SAR Investigation and Discovery of Water-Soluble 1-Methyl-1,4-dihydroindeno[1,2- c]pyrazoles as Potent Tubulin Polymerization Inhibitors

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SAR Investigation and Discovery of Water-Soluble 1‑Methyl-1,4-

dihydroindeno[1,2‑c]pyrazoles as Potent Tubulin Polymerization

Inhibitors

Ying-Jie Cui,^§Chao Liu,^§Chen-Chen Ma, Ya-Ting Ji, Yi-Li Yao, Long-Qian Tang, Cheng-Mei Zhang,

Jing-De Wu,* and Zhao-Peng Liu *

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ABSTRACT: Taking the previously discovered 1-methyl-1,4-

dihydroindeno[1,2c]pyrazol derivative LL01 as a lead, systematic

structural modiﬁcations were made at the phenolic 6- and 7-

positions and the aniline at the 3-position of the indenopyrazole

core to investigate the SARs and to improve water solubility.

Among the designed indenopyrazoles ID01−ID33, a series of

potent MTAs were identiﬁed. As the hydrochloride salt(s), ID09

and ID33 showed excellent aqueous solubility and favorable Log P value and displayed noteworthily low nanomolar potency against

a variety of tumor cells, including those taxol-resistant ones. They inhibited tubulin polymerization, disrupted cellular microtubule

networks by targeting the colchicine site, and promoted HepG2 cell cycle arrest and cell apoptosis. In the HepG2 xenograft mouse

model, ID09 and ID33 eﬀectively inhibited tumor growth at an oral dose of 25 mg/kg. At an intravenous (iv) injection dose of 10

mg/kg every other day, ID09 suppressed tumor growth by 68% without obvious toxicity.

due to low water solubility.^27,28In addition, the appearance of

INTRODUCTION

■

multidrug resistance also limits their antitumor eﬃciency.²⁹To

Microtubules are formed by the polymerization of α- and β-

reduce the side eﬀects of MTAs and overcome their drug

resistance, highly potent and cytotoxic MTAs, such as

dolastatin 10 analogues, monomethyl auristatin E, maytansine

derivatives, cryptophycins, and tubulysins, are used as powerful

drug warheads and linked to cancer-targeted monoclonal

antibodies to make antibody−drug conjugates (ADCs), where

the monoclonal antibodies recognize the tumor and deliver the

MTAs into the target tissue.³⁰⁻³³The FDA approved the ADC

drugs trastuzumab emtansine for HER2-positive metastatic

breast cancer and brentuximab vedotin for the treatment of

CD30-expressing T-cell lymphoma, primary cutaneous ana-

plastic large cell lymphoma, and Hodgkin’s lymphoma.^34,35

The success of ADCs for cancer treatment has expanded the

applications of MTAs. Besides the traditional roles of MTAs as

antimitotic agents, the multiple-hit mode of action of MTAs in

nondividing cells, including the inhibition of cellular transport,

the inhibition of metabolism, and the impairment of cellular

signaling important for carcinogenesis, is of major con-

tubulin heterodimers and serve as an integral part of the

cytoskeleton.¹⁻⁴The dynamic features of microtubule

polymerization/depolymerization play critical roles in multiple

cell functions, including cell division, cell cytoskeleton

maintenance, cell signaling, and organelle transport.⁵⁻¹⁰

Interfering with tubulin dynamics becomes an eﬀective strategy

in antitumor drug discovery.¹¹⁻¹³Microtubule-targeting agents

(MTAs) are categorized as microtubule stabilizers and

microtubule destabilizers. The microtubule-stabilizing agents

promote the α- and β-tubulin assembly, thereby increasing the

density of microtubules, while the microtubule-destabilizing

agents initiate microtubule depolymerization, leading to

microtubule disassembly and a decrease in microtubule

density. Until now, six well-known tubulin-binding sites have

been identiﬁed.¹⁴The colchicine,¹⁵vinca,¹⁶maytanine,¹⁷and

pironetin^18,19site-binding agents are microtubule destabilizers,

while the taxane and laulimalide/peloruside²⁰site-binding

agents are microtubule stabilizers. Currently, all of the Food

and Drug Administration (FDA)-approved MTAs are natural

products or their derivatives,²¹⁻²³including the vinca site-

destabilizing MTAs,²⁴⁻²⁶vinblastine, vincristine, vinorelbine,

vindesine, vinﬂunine, and eribulin, and the taxane-site

stabilizing MTAs, paclitaxel (taxol), docetaxel, cabazitaxel,

and ixabepilone.²¹These drugs are widely used and highly

eﬀective for the treatment of solid and hematological tumors.

However, these MTAs suﬀer from some neurotoxicity and

myelosuppression-related toxicity as well as formulation issues

Received: August 1, 2020

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Figure 1. Colchicine site ligands.

Figure 2. Design of indenopyrazole derivatives.

cern.³⁶⁻⁴²Therefore, the continued discovery and develop-

ment of new MTAs will contribute to cancer therapy.

types of inhibitors prompted us to elucidate the structure−

activity relationships (SARs) as well as to improve the water

solubility of these indenopyrazole analogues to develop

potential new drug candidates. Therefore, we maintained the

1-methyl-1,4-dihydroindeno-[1,2-c]pyrazole core and made

modiﬁcations on the phenolic 6- and 7-positions and the 3-

aniline portion (Figure 2). We ﬁrst changed the ethyl group in

6-ethoxy of LL01 from hydrogen and methyl to other larger

groups (Pr, Bu, p-methoxybenzyl) to determine the inﬂuences

of these groups on the interaction with zone 1. A reversion of

the positions of 7-oxaacetaminde and 6-alkyloxy was also made

to conﬁrm the importance of the interactions with Serα178 on

the interfacial surface. Next, we made modiﬁcations at the

phenolic 7-position by introducing diﬀerent substituents with

terminal hydroxyl or amino groups to maintain the interaction

with Serα178 and to increase the solubility of these

indenopyrazole analogues. Finally, keeping the optimized

substituents at both the 6- and 7-positions, we made

modiﬁcations at 3-aniline through introducing diﬀerent groups

at the meta- or para-position as well as replacing the phenyl

with a pyridyl to ﬁnd the suitable groups for the interaction

with zone III. Herein, we report the SAR investigation and the

discovery of water-soluble 1-methyl-1,4-dihydroindeno[1,2-

c]pyrazoles as potent tubulin polymerization inhibitors

targeting the colchicine site with good in vitro and in vivo

antitumor eﬃciency.

