and correlation of eﬃcacy with oral exposures. Phase

1

clinical studies have been completed and projected eﬃcacious concentration was achieved without any major safety concerns.

Phase 2 enabling long-term safety studies have been completed with no concerns for further development.

6

INTRODUCTION

gastroparesis. The 5-HT receptor is present at a high density

4

■

in brain regions like the hippocampus, amygdala, and cerebral

cortex, suggesting the possible involvement of this receptor in

cognitive processes. The 5-HT R agonists modulate amyloid

precursor protein (APP)-derived peptides, amyloid-beta (Aβ),

and soluble amyloid precursor protein alpha (sAPPα). The

sAPPα is a soluble nonamyloidogenic protein, and it is

reported to have a potent neuroprotective role against the

neurotoxic eﬀects of glutamate and β-amyloid. Human 5-

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

degenerative disorder, aﬀecting mostly the aging population.

AD is characterized by the presence of Aβ plaques and

pathologic tau deposits with clinical symptoms like confusion,

memory loss, and dementia. Dysfunction of acetylcholine

containing neurons in the brain contributes substantially to the

symptoms. The current pharmacotherapy provides only

symptomatic relief through augmentation of cholinergic

function with several undesired side eﬀects such as nausea,

vomiting, and bradycardia. Moreover, no disease-modifying

therapy is available to the patients to date. Thus, there is a

need to explore new therapies through diﬀerent modes of

7

4

8

9

HT R isoforms are positively coupled to adenylyl cyclase

4

production. Thus, activation of this G-protein coupled receptor

activates adenylyl cyclase, consequently increasing the

production of cyclic adenosine monophosphate (cAMP).

action. 5-Hydroxytryptamine-4 receptor (5-HT R) is one of

4

such targets which have attracted the attention of scientiﬁc

1

Received: April 17, 2021

community. The 5-HT R belongs to a superfamily of seven

4

transmembrane G-protein coupled receptors (GPCRs) and is

2

coupled to a G-protein containing Gαs subunit. The 5-HT R

4

is reported to have a potential role in many central nervous

3

,4

system (CNS) disorders such as AD

and peripherally

5

mediated disorders such as irritable bowel syndrome and

©

XXXX American Chemical Society

https://doi.org/10.1021/acs.jmedchem.1c00703

A

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Drug Annotation

Figure 1. Reported 5-HT R ligands along with the current series of compounds.

4

The increased cAMP, in turn, increases sAPPα release. The

Previous work from our group disclosed a series of imidazo-

[1,5-a]pyridine and pyrazolo[1,5-a]pyridine derivatives

2

4

27

stimulatory eﬀect of 5-HT R on sAPPα release is mimicked by

4

forskolin, a direct activator of adenylyl cyclase, and 8-bromo-

cAMP (membrane-permeant cAMP analog), suggesting the

involvement of adenylyl cyclase and cAMP in modulating

represented by compounds 5a and 4o (Figure 1), respectively,

as 5-HT R partial agonists with procognitive eﬀects in animal

4

models. While selected compounds from these series are at

various stages of development, we focused on earlier reported

9

sAPPα by 5-HT R. The 5-HT R also increases the neuronal

4

10

28

acetylcholine (ACh) levels in the brain. However, prolonged

exposure of GPCRs to a full agonist can result in receptor

desensitization, thereby limiting the compound’s therapeutic

usefulness. Partial agonists have shown to minimize the

potential for desensitization while retaining the desired

indazole derivatives represented by compound 11ab (Figure

2). Compound 11ab was reported as a 5-HT R antagonist, and

4

its structure can also be envisaged from 5-HT R partial agonist

4

24

5a based on the structural similarity (Figure 2). We aimed to

identify a structurally diverse 5-HT R partial agonist as a lead

4

1

activity. Thus, 5-HT R partial agonists may be of use for

compound with an adequate brain penetration, acceptable

pharmacokinetics, robust nonclinical eﬃcacy, and suﬃcient

safety margin for the treatment of AD and associated disorders.

4

both disease modifying and symptomatic treatment of

12

cognitive disorders associated with AD. Several structurally

13−18

diverse heteroaromatic derivatives

as 5-HT R agonists/

Selective 5-HT R partial agonists are reported to have the

4

partial agonists/antagonists have been explored for CNS and

potential to modulate the centrally located 5-HT receptors

4

peripheral indications. The 5-HT R partial agonist PRX-03140

while showing minimal eﬀects on the gastrointestinal system

4

showed signiﬁcant improvement in cognition assessed by the

Alzheimer’s Disease Assessment Scale-Cognition (ADAS-Cog)

through peripheral 5-HT R. Activation of peripheral 5-HT R is

implicated in modulating gastric intestinal motility and cardiac

function. The structural modiﬁcations were done keeping in

4

19,20

score in a phase 2a study for AD.

Apart from this

compound, several other 5-HT R compounds such as BIMU-

mind the 5-HT R pharmacophore requirements such as an

4

2

1

22

11

23

1

,

RS-67333, prucalopride, capeserod (SL65.0155),

aromatic moiety, a coplanar carbonyl group, or its bioisosteres

like oxadiazole, and a voluminous substituent in the basic

13

and PF-04995274 are in development (Figure 1).

2

9,30

In our continuous eﬀorts of discovering novel small

amino framework of the molecule.

The basic amino

28

molecules for the potential treatment of AD with a diverse

framework part of reported indazole derivative 11ab was

modiﬁed appropriately by implementing earlier understand-

24−27

mechanism of actions,

the 5-HT R agonists attracted our

4

24

24,27

attention as they have a dual mechanism of action in treating

ings. The well-optimized

N-isopropyl group on indazole

1

0

AD and availability of positive proof-of-concept phase 2

clinical results of PRX-03140. Most of the reported 5-HT₄R

ligands which have been explored for both CNS and peripheral

indications belong to benzamide, imidazole, imidazopyridine,

pyrazolopyridine, 2-oxoquinoline, and indazole derivatives.

nitrogen was kept constant throughout the structure−activity

relationship (SAR) (Figure 2). Modiﬁcations in the basic

amino framework led to chemical scaﬀolds showing desired

partial agonistic activity at 5-HT R. The successive mod-

4

iﬁcations led to 1-isopropylindazole-carboxamide derivatives

B

https://doi.org/10.1021/acs.jmedchem.1c00703

J. Med. Chem. XXXX, XXX, XXX−XXX

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Drug Annotation

Figure 2. Genesis of ligands.

a

(

7a−7ai), 1-isopropylindazole-1,3,4-oxadiazole derivatives

12a−12ad), and 1-isopropylindazole-1,2,4-oxadiazole deriva-

Scheme 1. Synthesis of Compounds 7a−7ai

tives (17a−17c and 23a−23c). The SAR covering in vitro

binding aﬃnity, hERG aﬃnity, metabolic stability, CYP

inhibition, pharmacokinetics, receptor occupancy, and in vivo

eﬃcacy resulted in clinical candidate 1-isopropyl-3-{5-[1-(3-

methoxypropyl) piperidin-4-yl]-[1,3,4]oxadiazol-2-yl}-1H-in-

dazole oxalate (12l, Usmarapride). Compound 12l has

completed phase 1 clinical studies and achieved projected

eﬃcacious concentrations within the tested doses. Phase 2

enabling nonclinical safety studies for compound 12l in rats

and dogs were completed with no concerns for further

development.

Chemistry. The general synthetic strategy used for the

preparation of the present series of compounds is summarized

in Schemes 1−4. The starting material indazole-3-carboxylic

acid was sourced from a commercial vendor, and other

building blocks were synthesized as per the earlier

2

4,27,31,32

reported

protocols. Synthesis of compounds 7

commenced (Scheme 1) with indazole-3-carboxylic acid 1

a

t

Reagents and conditions: (a) NaO Bu, 2-iodopropane, DMF, r.t.,

16.0 h, 60%. (b) SOCl , 1,2-dichloroethane, 0 °C-reﬂux, 1.0 h, 100%.

c) K CO , Boc protected amine 4a−4h, DCM, 0 °C-r.t., 16.0 h. (d)

which on N-isopropylation reaction with 2-iodopropane in the

t

2

presence of NaO Bu aﬀorded N-isopropylindazole-3-carboxylic

(

2

3

acid 2 as a single isomer. The carboxylic acid 2 was then

converted to acid chloride 3 by treating it with thionyl

chloride. The acid chloride 3 was then reacted with diﬀerent

N-Boc protected cyclic amines 4 in the presence of K CO to

TFA, DCM, 0 °C-r.t., 16.0 h, H O, NaHCO . (e) K CO , acetonitrile,

2

3

2

3

R−X, r.t.-reﬂux, 7.0−16.0 h.

2

3

aﬀord N-Boc protected carboxamide derivatives 5. Depro-

tection of Boc in compounds 5 in the presence of

triﬂuoroacetic acid followed by basiﬁcation with NaHCO₃

yielded compounds 6. The N-alkylation of compounds 6 by

C

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J. Med. Chem. XXXX, XXX, XXX−XXX

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Drug Annotation

a

Scheme 2. Synthesis of Compounds 12a−12ad

a

Reagents and conditions: (a) Boc O, Et N, DMAP, DCM, 0 °C-r.t., 16 h. (b) Hydrazine-hydrate, MeOH, r.t.-reﬂux, 10.0 h. (c) (i) DCM, acid

2

3

chlroride 3, 0 °C-r.t., 4.0 h; (ii) H O, NaHCO , 2 h. (d) SOCl , 1,2-dichloroethane, r.t.-reﬂux, 6.0−10.0 h. (e) K CO , acetonitrile, R−X, r.t-reﬂux,

7

2

3

2

3

.0−16.0 h.

a

reacting with alkyl halides yielded N-alkylated carboxamide

derivatives 7.

Scheme 3. Synthesis of Compounds 17a−17c

The 1,3,4-oxadiazole derivatives 12 were prepared as

mentioned in Scheme 2. The hydrazides 9 were prepared

from the corresponding esters 8 by reﬂuxing with hydrazine in

methanol. The esters 8 were either sourced commercially or

prepared by reported procedures. The hydrazides 9 were then

reacted with acid chloride 3 in DCM to aﬀord compounds 10

after basiﬁcation with NaHCO . Cyclization of compounds 10

3

with thionyl chloride in 1,2-dichloroethane at reﬂux temper-

ature aﬀorded compounds 11 with concomitant Boc

deprotection. The N-alkylation of compounds 11 with

appropriate alkyl halides in the presence of K CO aﬀorded

2

3

1

,3,4-oxadiazole derivatives 12.

The 1,2,4-oxadiazoles 17 were prepared as mentioned in

a

Scheme 3. Commercially available 4-cyanopiperidine 13 was

alkylated using a standard N-alkylation procedure to obtain

compounds 14. Compounds 14 on treatment with hydroxyl-

amine in ethanol in the presence of K CO aﬀorded

Reagents and conditions: (a) K CO , R−X, acetonitrile, r.t-reﬂux,

2

3

7.0−16.0 h. (b) NH -OH·HCl, K CO , EtOH, r.t.-reﬂux, 16.0 h. (c)

SOCl , CH OH, 0 °C-r.t., 6.0 h. (d) NaH, DMF, 120 °C 0.5 h.

2

3

2

3

2

3

compounds 15. The N-isopropylindazole-3-carboxylic acid 2

was converted to its methyl ester 16 by reacting with methanol

in the presence of dry HCl. The desired 1,2,4-oxadiazole

derivatives 17 were obtained upon treatment of compounds 15

with ester 16 in DMF in the presence of NaH.

alkylated ith appropriate alkyl halides using a standard N-

alkylation procedure to obtain compounds 22a−22c. The

desired 1,2,4-oxadiazole derivatives 23a−23c were obtained

upon treatment of compounds 22 with compound 20 in DMF

in the presence of NaH.

1

,2,4-Oxadiazoles 23 were prepared as mentioned in Scheme

RESULTS AND DISCUSSION

4

. The N-isopropyl-indazole-3-carboxylic acid 2 was converted

to N-isopropyl-indazole-3-carboxamide 18 with Boc anhydride

and NH HCO . The carboxamide derivative 18 was subjected

to dehydration using TFAA and triethylamine to aﬀord nitrile

9. The commercially available ethyl isonipecotate 21 was

■

The newly synthesized compounds were screened in the cell-

based luciferase reporter gene assay that measures the levels of

cAMP inside cells upon activation or inhibition of the receptor,

the results of which are shown in Table 1. Among the ﬁrst

4

3

1

D

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Drug Annotation

a

Scheme 4. Synthesis of Compounds 23a−23c

albeit with weak agonist activity (E_m_a_x< 20%). The bicyclic 3-

aza-bicyclo[3.1.0]hexane group was replaced with 8-aza-

bicyclo[3.2.1]octan (4h) which resulted in potent compounds

(

7aa−7ai) with EC values ranging from 3 nM to 62 nM and

5

0

the agonist activity ranging from partial (E , 64%) to full

max

agonist (E_m_a_x, 98%). Noteworthy in these compounds is

barring branched N-alkyl derivatives (7aa, 7ad, 7ae, and 7ai),

all other compounds showed <10 nM EC₅₀values with >50%

agonist activity. Compound 7ac showing a EC value of 3 nM

5

0

with 66% E_m_a_xvalue stands out to be the most potent partial

agonist from the indazole 3-carboxamide derivatives synthe-

sized.

Bioisosteres have been explored in the past to improve

potency, enhance selectivity, and optimize pharmacokinetic

(

PK) proﬁles or alter toxicophores in addition to being a

33

source of novel intellectual property. To have compounds

with improved potency and favorable pharmacokinetics, the

symmetrical 1,3,4-oxadiazole and asymmetrical 1,2,4-oxadia-

34

a

zole derivatives were envisaged as carboxamide bioisosteres

(Table 2). Compound 12a without any alkyl substitution on

piperidine nitrogen showed very weak activity with EC₅₀of

1507 nM, however; it retained partial agonist activity showing

an E_m_a_xvalue of 43% (Table 2). The alkyl-substituted

derivatives 12b−12i showed EC values between 23 nM

Reagents and conditions: (a) Boc O, pyridine, NH HCO ,

2

4

3

acetonitrile, r.t., 16.0 h, 83%. (b) TFAA, Et N, THF, 0 °C-r.t., 2.0

h, 86%. (c) NH -OH·HCl, K CO , EtOH, r.t.-reﬂux, 16.0 h, 100%.

3

2

3

(

1

d) K CO , R−X, acetonitrile, r.t-reﬂux, 7.0−16.0 h. (e) NaH, DMF,

2

3

20 °C, 16.0 h.

5

0

and 199 nM with moderate E values ranging from 28% to

prepared 1-isopropylindazole-carboxamide derivatives, 7a−7c,

consisting of aminopiperidine 4a, 1-hydroxypropyl derivative

max

5

6%. Among the lot, the isopropyl derivative 12b and

^p^h^eⁿ^e^t^h^y^l^d^e^rⁱ^v^a^tⁱ^v^e¹²^d^s^h^o^w^e^d^p^o^t^eⁿ^t^E^C50 values of 23

nM and 24 nM, respectively. The ﬂuoroethyl derivative 12j

^s^h^o^w^e^d^v^e^r^y^w^e^a^k^a^c^tⁱ^vⁱ^t^y^wⁱ^t^h^aⁿ^E^C50 value of 1814 nM, as

against its carboxamide counterpart 7c which showed a potent

^E^C50 value of 5.8 nM. The other heteroalkyl derivatives 12k−

7

a and 1-ﬂuoroethyl derivative 7c showed potent in vitro

eﬃcacies (EC ) of 6.4 and 5.8 nM with intrinsic activity

5

0

(E_m_a_x) values of 60 and 71%, respectively. Compound 7b with

a 1-hydroxyethyl group showed a modest EC₅₀of 38 nM and

E_m_a_xof 39%. The observed partial agonist activity was in

contrast with the antagonist activity reported for structurally

28

1

2t showed moderate EC values ranging between 22 nM and

50

28

similar types of indazole derivatives (11ab) with bigger alkyl

groups on the piperidine ring (Figure 2). Encouraged by these

ﬁndings, additional analogs (7d−7g) with branched alkyl

groups with ﬂuoro, hydroxy, or methoxy group substitutions

were synthesized and evaluated. These compounds were 5−7-

fold less potent as compared to normal alkyl group derivatives

172 nM. These compounds showed a weak agonist activity

ranging from (Emax, 17%) to moderate (Emax, 53%) partial

agonist activity. The heteroalicyclic-substituted derivatives 12o

and 12p showed EC50 values of 79 nM and 84 nM,

respectively, whereas compound 12u having a methylpyranyl

group with junction hydroxy showed a weak EC50 value of 210

nM. The higher EC50 value for compound 12u is in sharp

contrast to the corresponding carboxamide derivative 7j with

the same substitution that showed a potent EC50 value of 15

nM. Compound 12v showed very weak in vitro potency with

an EC50 value of 675 nM with a hydroxy group on the junction

of piperidine. The introduction of an additional methylene

group between 1,3,4-oxadiazole and piperidine resulted in

compounds 12w−12y with weak in vitro potency. The bicyclic

3-aza-bicyclo[3.1.0]hexane 1,3,4-oxadiazole derivatives 12z−

12ab turned out to be inactive. The similar carboxamide

derivative 7v was potent albeit with weak partial agonist

activity as compared to inactive 1,3,4-oxadiazole derivative

12ab. The tropane derivatives 12ac and 12ad showed weak

potency with an EC₅₀value of 695 nM and 1060 nM

respectively.

The 1,2,4-oxadiazole derivatives were made to compare their

activity with that of 1,3,4-oxadiazole counterparts. The 1,2,4-

oxadiazole derivative 17a having a methoxypropyl group on

the piperidine ring which is at the 5-position of oxadiazole

showed an EC₅₀value of 156 nM which is ∼3-fold less than

that of 1,3,4-oxadiazole derivative 12l. However, the other

synthesized derivatives 17b and 17c showed 2−4-fold more

potency than their corresponding 1,3,4-oxadiazole deriva-

tives12p and 12b respectively. On the other hand, 1,2,4-

(

7a−7c). Compounds with cyclic ethers such as pyran,

methylpyranyl groups (7h−7k) showed 2−3-fold more

potency than the branched alkyl-substituted compounds

(7d−7g) but were 2−4-fold less potent than the alkyl-

substituted derivatives (7a and 7c). Overall, the straight

alkoxyalkyl or ﬂuoroalkyl-substituted piperidine derivatives

were more potent than other branched alkyl or oxy-cyclic-

substituted piperidine derivatives. In the next change in the

compounds, a methylene linker was introduced between

piperidine and indazolyl-3-carboxamide moiety. The methyl-

piperidine derivatives 7l and 7n consisting of aminopiperidine

4

b showed very weak activity with higher EC and lower E

50 max

(

∼10%) values, indicating the linker was not tolerated.