Colchicine (Figure 1) is the ﬁrst MTA to be identiﬁed. As a

destabilizing MTA, it does not have an acceptable therapeutic

window for the treatment of cancer. Unlike the taxane and

vinca site MTAs, the colchicine site-binding agents in general

are not the substrate of the MDR eﬄux pump and can

overcome the multidrug resistance mediated by P-gp.^43,44

Moreover, the colchicine site-binding agents may serve as

vascular disrupting agents,⁴⁵⁻⁴⁸represented by the nature-

derived combretastatin A-4 (CA-4, Figure 1). In our previous

studies, we applied the privileged indenopyrazole scaﬀold in

the rational design of the colchicine site-binding agents and

discovered the lead compounds LL01 and LL02 (Figure 1)

that displayed strong inhibition (∼nM) against a variety of

tumor cells.⁴⁹As a hydroxamic acid, LL02 was found unstable

in the liver microsome stability assay, while the acetamide

LL01 was metabolically stable with acceptable pharmacoki-

netic (PK) properties.⁵⁰Further studies conﬁrmed that LL01

was not a P-gp substrate and eﬀective for multidrug-resistant

tumor cells. In HepG2 and KB/V tumor models, LL01

eﬀectively inhibited tumor growth via oral or subcutaneous

administration.^50,51Molecular docking simulations indicated

that LL01 may have a distinct binding mode with the

colchicine site,⁴⁹in that, the 1-methyl-1,4-dihydroindeno-[1,2-

c]pyrazole portion made hydrophobic interactions with zone II

pocket and the m-ethoxyaniline group penetrated into zone III

and formed hydrogen bonding with Tyrβ202 and Asnβ167.

The 6-ethoxy group of LL01 interacted with the hydrophobic

zone I only partially; however, the 7-acetamide substituent

protruded into a new region on the interfacial surface and

formed a critical hydrogen bond with Serα178. This unique

binding pattern of LL01 may be closely related to its high

antitumor activity. These promising results with these new

RESULTS AND DISCUSSION

■

Chemistry. Depending on the modiﬁcations at the diﬀerent

positions and the nature of the diﬀerent substituents, ﬁve

synthetic routes were designed to prepare these indenpyrazole

derivatives. The ﬁrst series of compounds ID01−ID05 were

made by modiﬁcations at the 6-phenolic position while

keeping the other portions of LL01 intact. The preparation

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a

Scheme 1. Synthesis of 1-Methyl-1,4-dihydroindeno[1,2-c]pyrazoles ID01−ID06

a

Reagents and conditions: (a) MOMCl, DIPEA, DMF, rt, 12 h; (b) TBSCl, imidazole, DMF, rt, 4 h; (c) (1) LiHMDS, 3-EtOPhNCS, THF, −78

°C to rt, 17 h; (2) NH₂NH₂·H₂O, 1,4-dioxane/EtOH, 55 °C; 36 h; (3) (CH₃)₂SO₄, DIPEA, DCM, rt, 7 h; (d) TBAF, THF, rt, 0.5 h; (e)

ClCH₂CO₂Me, K₂CO₃, acetone, 65 °C, 6 h; (f) NH₃·H₂O, MeOH, reﬂux, 1.5 h; (g) CSA, MeOH, 40 °C, 2 h; (h) (CH₃)₂SO₄or PrBr or BuBr or

p-MeOBnCl, K₂CO₃, DMF, 40 °C, 2.5−8 h; (i) PrBr, K₂CO₃, acetone, 65 °C, 8 h; (j) CSA, MeOH, 40 °C, 2 h.

of these analogues is shown in Scheme 1. 5,6-Dihydroxy-1-

indanone (1) was ﬁrst reacted with chloromethyl methyl ether

(MOMCl) in the presence of N,N-diisopropylethylamine

(DIPEA) in N,N-dimetylformamide (DMF) to selectively

protect the 5-phenolic hydroxyl group (65%) and then with

tert-butyldimethylsilyl chloride (TBSCl) to protect the 6-

phenolic hydroxyl group (86%). After the treatment of

indanone 3 with lithium hexamethyldisilazide (LiHMDS) in

anhydrous tetrahydrofuran (THF), the following reaction with

3-ethoxyphenylisothiocyanate generated a thioamide inter-

mediate, which underwent cyclization with hydrazine hydrate

and subsequent NH methylation with dimethyl sulfate in

dichloromethane (DCM) to give 1-methyl-1,4-dihydroindeno-

[1,2-c]pyrazole intermediate 4 (18%, three steps). The TBS

group in 4 was removed with tetrabutylammonium ﬂuoride

(TBAF), and the resulting indenopyrazole 5 (75%) was

reacted with methyl chloroacetate using K₂CO₃as a base under

the reﬂux conditions to provide methyl ester 6 (84%). After

the aminolysis of ester 6 with ammonia, amide 7 was obtained

(83%). The subsequent removal of the MOM protective group

in 7 with camphorsulfonic acid (CSA) provided the 6-

hyroxyindenopyrazole derivative ID01 in 90% yield. The

alkylation of 6-phenolic hydroxyl group in ID01 with dimethyl

sulfate, n-bromopropane, n-bromobutane, and p-methoxychlor-

obenzyl in DMF using K₂CO₃as a base generated the

corresponding indenopyrazole derivatives ID02−ID05 (66−

82%).