Introducing hydroxy and ﬂuoro groups at the fourth position

of the piperidine ring (aminopiperidine 4c and 4d) yielded

compounds 7o−7q. Though this modiﬁcation resulted in

compounds with potent EC₅₀values (<50 nM), the E was

max

on the lower side as compared to 7a and 7c. The

methylpiperidine was then replaced with morpholine methyl-

amine (4e) derivative 7r with an EC₅₀value of 3906 nM.

Introducing the bicyclic 3-aza-bicyclo[3.1.0]hexane ring in

place of piperidine resulted in less potent derivatives 7s−7u.

Bringing the methylene linker in 3-aza-bicyclo[3.1.0]hexane

group (4g) dramatically increased the potency of the

synthesized compounds (7v−7z) with EC₅₀values <12 nM,

oxadiazole derivative 23a with an EC value of 21 nM showed

50

E

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Drug Annotation

a

Table 1. 5-HT R Functional Activity of Indazole Carboxamide Derivatives 7a−7ai

4

a

Data represent mean ± SD. Functional activity determined in CHO cells stably expressing the human 5HT₄_Eisoform and a reporter gene was

used. EC₅₀values were determined from average of two independent experiments tested in duplicate. The in-house data are consistent with

F

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Drug Annotation

Table 1. continued

reported values of PRX-03140 (17−52 nM/30−61%), PF-04995274 (0.26 nM/7%), and prucalopride (26 nM/52%) (Supporting Information);

NA, not applicable.

2

-fold more potency than 1,3,4-oxadiazole derivative 12l. The

Evaluation of the compound’s potential to inhibit a speciﬁc

P450 enzyme is important, as coadministration of compounds

may result in one or both inhibiting the other’s metabolism.

This may aﬀect the compound’s plasma exposures and

potentially lead to adverse drug−drug interactions (DDI) or

toxicity. The CYP enzyme inhibition liability of the selected

compounds is remote, as most of them showed IC₅₀values of

>45 μM. The TDI of CYP enzyme refers to a change in

compound’s potency during an in vitro incubation or in vivo

dosing period. Potential mechanisms include the formation of

inhibitory metabolites and mechanism-based inhibition. TDI is

of particular concern as it is irreversible, and typically de novo

synthesis of the enzyme is required to restore the activity. The

consequences of TDI can be drug withdrawal or serious

restrictions of use. Gratifyingly, the tested compounds either

showed minimal or no TDI against CYP3A4 enzyme, therefore

discharging them from the potential for DDI liability in a

clinical setup. There is a literature precedent that disclosed the

other synthesized derivatives 23b and 23c from this series

showed a weaker potency with EC values of 106 and 297 nM,

respectively.

50

Several in vitro potent compounds which represent structural

diversity during the SAR were further evaluated for their rat

and human liver microsomal metabolic stability, metabolizing

enzymes cytochrome P450 (CYP) 3A4 and 2D6 inhibition,

time-dependent inhibition (TDI) for CYP 3A4 enzyme, and

the human ether-a-go-go related gene (hERG) inhibition and

in vivo rat pharmacokinetics, bioavailability, and brain

penetration (Table 3).

A compound’s metabolic stability is an important parameter

to consider during the early stages of SAR. The metabolic

stability of the compound is its susceptibility to biotransfor-

mation, which helps in predicting the compound’s PK

properties which in turn helps in designing compounds with

favorable PK properties. The metabolic stability results are

usually reported as measures of intrinsic clearance, from which

secondary PK parameters such as bioavailability and half-life

can be calculated if the data on the volume of distribution and

fraction absorbed are available. Since these parameters are very

important in deﬁning the PK and toxicological proﬁle of the

compound, the researchers need to give more emphasis on the

metabolic stability during SAR optimization. The metabolic

stability of the compounds reported here both in rat and

human liver microsomes was found to be low (∼5%) to high

cardiovascular adverse events associated with 5-HT R agonists.

4

For instance, Cisapride, a 5-HT R agonist, was withdrawn

4

from the market due to the risk of fatal cardiac

35−37

arrhythmias.

The hERG aﬃnity may have detrimental

eﬀects on cardiac safety which prompted us to identify 5-HT₄R

agonists having minimal hERG liability. Compounds 7d and

12k showed hERG IC₅₀values >10 μM, compounds 12p and

12l showed IC values of 8.5 μM and 3.0 μM respectively, and

5

0

compound 12h showed an IC₅₀of 2.4 μM. Compounds 7d,

12h, 12k, 12l, and 12p, based on their moderate to good

metabolic stability, good in vivo exposures in rats, and modest

aﬃnity for hERG channel, were evaluated in the object

recognition test (ORT), long-term memory deﬁcit assay. The

(

>70%). The carboxamide derivatives showed suboptimal

ADME properties, as given in Table 3. The carboxamides 7c

with ﬂuoroethyl group 7f, 7ad with a branched hydroxy-alkyl

group, and 7ag, 7ah with a pyranyl-methyl group on piperidine

nitrogen were extensively metabolized in rat liver microsomes;

whereas, in human liver microsomes, the metabolism was low

to moderate. The high metabolism of these compounds in rat

liver microsomes is very well reﬂected in their poor in vivo

exposures and bioavailability in rats for the tested analogs 7c,

38,39

ORT

is regarded as a spontaneous delayed nonmatching-

to-sample (DNMS) test. The test is based on spontaneous

behavior. The main assumption of this test is that access to

novelty (object or environment) can elicit approach behaviors

in animals. The “unconditioned preference” of animals for

novelty has been used in the ORT to study memory functions,

assessing the ability of animals to recognize a novel object in a

familiar environment. ORT does not involve reference memory

components, thus it can be considered as a “pure” recognition

memory test and a valid task to assess working memory.

Additionally, ORT does not involve positive or negative

reinforces (food or electric shocks), and this makes it

comparable to memory tests currently used in the clinic.

These advantages make the ORT eﬃcacy model, quick, and

simple.Thus, the ORT model has been widely used for

assessing procognitive eﬀects in preclinical models. In this set

of experiments, the rats were treated with compounds 7d, 12h,

7

f, and 7ah. Low to moderate microsomal metabolism

observed with the select carboxamide derivatives such as 7d,

v, 7w, and 7x is well correlated with modest oral exposure

7

and bioavailability in rats. Compound 7o, however, was the

exception, as its good metabolic stability both in rat and

human microsomes did not translate into good in vivo

exposures and bioavailability in rats.

The 1,3,4-oxadiazole derivatives 12h, 12l, and 12p showed

good microsomal stability both in rats and humans that

translated into good oral exposures, good bioavailability, and

good brain exposures in rats. The 1,2,4-oxadiazole derivatives

1

7c and 23a showed poor rat microsomal metabolic stability.

Rat PK data for 17c indicated poor oral exposures and

bioavailability, but that was not the case for 23a. The 1,2,4-

oxadiazole derivative 23a showed moderate oral exposures and

bioavailability. Good brain penetration property is essential for

the compounds intended to treat brain disorders. Most of the

compounds such as 7d, 12d, 12f, 12h, 12l, 12p, and 23a

showed very good brain penetration properties. Selected

compounds were further evaluated for their CYP and hERG

inhibiting properties. Cytochrome P450 (P450) is a family of

enzymes that play a major role in the metabolism of drugs.

12k, 12l, and 12p which also included reported 5-HT R partial

4

22

agonist RS-67333 as a positive control (Figure 3). The

reference compound, RS-67333, evaluated at 0.3, 1.0, and 3.0

mg/kg i.p. showed statistically signiﬁcant activity at 0.3 and 1.0

mg/kg, and the eﬀects faded at 3.0 mg/kg dose, which is in line

with literature reports. Compound 7d showed activity only at

10 mg/kg p.o. dose. Compound 12h showed activity at all

tested doses; however, procognitive activity reduced as the

dose increased. Compound 12k did not show activity at any of

the tested doses. Compound 12l showed activity at 1 and 3

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Drug Annotation

a

Table 2. 5-HT R Functional Activity of Indazole Carboxamide Derivatives 12a−12ad, 17a−17c, 23a−23c

4

a

Data represent mean ± SD. Functional activity determined in CHO cells stably expressing the human 5HT₄_Eisoform, and a reporter gene was

used. EC₅₀values were determined from average of two independent experiments tested in duplicate. The in-house data are consistent with

reported values of PRX-03140 (17−52 nM/30−61%), PF-04995274 (0.26 nM/7%), and prucalopride (26 nM/52%) (Supporting Information);

NA, not applicable.

H

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Drug Annotation

I

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Drug Annotation

Figure 3. Eﬀect of compounds 7d, 12h, 12k, 12l, 12p, and RS-67333 on attenuation of long-term memory deﬁcits in ORT. Data represent mean ±

SEM of exploration time in seconds; (N = 7−8); *p < 0.05, **p < 0.01, ***p < 0.001. Paired−t-test comparison of novel vs familiar object.

mg/kg p.o. doses. Compound 12p showed activity at 3 and 10

mg/kg p.o. doses. Compound 12k did not show activity

probably due to its poor brain penetration and lower plasma

exposures. The diﬀerence in the eﬃcacy for the other tested

compounds could be due to diﬀerences in the free fractions

which could have led to a diﬀerence in receptor occupancy

thereby a diﬀerence in the ORT.

reversed the scopolamine-induced amnesia at the tested

doses of 1, 3, and 10 mg/kg p.o. (Figure 4).

In order to see correlation between dose and eﬃcacy,

compound 12l was evaluated in ORT at doses 0.03, 0.1, 0.3,

1.0, 3.0, and 10.0 mg/kg p.o. In this study, compound 12l did

not show activity at 0.03 and 0.1 mg/kg, however did show

signiﬁcant eﬃcacy from 0.3 to 3.0 mg/kg. The eﬃcacy of

compound 12l in ORT assay at doses of 0.1−10 mg/kg, p.o.

along with the recognition index is depicted in Figure 5A,B

(exploration time and recognition index, respectively).

Compound 12l showed a statistically signiﬁcant eﬀect at 3.0

mg/kg on both exploration time and recognition index.

An inverted U-shaped dose−response curve has been

observed with procognitive compounds and approved

Based on eﬃcacy observed in ORT assay and good PK

properties, compounds 12h and 12l were shortlisted for further

evaluation. Compounds 12h and 12l were evaluated in another

40

cognitive assay, the radial arm maze task (RAM). The RAM

is a popular test of working memory in rodents. RAM exploits

the natural tendency of rodents to explore, learn, and

remember diﬀerent locations of food reinforcement. RAM in

rodents requires rats to explore a series of visually identical

arms arranged around a central start location. Each arm will be

baited with a reward, and the rat is required to remember

which arms it has visited. A rat returning to a previously visited

arm is recorded as an error, and one can infer how the rat is

using various cues based on the pattern of navigation and

errors. In this assay, compound 12h at tested doses of 1, 3, and

41

procognitive drugs. The exact reason for such a phenomenon

is not quite well understood. One school of thought is that an

optimal level of acetylcholine is required for cognition. An

increase in acetylcholine levels beyond optimal levels did not

show improvement in cognition. One reason could be that

moderate arousal is beneﬁcial to cognition; whereas too much

42

activation leads to cognitive impairment. Another reason

could be due to an oﬀ-target activity at higher doses which may

make the rat uncomfortable. It is believed that in case there is

1

0 mg/kg p.o. did not reverse the scopolamine-induced

cognitive deﬁcits; whereas, compound 12l signiﬁcantly

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Drug Annotation

Figure 4. Eﬀect of compounds 12h and 12l in RAM. Data represent mean ± SEM of % choice accuracy or total error compared to vehicle (F₄_,₁₀₃

9.54 for choice accuracy and F₄_,₁₀₃= 14.81 for total error for compound 12h and F₄_,₁₀₅= 36.16 for choice accuracy and F₄_,₁₀₅= 32.88 for total

error for compound 12l); n = 7−8; *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001 vs scopolamine (ANOVA followed by Dunnett’s test).

=

1

an activation of presynaptic autoreceptors, it may result in

amyloidogenic to nonamyloidogenic form by generating

41

reducing the eﬃcacy at higher doses. This could be the

reason that 12l did not show eﬃcacy at 10 mg/kg dose.

To show the relationship between compound 12l eﬃcacy

sAPPα which is reported to provide neuroprotective

9

,46,47

eﬀects.

The 5-HT R-mediated regulation of sAPPα

4

secretion has been demonstrated in vitro using primary

48

and its target engagement at 5-HT R, it was evaluated for 5-

neuronal cultures and recombinant cells. It is reported that

the endogenous ligand serotonin (5-HT) enhances the level of

secreted sAPPα in a time and dose-dependent manner in

4

43

HT receptor occupancy (R.O.). Gratifyingly, compound 12l

4

showed signiﬁcant R.O. at tested doses ranging from 0.1 mg/

kg to 30 mg/kg. The absolute ED value was found to be 2.75

Chinese hamster ovary (CHO) cells stably expressing the h5-

50

9

mg/kg, p.o. Brain EC₅₀was found to be 184.8 ng/g, and

plasma EC₅₀was found to be 59.3 ng/mL (Figure 6). The

observed R.O. suggests that the in vivo eﬃcacy for this

compound may be mediated through 5-HT R. The 5-HT R

HT₄_Ereceptor isoform. It is also reported that the 5-HT R

4

agonists increase sAPPα levels in the cortex and hippocampus

of male C57BL/6j mice and the eﬀects were blocked with

46

selective 5-HT R antagonist GR-113808. In agreement with

4

partial agonist PF-04995274 showed dose-dependent R.O.

with ED₅₀value of 0.033 mg/kg, s.c. which was comparable

the published results, activation of the 5-HT₄_Ereceptor isoform

dose dependently increased expression levels of nonamyloido-

genic sAPPα in tsa201 cells transiently transfected with the 5-

HT₄_Ereceptor and APP695. At 10 μM concentration, 5-HT,

RS-67333, and compound 12l increased sAPPα secretion by

2.9 and 1.5 and 1.8 fold, respectively. The increase in sAPPα

secretion by 5-HT and compound 12l was completely blocked

by GR-113808 (Figure 7), suggesting compound 12l has

disease-modifying potential and the activity is arising from the

44

with the reported value (0.01 mg/kg, s.c.). Another 5-HT₄R

partial agonist RS-67333 showed an ED₅₀value of 15.15 mg/

kg, p.o. which was also in close agreement with the reported

1

1,43

value.

The in vivo eﬃcacy doses of compound 12l were

very well correlated with observed R.O. data. The eﬀective

doses (0.3, 1.0, and 3 mg/kg, p.o.) of 12l showed R.O. from

2

7% to 60% (Figure 6).

The 5-HT R agonists can shift the equilibrium of amyloid

selective activation of the 5-HT receptor. RS-67333 was not

subjected to blockade with GR-113808.

The protein binding experiment was done in rat, dog, and

human liver microsomes to ascertain the free fraction available

4

precursor protein (APP) processing from an amyloidogenic to

nonamyloidogenic form, showing disease-modifying poten-

4

5

tial. The APP processing shifts equilibrium from an

K

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Drug Annotation

Figure 5. (A) Attenuation of long-term memory deﬁcits in ORT by compound 12l. Data represent (mean ± SEM) of exploration time compared

to vehicle (n = 8−11/group). **p < 0.01, ***p < 0.001 (Paired−t-test comparison of novel vs familiar object). (B) Eﬀect of compound 12l on

attenuation of long-term memory deﬁcits in ORT assay. Data represent mean ± SEM of discrimination index. **P < 0.01 vs vehicle group (H

4.12; Kruskal−Wallis test followed by Dunn’s post hoc).

=

,74

7

1

for 12l. Compound 12l showed 11.3, 9.2, and 7.3% free

fraction in rat dog and human plasma, respectively. The free

fraction in rat brain homogenate for 12l was 5.9% (Table 4).

Compound 12l showed comparable potencies at tested 5-

HT R isoforms (5-HT , 5-HT , and 5-HT ) with EC

nonhygroscopic and crystalline oxalate salt with excellent water

solubility (17.1 mg/mL). The solubility of 12l in buﬀer

solutions from pH 2 to 7.0 is about 20−75 mg/mL at 37 °C.

The highest possible dose of 12l is soluble in 250 mL in the

entire pH range. Compound 12l demonstrated high

permeability in Caco-2 experiments at a tested concentration

4

4A

4D

4E

50

values ranging from 32 to 114 nM. Compound 12l showed

excellent selectivity against 5-HT receptors (5-HT , 5-HT ,

(10 μM) with an apparent permeability coeﬃcient (P ) of 38

1

A

2A

app

−

6

−1

5

-HT , 5-HT , 5-HT ), adrenergic receptors (Adα and

× 10 cm·s . The high solubility and high permeability

suggest that 12l belongs to class I of the Biopharmaceutics

Classiﬁcation System (BCS). Compound 12l showed an acid

3

6

7

1B

Adα ), adenosine receptor A , cannabinoid receptors (CB

2

C

2A

1

and CB ), dopamine (D , D , D , and D ), histamine (H and

2

1

2S

3

5

1

H ), muscarinic receptors (M , M , M , M , and M ), and

dissociation constant (pK ) value of 8.4 (Supporting

3

1

2

3

4

5

a

serotonin transporter (SERT). Compound 12l did not show

any signiﬁcant binding up to the highest tested concentration

of 10 μ M toward GPCRs, ion channels, and transporters (see

Supporting Information ). Compound 12l did not show any

agonist activity at the 5-HT receptor when evaluated in rat

fundus assay (See Supporting Information). Compound 12l

did not show any activity at GPCRs or ion channels other than

Information ).

Pharmacokinetics of compound 12l was assessed in male

beagle dogs (n = 3) at 1 mg/kg, p.o., and 1 mg/kg, i.v., doses.

Compound 12l showed rapid oral absorption (T_m_a_xin the

range of 0.5−0.75 h) with oral exposures (AUC = 711 ±

2B

0−24h

4

9

369 ng·h/mL) and showed longer terminal i.v. half-life (6.0 ±

1.5 h). The moderate intravenous clearance (18 ± 6 mL/min/

kg) and volume of distribution (5.1 ± 0.6 L/kg) indicate a

5

-HT R, which are implicated in cognition. Compound 12l is a

4

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Drug Annotation

Compound 12l was found to be nonmutagenic in all four

strains of Salmonella typhimurium TA1537, TA1535, TA98,

and TA100 and Escherichia coli WP2 uvrA both in the absence

and presence of metabolic activation. It was found to be

nonclastogenic in in vitro chromosomal aberration tests in

human peripheral blood lymphocytes and in in vivo micro-

nucleus assay done on mice bone marrow. These studies were

conducted per the standard protocols and in line with

regulatory guidance documents (OECD 471 and 474 and

ICH S2R1). The CYP enzyme inhibition, CYP3A4 TDI assay,

CYP induction, and transporter assays were performed as

50

described earlier. Compound 12l was not an inhibitor of

P450 enzymes with IC₅₀values >45 μM against major P450

isoforms. It did not show any TDI liability at CYP3A4 or CYP

induction liability at CYP1A2, CYP2B6, and CYP3A4.

Compound 12l was neither an inhibitor (IC₅₀> 100 μM)

nor substrate of OAT1, OAT3, OCT1, OCT2, OATP1B1,

OATP1B3 uptake transporters. The likelihood of drug−drug

interaction potential of compound 12l as an inhibitor/inducer

is remote.

Figure 6. 5-HT R occupancy of compound 12l and correlation with

plasma and brain concentrations. Data shown are the mean ± SEM (n

4

Compound 12l as a clinical candidate has completed

regulatory safety assessment studies in rats and dogs with

excellent safety margins which supported its progression to ﬁrst

in human phase 1 clinical studies including pharmacokinetics,

safety, and tolerability under US IND (ClinicalTrials.gov ,

=

4 −6 animals).

51

identiﬁers: NCT02575482 and NCT03031574). Compound

2l showed a favorable safety and PK proﬁle in a single

1

ascending dose in healthy male and multiple ascending doses

for 14 days in healthy male subjects. Gender, age, and food did

not have any signiﬁcant eﬀect on the pharmacokinetics. It was

well-tolerated in humans with adequate plasma exposures and

a favorable half-life for once-a-day dosing. Phase 2 enabling

long-term animal safety evaluation to support repeat dosing

has also been completed. Compound 12l will be evaluated in

phase 2 proof of concept studies in patients with cognitive

deﬁcits associated with AD in due course of time.

Figure 7. Eﬀect of compound 5-HT, 12l, and RS-67333 on sAPPα

secretion in the absence or presence of selective 5-HT R antagonist

4

GR-113808 by Western blot. Data represent mean of duplicates of

CONCLUSIONS

In summary, indazole-3-carboxamide, indazole 1,2,4-oxadia-

zole, and indazole 1,3,4-oxadiazole derivatives have been

fold over basal of sAPPα levels. Compound 5-HT and 5-HT receptor

4

■

partial agonist compound 12l and RS-67333 evoked stimulation of

sAPPα secretion in vitro were monitored using tsa201 cells transiently

transfected with human 5-HT₄_Ereceptor and human APP695.

synthesized and tested for their in vitro aﬃnity at 5-HT R.

4

The focused structure−activity relationships for in vitro aﬃnity

Table 4. In Vitro Plasma Protein Binding for Compound

at 5-HT R combined with microsomal metabolic stability, PK

a

4

12l

evaluation, eﬃcacy in cognitive models, target engagement,

%unbound

%bound

and safety assessments resulted in a safe, potent, and selective

a

5-HT R clinical candidate (12l, Usmarapride). Compound 12l

4

species

replicate 1 replicate 2 replicate 1 replicate 2 %bound

Plasma Binding

showed high selectivity against ganglionic 5-HT R and other

3

closely related receptors and good pharmacokinetics in rats,

dogs, and healthy humans. It also showed promising in vivo

eﬃcacy in animal models of cognition. Additionally, compound

human

beagle dog

Wistar rat

6.4

8.3

11.8

8.2

10.0

10.9

93.6

91.7

88.2

91.8

90.0

89.1

92.7

90.8

88.7

1

2l increased neuroprotective sAPPα levels and may possess

Brain Homogenate Binding

6.2 94.5

disease-modifying potential. Further, 12l showed a dose-

dependent receptor occupancy which correlates well with

eﬃcacy doses in tested cognition models. When evaluated in

safety models, 12l did not show any cardiotoxicity in dog

telemetry studies. Based on its robust eﬃcacy in cognition

models and a wide margin of safety in preclinical species, it was

evaluated in healthy human subjects in phase 1 clinical trials

for tolerability and PK properties. Compound 12l was found to

be well tolerated and showed good pharmacokinetics. Further

evaluation of compound 12l for its eﬃcacy in cognitive deﬁcits

associated with AD in a phase 2 proof-of-concept study is

being planned.

Wistar rat

5.5

93.8

94.1

a

Data represent mean, and experiment was done in duplicates.

higher tissue distribution. The observed absolute oral

bioavailability (%F) was 72 ± 11%. Compound 12l was

evaluated for cardiovascular toxicity using dog telemetry assay

as per ICH 7A guidelines. In this study, compound 12l was

administered orally to freely moving conscious male dogs.

Gratifyingly, it did not show any adverse eﬀect on QT interval

prolongation up to the tested dose of 24 mg/kg p.o.

M

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Drug Annotation

obtain crude carboxamide derivative, which was puriﬁed by silica gel

column chromatography with appropriate solvent system.

EXPERIMENTAL SECTION

■

Unless stated otherwise, all reagents, commercially available building

blocks, and solvents were purchased from common commercial

suppliers and were used without further puriﬁcation. Synthesized

compounds were puriﬁed wherever required by passing through 100−

00 mesh silica gel using appropriate solvent systems. H NMR

spectra were recorded at 400 MHz and C NMR at 100 MHz on a

Bruker NMR spectrometer instrument (Fallanden, Switzerland). All

HNMR shifts are reported in δ units (ppm) relative to the signals for

chloroform (7.27 ppm), DMSO (2.50 ppm), and MeOH (3.31 ppm).

All coupling constants (J values) are reported in hertz (Hz). NMR

abbreviations are as follows: bs, broadened singlet; s, singlet; d,

doublet; t,triplet; q, quartet; m, multiplet and dd, doublet of doublets.

Thin-layer chromatography (TLC) was performed on Merck silica gel

General Procedure 5 (Hydrazide Formation). To a stirred

solution of ester 8a−8f (1.0 mmol) in methanol (2.0 mL) at r.t.,

hydrazine hydrate (80% w/v, 3.0 mmol) was added. The reaction

mixture was heated to reﬂux for 4 h. An additional amount of

hydrazine (80% w/v, 3.0 mmol) was added, and the reaction was

heated until (6 h) all of the ester was consumed. Solvent methanol

was evaporated under reduced pressure, and the residue was dissolved

in DCM. The two layers were separated, the organic layer was dried

over anhydrous Na SO , and the solvent was evaporated under

1

2

1

3

1

2

4

vacuum to obtain hydrazide derivative (9a−9f), which was used in the

next reaction without further puriﬁcation.

General Procedure 6 (Acid Chloride-Hydrazide Coupling).

To a stirred solution of hydrazide 9a−9f (1.0 mmol) in DCM (2.0

mL) cooled at 0 °C, a solution of 1-isopropyl-1H-indazole-3-carbonyl

chloride 3 (1.1 mmol) in DCM (2.0 mL) was added over a period of

6

⁰^F²54 plates. Electrospray ionization mass spectra were recorded on

API 4000 triple quadrupole instrument (MDS-SCIEX, Concord,

Ontario, Canada). Infrared spectra were recorded on KBr disc and in

solid state using PerkinElmer model 1600 FT-IR spectrophotometer

5

min. The reaction was gradually warmed to r.t. and was stirred for 2

h. The progress of the reaction was monitored by TLC, which showed

an absence of hydrazide. The reaction mixture was then diluted with

water, and the two layers were separated. The aqueous layer was

washed with EtOAc, cooled to 0 °C, and cautiously basiﬁed to pH 7.6

(

PerkinElmer, Norwalk, CT, USA). Elemental analyses were carried

out in an “Elementar” GmbH, Vario microcube instrument, and the

analyses indicated by the symbols of the elements were within ±0.4%

of the theoretical values. Column chromatography was performed

using 100−200 mesh silica gel. The purity of all ﬁnal compounds was

established by HPLC (Agilant, Model-1100 series). All ﬁnal

compounds’ HPLC purity was found to be ≥95%. DSC was recorded

on Waters DSC Q100 instrument. Melting points of synthesized

compounds were determined using an Electro Derman open capillary

apparatus and are uncorrected. The reference standards PRX-03140,

RS-67333, PF-04995274, GR-113808, prucalopride, and others were

synthesized and characterized in-house using respective reported

procedures. The purity of all ﬁnal compounds was determined using

HPLC methods unless stated otherwise.

with 10% aqueous NaHCO solution. The basiﬁed aqueous layer was

3

then extracted with DCM. The combined organic layer was dried over

anhydrous Na SO , and the solvent was removed under reduced

2

4

pressure. The residue was then cooled to r.t. and diluted with hexane.

The slurry, thus obtained, was ﬁltered at r.t. under nitrogen

atmosphere, and the wet product cake was washed with hexanes to

obtain respective compounds 10a−10f in quantitative yields.

General Procedure 7 (1,3,4-Oxadiazole Formation). To a

stirred solution of compounds 10a−10f (1.0 mmol) in 1,2-

dichloroethane (4.0 mL) under nitrogen atmosphere at r.t., thionyl

chloride (2.0 mmol) was added over a period of 15 min. The reaction

temperature was then gradually raised, and the reaction mass was

reﬂuxed for 6−10 h until all starting material was consumed. The

excess thionyl chloride and solvent 1,2-dichloroethane were distilled

oﬀ under reduced pressure. The reaction mass was cooled to r.t. and

diluted with water and solvent ether. The resulting mass was stirred

for 15 min, and the two layers were separated. The pH of the aqueous

layer was adjusted to 9−10 by adding an aqueous NaOH (2.5 N).

The basiﬁed aqueous layer was then extracted multiple times with

DCM. The combined organic layer was washed with cold (5−10 °C)

aqueous NaOH (0.6 N) and dried over anhydrous Na SO , and the

General Procedure 1 (Boc Protection). To the solution of

amine/amide (1.0 mmol) in DCM (2.0 mL) stirred at room

temperature (r.t.), Boc anhydride (1.1 mmol), DMAP (0.05 mmol),

and triethylamine (1.5 mmol) were added. The reaction mixture was

stirred for 16 h at r.t. Upon completion of reaction, it was diluted with

EtOAc, washed with water and brine, and dried over anhydrous

Na SO . Volatiles were removed under reduced pressure to obtain the

2

4

crude product, which was triturated with hexanes to obtain titled

compound.

General Procedure 2 (Boc Deprotection). To a Boc protected

compound (1.0 mmol) in DCM (4.0 mL) under nitrogen at 0 °C,

TFA (5.0 mmol) was added. The reaction was gradually warmed to

r.t. and stirred for 16 h. The mixture was concentrated under vacuum

to obtain a crude product, which was basiﬁed with aqueous NaHCO₃

solution. The reaction mass was diluted with DCM (10 mL), and the

two layers were separated. The organic layer was dried over

anhydrous Na SO and concentrated under vacuum which aﬀorded

2

4

solvent was removed under reduced pressure to aﬀord 1,3,4-

oxadiazole derivatives 11a−11f.

General Procedure 8 (Salt formation). To the stirred solution

of N-alkylated carboxamide derivative/oxadiazole derivative (1.0

mmol) in isopropanol (8.0 mL), respective acid (1.0 mmol) was

added. The reaction mass was stirred for 1 h before the volatiles were

evaporated under reduced pressure. The crude salt was triturated with

copious amounts of ether and ethyl acetate to obtain the respective

salts. The salts were recrystallized from isopropanol or mixture of

solvents wherever required to obtain the desired purity.

2

4

amino compound.

General Procedure 3 (N-Alkylation). To the stirred solution of

amino compound (1.0 mmol) in acetonitrile (4.0 mL) at r.t., K CO

2

3

(

1.5 mmol) and alkyl halide (1.05 mmol) were added. The reaction

General Procedure 9 (Reductive Amination). To a stirred

mixture of carboxamide derivatives (1.0 mmol), aldehyde/ketone (1.5

mmol), and acetic acid (0.1 mL) in DCM (4.0 mL) cooled at 0 °C,

sodium triacetoxyborohydride (2.0 mmol) was added. The reaction

mass was gradually warmed to r.t and stirred for 16 h. The reaction

mixture was gradually heated to reﬂux (82−85 °C) for 7−16 h. After

completion of the reaction, volatiles were removed under reduced

pressure. The crude mass was diluted with DCM and water and

allowed to stir for 15 min, then two layers were separated. The

organic layer was washed with brine,and dried over anhydrous

Na SO , and volatiles were removed under pressure to get gummy

mass was diluted with saturated aqueous NaHCO

extracted with DCM. The combined organic layer was dried over

anhydrous Na SO , and the solvent was removed under reduced

solution and

3

2

4

mass of N-alkylated compound.

2

4

General Procedure 4 (Acid Chloride and Amine Coupling

Reaction). To a stirred solution of 1-isopropyl-1H-indazole-3-

carbonyl chloride (3, 1.0 mmol) in DCM (2.0 mL) under nitrogen

atmosphere at 0 °C, triethylamine (1.5 mmol) and a solution of amine

pressure. The crude product was puriﬁed by silica gel column

chromatography to obtain the N-alkylated derivatives.

1-Isopropyl-lH-indazole-3-carboxylic Acid (2). To the stirred

DMF (50.0 mL) under nitrogen atmosphere at r.t., sodium tert-

butoxide (6.0 g, 62.43 mmol) was added over a period of 15 min. The

reaction mixture was stirred for 10 min after which it was cooled to 0

to 5 °C. A solution of indazole-3-carboxylic acid (4.0 g, 24.67 mmol)

in DMF (50 mL) was added slowly into the reactor over a period of

45 min, maintaining the reaction mass temperature between 0 to 5

(

4, 1.02 mmol) in DCM (2.0 mL) were added. The reaction mixture

was gradually warmed to r.t. and stirred for 16 h. After completion of

the reaction, the reaction mixture was diluted with water and DCM.

The separated organic layer was washed with brine and dried over

anhydrous Na SO , and the solvent was evaporated under vacuum to

2

4

N

https://doi.org/10.1021/acs.jmedchem.1c00703

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

°

C. The cooling was removed, and the reaction temperature was

4-Fluoro-4-{[(1-isopropyl-1H-indazole-3-carbonyl)-amino]-

methyl}-piperidine-1-carboxylic Acid tert-Butyl Ester (5d). By

following general procedure 4, 1-isopropyl-1H-indazole-3-carbonyl

chloride 3 (500 mg, 2.25 mmol) was reacted with 4-aminomethyl-4-

ﬂuoro-piperidine-1-carboxylic acid tert-butyl ester 4d (522.5 mg, 2.25

gradually raised to r.t. over a period of 30 min. After stirring at this

temperature for 1 h, the reaction mixture was cooled to 0 °C, and

isopropyl iodide (37.18 mmol) was added over a period of 30 min.

The cooling was removed, and the reaction temperature was warmed

to r.t. After 16 h of stirring, the HPLC analysis of the reaction mixture

revealed l0% of indazole-7-carboxylicacid remaining. The reaction

mass was quenched cautiously by adding cold water and extracted

with EtOAc. The separated aqueous layer was acidiﬁed to pH 4.0 with

aqueous HCl solution (6 N). The acidiﬁed reaction mass was then

extracted with EtOAc. The combined organic layer was washed with

water, brine, and dried over anhydrous Na SO , and the solvent was

mmol) to obtain title compound 5d (904.2 mg) in 96.1% yield with

1

HPLC purity 94.0%. H NMR (CDCl ): δ 8.42 (d, J = 8.0 Hz, 1H),

3

7

1

.42 (d, J = 8.4 Hz, 2H), 7.21 (t, J = 8.4 Hz, 1H), 7.14 (t, J = 7.6 Hz,

H), 4.25 (bs, 3H), 3.49 (bs, 2H), 2.71 (t, J = 12.0 Hz, 2H), 1.84 (d, J

=

17.6 Hz, 4H), 1.64 (d, J = 6.8 Hz, 6H), 1.46 (s, 9H); mass (m/z):

+

419.2 (M + H) .

6

-{[(1-Isopropyl-1H-indazole-3-carbonyl)-amino]-methyl}-[1,3]-

2

4

oxazinane-3-carboxylic Acid tert-Butyl Ester (5e). By following

general procedure 4, 1-isopropyl-1H-indazole-3-carbonyl chloride 3

removed under reduced pressure to obtain a crude mass. The

obtained crude mass was diluted with DCM (28.0 L) and stirred for

(

4

750.0 mg, 3.37 mmol) was reacted with 2-aminomethyl-morpholine-

-carboxylic acid tert-butyl ester 4e (729.7 mg, 3.37 mmol) to obtain

1

5 min. The solids precipitated (unreacted indazole-7-carboxylic acid)

were ﬁltered, and the ﬁlter bed was washed once with DCM. The

combined ﬁltrate was dried over anhydrous Na SO and distilled

title compound 5e (1.2 g) in 90.1% yield with HPLC purity 90.0%.

2

4

1

H NMR (DMSO-d ): δ 8.15 (d, J = 7.6 Hz, 2H), 7.76 (d, J = 8.4 Hz,

6

under reduced pressure which yielded a crude mass. The crude mass

was triturated with ether, and the solids were ﬁltered to obtain the wet

cake which was dried further under reduced pressure to obtain the

title compound (3.0 g) in 60% yield. HPLC purity: 99.86%. H NMR

(

7

1

H), 7.43 (t, J = 7.6 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 5.07−5.04 (m,

1

H), 3.80−3.44 (m, 3H), 2.65−2.62 (m, 2H), 2.37−2.29 (m, 2H),

1

1.53 (d, J = 6.8 Hz, 6H), 1.44 (s, 9H), 1.22 (m, 2H); mass (m/z):

+

403.2 (M + H) .

400 MHz, CDCl ): 8.27 (d, J = 8.1 Hz, l H), 7.55 (d, J = 8.4 Hz, lH),

3

6

-[(1-Isopropyl-1H-indazole-3-carbonyl)-amino]-3-aza-bicyclo-

.46 (t, J = 7.6 Hz, lH), 7.34 (t, J = 7.4 Hz, lH), 5.01−4.95 (m, lH),

1

3

[3.1.0]hexane-3-carboxylic Acid tert-Butyl Ester (5f). By following

general procedure 4, 1-isopropyl-1H-indazole-3-carbonyl chloride 3

(1.0 g, 4.50 mmol) was reacted with 6-amino-3-aza-bicyclo[3.1.0]-

hexane-3-carboxylic acid tert-butyl ester 4f (892.3 mg, 4.50 mmol) to

.68 (d, J = 6.6 Hz, 6H); C NMR (100 MHz, CDCl ): δ 167.6,

3

39.9, 133.7, 126.6, 124.1, 123.4, 122.3, 109.8, 51.8, 22.0; DSC:

+

(

RAMP 5 °C/min): 163.0 °C; mass (m/z): 205.1 (M + H) .