The indenopyrazole derivative ID06 featured with a position

reversion of 7-oxaacetaminde and 6-alkyloxy to conﬁrm the

importance of the supposed interactions with Serα178 on the

interfacial surface. The synthesis of ID06 is shown in Scheme

1. Propyl ether formation was achieved by the reaction of 5

with n-bromopropane to give 7-propoxyindenopyrazole 8

(90%). Following similar procedures to those for the

preparation of ID02−ID05 via the removal of the MOM

protective group (80%), the Williamson etheriﬁcation with

methyl chloroacetate (87%), and the subsequent ammonolysis

with ammonia (76%), ID06 was prepared.

To further verify the contribution of 7-oxaacetaminde in

LL01 to the tubulin polymerization inhibition, this substituent

was changed to 7-oxaacetic acid (ID07). As shown in Scheme

2, following similar procedures to those for the construction of

the 1-methyl-1,4-dihydroindeno[1,2-c]pyrazole scaﬀold, the 7-

hydroxyl and methyl 7-oxaacetic acid intermediates 13a and

14a were transformed from indanone 11.⁴⁹Methyl 7-oxaacetic

acid 14a⁴⁹was hydrolyzed with lithium hydroxide (LiOH) in

an aqueous methanol solution to give 7-oxaacetic acid

derivative ID07 in 85% yield. Next, we changed 7-

oxaacetaminde in LL01 to 7-(3-hydroxyproxy) (ID08) and

7-(3-aminoproxy) (ID09) to investigate the eﬀects of the

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a

Scheme 2. Synthesis of 1-Methyl-1,4-dihydroindeno[1,2-c]pyrazoles ID07−ID11

a

Reagents and conditions: (a) (1) LiHMDS, 3-EtOPhNCS, THF, −78 °C to rt; 16 h; (2) NH₂NH₂·H₂O, 1,4-dioxane/EtOH, 55 °C, 26 h; (3)

(CH₃)₂SO₄, DIPEA, DCM, rt; 6 h; (b) TBAF, THF, rt, 1 h; (c) ClCH₂CO₂Me, K₂CO₃, acetone, 65 °C, 6 h; (d) LiOH, MeOH/H₂O, 30 °C, 2 h;

(e) 3-bromo-1-propanol, K₂CO₃, DMF, 75 °C, 10 h; (f) MOMCl, DIPEA, DMF, rt, 8 h; (g) CSA, MeOH, 40 °C, 2 h; (h) BocNHCH₂CH₂CH₂Br,

K₂CO₃, acetone, 65 °C, 8 h; (i) CH₃COCl, EtOH, rt, 0.5 h; (j) (CH₃CO)₂O, Et₃N, DCM, rt, 4 h; (k) PrBr, K₂CO₃, acetone, 65 °C, 12 h.

terminal amino or hydroxyl group on the interaction with α-

tubulin. The reaction of 5-ethoxy-6-hydroxy-1-indanone 15⁴⁹

with 3-bromopropanol in the presence of K₂CO₃provided 5-

ethoxy-6-(3-hydroxypropoxy)-1-indanone 16 (79%). After the

protection of the primary hydroxyl group with the MOM

group, indanone 17 was similarly transformed into 1-methyl-

1,4-dihydroindeno[1,2-c]pyrazole intermediate 18a (16%,

three steps). Deprotection of the MOM group in 18a with

CSA furnished the synthesis of ID08 (75%). 7-Hydroxyinde-

nopyrazole 13a was reacted with N-Boc-3-bromopropylamine

to give 19a (87%). After the removal of the Boc protective

group in a mixed solution of acetyl chloride and ethanol, ID09

was obtained as a hydrochloride salt in 88% yield. Acetylation

of the terminal amino group in ID09 gave indenopyrazole

derivative ID10 (92%). To further conﬁrm the importance of

the terminal hydroxyl or amino group, the 7-propoxy derivative

ID11 was designed for comparison. ID11 was prepared from

13a via the propyl ether formation by the reaction with propyl

bromide (64%) (Scheme 2).

amine. The synthesis of these indenopyrazole derivatives is

shown in Scheme 3.

5-Ethoxy-6-tert-butyldimethylsiloxy-1-indanone 11 was

transformed into 1-methyl-1,4-dihydroindeno[1,2-c]pyrazoles

12b−j (13−22%) via the reaction with a variety of substituted

aryl isothiocyanate isothiocyanates following the general

protocols. The removal of the TBS protective group by

TBAF aﬀorded 7-phenolic derivatives 13b−g and 13j (71−

91%). The simultaneous deprotection of the TBS and Boc or

the acetal groups by acetyl chloride in EtOH generated

compounds 13h and 13i in 98 and 89% yields, respectively.

These phenolic intermediates 13b−j underwent the William-

son etheriﬁcation with methyl chloroacetate and subsequent

ammonolysis with ammonia to give indenopyrazole derivatives

ID12−ID19 and ID22 (73−95%). Aminolysis of the methyl

esters 14c and 14d with methylamine provided the

corresponding N-methylamides ID20 (83%) and ID21

(92%) (Scheme 3).

Following the general protocols to build up the 1-methyl-

1,4-dihydroindeno[1,2-c]pyrazole core and similar transforma-

tions, indeopyrazoles ID23−ID30 with 3-hydroxyprpoxy at the

7-position and variations at the 3-position were prepared

(Scheme 4).

Further structural modiﬁcations of LL01 were made at the

indenopyrazole 3-aniline position by changing the meta-ethoxy

to the para-position or by introducing diﬀerent substituents at

the meta-position as well as replacing 3-aniline with pyridyl-2-

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a

Scheme 3. Synthesis of 1-Methyl-1,4-dihydroindeno[1,2-c]pyrazoles ID12−ID22

a

Reagents and conditions: (a) (1) LiHMDS, R-Ph-NCS, THF, −78 °C to rt, 16−20 h; (2) NH₂NH₂·H₂O, 1,4-dioxane/EtOH, 55 °C, 20−36 h;

(3) (CH₃)₂SO₄, DIPEA, DCM, rt, 8−16 h; (b) TBAF, THF, rt, 1 h; (c) ClCH₂CO₂Me, K₂CO₃, acetone, 65 °C, 8−12 h; (d) NH₃·H₂O, MeOH,

reﬂux, 2−4 h; (e) CH₃COCl, EtOH, rt, 1 h; (f) CH₃NH₂, MeOH, reﬂux, 4 h; (g) (1) LiHMDS, 6-EtO-Py-2-NCS, THF, −78 °C to rt, 18 h; (2)

NH₂NH₂·H₂O, 1,4-dioxane/EtOH, 55 °C, 30 h; (3) (CH₃)₂SO₄, DIPEA, DCM, rt, 14 h.