1

-Isopropyl-1H-indazole-3-carbonyl Chloride (3). To a stirred

obtain title compound 5f (1.5 g) in 90.1% yield with HPLC purity

solution of 1-isopropyl-lH-indazole-3-carboxylic acid 2 (1.0 g, 4.90

mmol) in 1,2-dichloroethane (19.6 mL) cooled at 0 °C under

nitrogen atmosphere, thionyl chloride (0.42 mL, 5.88 mmol) was

added. The reaction mixture was gradually raised to r.t. and then

reﬂuxed for 1 h. After completion of the reaction, volatiles were

evaporated which yielded title compound 3 (1.0 g) in quantitative

yield. H NMR (CDCl ): δ 8.21 (d, J = 8.4 Hz, l H), 7.57 (d, J = 8.4

Hz, l H), 7.49 (t, J = 6.8 Hz, l H), 7.41 (t, J = 7.6 Hz, l H), 5.01−4.96

(

H) .

1

9

8

5.3%. H NMR (DMSO-d ): δ 8.15 (d, J = 8.0 Hz, 1H), 7.95 (d, J =

6

.4 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.25

(

=

9

t, J = 7.6 Hz, 1H), 5.07−5.04 (m, 1H), 2.49−2.42 (m 1H), 2.20 (t, J

11.2 Hz, 2H), 1.68−1.63 (m, 2H), 1.54 (d, J = 6.4 Hz, 6H), 1.44 (s,

+

H), 1.23 (m, 2H); mass (m/z): 385.2 (M + H) .

6

-{[(1-Isopropyl-1H-indazole-3-carbonyl)-amino]-methyl}-3-aza-

1

3

bicyclo[3.1.0]hexane-3-carboxylic Acid tert-Butyl Ester (5g). By

following general procedure 4, 1-isopropyl-1H-indazole-3-carbonyl

chloride 3 (500 mg, 2.25 mmol) was reacted with 6-aminomethyl-3-

aza-bicyclo[3.1.0]hexane-3-carboxylic acid tert-butyl ester 4g (477.4

m, l H), 1.68 (d, J = 6.6 Hz, 6H); mass (m/z): 223.1, 225.1 (M +

+

4

-[(1-Isopropyl-1H-indazole-3-carbonyl)-amino]-piperidine-1-

mg, 2.25 mmol) to obtain title compound 5g (806.7 mg) in 90.1%

carboxylic Acid tert-Butyl Ester (5a). By following general procedure

4

mmol) was reacted with 4-amino-piperidine-1-carboxylic acid tert-

butyl ester 4a (450.0 mg, 2.25 mmol) to obtain title compound 5a

(

J = 8.4 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 4.90−4.84 (m, 1H), 4.13

(

J = 6.8 Hz, 6H), 1.46 (s, 9H); mass (m/z): 387.2 (M + H) .

1

yield with HPLC purity 93.4%. H NMR (DMSO-d ): δ 8.15 (d, J =

6

, 1-isopropyl-1H-indazole-3-carbonyl chloride 3 (500 mg, 2.25

8

.0 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.43

(

2

6

t, J = 7.6 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 5.07−5.04 (m, 1H), 2.91

d, J = 11.6 Hz, 2H), 2.71 (d, J = 12.0 Hz, 2H), 2.49−2.42 (m, 1H),

1

825.8 mg) in 95.1% yield with HPLC purity 97.0%. H NMR

.20 (t, J = 11.2 Hz, 2H), 1.68−1.63 (m, 2H), 1.53 (d, J = 6.4 Hz,

CDCl ): δ 8.37 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.39 (t,

+

3

H), 1.44 (s, 9H); mass (m/z): 399.1 (M + H) .

3

-[(1-Isopropyl-1H-indazole-3-carbonyl)-amino]-8-aza-bicyclo-

3.2.1]octane-8-carboxylic Acid tert-Butyl Ester (5h). By following

general procedure 4, 1-isopropyl-1H-indazole-3-carbonyl chloride 3

500 mg, 2.25 mmol) was reacted with 3-amino-8-aza-bicyclo[3.2.1]-

bs, 3H), 2.75 (t, J = 12.0 Hz, 2H), 1.88 (d, J = 17.6 Hz, 4H), 1.64 (d,

[

+

4

-{[(1-Isopropyl-1H-indazole-3-carbonyl)-amino]-methyl}-piperi-

(

dine-1-carboxylic Acid tert-Butyl Ester (5b). By following general

procedure 4, 1-isopropyl-1H-indazole-3-carbonyl chloride 3 (200 mg,

0

octane-8-carboxylic acid tert-butyl ester 4h (509.6 mg, 2.25 mmol) to

obtain title compound 5h (750.7 mg) in 80.8% yield with HPLC

.9 mmol) was reacted with 4-aminomethylpiperidine-1-carboxylic

1

purity 92.4%. H NMR (CDCl ): δ 8.37 (d, J = 8.4 Hz, 1H), 7.52 (d,

3

acid tert-butyl ester 4b (192.6 mg, 0.9 mmol) to obtain title

J = 6.0 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 6.8 Hz, 1H),

compound 5b (324.0 mg) in 90.1% yield with HPLC purity 95.0%.

4

.91−4.86 (m, 1H), 4.35−4.34 (m, 1H), 3.65 (bs, 1H), 2.85−2.75

1

H NMR (CDCl ): δ 8.39 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.4 Hz,

3

(m, 1H), 2.72−2.65 (m, 1H), 2.41−2.25 (m, 2H), 2.21−2.08 (m,

2

1

=

H), 7.39 (t, J = 8.4 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 4.90−4.84 (m,

H), 4.13 (bs, 3H), 3.49 (bs, 2H), 2.71 (t, J = 12.0 Hz, 2H), 1.84 (d, J

2H), 2.05−1.9 (m, 2H), 1.83−1.80 (m, 2H), 1.62 (d, J = 6.4 Hz, 6H),

1.44 (s, 9H); mass (m/z): 413.4 (M + H) .

+

17.6 Hz, 4H), 1.61 (d, J = 6.8 Hz, 6H), 1.45 (s, 9H); mass (m/z):

1

-Isopropyl-1H-indazole-3-carboxylic Acid Piperidin-4-ylamide

+

4

01.2 (M + H) .

(6a). Following the procedure of Boc deprotection as mentioned in

general procedure 2, compound 4-[(1-isopropyl-1H-indazole-3-

carbonyl)-amino]-piperidine-1-carboxylic acid tert-butyl ester 5a

4

-Hydroxy-4-{[(1-isopropyl-1H-indazole-3-carbonyl)-amino]-

methyl}-piperidine-1-carboxylic Acid tert-Butyl Ester (5c). By

following general procedure 4, 1-isopropyl-1H-indazole-3-carbonyl

chloride 3 (1.0 g, 4.50 mmol) was reacted with 4-aminomethyl-4-

hydroxy-piperidine-1-carboxylic acid tert-butyl ester 4c (1.03 g, 4.50

mmol) to obtain title compound 5c (1.7 g) in 95.0% yield with HPLC

purity 97.0%. H NMR (CDCl ): δ 8.37 (d, J = 8.1 Hz, 1H), 7.44 (d,

(

825.8 mg, 2.13 mmol) was converted to title compound 6a (580.7

1

mg) in 95.1% yield with HPLC purity 91.4%. H NMR (CDCl ): δ

3

8

.39 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.39 (t, J = 8.4 Hz,

1H), 7.28 (t, J = 7.6 Hz, 1H), 5.01- 4.97 (m, 1H), 4.13 (bs, 2H), 2.97

(t, J = 12.0 Hz, 3H), 2.05 (m, 2H), 1.92 (d, J = 17.6 Hz, 4H), 1.61 (d,

J = 6.8 Hz, 6H) ; mass (m/z): 287.4 (M + H) .

1

3

+

J = 8.3 Hz, 2H), 7.38 (t, J = 8.4 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H),

4

1

4

.20 (bs, 3H), 3.54 (bs, 2H), 2.73 (t, J = 12.0 Hz, 2H), 1.81 (d, J =

1-Isopropyl-1H-indazole-3-carboxylic Acid (Piperidin-4-ylmeth-

yl)-amide (6b). Following the procedure of Boc deprotection as

mentioned in general procedure 2, compound 4-{[(1-isopropyl-1H-

7.6 Hz, 4H), 1.64 (d, J = 6.8 Hz, 6H), 1.46 (s, 9H); mass (m/z):

+

17.2 (M + H) .

O

https://doi.org/10.1021/acs.jmedchem.1c00703

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

indazole-3-carbonyl)-amino]-methyl}-piperidine-1-carboxylic acid

tert-butyl ester 5b (260.0 mg, 0.65 mmol) was converted to title

1H), 4.35−4.34 (m, 1H), 3.65 (bs, 1H), 2.85−2.75 (m, 1H), 2.72−

2.65 (m, 1H), 2.41−2.25 (m, 2H), 2.21−2.08 (m, 2H), 2.05−1.9 (m,

2H), 1.83−1.80 (m, 2H), 1.62 (d, J = 6.4 Hz, 6H); mass (m/z): 313.4

compound 6b (190.0 mg) in 97.4% yield with HPLC purity 95.21%.

1

+

H NMR (CDCl ): δ 8.39 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 8.4 Hz,

(M + H) .

3

2

1

=

H), 7.39 (t, J = 8.4 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 5.01- 4.97 (m,

H), 4.13 (bs, 3H), 2.97 (t, J = 12.0 Hz, 3H), 2.05 (m, 1H), 1.92 (d, J

1₊7.6 Hz, 4H), 1.61 (d, J = 6.8 Hz, 6H); mass (m/z): 301.4 (M +

1-Isopropyl-1H-indazole-3-carboxylic Acid [1-(3-Hydroxy-prop-

yl)-piperidin-4-yl]-amide Tartarate (7a). 1-Isopropyl-1H-indazole-3-

carboxylic acid piperidin-4-ylamide 6a (580.0 mg, 2.02 mmol) was N-

alkylated with 3-bromo-propan-1-ol by following general procedure 3

to obtain title compound 7a free base which was converted to its

tartarate salt by using the general procedure 8 to obtain title

H) .

1

-Isopropyl-1H-indazole-3-carboxylic Acid (4-Hydroxy-piperidin-

4

-ylmethyl)-amide (6c). Following the procedure of Boc depro-

tection as mentioned in general procedure 2, compound 4-hydroxy-4-

[(1-isopropyl-1H-indazole-3-carbonyl)-amino]-methyl}-piperidine-

-carboxylic acid tert-butyl ester 5c (1.7 g, 4.08 mmol) was converted

to title compound 6c (1.1 g) in 98.0% yield with HPLC purity

compound (685.6 mg) as cream color solid in 88.2% yield with HPLC

1

{

1

purity 95.7%. H NMR (DMSO-d

6

): δ 8.15 (t, J = 8.0 Hz, 2H), 7.78

(d, J = 8.8 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H),

5.10−5.03 (m,1H), 4.05 (s, 2H), 3.98−3.96 (m, 1H), 3.90 (bs, 1H),

3.49 (t, J = 7.2 Hz, 2H), 3.20 (d, J = 11.2 Hz, 2H), 2.72 (d, J = 6.4 Hz,

2H), 2.52 (bs, 2H), 1.91 (d, J = 10.8 Hz, 2H), 1.81 (d, J = 11.2 Hz,

1

9

8

7.15%. H NMR (CDCl ): δ 8.37 (d, J = 8.1 Hz, 1H), 7.44 (d, J =

3

.3 Hz, 2H), 7.38 (t, J = 8.4 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 4.21

1

3

(bs, 3H), 3.54 (bs, 2H), 2.73 (t, J = 12.0 Hz, 2H), 1.81 (d, J = 17.6

2H), 1.67 (t, J = 6.0 Hz, 2H), 1.54 (d, J = 6.8 Hz, 6H); C NMR

(100 MHz, CDCl ): δ 174.4, 167.3, 160.6, 139.6, 129.1, 127.0, 122.8,

+

Hz, 4H), 1.64 (d, J = 6.8 Hz, 6H); mass (m/z): 317.4 (M + H) .

-Isopropyl-1H-indazole-3-carboxylic Acid (4-Fluoro-piperidin-

-ylmethyl)-amide (6d). Following the procedure of Boc depro-

tection as mentioned in general procedure 2, compound 4-ﬂuoro-4-

[(1-isopropyl-1H-indazole-3-carbonyl)-amino]-methyl}-piperidine-

-carboxylic acid tert-butyl ester 5d (1.32 g, 3.15 mmol) was

3

1

122.5, 121.9, 109.6, 81.0, 62.7, 57.9, 52.1, 51.6, 31.9, 28.2, 27.0, 22.0;

+

4

DSC: (RAMP 5 °C/min): 143.2 °C; mass (m/z): 345.1 (M + H) ;

C H N O ; % C, H, N: calculated C; 55.86, H; 6.93, N; 11.33;

2

3

34

4

8

{

1

found C; 55.62, H; 6.93, N; 11.52.

1-Isopropyl-1H-indazole-3-carboxylic Acid [1-(3-Methoxy-prop-

yl)-piperidin-4-ylmethyl]-amide Oxalate (7l). By following general

procedure 3, 1-isopropyl-1H-indazole-3-carboxylic acid (piperidin-4-

ylmethyl)-amide 6b (190.0 mg, 0.63 mmol) was N-alkylated with 1-

bromo-3-methoxypropane to obtain 7l free base as a gummy mass

which was converted to its oxalate salt by using the general procedure

converted to title compound 6d (0.97 g) in 97.7% yield with HPLC

purity 96.2%. H NMR (CDCl ): δ 8.36 (d, J = 8.2 Hz, 1H), 7.45 (d,

J = 8.1 Hz, 2H), 7.38 (t, J = 8.4 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H),

1

3

4

1

.26 (bs, 3H), 3.55 (bs, 2H), 2.73 (t, J = 12.0 Hz, 2H), 1.81 (d, J =

7.6 Hz, 4H), 1.64 (d, J = 6.8 Hz, 6H); mass (m/z): 319.2 (M + H) .

+

1

-Isopropyl-1H-indazole-3-carboxylic Acid (Morpholin-2-yl-

8 to obtain title compound 7l (188.6 mg) as oﬀ-white solid in 80.0%

1

methyl)-amide (6e). Following the procedure of Boc deprotection

as mentioned in general procedure 2, compound 2-{[(1-isopropyl-

yield with HPLC purity 98.7%. H NMR (DMSO-d ): δ 8.16 (d, J =

6

7.6 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.26

(t, J = 7.6 Hz, 1H), 5.10−5.06 (m, 1H), 4.11−3.99 (m, 1H), 3.80 (d,

J = 10.8 Hz, 1H), 3.62−3.50 (m, 1H), 3.34 (t, J = 9.6 Hz, 2H), 3.18

(s, 3H), 2.79 (d, J = 10.4 Hz, 2H), 2.69 (d, J = 10.4 Hz, 4H), 2.29 (bs,

2H), 1.97−1.76 (m, 2H), 1.64−1.61 (m, 1H), 1.53 (d, J = 6.8 Hz,

1

H-indazole-3-carbonyl)-amino]-methyl}-morpholine-4-carboxylic

acid tert-butyl ester 5e (1.2 g, 2.98 mmol) was converted to title

1

compound 6e (827.4 g) in 93.4% yield with HPLC purity 94.15%. H

NMR (DMSO-d ): δ 8.15 (d, J = 7.6 Hz, 2H), 7.76 (d, J = 8.4 Hz,

6

1

3

1

2

H), 7.43 (t, J = 7.6 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 5.07−5.04 (m,

6H); C NMR (100 MHz, CDCl ): δ 167.9, 160.6, 139.6, 129.1,

3

H), 3.80−3.44 (m, 3H), 2.71 (t, J = 12.0 Hz, 2H), 2.65−2.62 (m,

127.0, 122.8, 122.5, 121.9, 109.6, 69.9, 57.9, 52.1, 51.6, 31.9, 28.2,

H), 2.37−2.29 (m, 2H), 1.53 (d, J = 6.8 Hz, 6H); mass (m/z): 303.1

27.0, 22.0; DSC: (RAMP 5 °C/min): 157.1 °C; mass (m/z): 373.0

+

(M + H) .

(M + H) ; C H N O ; % C, H, N: calculated C; 59.72, H; 7.41, N;

2

3

34

4

6

1

-Isopropyl-1H-indazole-3-carboxylic Acid (3-Aza-bicyclo[3.1.0]-

12.11; found C; 59.81, H; 7.61, N; 12.23.

hex-6-yl)-amide (6f). Following the procedure of Boc deprotection as

mentioned in general procedure 2, compound 6-[(1-isopropyl-1H-

indazole-3-carbonyl)-amino]-3-aza-bicyclo[3.1.0]hexane-3-carboxylic

acid tert-butyl ester 5f (1.5 g, 3.90 mmol) was converted to title

1

-Isopropyl-1H-indazole-3-carboxylic Acid [4-Hydroxy-1-(3-me-

thoxy-propyl)-piperidin-4-ylmethyl]-amide Oxalate (7o). 1-Isoprop-

yl-1H-indazole-3-carboxylic acid (4-hydroxy-piperidin-4-ylmethyl)-

amide 6c (1.1 g, 3.47 mmol) was N-alkylated with 1-bromo-3-

methoxy-propane by following general procedure 3 to obtain 7o free

base as a gummy mass which was converted to its oxalate salt by using

1

compound (998.0 mg) 6f in 90.0% yield with HPLC purity 94.1%. H

NMR (DMSO-d ): δ 8.15 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.4 Hz,

6

1

H), 7.77 (d, J = 8.8 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.25 (t, J = 7.6

Hz, 1H), 5.07−5.04 (m, 1H), 2.91 (d, J = 11.6 Hz, 2H), 2.49−2.42

m 1H), 2.20 (t, J = 11.2 Hz, 2H), 1.68−1.63 (m, 2H), 1.53 (d, J =

the general procedure 8 to obtain title compound in 92.1% yield (1.32

g) with HPLC purity 97.5%. H NMR (CD OD): δ 8.31 (bs, 1H),

8.22 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.47 (t, J = 7.6 Hz,

1

3

(

6

+

.4 Hz, 6H); mass (m/z): 285.4 (M + H) .

1H), 7.28 (t, J = 7.6 Hz, 1H), 5.08−5.02 (m, 1H), 3.65 (bs, 2H), 3.54

1

-Isopropyl-1H-indazole-3-carboxylic Acid (3-Aza-bicyclo[3.1.0]-

(s, 3H), 3.51- 3.46 (m, 6H), 3.23−2.219 (m, 2H), 2.02−1.95 (m,

+

hex-6-ylmethyl)-amide (6g). Following the procedure of Boc

6

H), 1.62 (d, J = 6.4 Hz, 6H); mass (m/z): 389.2 (M + H) ;

deprotection as mentioned in general procedure 2, compound 6-

C H N O ; % C, H, N: calculated C; 59.72, H; 7.41, N; 12.11;

2

3

34

4

6

{

[(1-isopropyl-1H-indazole-3-carbonyl)-amino]-methyl}-3-aza-

bicyclo[3.1.0]hexane-3-carboxylic acid tert-butyl ester 5g (806.7 mg,

.90 mmol) was converted to title compound 6g (555.0 mg) in 92.0%

found C; 59.39, H; 7.61, N; 12.23.

-Isopropyl-1H-indazole-3-carboxylic Acid [4-Fluoro-1-(3-me-

1

3

thoxy-propyl)-piperidin-4-ylmethyl]-amide (7q). 1-Isopropyl-1H-

indazole-3-carboxylic acid (4-hydroxy-piperidin-4-ylmethyl)-amide

6d (0.5 g, 1.67 mmol) was N-alkylated with 1-bromo-3-methoxy-

propane by following general procedure 3 to obtain 7q as oﬀ-white

1

yield with HPLC purity 96.8%. H NMR (DMSO-d ): δ 8.15 (d, J =

6

8

.0 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H), 7.43

(

t, J = 7.6 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 5.07−5.04 (m, 1H), 2.91

1

d, J = 11.6 Hz, 2H), 2.71 (d, J = 12.0 Hz, 2H), 2.49−2.42 (m, 1H),

powder in 88.2% yield (0.49 g) with HPLC purity 95.8%. H NMR

(CDCl ): δ 8.36 (d, J = 8.2 Hz, 1H), 7.45 (d, J = 8.1 Hz, 2H), 7.38 (t,

2

6

.20 (t, J = 11.2 Hz, 2H), 1.68−1.63 (m, 2H), 1.53 (d, J = 6.4 Hz,

3

+

H); mass (m/z): 299.1 (M + H) .

J = 8.4 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 4.26 (bs, 3H), 3.55−3.43

1

-Isopropyl-1H-indazole-3-carboxylic Acid (8-Aza-bicyclo[3.2.1]-

(m, 4H), 3.54 (s, 3H), 2.73 (m, 2H), 2.04−1.88 (m, 6H), 1.64 (d, J =

+

oct-3-yl)-amide (6h). Following the procedure of Boc deprotection as

mentioned in general procedure 2, compound 3-[(1-isopropyl-1H-

indazole-3-carbonyl)-amino]-8-aza-bicyclo[3.2.1]octane-8-carboxylic

acid tert-butyl ester 5h (340.7 mg, 1.82 mmol) was converted to title

6

.8 Hz, 6H); mass (m/z): 391.1 (M + H) ; C H FN O ; % C, H,

2

1

31

4

2

N: calculated C; 64.59, H; 8.0, N; 14.35; found C; 64.02, H; 7.69, N;

4.26.

-Isopropyl-1H-indazole-3-carboxylic Acid [4-(3-Methoxyprop-

1

compound 6h (340.7 mg) in 90.0% yield with HPLC purity 97.1%.

yl)-morpholin-2-ylmethyl]-amide Tartarate (7r). 1-Isopropyl-1H-

indazole-3-carboxylic acid (4-ﬂuoro-piperidin-4-ylmethyl)-amide was

N-alkylated with 1-bromo-3-methoxy-propane by following general

1

H NMR (CDCl ): δ 8.37 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 6.0 Hz,

3

1H), 7.43 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 6.8 Hz, 1H), 4.91−4.86 (m,

P

https://doi.org/10.1021/acs.jmedchem.1c00703

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

procedure 3 to obtain compound 7r free base as a gummy mass which

was converted to its tartarate salt using general procedure 8 to obtain

4-Hydrazinocarbonyl-piperidine-1-carboxylic Acid tert-Butyl

Ester (9a). By following general procedure 5, compound 8a (5.0 g,

19.45 mmol) was converted to the titled compound 9a (4.1 g) as an

title compound 7r (851.8 mg) in 83% yield with HPLC purity 95.5%.

1

H NMR (DMSO-d ): δ 8.16 (d, J = 7.6 Hz, 2H), 7.79 (d, J = 8.4 Hz,

oﬀ-white crystalline powder in 87.2% yield with GC purity 99.79%.

6

1

H NMR (CDCI ): δ 8.12 (bs, 1H), 2.86 (t, J = 12.0 Hz, 2H), 2.43−

1

2

H), 7.44 (t, J = 7.6 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 5.08−5.05 (m,

3

H), 3.80−3.44 (m, 3H), 3.35−3.29 (m, 2H), 3.18 (s, 3H), 2.79−

2.43 (m, 1H), 2.05 (bs, 2H), 1.88−1.85 (bs, 2H), 1.67−1.57 (m,

4H), 1.47 (s, 9H); C NMR (100 MHz, CDCl ): δ 177.9, 159.3,

1

3

.76 (m, 2H), 2.65−2.62 (m, 4H), 2.37−2.29 (m, 2H), 1.64−1.61

3

+

(m, 2H), 1.53 (d, J = 6.8 Hz, 6H); mass (m/z): 375.1 (M + H) ;

69.2, 51.0, 30.5, 28.4, 26.5; DSC: (RAMP 5 °C/min): 102.1 °C; mass

+

C H N O ; % C, H, N: calculated C; 54.95, H; 6.92, N; 10.68;

(m/z): 244.4 (M + H) .

24

36

4

9

found C; 55.01, H; 6.73, N; 10.43.

-Isopropyl-1H-indazole-3-carboxylic Acid [3-(3-Methoxy-prop-

yl)-3-aza-bicyclo[3.1.0]hex-6-yl]-amide Oxalate (7s). 1-Isopropyl-

H-indazole-3-carboxylic acid (3-aza-bicyclo[3.1.0]hex-6-yl)-amide 6f

998.4 mg, 3.51 mmol) was N-alkylated with 1-bromo-3-methoxy-

propane by following general procedure 3 to obtain compound 7s free

base as a gummy mass which was converted to its oxalate salt using

4-[N′-(1-Isopropyl-1H-indazole-3-carbonyl)-hydrazinocarbonyl]-

piperidine-1-carboxylic Acid tert-Butyl Ester (10a). By following

general procedure 6, 4-hydrazinocarbonyl-piperidine-1-carboxylic acid

tert-butyl ester 9a (3.2 g, 13.48 mmol) was reacted with 1-isopropyl-

1H-indazole-3-carbonyl chloride 3 (3.0 g, 13.48 mmol) to obtain the

1

(

above titled compound 10a (5.3 g) as an oﬀ-white crystalline powder

1

in 92% yield with HPLC purity 98.75%. H NMR (DMSO-d

): δ

6

general procedure 8 to obtain title compound 7s (1.1 g) as oﬀ-white

10.02 (bs, 1H), 9.86 (bs, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.82 (d, J =

8.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 5.13−

5.06 (m, 1H), 2.80 (bs, 2H), 2.71 (bs, 3H), 1.76 (d, J = 6.8 Hz, 2H),

1

powder in 93% yield with HPLC purity 95.5%. H NMR (DMSO-d ):

6

δ 8.37 (d, J = 3.2 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.4

Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 5.07−5.04

1

3

1.55 (d, J = 6.4 Hz, 6H), 1.40 (s, 9H), 1.28 (t, J = 10.8 Hz, 2H);

C

(

m, 1H), 3.75 (s, 3H), 3.68 (t, J = 9.2 Hz, 2H), 3.52−3.33 (m, 4H),

NMR (100 MHz, DMSO-d ): δ 177.9, 167.3, 159.3, 139.9, 128.9,

6

3

2

1

5

1

.22- 3.14 (m, 6H), 3.14−2.99 (m, 2H), 2.06 (s, 2H), 1.81−1.78 (m,

127.5, 123.5, 121.9, 121.3, 111.1, 69.2, 53.7, 51.0, 30.5, 28.4, 26.5,

1

3

H), 1.52 (d, J = 6.8 Hz, 6H); C NMR (100 MHz, CDCl ): δ

22.2; DSC: (RAMP 5 °C/min): 123.2 °C; mass (m/z): 430.5 (M +

3

+

63.2, 163.0, 139.9, 136.4, 126.3, 122.9, 122.6, 122.5, 109.3, 69.0,

H) .

8.7, 55.9, 54.3, 51.0, 39.8, 26.1, 22.1, 21.1; DSC: (RAMP 5 °C/min):

4-Hydroxy-4-[N′-(1-isopropyl-1H-indazole-3-carbonyl)-hydrazi-

nocarbonyl]-piperidine-1-carboxylic Acid tert-Butyl Ester (10b). By

following general procedure 6, 4-hydrazinocarbonyl-4-hydroxy-piper-

idine-1-carboxylic acid tert-butyl ester 9b (2.0 g, 8.98 mmol) was

reacted with 1-isopropyl-1H-indazole-3-carbonyl chloride 3 (2.3 g,

8.98 mmol) to obtain the above titled compound 10b (1.2 g) as an

+

72.6 °C; mass (m/z): 357.2 (M + H) ; C H N O ; % C, H, N:

2

30

4

6

calculated C; 59.18, H; 6.77, N; 12.55; found C; 59.01, H; 6.71, N;

1

yl)-3-aza-bicyclo[3.1.0]hex-6-ylmethyl]-amide Oxalate (7v). 1-Iso-

propyl-1H-indazole-3-carboxylic acid (3-aza-bicyclo[3.1.0]hex-6-yl-

methyl)-amide 6g (555.0 mg, 1.86 mmol) was N-alkylated with 1-

bromo-3-methoxy-propane by following general procedure 3 to obtain

compound 7v free base as a gummy mass which was converted to its

oxalate salt using general procedure 8 to obtain title compound 7v

oﬀ-white crystalline powder in 63.1% yield with HPLC purity 98.7%.

1

H NMR (DMSO-d ): δ 10.02 (bs, 1H), 9.86 (bs, 1H), 8.12 (d, J =

6

8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.30

(t, J = 7.6 Hz, 1H), 5.13−5.06 (m, 1H), 2.80 (bs, 2H), 2.71 (bs, 4H),

1.76 (d, J = 6.8 Hz, 2H), 1.55 (d, J = 6.4 Hz, 6H), 1.40 (s, 9H), 1.28

(t, J = 10.8 Hz, 2H); mp: 106.1−107.2 °C; mass (m/z): 446.3 (M +

(

640.0 mg) as a gummy mass in 91.7% yield with HPLC purity 97.2%.

1

+

H NMR (DMSO-d ): δ 8.41 (bs, 1H), 8.17 (d, J = 8.0 Hz, 1H), 7.80

H) .

6

(

5

d, J = 8.3 Hz, 1H), 7.45 (t, J = 7.21 Hz, 1H), 7.27 (t, J = 7.5 Hz, 1H),

4-{2-[N′-(1-Isopropyl-1H-indazole-3-carbonyl)-hydrazino]-2-

oxo-ethyl}-piperidine-1-carboxylic Acid tert-Butyl Ester (10c). By

following general procedure 6, 4-hydrazinocarbonylmethylpiperidine-

1-carboxylic acid tert-butyl ester 9c (3.4 g, 13.47 mmol) was reacted

with 1-isopropyl-1H-indazole-3-carbonyl chloride 3 (3.0 g, 13.47

mmol) to obtain the above titled compound 10c (4.7 g) as brown

.09−5.06 (m, 1H), 3.32 (t, J = 11.0 Hz, 4H), 3.19 (s, 6H), 3.05 (bs,

1

3

4

H), 1.75 (bs, 4H), 1.54 (d, J = 6.4 Hz, 6H); C NMR (100 MHz,

DMSO-d ): δ 163.9, 163.1, 139.9, 136.4, 126.3, 122.9, 122.6, 122.5,

6

1

(

09.3, 69.0, 58.7, 55.9, 54.3, 51.0, 47.1, 39.8, 26.1, 22.1, 21.0; DSC:

+

RAMP 5 °C/min): 163.8 °C; mass (m/z): 371.2 (M + H) ;

1

C H N O ; % C, H, N: calculated C; 59.99, H; 7.00, N; 12.17;

found C; 60.20, H; 6.98, N; 12.23.

color gummy mass in 80.0% yield with HPLC purity 95.6%. H NMR

22

33

4

6

(DMSO-d ): δ 10.02 (bs, 1H), 9.86 (bs, 1H), 8.12 (d, J = 8.4 Hz,

6

1

-Isopropyl-1H-indazole-3-carboxylic Acid [8-(2-Fluoro-ethyl)-8-

1H), 7.82 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.6

Hz, 1H), 5.13−5.06 (m, 1H), 3.98 (d, J = 12.0 Hz, 2H), 2.80 (bs,

2H), 2.71 (bs, 4H), 2.45 (bs, 2H),1.76 (d, J = 6.8 Hz, 2H), 1.55 (d, J

= 6.4 Hz, 6H), 1.40 (s, 9H), 1.28 (t, J = 10.8 Hz, 2H); mass (m/z):

aza-bicyclo[3.2.1]oct-3-yl]-amide (7aa). 1-Isopropyl-1H-indazole-3-

carboxylic acid (8-aza-bicyclo[3.2.1]oct-3-yl)-amide 6h (340.0 mg,

1

.08 mmol) was N-alkylated with 1-bromo-3-methoxy-propane by

following general procedure 3 to obtain title compound 7aa (351.0

mg) as oﬀ-white color powder in 90% yield with HPLC purity 96.7%.

H NMR (CDCl ): δ 8.38 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 6.0 Hz,

H), 7.46 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 6.8 Hz, 1H), 4.91−4.86 (m,

H), 4.70−4.60 (m, 1H), 4.55−4.48 (m, 1H), 4.35−4.34 (m, 1H),

.65 (bs, 1H), 3.34 (bs, 2H), 2.85−2.75 (m, 1H), 2.72−2.65 (m,

H), 2.41−2.25 (m, 2H), 2.21−2.08 (m, 2H), 2.05−1.9 (m, 2H),

.83−1.80 (m, 2H), 1.62 (d, J = 6.4 Hz, 6H); C NMR (100 MHz,

CDCl ): δ 166.9, 139.8, 136.4, 126.3, 122.9, 122.6, 122.5, 109.3, 84.1,

+

444.2 (M + H) .

6-[N′-(1-Isopropyl-1H-indazole-3-carbonyl)-hydrazinocarbonyl]-

3-aza-bicyclo[3.1.0]hexane-3-carboxylic Acid tert-Butyl Ester (10d).

By following general procedure 6, 6-hydrazinocarbonyl-3-aza-

bicyclo[3.1.0]hexane-3-carboxylic acid tert-butyl ester 9d (1.01 g,

4.50 mmol) was reacted with 1-isopropyl-1H-indazole-3-carbonyl

chloride 3 (1.0 g, 4.50 mmol) to obtain the above titled compound

1

3

1

3

1

3

10d (1.5 g) as crystalline solid in 78% yield with HPLC purity 95.2%.

1

H NMR (DMSO-d ): δ 10.02 (bs, 1H), 9.86 (bs, 1H), 8.12 (d, J =

3

6

5

8.7, 55.9, 54.3, 51.0, 39.8, 26.1, 22.1, 21.8; DSC: (RAMP 5 °C/min):

8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.30

(t, J = 7.6 Hz, 1H), 5.13−5.06 (m, 1H), 3.98 (d, J = 12.0 Hz, 2H),

2.80 (bs, 2H), 2.71 (bs, 2H), 1.55 (d, J = 6.4 Hz, 6H), 1.40 (s, 9H),

1.28 (t, J = 10.8 Hz, 2H); mp: 152.3−154.1 °C; mass (m/z): 428.4

+

1

59.2 °C; mass (m/z): 359.2 (M + H) ; C H FN O; % C, H, N:

2

0

27

4

calculated C; 67.01, H; 7.59, N; 15.63; found C; 67.18, H; 7.23, N;

1

5.61.

Piperidine-1,4-dicarboxylic Acid 1-tert-Butyl Ester 4-Ethyl Ester

8a). By following general procedure 1, ethyl isonipecotate (6.5 g,

1.35 mmol) was converted to the title compound 8 (9.54 g) in 90%

+

(M + H) .

(

4

3-[N′-(1-Isopropyl-1H-indazole-3-carbonyl)-hydrazinocarbonyl]-

8-aza-bicyclo[3.2.1]octane-8-carboxylic Acid tert-Butyl Ester (10e).

By following general procedure 6, 3-hydrazinocarbonyl-8-aza-

bicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester 9d (2.4 g, 8.98

mmol) was reacted with 1-isopropyl-1H-indazole-3-carbonyl chloride

3 (2.4 g, 8.98 mmol) to obtain the above titled compound 10e (3.89

g) as an oﬀ-white crystalline powder in 95.1% yield with HPLC purity

1

yield with GC purity 98.8%. H NMR (CDCI ): δ 4.16−4.11 (m,

2

NMR (100 MHz, CDCl ): δ 175.0, 159.3, 71.1, 51.2, 30.9, 28.4, 26.5;

mass (m/z): 258.2 (M + H) .

3

H), 2.86 (t, J = 12.0 Hz, 2H), 2.43−2.43 (m, 1H), 1.88−1.85 (bs,

13

H), 1.67−1.57 (m, 4H), 1.47 (s, 9H), 1.27 (t, J = 7.2 Hz, 3H);

C

3

+

Q

https://doi.org/10.1021/acs.jmedchem.1c00703

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

1

9

8.7%. H NMR (DMSO-d ): δ 10.02 (bs, 1H), 9.86 (bs, 1H), 8.12

(DMSO-d ): δ 10.02 (bs, 1H), 9.86 (bs, 1H), 8.12 (d, J = 8.4 Hz,

6

(

d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H),

1H), 7.82 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.6

Hz, 1H), 5.13−5.06 (m, 1H), 2.71 (bs, 4H), 1.76 (d, J = 6.8 Hz, 2H),

1.55 (d, J = 6.4 Hz, 6H), 1.35−1.28 (m, 3H); mp: 123.1−124.1 °C;

7

2

1

4

.30 (t, J = 7.6 Hz, 1H), 5.13−5.06 (m, 1H), 3.98 (d, J = 12.0 Hz,

H), 2.71 (bs, 4H), 1.76 (d, J = 6.8 Hz, 2H), 1.55 (d, J = 6.4 Hz, 6H),

.40 (s, 9H), 1.35−1.28 (m, 3H); mp: 117.9−118.8 °C; mass (m/z):

+

mass (m/z): 339.5 (M + H) .

+

56.2 (M + H) .