Indenpyrazole derivatives ID30−ID33 are characterized by

a 3-aminoprpoxy group at the 7-position and variations at the

3-position. The preparation of these derivatives is shown in

Scheme 5. 7-Phenolic intermediates 13c, 13i, and 13j were

reacted with N-Boc-3-bromopropylamine to give 19c, 19i, and

19j (70−76%), respectively. The removal of the Boc protective

group in 19c with triﬂuoroacetic acid (TFA) provided

indenopyazole ID31 (90%) with a meta-cyanoaniline at the

3-position. Using acetyl chloride in ethanol to remove the Boc

protective groups in 19i and 19j gave the meta-propionylani-

line derivative ID32 (88%) and the 6-ethoxypyridyl-2-amine

derivative ID33 (90%), respectively, as hydrochloride salts.

In Vitro Tubulin Polymerization and Tumor Cell

Growth Inhibitory Activity and SAR Analysis. The

newly synthesized indenopyrazole derivatives ID01−ID33

were evaluated for their tubulin polymerization inhibitory

activity and antiproliferative activity against HepG2 (human

liver carcinoma), A549 (human lung adenocarcinoma), and

HCT116 (human colorectal carcinoma) cells using LL01 and

CA-4 as positive controls. The results are given in Table 1.

To establish SAR of LL01, we ﬁrst made variations at the

indenopyrazole 6-position. The 6-hydroxy derivative ID01 lost

its tubulin polymerization inhibitory activity as well as weak

antitumor cell proliferative activity. The 6-methoxy analogue

ID02 was more than threefold less potent in the tubulin assay

but maintained a similar cellular activity as that of LL01. The

6-propoxy derivative ID03 was more than twofold less potent

in tubulin activity and more than ﬁvefold less potent in the

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a

Scheme 4. Synthesis of 1-Methyl-1,4-dihydroindeno[1,2-c]pyrazoles ID23−ID30

a

Reagents and conditions: (a) (1) LiHMDS or NaH, R-Ph-NCS, THF, −78 °C to rt, 16−20 h; (2) NH₂NH₂·H₂O, 1,4-dioxane/EtOH, 55 °C, 20−

34 h; (3) (CH₃)₂SO₄, DIPEA, DCM, rt, 8−12 h; (b) CSA, MeOH, rt, 2 h; (c) LiOH, MeOH, H₂O, 40 °C, 2 h; (d) (1) LiHMDS, 6-EtO-Py-

2NCS, THF, −78 °C to rt, 18 h; (2) NH₂NH₂·H₂O, 1,4-dioxane/EtOH, 55 °C, 26 h; (3) (CH₃)₂SO₄, DIPEA, DCM, rt, 12 h.

a

Scheme 5. Synthesis of 1-Methyl-1,4-dihydroindeno[1,2-c]pyrazoles ID31−ID33

a

Reagents and conditions: (a) BocNHCH₂CH₂CH₂Br, K₂CO₃, acetone, 65 °C, 12−18 h; (b) TFA, DCM, rt, 2 h; (c) CH₃COCl, EtOH, rt, 0.5 h.

cellular assay than LL01. With the increase of the length and

bulkiness of the substituents at this position, 6-butoxy (ID04)

and 6-p-methoxybenzyloxy (ID05) derivatives lost both the

tubulin potency and cellular potency. Therefore, the 6-ethoxy

group was the best for the high tubulin inhibitory activity. The

indenopyrazole derivative ID06 with a position reversion of

the substituents in LL01, 6-oxaacetamide, and a propoxy to

maintain the proper chain length at the 7-position lost its

activity. This result is in agreement with the docking

simulations that the interaction of the 7-terminal amide in

LL01 with Serα178 at the α/β interface might be very

important for binding with the tubulin colchicine site.

Next, we tried to ﬁnd more suitable substituents at the

indenopyrazole 7-phenolic position. When 7-oxaacetamide was

changed to 7-oxaacetic acid, compound ID07 lost its tubulin

and cellular potency, indicating that a terminal acid group was

not favored for interaction with tubulin. The 7-(3-

hydroxypropxy)indenopyrazole derivative ID08 showed sim-

ilar tubulin and cellular potency as that of LL01. As a

hydrochloride salt, 7-(3-aminopropxy)indenopyrazole ID09

was a more potent tubulin polymerization inhibitor than LL01

and exhibited more potent or similar antiproliferative activity

as that of CA-4. When the terminal amine in ID09 was

acetylated, the resulting ID10 lost tubulin activity and its

tumor cell inhibitory potency was greatly reduced. Without the

terminal amino or hydroxyl group, the 7-propoxy derivative

ID11 had very weak tubulin polymerization and tumor cell

proliferative inhibitory activity, indicating that the hydrophobic

interaction with tubulin in this area also played some roles, but

a terminal functional group as a hydrogen bond donor at the

indenopyrazole 7-position was much more important for

potent tubulin polymerization inhibition, possibly through

hydrogen binding with Serα178 at the α/β interface.

In LL01, indenpyrazole 3-aniline was supposed to occupy

zone III at the colchicine site and meta-ethoxy acted as a

hydrogen acceptor to interact with Tyrβ202 and Asnβ167.

When the ethoxy group was transferred from the meta- to the

para-position, compound ID12 lost its activity, indicating the

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Table 1. Tubulin Polymerization and Tumor Cell Growth Inhibitory Activities of ID01−ID33

a

b

SD, standard deviation. Hydrochloride salt.

substitution at the para-position was not tolerated. Therefore,

we focused on the structural modiﬁcations at the aniline meta-

position. A cyano at the meta-position (ID13) decreased its

tubulin potency by more than twofold. When a carbonyl

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Table 2. Growth Inhibitory Eﬀects of ID09 and ID33 on LO2 and A549/Tax Cells

a

b

a

c

GI₅₀SD (nM)

SF

GI₅₀SD (nM)

RF

compound

HepG2

LO2

A549

A549/Tax

ID09

ID33

CA-4

paclitaxel

2.4 0.1

3.2 0.3

6.1 0.3

2.5 0.04

283.9 25.9

218.0 31.4

332.6 67.6

30.8 3.5

118

68

55

3.7 1.5

5.5 0.9

4.8 1.6

3.8 0.4

14.7 0.3

10.7 0.3

20.4 2.4

4

2

4

12

935.3 143.6

246

a

b

c

SD, standard deviation. Selectivity factor = GI₅₀human normal cells/GI₅₀HepG2. RF (resistant fold) = GI₅₀against resistant cells/GI₅₀against

sensitive cells.

oxygen was introduced to act as a hydrogen bond acceptor,

ethyl ester ID15 and methyl ester ID14 as well as ethyl ketone

ID19 showed similar or slightly increased tubulin polymer-

ization inhibitory activity as well as antiproliferative activity as

that of LL01, while iso-propyl ester ID16 was much less potent

than LL01, stressing the steric requirements in this region. In

contrast, the N-methylbenzamide ID17 was not active,

possibly due to the diﬀerent orientation of the carbonyl

group. Moreover, a meta-ethylamine (ID18) was not favored,

leading to a more than twofold decrease in tubulin and a

tenfold decrease in cellular potency. To increase the water

solubility of LL01, the aniline was replaced by pyridyl-2-amine.

The 6-ethoxypyridin-2-amine derivative ID22 maintained the

high tubulin and antiproliferative activity of LL01. In

comparison with the parent compounds ID13 and ID14, the

N-methylation of 7-oxaacetamide decreased the potency but

was somewhat tolerated since the N-methyl 7-oxaacetamide

derivative ID21 was still a potent tubulin polymerization

inhibitor (IC₅₀= 6.33 μM) and highly active against tumor

cells.

A combination of the 7-(3-hydroxypropoxy) group with the

variation of the substituents at the indenopyrazole 3-position

showed similar SARs to the 7-oxaacetamide derivatives. A

meta-cyano at 3-aniline (ID23) decreased the tubulin potency,

a N-methyl amide (ID27) was detrimental for the activity, and

a meta-propionyl (ID28) or replacement of 3-aniline by

pyridin-2-amine (ID30) maintained the high potency of the

parent compound ID08. Among the esters, ethyl ester (ID25)

was the most favored with improved potency in comparison

with ID08, methyl ester (ID24) maintained high potency,

while isopropyl ester (ID26) exhibited reduced activity. In

addition, the hydrolysis of the ester into a carboxylic acid

(ID29) led to the loss of activity.

substituents were in the order −CH₂CH₂CH₂NH₂>

−CH₂CONH₂> −CH₂CH₂CH₂OH > −CH₂CONHOH >

−CH₂CONHCH₃; (3) a substitution at the para-position of

the indenopyrazole 3-aniline was not tolerated; and (4) a

functional group, such as an ethoxy, a propionyl, an ethyl ester,

and a methyl ester, as a hydrogen bond acceptor at the

indenopyrazole 3-aniline meta-position was favored for

interaction with zone III at the colchicine site, a cyano group

and ethylamine were moderately tolerated, while carboxylic

acid and N-methyl amide were detrimental for the activity; and

(5) the replacement of meta-ethoxyaniline by 6-ethoxypyridin-

2-amine was also well tolerated and favorable for the water

solubility improvement of these indenopyrazoles.

As the hydrochloride salt, indenopyrazoles ID09 and ID33

showed good tubulin polymerization inhibitory activity as well

as excellent antiproliferative activity against three tested tumor

cell lines, and their eﬀects on normal liver LO2 cells and taxol-

resistant A549 cells (A549/Tax) were further investigated. As

shown in Table 2, compounds ID09 and ID33 showed lower

toxicity toward normal liver LO2 cells with GI₅₀values of

283.9 and 218.0 nM, respectively. In comparison with

paclitaxel and CA-4, ID09 and ID33 were more selective for

the tumor HepG2 cells over the normal liver LO2 cells. For the

A549/Tax cells, ID09 and ID33 were still highly potent with

only two- to ﬁvefold decrease in activity (vs A549 cell),

comparable to that of CA-4. Therefore, ID09 and ID33 were

eﬀective against taxol-resistant tumor cells and had the

potential to overcome multidrug resistance.

ID09 and ID33 Interrupted Cellular Microtubule

Network and Bound to the Tubulin Colchicine-Binding

Site. The eﬀects of ID09 and ID33 on microtubule

cytoskeletons were further evaluated by the immunoﬂuor-

escence staining assay in HepG2 cells using colchicine as a

positive control. HepG2 cells were treated with ID09 or ID33

at 2.5, 5, and 10 nM or colchicine at 10 nM for 24 h. An anti-

α-tubulin antibody (red) and Hoechst 33342 (blue) were used

for the microtubule and cell nucleus visualization, respectively.

As shown in Figure 3, in the absence of drug treatment, the

microtubule network in HepG2 cells exhibited a normal

arrangement and tissue morphology, which permeated the

whole cell and maintain the integrity of cell structure. The

treatment with colchicine at 10 nM led to dramatic changes in

the microtubule network; the microtubule cytoskeleton was

destroyed and shrunk to the cell border. Similar results were

observed when the cells were treated with ID09 or ID33.

Almost no complete microtubules were found in the cytosol in

HepG2 cells treated with ID09 or ID33 at the concentration

of 10 nM. These results were in agreement with the potent in

vitro tubulin polymerization inhibitory activity of ID09 and

ID33.