1-Isopropyl-3-{5-[1-(3-methoxypropyl)-piperidin-4-yl]-[1,3,4]-

oxadiazol-2-yl}-1H-indazole oxalate (12l). By following general

procedure 3, 1-isopropyl-3-(5-piperidin-4-yl-[1,3,4]oxadiazol-2-yl)-

1H-indazole 11a (1.8 g, 6.07 mmol) was N-alkylated with 1-bromo-

3-methoxypropane to obtain 1-isopropyl-3-{5-[1-(3-methoxypropyl)-

piperidin-4-yl]-[1,3,4]oxadiazol-2-yl}-1H-indazole (1.84 g) as light

yellow gummy mass in 80.0% yield with HPLC purity 99.0%. The

obtained free base was converted to oxalate salt by following general

procedure 8 to obtain the titled compound 12l as oﬀ-white crystalline

1

-Aza-bicyclo[2.2.2]octane-3-carboxylic Acid N′-(1-Isopropyl-

1

H-indazole-3-carbonyl)-hydrazide (10f). By following general

procedure 6, 1-aza-bicyclo[2.2.2]octane-3-carboxylic acid hydrazide

f (3.0 g, 12.34 mmol) reacts with 1-isopropyl-1H-indazole-3-

9

carbonyl chloride 3 (2.7 g, 12.34 mmol) to obtain the above titled

compound 10f (4.24 g) as an oﬀ-white crystalline powder in 74.5%

yield with HPLC purity 96.2%. H NMR (DMSO-d ): δ 10.02 (bs,

1

7

2

Hz, 6H), 1.28 (m, 3H); mp: 137.3−138.8 °C; mass (m/z): 356.4 (M

+

1

6

H), 9.86 (bs, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H),

.45 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 5.13−5.06 (m, 1H),

.80 (m, 4H), 2.71 (m, 3H), 1.76 (d, J = 6.8 Hz, 2H), 1.55 (d, J = 6.4

−1

powder with HPLC purity 99.83%. IR (cm ): 3435, 2974, 2932,

1

2890, 2697, 2537, 1710, 1604, 1564, 1465, 1193, 1113, 992, 750; H

NMR (DMSO-d ): δ 8.20 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.4 Hz,

6

+

H) .

-Isopropyl-3-(5-piperidin-4-yl-[1,3,4]oxadiazol-2-yl)-1H-inda-

1H), 7.55 (t, J = 7.2 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 5.25−5.12 (m,

1H), 3.50−3.37 (m, 5H), 3.24 (s, 3H), 3.08−2.97 (m, 4H), 2.37−

2.27 (m, 2H), 2.18−2.05 (m, 2H), 1.95−1.85 (m, 2H), 1.56 (d, J =

1

zole (11a). By following general procedure 7, 4-[N′-(1-isopropyl-1H-

indazole-3-carbonyl)-hydrazinocarbonyl]-piperidine-1-carboxylic acid

tert-butyl ester 10a (5.3 g, 12.33 mmol) was converted to above titled

compound 11a (3.6 g) as brown color solid in 95% yield with HPLC

purity 99.3%. H NMR (DMSO-d ): δ 8.19 (d, J = 8.0 Hz, 1H), 7.91

1

3

6.8 Hz, 6H); C NMR (100 MHz, DMSO-d ): δ 167.2, 164.9 (2C),

6

160.3, 139.9, 128.8, 127.6, 123.5, 121.9, 121.3, 111.2, 69.5, 58.3, 54.1

(2C), 51.0, 50.7, 30.5, 26.6 24.3 (2C), 22.4 (2C); DSC: (RAMP 5

1

+

6

°C/min): 208.2 °C; mass (m/z): 384.2 (M + H) ; oxalic acid content

(d, J = 8.8 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H),

18.34% (average of two titration experiments; corresponds to mono

oxalate); C H N O ; % C, H, N: calculated C; 58.34, H; 6.60, N;

5

2

1

3

.20−5.13 (m,1H), 3.22−3.16 (m, 1H), 3.02 (d, J = 12.4 Hz, 2H),

2

3

31

5

6

.67 (d, J = 8.8 Hz, 2H), 2.01(d, J = 10.8 Hz, 2H), 1.73−1.65 (m,

14.79; found C; 58.49, H; 6.49, N; 14.54.

1

3

H), 1.56 (d, J = 6.4 Hz, 6H); C NMR (100 MHz, DMSO-d ): δ

4-[5-(1-Isopropyl-1H-indazol-3-yl)-[1,3,4]oxadiazol-2-yl]-1-(3-

methoxypropyl)-piperidin-4-ol (12v). By following general proce-

dure 3, 4-[5-(1-isopropyl-1H-indazol-3-yl)-[1,3,4]oxadiazol-2-yl]-pi-

peridin-4-ol 11b (705.0 mg, 2.15 mmol) was N-alkylated with 1-

bromo-3-methoxypropane to obtain above titled compound 12v

6

60.1, 139.8, 128.6, 127.5, 123.4, 121.9, 121.3, 111.0, 51.0, 45.4, 33.4,

0.3, 22.2; DSC: (RAMP 5 °C/min): 109.6 °C; mass (m/z):

+

12.1(M + H) .

4

-[5-(1-Isopropyl-1H-indazol-3-yl)-[1,3,4]oxadiazol-2-yl]-piperi-

din-4-ol (11b). By following general procedure 7, 4-hydroxy-4-[N′-(1-

isopropyl-1H-indazole-3-carbonyl)-hydrazinocarbonyl]-piperidine-1-

carboxylic acid tert-butyl ester 10b (1.2 g, 2.69 mmol) was converted

to above titled compound 11b (705.4 mg) which was isolated as a

(585.6 mg) as cream color powder in 68% yield with HPLC purity

1

98.2%. H NMR (CDCl ): δ 8.37 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 8.4

3

Hz, 1H), 7.47 (t, J = 6.9 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 5.0−4.93

(m, 1H), 3.10−2.98 (m, 2H), 1.67 (d, J = 6.6 Hz, 6H), 1.45−1.40 (m,

1

13

gummy mass in 90.0% yield with HPLC purity 92.3%. H NMR

2H), 0.92 (d, J = 6.5 Hz, 6H); C NMR (100 MHz, CDCl ): δ

3

(

DMSO-d ): δ 10.12 (bs, 1H), 9.84 (bs, 1H), 8.10 (d, J = 8.4 Hz,

167.1, 164.9, 160.4, 139.8, 128.8, 127.6, 123.5, 121.9, 121.3, 111.0,

6

1

H), 7.82 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.6

Hz, 1H), 5.13−5.06 (m, 1H), 2.80 (bs, 2H), 2.71 (bs, 2H), 1.76 (d, J

6.8 Hz, 2H), 1.55 (d, J = 6.4 Hz, 6H), 1.28 (t, J = 10.8 Hz, 2H);

74.1, 69.5, 58.3, 54.1, 51.0, 50.7, 26.6 24.3, 22.4; DSC: (RAMP 5 °C/

+

min): 197.3 °C; mass (m/z): 400.1 (M + H) ; C H N O ; % C, H,

2

1

29

5

3

=

N: calculated C; 63.14, H; 7.32, N; 17.53; found C; 62.91, H; 7.51, N;

17.64.

+

mass (m/z): 328.1 (M + H) .

1

-Isopropyl-3-(5-piperidin-4-ylmethyl-[1,3,4]oxadiazol-2-yl)-1H-

3-[5-(1-Cyclobutyl-piperidin-4-ylmethyl)-[1,3,4]oxadiazol-2-yl]-

1-isopropyl-1H-indazole Oxalate (12x). By following general

procedure 3, 1-isopropyl-3-(5-piperidin-4-ylmethyl-[1,3,4]oxadiazol-

2-yl)-1H-indazole 11c (3.1 g, 9.53 mmol) was N-alkylated using

general reductive amination procedure 9 with cyclobutane to obtain

compound 12x free base which was converted to its oxalate salt by

following the general procedure 8 to obtain above titled compound

indazole (11c). By following general procedure 7, 4-{2-[N′-(1-

isopropyl-1H-indazole-3-carbonyl)-hydrazino]-2-oxo-ethyl}-piperi-

dine-1-carboxylic acid tert-butyl ester 10c (4.7 g, 10.6 mmol) was

converted to above titled compound 11c (3.1 g) as brown color solid

1

compound in 89% yield with HPLC purity 99.3%. H NMR (DMSO-

d₆): δ 10.02 (bs, 1H), 9.86 (bs, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.82 (d,

J = 8.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H),

.13−5.06 (m, 1H), 2.80 (bs, 2H), 2.71 (bs, 2H), 2.45 (bs, 2H),1.76

d, J = 6.8 Hz, 2H), 1.55 (d, J = 6.4 Hz, 6H), 1.28 (t, J = 10.8 Hz,

12x (2.5 g) as light yellow color powder in 72.0% yield with HPLC

1

5

(

2

purity 99.1%. H NMR (DMSO-d

6

): δ 8.19 (d, J = 8.0 Hz, 1H), 7.91

(d, J = 8.4 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H),

5.19−5.15 (m, 1H), 3.15−3.09 (m, 5H), 3.01 (d, J = 6.8 Hz, 2H),

2.06−1.99 (m, 5H), 1.88−1.85 (m, 2H), 1.69−1.62 (m, 2H), 1.55 (d,

+

H); mp: 134.6−134.9 °C; mass (m/z): 325.1 (M + H) .

-[5-(3-Aza-bicyclo[3.1.0]hex-6-ylmethyl)-[1,3,4]oxadiazol-2-yl]-

-isopropyl-1H-indazole (11d). By following general procedure 7, 6-

3

1

3

1

[

J = 6.8 Hz, 6H), 1.48−1.42 (m, 2H); C NMR (100 MHz, DMSO-

N′-(1-isopropyl-1H-indazole-3-carbonyl)-hydrazinocarbonyl]-3-aza-

bicyclo[3.1.0]hexane-3-carboxylic acid tert-butyl ester 10d (1.5 g, 3.51

mmol) was converted to above titled compound 11d (1.0 g) as a

gummy mass in 93.2% yield with HPLC purity 95.2%. H NMR

DMSO-d ): δ 10.02 (bs, 1H), 9.86 (bs, 1H), 8.12 (d, J = 8.4 Hz,

d ): δ 174.4, 170.6, 165.0, 161.5, 160.3, 139.9, 139.8, 135.5, 128.8,

6

127.6, 126.9, 123.5, 123.0, 122.9, 121.8, 121.8, 121.3, 111.2, 110.9,

72.5, 58.5, 51.0, 50.7, 48.4, 48.2, 32.0, 31.2, 30.7, 28.4, 25.4, 25.3,

22.4, 22.4, 13.7; DSC: (RAMP 5 °C/min): 179.1 °C; mass (m/z):

1

+

(

380.2 (M + H) ; C H N O ; % C, H, N: calculated C; 61.39, H;

6

24 31

5

1

H), 7.82 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.6

Hz, 1H), 5.13−5.06 (m, 1H), 2.80 (bs, 2H), 2.71 (bs, 4H), 1.55 (d, J

6.4 Hz, 6H), 1.28 (t, J = 10.8 Hz, 2H); mp: 127.3−128.9 °C; mass

6.65, N; 14.92; found C; 61.74, H; 6.49, N; 14.78.

1-Isopropyl-3-[5-(3-isopropyl-3-aza-bicyclo[3.1.0]hex-6-yl)-

[1,3,4]oxadiazol-2-yl]-1H-indole Oxalate (12z). By following general

procedure 3, 3-[5-(3-aza-bicyclo[3.1.0]hex-6-yl)-[1,3,4]oxadiazol-2-

yl]-1-isopropyl-1H-indole 11d (1.0 g, 3.26 mmol) was N-alkylated

with 2-iodo propane compound 12z free base which was converted to

its oxalate salt by following the general procedure 8 to obtain above

=

+

(

m/z): 324.4 (M + H) .

-[5-(8-Aza-bicyclo[3.2.1]oct-3-yl)-[1,3,4]oxadiazol-2-yl]-1-iso-

propyl-1H-indazole (11e). By following general procedure 7, 3-[N′-

1-isopropyl-1H-indazole-3-carbonyl)-hydrazinocarbonyl]-8-aza-

bicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester 10e (3.8 g,

.342 mmol) was converted to above titled compound 11e (2.71 g) as

3

(

titled compound 12z (758.6 mg) as pale yellow color powder in 70.0

1

8

yield with HPLC purity 96.8%. H NMR (DMSO-d ): 8.16 (d, J = 8.0

6

1

white color solid in 98% yield with HPLC purity 95.2%. H NMR

Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.39 (t, J =

R

https://doi.org/10.1021/acs.jmedchem.1c00703

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

7

4

1

.6 Hz, 1H), 5.18−5.15 (m, 1H), 3.79−3.73 (m, 1H), 3.28−3.23 (m,

(20.0 mL) cooled at 0 °C, thionyl chloride (1.2 mL, 17.22 mmol) was

added. The reaction temperature was gradually warmed to r.t., and the

reaction was stirred for 6 h. After completion of the reaction, the

volatiles were evaporated. The crude mass thus obtained was

H), 2.68 (bs, 1H), 1.54 (d, J = 6.4 Hz, 6H), 1.22 (d, J = 6.0 Hz, 6H),

.03 (d, J = 6.0 Hz, 2H); DSC: (RAMP 5 °C/min): 184.2 °C; mass

+

(m/z): 351.0 (M + H) ; C H N O ; % C, H, N: calculated C;

2

3

28

4

5

62.71, H; 6.41, N; 12.72; found C; 63.04, H; 6.23, N; 12.44.

quenched with aqueous NaHCO and diluted with ethyl acetate,

3

1

-Isopropyl-3-[5-(8-methyl-8-aza-bicyclo[3.2.1]oct-3-yl)-[1,3,4]-

and the two layers were separated. The organic layer was washed with

brine solution and dried over Na SO , and the solvent was evaporated

oxadiazol-2-yl]-1H-indazole Oxalate (12ac). By following general

procedure 3, 3-[5-(8-aza-bicyclo[3.2.1]oct-3-yl)-[1,3,4]oxadiazol-2-

yl]-1-isopropyl-1H-indazole 11e (2.7 g, 8.00 mmol) was N-alkylated

with iodomethane to obtain 12ac free base which was converted to its

oxalate salt by following the general procedure 8 to obtain above titled

2

4

under vacuum to aﬀord the title compound 16 in quantitative yield.

1

H NMR (CDCl ): δ 8.25 (d, J = 8.1 Hz, l H), 7.53 (d, J = 8.4 Hz, l

3

H), 7.45 (t, J = 7.1 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 5.02−4.93 (m, l

H), 4.04 (s, 3H) 1.67 (d, J = 6.7 Hz, 6H); mass (m/z): 219 (M +

+

compound 12ac (2.5 g) as oﬀ-white color powder in 90.0% yield with

HPLC purity 97.9%. H NMR (DMSO-d ): δ 8.20 (d, J = 8.0 Hz,

H) .

1

6

1-Isopropyl-3-{3-[1-(3-methoxy-propyl)-piperidin-4-yl]-[1,2,4]-

oxadiazol-5-yl}-1H-indazole (17a). To a stirred solution of

compound 15a (500.0 mg, 2.32 mmol) in DMF (10.0 mL) at r.t., a

DMF (5.0 mL) solution of sodium hydride (116.0 mg, 3.0 mmol)

followed by compound 16 (659.0 mg, 3.02 mmol) was added, and the

reaction mass was stirred at 120 °C for 16 h. After completion of the

reaction, the reaction mass was quenched with ice cold water and

extracted with ethyl acetate, and two layers were separated. The

organic layer was washed with brine solution and dried over Na SO ,

1

7

2

H), δ 7.92 (d, J = 8.8 Hz, 1H), 7.56 (t, J = 7.2 Hz, 1H), 7.40 (t, J =

.2 Hz, 1H), 5.19−5.16 (m, 1H), 2.67 (s, 3H), 2.53−2.49 (m, 2H),

.34−2.27 (m, 4H), 2.13−2.06 (m, 4H), 1.99−1.93 (m, 1H), 1.55 (d,

13

J = 6.8 Hz, 6H); C NMR (100 MHz, DMSO-d ): δ 175.4, 174.1,

6

1

3

1

72.7, 171.2, 161.4, 161.2, 139.8, 135.6, 126.8, 124.4, 123.1, 123.0,

22.9, 121.8, 10.8, 73.3, 72.3, 72.2, 62.5, 61.4, 61.2, 50.6, 32.3, 32.0,

1.5, 30.7, 24.4, 24.2, 24.0, 22.4, 17.1; DSC: (RAMP 5 °C/min):

78.1 °C; mass (m/z): 352.0 (M + H) ; C H N O ; % C, H, N:

calculated C; 59.85, H; 6.16, N; 15.86; found C; 60.01, H; 6.02, N;

+

2

27

5

2

4

and the solvent was evaporated under vacuum to aﬀord above titled

1

5.43.

compound 17a in quantitative yield. H NMR (CDCl ): δ 8.33 (d, J =

3

1

-(3-Methoxypropyl)-piperidine-4-carbonitrile (14a). By follow-

8.1 Hz, 1H), 7.57 (d, J = 8.5 Hz, 1H), 7.50 (t, J = 7.2 Hz, 1H), 7.38

(t, J = 7.6 Hz, 1H), 5.03−4.97 (m, 1H), 3.47 (t, J = 6.4 Hz, 2H), 3.35

(s, 3H), 3.06−3.03 (m, 2H), 2.88 (s, 1H), 2.43−2.40 (m, 2H), 2.17

(bs, 1H), 2.05 (bs, 2H), 1.85−1.84 (m, 2H), 1.76 (d, J = 6.4 Hz, 6H),

1.68−1.60 (m, 4H) in 17.9% yield with HPLC purity 98.1%. DSC:

ing general procedure 3, piperidine-4-carbonitrile 13 (2.0 g, 18.18

mmol) was N-alkylated using 1-bromo-3-methoxy propane (3.0 g,

0.01 mmol) to obtain titled compound 14a in 63.6% yield. H NMR

CDCl ): δ 3.42 (t, J = 6.3 Hz, 2 H), 3.32 (s, 3H), 2.64 (bs, 3H), 2.43

t, J = 7.3 Hz, 2 H), 1.96−1.83 (m, 4 H), 1.77−1.70 (m, 2 H); mass

m/z): 183.1 (M + H) .

1

2

(

3

+

(RAMP 5 °C/min): 154.7 °C; mass (m/z): 384.1 (M + H) ;

+

C H N O ; % C, H, N: calculated C; 65.77, H; 7.62, N; 18.26;

2

1

29

5

2

1

-(Tetrahydropyran-4-ylmethyl)-piperidine-4-carbonitrile (14b).

By following general procedure 3, piperidine-4-carbonitrile 13 (1.0 g,

.09 mmol) was N-alkylated using 4-bromomethyltetrahydropyran

found C; 65.39, H; 7.33, N; 18.18.