A combination of the 7-(3-aminopropoxy) group with

variation of the substituents at the indenopyrazole 3-position

led to the discovery of two very potent tubulin polymerization

inhibitors, a meta-propionyl derivative ID32 and a 6-

ethoxypyridin-2-amine derivative ID33.

In summary, through the systematic modiﬁcation of LL01 at

the indenopyrazole 6-, 7-, and 3-positions, a series of potent

tubulin polymerization inhibitors with strong antitumor cell

proliferative activity were identiﬁed. Among them, compounds

ID09, ID14, ID15, ID25, ID32, and ID33 were more potent

than LL01 and as potent as CA-4 in the inhibition of tumor

cell proliferation. The SARs supported the predicted binding

mode of LL01 and were summarized as follows: (1) an ethoxy

group at the indenopyrazole 6-position was the best for high

tubulin polymerization inhibition, followed by n-propyl and

methyl groups; (2) the substituents at indenopyrazole 7-

phenolic hydroxyl should be of proper length and contain a

terminal functional group as a hydrogen bond donor to

interact with α-tubulin at the α/β interface, and the preferred

To verify whether compounds ID09 and ID33 directly

targeted the tubulin colchicine-binding site, the EBI competi-

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tubulin itself.^52,53In the presence of the colchicine site

targeting agents, the formation of the EBI/β-tubulin adduct is

inhibited. As shown in Figure 4, in the EBI (100 μM)

treatment group, a second band below the β-tubulin band

appeared. Coincubation with ID09 or ID33 (0.2, 1, 5, and 25

μM) inhibited the β-tubulin adduct formation in a dose-

dependent way, resulting in the disappearance of the adduct

band, which was consistent with the eﬀect of colchicine at 5

μM. In contrast, the vinca site-binding agent, vincristine

(VCR), had little eﬀects on the formation of the β-tubulin

adduct, supporting that these indenopyrazole derivatives

directly targeted colchicine-binding sites and inhibited tubulin

polymerization.

ID09 and ID33 Induced HepG2 Cell G2/M Arrest and

Apoptosis. To investigate whether the cytotoxicity of ID09

and ID33 was due to the cell cycle arrest, we ﬁrst examined

their eﬀects on HepG2 cell cycle progression using propidium

iodide (PI) staining by ﬂow cytometry analysis. As shown in

Figure 5, the treatment with ID09 or ID33 for 24 h caused a

remarkable and concentration-dependent G2/M arrest in

HepG2 cells, whereas the control cells were primarily in the

G1 and S phases. ID09 and ID33 induced the obvious G2/M

arrest at 20 nM. The percentage of HepG2 cells in G2/M was

12.6%; when cells were exposed to 20 nM ID09 or ID33, the

percentage of G2/M phase increased to 88.5 or 85.2%,

respectively.

To further verify the eﬀects of ID09 and ID33 on cell

apoptosis, HepG2 cells treated with ID09 or ID33 were

stained with annexin V-PE and 7-aminoactinomycin D (7-

AAD) and analyzed by ﬂow cytometry. Dual staining for

annexin V-PE and 7-ADD permitted the discrimination among

live cells (annexin V-PE⁻/7-AAD⁻), early apoptotic cells

(annexin V-PE⁻/7-AAD⁻), late apoptotic cells (annexin V-

PE⁻/7-AAD⁻), and necrotic cells (annexin V-PE⁻/7-AAD⁻).

As shown in Figure 6, the percentages of apoptotic HepG2

cells were 9.2, 13.9, and 20.9% for ID09 at the concentrations

of 5, 10, and 20 nM at 12 h and increased to 14.1%, 23.9%, and

Figure 3. Eﬀects of ID09 and ID33 on cellular microtubule networks.

HepG2 cells were treated with dimethyl sulfoxide (DMSO), ID09

(2.5, 5, and 10 nM), ID33 (2.5, 5, and 10 nM), and colchicine (10

nM) for 24 h. Microtubules were visualized with an anti-α-tubulin

antibody (red), and the cell nucleus was visualized with Hoechst

33342 (blue).

tion assay was carried out in HepG2 cells. N,N′-Ethylene-

bis(iodoacetamide) (EBI) is an alkylating agent that can

speciﬁcally cross-link the Cys239 and Cys354 residues of β-

tubulin at the colchicine-binding site to form a β-tubulin

adduct that is easily detected by western blotting as a second

immunoreactive β-tubulin band that migrates faster than the β-

Figure 4. EBI assay conﬁrmed that ID09 and ID33 targeted the colchicine-binding site: the competition assay of ID09 and ID33 (0.2, 1, 5, 25

μM), VCR (5 μM), and colchicine (5 μM) with EBI (100 μM) on HepG2 cells.

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Figure 5. Eﬀects of ID09 and ID33 on HepG2 cell cycle progress. Cells were treated with ID09 or ID33 at 5, 10, and 20 nM for 24 h and then

analyzed by ﬂow cytometry.

47.3%, respectively, at 24 h. Similar tendency were observed

for ID33. At the concentrations of 5, 10, and 20 nM, ID33

induced cell apoptosis from 7.0 to 17.2% at 12 h and 10.9 to

39.0% for 24 h. Therefore, ID09 and ID33 induced cell

apoptosis in both time- and concentration-dependent manners.

Bcl family proteins play important roles in cell apoptosis

through the regulation of the mitochondria-mediated intrinsic

pathway, in which the proapoptotic (Bax and others) and

antiapoptotic (Bcl-2 and others) proteins are activated or

inhibited depending on upstream signaling.⁵⁴Cells undergoing

intrinsic mitochondrial apoptosis show downregulation of Bcl-

2 and upregulation of Bax. To investigate the pathway involved

in these indenopyrazole-mediated apoptosis pathways, the

HepG2 cells were treated with ID09 at diﬀerent concen-

trations (2.5, 5, 10, 20 nM) for 24 followed by the apoptotic

hallmark analysis.

As shown in Figure 7, ID09 caused dose-dependent

increases in proapoptotic protein Bax and decreases in

antiapoptotic protein Bcl-2, indicating that ID09 perturbed

the key proteins involved in the intrinsic apoptosis pathway.