1-Isopropyl-1H-indazole-3-carboxylic Acid Amide (18). To a

stirred solution of compound 2 (1.0 g, 4.91 mmol) in acetonitrile

(20.0 mL) at r.t., pyridine (425.9 mg, 5.39 mmol) and Boc anhydride

(1.28 g, 5.88 mmol) were added. After stirring for 1 h at r.t.,

NH HCO (627.0 mg, 7.93 mmol) was added to the reaction

9

1

(

1.7 g, 9.54 mmol) to obtain titled compound 14b in 59.2% yield. H

NMR (CDCl ): δ 3.92−3.86 (m, 2H), 3.86−3.82 (m, 2H), 2.69−

3

2

(

.63 (m, 3H), 2.46−2.42 (m, 4H), 2.42−2.39 (m, 1H), 1.98−1.81

4

3

+

m, 4H) 1.77−1.70 (m, 4H); mass (m/z): 209.2 (M + H) .

mixture, and the reaction was stirred for 16 h. The reaction mass was

diluted with ethylaceate and water, and the two layers were separated.

The organic layer was washed with 1 N HCl followed by 1 N NaOH

solution and dried over anhydrous Na SO , and the solvent was

1

-Isopropyl-piperidine-4-carbonitrile (14c). By following general

procedure 3, piperidine-4-carbonitrile 13 (1.0 g, 9.09 mmol) was N-

alkylated using 2-iodopropane (1.62 g, 9.54 mmol) to obtain titled

2

4

1

compound 14c in 77.8% yield. H NMR (CDCl ): δ 4.32−4.22 (m,

evaporated under vacuum to obtain title compound 18 (830.0 mg) in

3

1

+

H), 2.71−2.65 (m, 3H), 2.49−2.43 (m, 1H), 1.96−1.83 (m, 3H),

83.4% yield. H NMR (DMSO-d ): δ 8.17 (d, J = 8.2 Hz, l H), 7.89

6

.76−1.71 (m, 2H), 1.23 (d, J = 6.2 Hz, 6H); mass (m/z): 153.1 (M

(d, J = 8.6 Hz, 1H), 7.57 (bs, 1H), 7.44 (t, J = 7.4 Hz, 1H), 7.34 (bs,

1 H), 7.24 (t, J = 7.3 Hz, 1H), 5.09−5.03 (m, 1H), 1.53 (d, J = 6.5

Hz, 6H); mass (m/z): 204.0 (M + H) .

+

H) .

+

N-Hydroxy-1-(3-methoxypropyl)-piperidine-4-carboxamidine

15a). To the stirred solution of compound 14a (800.0 mg, 4.39

mmol) in ethanol (20.0 mL) at r.t., hydroxylamine hydrochloride

636.0 mg, 9.21 mmol) and K CO (1.27 g, 9.21 mmol) were added.

The reaction was gradually heated to reﬂux for 16 h. After completion

of the reaction, it was cooled to r.t. and ﬁltered through a Celite bed.

The ﬁltrate was concentrated under vacuum to aﬀord title compound

5a in quantitative yield. H NMR (CDCl ): δ 8.77 (bs, 1H), 5.32 (s,

H), 4.02−3.99 (m, 1H), 3.29 (bs, 2 H), 3.20 (s, 3 H), 2.85 (d, J =

1.2 Hz, 3H), 2.70 (t, J = 10.4 Hz, 3 H), 1.92 (d, J = 3.6 Hz, 2H),

(

1-Isopropyl-1H-indazole-3-carbonitrile (19). To a stirred solution

of compound 18 (1.0 g, 5.71 mmol) in THF (22.0 mL) at 0 °C,

trimethylamine (1.73 g, 17.1 mmol) followed by triﬂuoroacetic

anhydride (1.79 g, 8.55 mmol) were added. The reaction temperature

was gradually warmed to r.t., and the reaction was stirred for 2 h. The

reaction was quenched by adding ice cold water and ethyl acetate.

The two layers were separated and the organic layer was washed with

sat. NaHCO₃solution and dried over Na SO . The solvent was

(

2

3

1

2

1

3

2

4

evaporated under vacuum to obtain title compound 19 (933.0 mg) in

+

1

.83 (t, J = 1.8 Hz, 4H); mass (m/z): 216.4 (M + H) .

N-Hydroxy-1-(tetrahydropyran-4-ylmethyl)-piperidine-4-carbox-

amidine (15b). By following the procedure as mentioned for

86% yield. H NMR (CDCl ): δ 7.85 (d, J = 8.1 Hz, l H), 7.57 (d, J =

3

8.5 Hz, l H), 7.50 (t, J = 7.0 Hz, l H), 7.36 (t, J = 7.5 Hz, l H), 5.09−

5.03 (m, l H), 1.64 (d, J = 6.6 Hz, 6H); mass (m/z): 186.1 (M + H) .

+

1

compound 15a, compound 15b was prepared. H NMR (CDCl ):

N-Hydroxy-1-isopropyl-1H-indazole-3-carboxamidine (20). To a

stirred solution of compound 19 (770.0 mg, 4.16 mmol) in ethanol

(16.0 mL) at r.t., hydroxylamine hydrochloride (603.0 mg, 8.74

mmol) and K CO (1.2 g, 8.74 mmol) were added. The reaction

3

δ 8.72 (bs, 1H), 5.34 (bs, 2H), 3.92−3.86 (m, 2H), 3.86−3.82 (m,

2

1

H), 2.69−2.63 (m, 3H), 2.46−2.42 (m, 4H), 2.42−2.39 (m, 1H),

.98−1.81 (m, 4H) 1.77−1.70 (m, 4H); mass (m/z): 242.2 (M +

2

3

+

H) .

mixture was gradually heated to reﬂux for 16 h. The reaction mass was

cooled to r.t. and ﬁltered through Celite, and the ﬁltrate was dried

over Na SO and evaporated under vacuum to aﬀord title compound

N-Hydroxy-1-(isopropyl)-piperidine-4-carboxamidine (15c). By

following the procedure as mentioned for compound 15a, compound

5c was prepared. H NMR (CDCl ): δ 8.92 (bs, 1H), 5.27 (bs, 2H),

2

4

1

4

1

20 (923.6 mg) in quantitative yield. H NMR (DMSO-d ): δ 9.76

(bs, 1H), 8.09 (d, J = 8.1 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.40 (t, J

= 7.3 Hz, 1H), 7.18 (t, J = 7.5 Hz, 1H), 5.60 (bs, 2H), 5.03- 4.96 (m,

1H), 1.51 (d, J = 6.5 Hz, 6H); mass (m/z): 219.0 (M + H) .

1-(3-Methoxypropyl)-piperidine-4-carboxylic Acid Ethyl Ester

(22a). By following general procedure 3, ethylisonipecotate 21 was

3

6

.32−4.22 (m, 1H), 2.71−2.65 (m, 3H), 2.49−2.43 (m, 1H), 1.96−

.83 (m, 3H), 1.76−1.71 (m, 2H), 1.23 (d, J = 6.2 Hz, 6H); mass (m/

+

z): 186.3 (M + H).

1

-Isopropyl-1H-indazole-3-carboxylic acid methyl ester (16). To

the stirred solution of compound 2 (1.0 g, 4.91 mmol) in methanol

S

https://doi.org/10.1021/acs.jmedchem.1c00703

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

alkylated using 1-bromo-3-methoxy propane to obtain compound 22a

England Biolabs (Ipswich, Massachusetts, USA). All other reagents

and common chemicals were purchased from Sigma-Aldrich (St.

1

as a gummy liquid. H NMR (CDCl ): δ 4.12 (q, 2H), 3.41 (t, J = 6.4

3

Hz, 2H), 2.90−2.85 (m, 2H), 2.38 (t, J = 7.4 Hz, 2H), 2.34−2.20 (m,

Louis, Missouri, USA). Human 5-HT R cDNA clone was purchased

4

1

H), 2.05−1.93 (m, 2H), 1.92−1.85 (m, 2H), 1.80−1.70 (m, 2H),

from Origene (Rockville, Maryland, USA). The coding sequence was

ampliﬁed by PCR using gene speciﬁc primers. Ampliﬁed DNA was

cloned in to mammalian expression vector pcDNA 3.1. The

authenticity of the cloned genes was determined by restriction

analysis and nucleotide sequencing. CHO cell line was purchased

from American Type Culture Collection, Manassas, Virginia, USA.

The cells were routinely cultured in Hams F12 medium supplemented

+

.23 (t, J = 7.1 Hz, 3H); mass (m/z): 230.2 (M + H) .

1

-(Tetrahydropyran-4-ylmethyl)-piperidine-4-carboxylic Acid

Ethyl Ester (22b). By following general procedure 3, ethyl-

isonipecotate 21 was alkylated using 4-bromomethyltetrahydropyran

1

to obtain compound 22b as a gummy liquid. H NMR (CDCl ): δ

3

4

.12 (q, 2H), 3.89 (t, J = 6.1 Hz, 2H), 3.81 (t, J = 6.1 Hz, 2H), 3.20−

3

.12 (m, 3H), 2.90−2.85 (m, 3H), 2.36−2.26 (m, 1H), 2.11−2.07,

with 10% fetal bovine serum (FBS) at 37 °C in 5% CO . The cells

2

(

2

m, 1H), 2.05−1.93 (m, 4H), 1.92−1.85 (m, 2H), 1.80−1.70 (m,

were transfected with 5-HT R isoforms along with 3-fold excess of

4

+

H), 1.25 (t, J = 6.5 Hz, 3H); mass (m/z): 256.3 (M + H) .

CRE-Luc reporter gene using lipofectamine as recommended by the

supplier. Cells were further cultured in the same medium

supplemented with 800 μg/mL G418. Individual colonies were

picked up after 2 weeks of selection and screened for serotonin-

induced luciferase activity. Colonies exhibiting maximum serotonin-

induced luciferase activity were selected and ampliﬁed for further

investigations. The recombinant CHO cells were plated in 96-well

clear bottom white plates (Corning) and cultured overnight as

described above. Cells were grown overnight in serum-free medium

before incubation with compounds to prevent receptor desensitization

due to presence of endogenous ligands in the serum.

1

-(Isopropyl)-piperidine-4-carboxylic Acid Ethyl Ester (22c). By

following general procedure 3, ethylisonipecotate 21 was alkylated

1

using 2-iodopropane to obtain compound 22c as a gummy liquid. H

NMR (CDCl ): δ 4.12 (q, 2H), 4.02−3.98 (m, 1H), 3.20−3.12 (m,

3

1

H), 2.90−2.85 (m, 1H), 2.36−2.26 (m, 1H), 1.92−1.85 (m, 2H),

.80−1.70 (m, 2H), 1.62 (d, J = 6.7 Hz, 6H), 1.21 (t, J = 6.5 Hz, 3H);

+

mass (m/z): 200.2 (M + H) .

1

-Isopropyl-3-{5-[1-(3-methoxypropyl)-piperidin-4-yl]-[1,2,4]-

oxadiazol-3-yl}-1H-indazole (23a). To a stirred solution of

compound 22a (685.0 mg, 2.9 mmol) in DMF (6.0 mL) at 0 °C, a

DMF solution of sodium hydride (119.0 mg, 2.9 mmol) followed by

compound 20 (500.0 mg, 2.29 mmol) was added. The reaction

temperature was gradually raised to 120 °C for 16 h. The reaction

mass was cooled to r.t. and ﬁltered through Celite, the ﬁltrate was

dried over Na SO , and the solvent was evaporated under vacuum to

The reference endogenous agonist serotonin and test compounds

in Opti-MEM medium at 11 diﬀerent test concentrations, starting

from 10 μM until 0.1 nM in 3-fold serial dilutions, were incubated

with the cells separately in individual wells for 4 h at 37 °C in 5%

2

4

CO . Compounds whose potency was <1 nM were tested in a dose−

2

1

aﬀord title compound 23a (105.6 mg) in 95% yield. H NMR

CDCl ): δ 8.31 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.46 (t,

response study with concentrations starting from 1 μM to 0.01 nM.

After incubation, the medium was removed, and the cells were washed

with phosphate buﬀered saline and lysed in lysis buﬀer (Tris, 10 mM

(pH-8.0); NaCl, 50 mM; DTT, 1 mM; protease inhibitor cocktail 1×,

NP 40−0.1%). The luciferase activity was measured in individual

wells using luciferin substrate in a Victor Light Luminometer,

PerkinElmer.

(

3

J = 7.1 Hz, 1H), 7.32 (t, J = 7.5 Hz, 1H), 5.01−4.96 (m, 1H), 3.46 (t,

J = 6.3 Hz, 2H), 3.35 (s, 3H), 3.01 (bs, 2H), 2.48 (t, J = 7.2 Hz, 2H),

2

6

1

6

.20−2.10 (m, 2H), 2.05 (bs, 1H), 1.83−1.76 (m, 2H), 1.69 (d, J =

1

3

.7 Hz, 6H), 1.25−1.23 (m, 4H); C NMR (100 MHz, CDCl ): δ

3

72.4, 170.7, 164.9, 139.9, 128.8, 127.7, 124.1, 122.4, 121.1, 111.5,

9.5, 58.3, 54.2, 51.3, 51.1, 31.3, 27.1, 24.4, 22.4; DSC: (RAMP 5 °C/

The maximum response produced by each drug was normalized to

the 5-HT-induced maximum response. Data were analyzed using

Graphpad prism software to derive EC₅₀values that corresponded to

the concentration of agonists required to obtain half-maximal

stimulation of adenylyl cyclase. Values were expressed as the mean

of two independent experiments performed in duplicates. Reference

+

min): 167.3 °C; mass (m/z): 384.4 (M + H) ; C H N O ; % C, H,

N: calculated C; 65.77, H; 7.62, N; 18.26; found C; 65.93, H; 7.84, N;

2

1

29

5

2

1

8.01.

Animals and Ethics. Adult male Wistar rats (8−12 weeks; 200−

00 g; in-house bred) were used in all the studies. Naive animals were

3

9

used for experimentation. Rats were housed in groups of 3−4 in

compound PF-04995274, a reported partial agonist , showed partial

hanging polycarbonate cages (dimension: length, 43.2 cm × width,

agonist activity in our 5-HT₄_Ecell-based reporter gene assay with an

EC₅₀value of 1.3 nM and E_m_a_x18%, which was very well in agreement

with published data of EC /E_m_a_x: 0.26 nM/7%. Some of other

2

5.4 in. × height, 21.2 cm) with a bedding of clean corn cob. For all

the experiments, animals were introduced to the animal house at least

days prior to the commencement of the study. The animal house

5

0

7

reported compounds EC₅₀values were also determined using our

assay and compared with the published data. The EC ₅ ₀ values

obtained versus reported values are tabulated in the Supporting

Information of this manuscript.

was maintained at 21 ± 3 °C, 30−70% relative humidity and 12 h

light/dark cycle (lights on at 7:00 AM and oﬀ at 7:00 PM). Food

(pelleted rodent feed, Rayan’s Biotechnologies Pvt. Ltd. Hyderabad)

and water were made available ad libitum unless speciﬁed otherwise

for the experiment. The test compound was administered orally, and

scopolamine was administered through the intraperitoneal route. For

the object recognition task, each group had 12 rats, and in the radial

arm maze, each group had 6 rats. All experimental procedures were

performed in accordance with the Institutional Animal Ethics

Committee of Suven Life Sciences Ltd., Hyderabad, constituted as

per the directions of the Committee for the Purpose of Control and

Supervision of Experiments on Animals (CPCSEA), India.

Microsomal Metabolic Stability. Compounds were incubated

with microsomes at 0.5 mg protein/mL in 100 mM phosphate buﬀer

pH 7.4 at 37 °C. Reactions were initiated by addition of NADPH. All

incubations were carried out for 30 min. At 30 min, an aliquot of the

sample was removed and transferred into 300 μL of acetonitrile. The

samples were stored at −20 °C until analysis. Samples were analyzed

using LC-MS/MS. Results were expressed as % metabolized at 30 min

postincubation.

Protein Binding. Unbound fractions of test compound in plasma,

brain homogenate, and liver microsomes were determined using high-

throughput dialysis (HT dialysis). Brieﬂy, dialysis membranes were

soaked in deionized water for 20 min, then in deionized water with

30% ethanol for 15 min, and ﬁnally in phosphate buﬀer until use.

Membranes were rinsed in phosphate buﬀer before assembling. The

membranes were layered between Teﬂon bars of dialysis assembly.

Stock solution of test compound was prepared at 10 mM in DMSO,

diluted to 1 mM in acetonitrile, and further diluted to 100 μM in

mixture of water and acetonitrile (1:1 v/v). Rat blood and brains were

isolated on the day of the study, and brains were homogenized with

two volumes of phosphate buﬀer. The blood was centrifuged at 4000

rpm for 10 min to obtain plasma. Human plasma (pool of 3) was

Determination of EC₅₀Values for 5-HT R. The compounds

4

were screened in the cell-based luciferase reporter gene luminescence

assay which measures the levels of cAMP inside cells upon activation

or inhibition of the receptor. Serotonin (5-HT) and luciferin was

purchased from Sigma-Aldrich (St. Louis, Missouri, USA). T4 DNA

ligase and high ﬁdelity Taq polymerase were procured from Roche

(

Basel, Switzerland). Superscript reverse transcriptase and mammalian

expression vector pcDNA3.1 were purchased from Invitrogen

Carlsbad, California, USA). CRE-Luc reporter gene construct was

(

supplied by Strata gene (La Jolla, California, USA). Cell culture media

and sera were procured from Invitrogen (Carlsbad, California, USA).

All other DNA restriction and modiﬁcation enzymes were from New

T

https://doi.org/10.1021/acs.jmedchem.1c00703

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

prepared from human blood (3 donors) by centrifuging at 4000 rpm

intervals for an intravenous route of administration, and food was

oﬀered after 4 h of postoral dose. On the day of dosing (day 0), each

dog was administered intravenously as 5 min infusion at 1 mg/kg and

oral administration as gavage at 3 mg/kg on day 7. Dose formulations

were prepared on the day of treatment. At each time point, 0.5 mL of

blood sample was collected from saphenous/cephalic vein at

designated time points, and collected blood samples were transferred

to prelabeled sodium heparin-coated sampling tubes. The time points

for blood collection from each animal were predose and 0.08 (only

i.v.), 0.16 (only i.v.), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h post dose (n = 3).