Caspases are the ﬁnal facilitator of apoptosis and are composed

of two distinct classes, the initiators and the executioner.

Caspase-3 is one of the major executive enzymes that is cleaved

and activated during apoptosis. ID09 induced dose-dependent

activation of caspase-3 through the upregulation of the cleaved

caspase-3 (Figure 7). The caspase-dependent cleavage of

poly(ADP-ribose) polymerase (PARP), a marker of apoptosis,

was also detected in ID09−treated HepG2 cells, similar to that

of colchicine. Therefore, ID09 induced HepG2 cell apoptosis

possibly through the mitochondria-mediated intrinsic apop-

totic pathway as well as the activation of caspase-3.

ID09 Inhibited HepG2 Cell Migration. Metastasis is

responsible for 90% of cancer patient deaths, and it remains a

major hurdle in cancer treatment. Tumor cells invade through

the basement membrane and migrate to distant sites to form

metastatic lesions during metastatic progression. A scratch-

migration or wound-healing assay was used to investigate the

eﬀects of ID09 on HepG2 cell migration using CA-4 as a

positive control. The typical images of the wound at the

beginning of the experiment (0 h) and the ﬁnal treatment with

ID09 (24 h) at diﬀerent concentrations (2.5, 5, and 10 nM)

are shown in Figure 8. In comparison with the vehicle

(DMSO) group, ID09 signiﬁcantly suppressed the wound

closure of the cell migration. At 10 nM, ID09 signiﬁcantly

inhibited cell migration by 58%. At the same concentration,

CA-4 caused 47% inhibition of HepG2 cell migration.

Therefore, ID09 may have the potential to inhibit tumor cell

migration and metastasis.

Druglike Properties of ID09 and ID33 and Their

Antitumor Activity in Vivo. One of the problems of the

MTAs used in clinics or under clinical investigations is their

low water solubility. The water solubility of a drug or drug

candidate is closely related to its druglike properties like

ADME/Tox (absorption, distribution, metabolism, elimina-

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Figure 6. ID09 and ID33 induce HepG2 cell apoptosis. Exposed to ID09 or ID33 for 12h (A) or 24 h (B), HepG2 cells were harvested and

stained with annexin V-PE/7-AAD for analysis. The diﬀerent cell stages were assigned as live (annexin V-PE⁻/7-AAD⁻), early apoptotic (annexin

V-PE⁺/7-AAD⁻), late apoptotic (annexin V-PE⁺/ 7-AAD⁺), and necrotic cells (annexin V-PE⁻/7-AAD⁺).

tion, toxicity) and its formulations. One of the goals of this

study is to improve the water solubility of the lead LL01;

therefore, we measured the aqueous solubility and log P values

of ID09 and ID33 at pH 7.4 according to the reported

methods.⁵⁵The results are summarized in Table 3. As the

hydrochloride salt, indenopyrazoles ID09 and ID33 showed

very good and much improved aqueous solubility (>1 mg/mL)

than LL01 (>200-fold). They also had lower log P values than

those of LL01 and fell into a desirable druglike range (0 <

log P < 3, pH = 7.4). Therefore, ID09 and ID33 have a good

balance between their high cellular as well as tubulin potency

and the improved aqueous solubility and druglike properties

and thus might be potential drug candidates for further

developments.

To further validate the antitumor eﬃciency in vivo, nude

mouse HepG2 xenograft models were established, and

compounds ID09 and ID33 were administered by intragastric

administration at a dose of 25 mg/kg (aqueous solution) one

time every day for 21 consecutive days. The results are

summarized in Figure 9. In comparison with the vehicle

(distilled water) group, the oral administration of ID09 and

ID33 at 25 mg/kg resulted in 57 and 54% reduction in tumor

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Figure 7. Eﬀects of ID09 on apoptosis-related proteins. HepG2 cells were incubated with ID09 (2.5, 5, 10, and 20 nM) and colchicine 20 nM for

24 h followed by the analysis of the expression of Bax, Bcl-2, cleaved caspase-3, and cleaved PARP by western blotting. *P < 0.05, **P < 0.01, ***P

< 0.001 vs control.

Figure 8. Eﬀect of ID09 on HepG2 cell migration. HepG2 cells (5 × 10⁵cells) suspended in free serum DMEM containing ID09 and CA-4 (2.5, 5,

and 10 nM) for 24 h were photographed by phase contrast microscopy (magniﬁcation, 200×). *P < 0.05, **P < 0.01, ***P < 0.001 vs control.

reduction in tumor volume in an established HepG2 xenograft

model.⁵⁰ID09 and ID33 exhibited slightly better orally

antitumor eﬃciency than that of LL01 despite the formulation

diﬀerences.

After the demonstration of the orally antitumor eﬃciency of

D09 and ID33, we further investigated the antitumor eﬃciency

of ID09 on HepG2 xenograft models via intravenous injection

(iv). The nude mice were randomly divided into three groups:

vehicle (distilled water) group and ID09 dose (aqueous

solution) groups (5 and 10 mg/kg). Five nude mice in each

group were administrated intravenously once every two days

for 21 consecutive days. The results are summarized in Figure

10.

Table 3. Aqueous Solubility and Log P of ID09 and ID33

pH 7.4

compd

aqueous solubility

Log P

ID09

ID33

LL01

>1 mg/mL

<5 μg/mL

1.70 0.04

0.97 0.02

3.18 0.13

volume, respectively. The treatment with ID09 or ID33 did

not cause signiﬁcant diﬀerences in body weight changes (Table

4) and had no other adverse eﬀects. The similar in vivo

antitumor eﬃciency of ID09 and ID33 is in agreement with

their similar in vitro tubulin and tumor cell growth inhibitory

activities and druglike properties. In previous investigations,

the oral administration of LL01 as a 0.5% MC (carbox-

ymethylcellulose sodium) suspension resulted in a 50%

ID09 dose-dependently inhibited the tumor growth. At the

doses of 5 and 10 mg/kg, ID09 caused 54 and 68% tumor

volume reduction, respectively. No other adverse signs were

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Figure 9. Orally antitumor eﬃciency of ID09 and ID33 on HepG2 xenografts. (A) Tumor images, (B) tumor volume changes, (C) body weight

changes, and (D) tumor weight changes. **P < 0.01, ***P < 0.001 vs vehicle.