The collected blood samples were centrifuged at 4000 rpm for 10 min

at 4 °C within 30 min of collection, and plasma was separated in

prelabeled tubes and stored at −70 °C until analysis. Compound 12l

concentrations in plasma samples were determined using a qualiﬁed

LC-MS/MS method with ACN protein precipitation technique and

quantiﬁed in the calibration range of 1.013−1013 ng/mL. The PK

evaluation was performed using noncompartmental analysis (Win-

NonLin version 5.0.3 Pharsight Corporation, Mountain View,

California 94040/USA) and using the linear trapezoidal with linear

interpolation calculation method.

for 10 min at 4 °C. Suspension of liver microsomes was prepared at

0.5 in phosphate buﬀer (100 mM, pH 7.4). The dialysate chambers

were loaded with 150 μL of 100 mM phosphate buﬀer (pH 7.4) in

triplicates. The matrix chambers were loaded with 150 μL of the

plasma or brain homogenate or microsomal suspension spiked with

test compound at a ﬁnal concentration of 1 μM. 50 μL of the sample

was removed from both of the chambers at 0 h. The plate was sealed

and incubated at 37 °C for 6 h at 100 rpm. After 6 h, 50 μL of the

sample was removed from both the chambers. Equal volumes of buﬀer

or matrix were added to the matrix and buﬀer samples, respectively, to

create identical sample matrices for analysis. The supernatants were

transferred to vials and were analyzed by LC-MS/MS.

Pharmacokinetic Study in Rats. Male Wistar rats (225 ± 25 g)

were used for evaluation of pharmacokinetics. Three animals were

housed in each cage. Two days prior to dosing day, male Wistar rats

were anesthetized with isoﬂurane for surgical placement of jugular

vein catheter. Animals were fasted overnight before oral dosing (p.o.),

and food pellets were allowed 2 h postdosing, whereas during

intravenous dosing food and water were provided as ad libitum. Three

rats were dosed with test compound orally (3 mg/kg) or

intravenously (i.v., 1 mg/kg). Dose formulation was prepared by

using water as a vehicle (10 mL/kg for oral and 2 mL/kg for

intravenous dosing). At each time point, 200 μL of blood was

collected through jugular vein cannula and immediately replenished

with an equivalent volume of normal saline in freely moving rats.

Collected blood was transferred into a labeled eppendorf tube

containing 10 μL of heparin as anticoagulant. The time points for

blood collection were predose and 0.08 (only i.v.), 0.25, 0.5, 1, 2, 4, 6,

5

0

CYP 3A4 and 2D6 Inhibition. CYP 3A4 and 2D6 inhibitory

potential of the test compounds was studied using human liver

microsomes. Inhibitory activity was evaluated by incubating the test

compound (0.1−45 μM) in duplicates for 2 (3A4) and 12 min (2D6)

with human liver microsomes in the presence of isoform-speciﬁc

substrate (midazolam-3A4 or dextromethorphan-2D6) and NADPH

regeneration system. Incubations were terminated by adding ice-cold

acetonitrile containing an internal standard, and metabolites were

quantiﬁed using LC-MS/MS. Peak area ratios of analyte versus

internal standards were used for calculating IC .

8

4

, and 24 h postdose (n = 3). The collected blood was centrifuged at

000 rpm for 10 min at 4 °C. Plasma was separated in prelabeled

5

0

5

2

hERG Patch Clamp Assay IC₅₀Determination.

The

tubes and stored below −70 °C until analysis. The test compound

concentrations were extracted from plasma samples by protein

precipitation using four volumes of acetonitrile (v/v) containing

predetermined internal standard. The precipitants were centrifuged

HEK293-hERG cells for patch clamp assay were cultured in

Dulbecco’s modiﬁed Eagle’s medium (DMEM):Ham’s F12 (1:1

ratio) with 10% FBS containing 0.5 mg/mL of selective antibiotic, G-

418 sulfate (Calbiochem), and 0.1% penicillin and streptomycin

(Thermoﬁsher, MA, USA) in a T-75 ﬂask. Cells were harvested at

70−80% conﬂuency by washing with HBSS lacking Mg and Ca

ions and subsequent addition of accumax (2.5 mL) (Sigma-Aldrich,

(at 11292×g and 10 °C for 10 min), and supernatant was transferred

2

+

2+

and injected in LC-MS/MS. The analytes of interest were quantiﬁed

using a suitable multiple reaction monitoring mode in the range of 2−

2000 ng/mL or ng/g against a set of calibration standards and quality

MO, USA) and incubated in the ﬂask at 5% CO and 37 °C until cells

2

control samples. Study samples were analyzed using calibration

samples in the batch and quality control samples spread across the

batch. Pharmacokinetic parameters were determined by using

standard noncompartmental analysis (Phoenix WinNonLin 6.2

version or higher software) using linear trapezoidal with linear

interpolation method.

start sliding on the surface. After cells have completely detached from

the surface, about 10 mL of complete media without selective

antibiotic was added and pipetted to detach the cell clumps.

Additional 7.5 mL of complete media was added to collect the

residual cells in the ﬂask, and cell suspension was incubated at 5%

CO and 37 °C for 30 min. Centrifuged at 200g for 2 min, the

2

Rodent Brain Penetration Study. Male Wistar rats (225 ± 25

g) were used as experimental animals. Three animals were housed in

each cage. Animals were given water and food ad libitum throughout

the experiment and maintained on a 12 h light/dark cycle. Brain

penetration was determined in a discrete manner in the rats. The

compounds were formulated in water and administered orally at 10

mg/kg (free base equivalent). Blood samples were removed via

cardiac puncture under isoﬂurane anesthesia at 1 h postdose. The

animals were sacriﬁced to collect the brain tissue. Plasma was

separated, and brain samples were homogenized and stored at −70 °C

until analysis. The concentrations of the compound in plasma and

brain were determined using a LC-MS/MS method. The test

compound was extracted from brain homogenate by protein

precipitation using four volumes of acetonitrile (v/v) containing

predetermined internal standard. The precipitants were centrifuged

supernatant was discarded, and the cell pellet was resuspended in

external solution which was used for the patch clamp assay. hERG

patch clamp assay was performed using the external solution of 140

mM NaCl, 4 mM KCl, 1 mM MgCl , 2 mM CaCl , 5 mM D-glucose

2

monohydrate, 10 mM HEPES/NaOH, pH 7.4, osmolarity: 298

mOsmol and internal solution of 50 mM KCl, 10 mM NaCl, 60 mM

KF, 20 mM EGTA, 10 mM HEPES/KOH, pH 7.2, osmolarity 285

mOsmol. Whole cell patch clamp recordings were performed by using

the I−V protocol of holding HEK293-hERG cells at −80 mV and

stepping to +40 mV for 500 ms and followed by 500 ms step to −40

mV before stepping back to the holding potential. The peak current is

measured at −40 mV tail currents using the Patchliner instrument.

The I−V pulse protocol was pulsed every 10 s, online analyses were

recorded by substituting the leak current from the peak current, and

IC₅₀values were calculated from IGOR PRO software (WaveMetrics,

Inc. Portland, OR, USA).

(at 11292g at 10 °C for 10 min), and the supernatant was transferred

and injected in LC-MS/MS. The compounds were quantiﬁed in the

calibration range of 1−2000 ng/mL in plasma and brain homogenate.

Study samples were analyzed using calibration samples in the batch

and quality control samples spread across the batch. The extent of

brain to plasma ratio was calculated (C_b_r_a_i_n/C_p_l_a_s_m_a).

In Vivo PK Proﬁle in Male Beagle Dogs. A single dose,

crossover study design with 7 days washout period between the

treatments was used to administer compound 12l to three male beagle

dogs. All dogs were fasted overnight, food was oﬀered at regular time

Determination of In Vivo Receptor Occupancy. Male Wistar

rats (n = 4−6/group) were administered with vehicle or test

compounds (compound 12l (0.1, 0.3, 1, 3, 10, and 30 mg/kg, p.o.),

PF-04995274 (0.003, 0.01, 0.03, 0.1, 0.3, 1, and 3 mg/kg, s.c.) or RS-

67333 (1, 3, 10, 20, and 30 mg/kg, p.o.). After 1 h of treatment with

test compounds or vehicle, rats were restrained and administered with

tracer (SB207145, 1 μg/kg) intravenously. After 30 min of tracer

administration, rats were sacriﬁced, and the brain was separated for

isolation of cerebellum (nonspeciﬁc region) and striatum (speciﬁc

U

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

region). Isolated brain tissues were transferred to prelabeled tubes and

stored on dry ice until quantiﬁcation of tracer concentrations using

LC-MS/MS. Blood and remaining brain were also collected from the

rats treated with compound 12l for measurement of test compound

concentrations. Percentage R.O. of test compound was calculated

using the concentrations of tracer in the striatum and cerebellum.

Object Recognition Task. Male Wistar rats 10−12 weeks old

were used. The arena was an open ﬁeld made up of acrylic (50 × 50 ×

expression constructs encoding the human APP695 gene and 5-HT₄_E

gene using lipofectamine 2000 according to the manufacturer’s

instructions. Brieﬂy, 50−60% conﬂuent cell cultures were incubated

for 4 h in a mixture of a cationic transfection reagent and expression

constructs. At the end of the incubation period, media were changed,

and cells were cultured for an additional period of 40 h before the

determination of sAPPα.

Measurement of sAPPα by Western Blot. Cells were cultured

as described above and 40 h prior to assay were seeded into 6-well

poly-D-lysine-coated multiwell dishes and incubated in a humidiﬁed

5

0 cm). Twenty-4 h prior to testing, the rats were habituated to

individual test arenas for 20 min in the absence of any objects.

Twenty-4 h after the habituation, rats (12 animals per group) were

administered respective treatments. After the post-treatment interval

of 30 min, rats were subjected to familiarization phase (T1). Rats

were placed individually in the open ﬁeld for 3 min, containing two

identical objects (a1 and a2). Choice trial (T2) was carried out after

5% CO incubator at 37 °C. Cells were grown to approximately 90%

2

conﬂuency and, following aspiration of the growth medium, incubated

in serum-free DMEM for 4 h. Cells then were washed with phosphate-

buﬀered saline prior to incubation with test compound (0.001−10

μM in DMEM) for 30 min at 37 °C. For antagonist study, the cells

24 h after the T1 trial. Rats were administered respective treatments.

were preincubated for 15 min and then treated with 5-HT R agonist

4

After the post treatment interval of 30 min rats were subjected to

choice trial (T2). Rats were allowed to explore the open ﬁeld for 3

min in the presence of one familiar object (a3) and one novel object

for 30 min. A buﬀer control and 5-HT (1 μM) were included as

controls on every plate. For each condition, determinations were

made in duplicate. At the end of the assay incubation period, an

aliquot of extracellular medium was transferred from the respective

wells into a tube containing protease inhibitor cocktail. The sample

was centrifuged, and the supernatant collected into a new tube and

stored at −80 °C prior to assay by Western blot. Samples were

separated on SDS-PAGE 10% and transferred to PVDF membrane

from Millipore (Burlington, Massachusetts, USA) in a mini trans blot

cell apparatus from Bio-Rad (Hercules, California, USA) with transfer

conditions of 10−15 V for overnight at 2−8 °C using transfer buﬀer

(25 mM Tris, 192 mM glycine and 10% methanol). Membrane was

preactivated in 100% methanol for 20 s and equilibrated in transfer

buﬀer for 5 min before subjecting for transfer. After transfer,

membrane was washed for 5 min in Tris buﬀered saline (TBS). The

membrane was blocked in blocking buﬀer (TBS containing 0.1%

Tween 20 and 5% nonfat dry milk) for 1 h at r.t. After blocking, the

membrane was incubated with primary antibody puriﬁed anti-β-

amyloid, 1−16 monoclonal antibody (clone 6E10) raised in a mouse

for overnight at 2−8 °C. Membrane was washed three times each for

5 min in wash buﬀer (TBS containing 0.1% Tween 20). Membrane

was incubated with secondary antibody antimouse IgG-alkaline

phosphatase raised in a goat for 1 h at r.t. Membrane was washed

three times each for 5 min in wash buﬀer (TBS containing 0.1%

Tween 20). Membrane was developed with BCIP/NBT detection

reagent, and images were captured in GelDoc imaging System (Bio-

Rad). Electrophoresis molecular weight markers were used to verify

the size of the protein, that is, sAPPα 98 kDa. Images were analyzed

using ImageJ software (NIH).

(b). Exploration time was noted. Recognition index was calculated as

time spent with the novel object and time spent with the familiar

object in the choice trial. Rats which did not explore the familiar

object for ≥15 s in the familiarization trial and ≥10 s in the

recognition trial were not considered for statistical evaluation. The

object was such that it could not be moved from its place. The

familiar object was a bright yellow colored bottle of dimension 12 × 4

cm, while the novel object was an ambered colored bottle of

dimension 11 × 3.5 cm. After each of the familiarization or

recognition trials, the bottles were cleaned with 20% alcohol to

mask olfactory cues.

Radial Arm Maze. Rats were fasted until they reached 85% of

their body weight and then trained to eat pelleted food in the home

cage. On days 1 and 2, the rats were habituated in the radial arm maze

to ﬁnd and eat the pellets. On the subsequent 4 days, the rats had to

ﬁnd and eat the pellets which were exclusively placed in the hopper of

the arm. Each hopper was baited once. Re-entry into an arm from

which the food was retrieved or exited from an arm without eating the

pellet was considered as an error. % Choice accuracy was calculated as

%choice accuracy = 100 − {(TE/TA) × 100}

where TE is the total error and TA is the total number of choices

made. A rat could make a total of 16 arm visits. The trial was ended

when all the pellets were retrieved or 16 arms were visited or a time of

1

0 min had elapsed. Total error was calculated as sum of number of

repeat entries into an arm after food retrieval + number of arms

omitted + exit from an arm without food retrieval. Following each

trial, the radial maze was cleaned with soap solution to mask olfactory

cues.

ASSOCIATED CONTENT

■

*

sı Supporting Information

Estimation of sAPPα Levels Secreted by Cell Line. The

measurement of sAPPα by Western blot was performed according to

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00703.

4

8

Fei Shen with slight modiﬁcations. 5-HT R agonist-evoked

4

stimulation of sAPPα secretion in vitro was monitored using tsa201

cells transiently transfected with h5-HT₄_Ereceptor and human

APP695. The amount of sAPPα released into the culture medium

was determined using a semiquantitative Western blot assay with an

antibody puriﬁed anti-β-amyloid, 1−16 monoclonal antibody (clone

Characterization data of ﬁnal compounds, comparative

in vitro aﬃnities of 5-HT R reference standards, data

4

from functional assays, selectivity proﬁle and elemental

analysis (PDF)

6

E10) to amino acid residues 1−17 of amyloid beta (Aβ) in the C

terminus of sAPPα.

Additional data (PDF)

Cell culture medium, serum, and transfection reagent were

purchased from Invitrogen (Carlsbad, California, USA). The human

kidney cell line tsa201 was purchased from ECACC Sigma-Aldrich

Molecular formula strings (CSV)

Additional data (PDF)

(

St. Louis, Missouri, USA). Primary antibody clone 6E10 was

purchased from Covance (Princeton, New Jersey, USA). Secondary

antibody antimouse IgG-alkaline phosphatase, detection reagent,

protease inhibitor, and chemicals were purchased from Sigma-Aldrich

AUTHOR INFORMATION

■

(

St. Louis, Missouri, USA).

Cell Culture and Transient Transfection. The human kidney

Corresponding Author

Ramakrishna Nirogi − Suven Life Sciences Limited,

Hyderabad 500 034, India; orcid.org/0000-0002-2045-

0784 ; Email: nvsrk@suven.com

cell line tsa201 was grown at 37 °C and 5% CO₂in DMEM

supplemented with 10% FBS, 2 mM glutamine, and antibiotics. For

transient transfection experiments, tsa201 cells were transfected with

V

https://doi.org/10.1021/acs.jmedchem.1c00703

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

Authors

receptor; p.o., per oral; s.c., subcutaneous; i.v., intravenous;

min, minute; mm, millimeter; rpm, rotations per minute; cps,

counts per second; mp, melting point; DSC, diﬀerential

Abdul Rasheed Mohammed − Suven Life Sciences Limited,

Hyderabad 500 034, India; orcid.org/0000-0001-7873-

4

600

scanning calorimetry; hERG, human ether-a-go-go-related

̀

Anil Karbhari Shinde − Suven Life Sciences Limited,

Hyderabad 500 034, India

Shankar Reddy Gagginapally − Suven Life Sciences Limited,

Hyderabad 500 034, India

gene; sAPPα, soluble amyloid precursor protein α; R.O.,

receptor occupancy; LC-MS/MS, liquid chromatography−

mass spectrometry; HPLC, high-performance liquid chroma-

tography; Ach, acetylcholine; EC , concentration giving a 50%

5

0

Durga Malleshwari Kancharla − Suven Life Sciences Limited,

response; ED , dose giving a 50% response; E_m_a_x, maximal

50

Hyderabad 500 034, India

eﬀect; GPCR, G-protein-coupled receptor; HEK, human

embryonic kidney; HLM, human liver microsome; RLM, rat

liver microsome; SEM, standard error of the mean; cAMP,

cyclic adenosine monophosphate; CHO, Chinese hamster

ovary; THP, tetrahydropyran; DCM, dichloromethane; TLC,

thin-layer chromatography; IPA, isopropanol; EtOAc, ethyl

acetate; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethy-

laminium tetraﬂuoroborate; HATU, 1-[bis(dimethylamino)-

methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexa-

ﬂuorophosphate; TFA, triﬂuoroacetic acid; TFAA, triﬂuoro-

acetic anhydride; DMAP, N,N-dimethylaminopyridine; ICH,

International Council for Harmonization; OECD, Organiza-

tion for Economic Cooperation and Development

Srinivasa Rao Ravella − Suven Life Sciences Limited,

Hyderabad 500 034, India

Narsimha Bogaraju − Suven Life Sciences Limited, Hyderabad

5

00 034, India

Vanaja Reddy Middekadi − Suven Life Sciences Limited,

Hyderabad 500 034, India

Ramkumar Subramanian − Suven Life Sciences Limited,

Hyderabad 500 034, India

Raghava Choudary Palacharla − Suven Life Sciences Limited,

Hyderabad 500 034, India

Vijay Benade − Suven Life Sciences Limited, Hyderabad 500

0

34, India

Nageswararao Muddana − Suven Life Sciences Limited,

Hyderabad 500 034, India

REFERENCES

■

Renny Abraham − Suven Life Sciences Limited, Hyderabad

(

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Hyderabad 500 034, India

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Hyderabad 500 034, India

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Surendra Petlu − Suven Life Sciences Limited, Hyderabad 500

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ACKNOWLEDGMENTS

■

The support of the scientists from Analytical Chemistry, In

Vitro and In Vivo Pharmacology, Drug Metabolism, and

Pharmacokinetics Departments and the moral and ﬁnancial

support from Mr. Venkateswarlu Jasti, CEO, Suven Life

Sciences Ltd., Hyderabad are greatly acknowledged.

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ABBREVIATIONS USED

■

AD, Alzheimer’s disease; SAR, structure−activity relationship;

CNS, central nervous system; 5-HT R, 5-hydroxytryptamine 4

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