Table 4. Eﬀects of ID09 and ID33 on Body Weight in Nude

Mice

substituents at the indenopyrazole 7-position should be of

proper length and contained a terminal functional group as a

hydrogen bond donor, such as 3-aminoproxy, oxaacetamide,

and 3-hydroxypropoxy groups. For the indenopyrazole 3-

aniline moiety, an ethoxy, a propionyl, and an ethyl or methyl

ester at the meta-position serving as a hydrogen bond acceptor

were favored. The replacement of the 3-meta-ethoxyaniline by

a 6-ethoxypyridin-2-amine was also well tolerated. As the

hydrochloride salt(s), indenpyrazole derivatives ID09 and

ID33 have excellent aqueous solubility and favorable Log P

values. ID09 and ID33 targeted the tubulin colchicine-binding

site, potently inhibited tubulin polymerization with the IC₅₀

values of 3.65 and 3.13 μM, respectively, and inhibited tubulin

assembly in HepG2 cells. They showed strong antiproliferative

activities for a variety of tumor cells including those with taxol

resistance, with the GI₅₀values ranging from 2.4 to 14.7 nM.

ID09 and ID33 caused the arrest at the G2/M phase and

induced the caspase-dependent and mitochondrial apoptosis in

HepG2 cells. ID09 also had the ability to inhibit HepG2 cell

migration. In HepG2 xenograft mouse models, ID09 and ID33

demonstrated their oral antitumor eﬃciency at the dose of 25

mg/kg, reducing the tumor growth by more than 50% without

obvious signs of toxicity. The iv injection of ID09 at the dose

of 10 mg/kg, one time for every other day, resulted in 68%

inhibition of tumor growth without obvious toxicity. There-

fore, ID09 and ID33 could serve as potential anticancer drug

candidates for further developments.

body weight (g) initial/

end

body weight

changes (%)

groups

vehicle

ID09 25

mg/kg

administration

po

20.3 0.8/16.8 0.5

19.4 0.5/15.8 0.6

−17.2

−18.5

ID33 25

mg/kg

po

19.9 0.5/16.2 0.5

−18.5

vehicle

ID09 5 mg/

kg

iv

20.6 0.3/22.2 0.6

20.3 0.3/22.0 0.5

+7.8

+8.4

ID09 10

mg/kg

iv

20.2 0.4/21.6 0.7

+6.9

observed during the experiments, and ID09 had no signiﬁcant

eﬀects on animal weight changes (Table 4).

CONCLUSIONS

■

Like that of LL01, a common drawback of most of the MTAs

is their low water solubility. To address this issue and to

investigate the SARs of the indenpyrazoles as tubulin

polymerization inhibitors targeting the colchicine-binding

site, we used LL01 as a lead, maintained the 1-methyl-1,4-

dihydroindeno-[1,2-c]pyrazole core, and made systematic

modiﬁcations on the phenolic 6- and 7-positions and the 3-

aniline portion. The indenopyrazole derivatives ID01−ID33

were designed, synthesized, and evaluated for their tubulin

polymerization and tumor cell growth inhibitory activities,

resulting in the discovery of a series of potent MTAs. The

SARs were in agreement with the predicted binding mode of

LL01 with tubulin. In general, an ethoxy group at the

indenopyrazole 6-position was the best choice, and the

EXPERIMENTAL SECTION

■

Chemistry. Melting points were determined on an RY-1 melting

point tester. ¹H and ¹³C NMR spectra were recorded at room

temperature on a Bruker-400 NMR or Bruker-600 NMR

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Figure 10. Antitumor eﬃciency of ID09 on HepG2 xenografts via iv injection injection. (A) Tumor images, (B) tumor volume changes, (C) body

weight changes, and (D) tumor weight changes. **P < 0.01, ***P < 0.001 vs vehicle.

spectrometer with DMSO-d₆or CDCl₃solution. MS spectra data

were acquired by an API 4000 instrument. High-resolution mass

spectrometry (HRMS) data were acquired by a Q-TOF6540

instrument. The compound purity was determined using an Agilent

1200 series equipment (column, Diamonil C18, 4.6 mm × 150 mm, 5

μm; mobile phase, methanol/H₂O/CF₃COOH (70:30:0.1%); ﬂow

rate, 1.0 mL/min; UV wavelength, 254 nm). All the compounds used

in the bioassay are of high purity (>95%). Column chromatography

was performed on silica gel (200−300 mesh). All chemicals were from

commercial sources and used without puriﬁcation. THF was distilled

from sodium/benzophenone immediately prior to use.

THF (15 mL) was cooled to −78 °C under a nitrogen atmosphere,

LiHMDS (3.26 mL, 3.26 mmol) was added dropwise and stirred for

2.5 h. The reaction mixture was warmed to −45 °C, and a solution of

1-ethoxy-3-isothiocyanatobenzene (580 mg, 3.26 mmol) in anhydrous

THF (8 mL) was added and stirred at room temperature overnight.

The reaction mixture was adjusted to pH 7 by the addition of the

saturated KHSO₄aqueous solution and then extracted with EtOAc

(50 mL × 3). The organic phase was washed with brine and dried

over Na₂SO₄. After ﬁltration and evaporation, the residue was puriﬁed

by column chromatography (ethyl acetate/hexanes = 1:15) to give the

resulting thioamide intermediate, which was dissolved in 1,4-dioxane

(15 mL) and ethanol (15 mL). Hydrazine hydrate (1.09 mL, 17.39

mmol) was added dropwise, and the reaction mixture was heated to

55 °C and stirred for 36 h. Distilled water (60 mL) was added, and

the mixture was extracted with EtOAc (80 mL × 3). The organic

phase was washed with brine and dried over Na₂SO₄. After ﬁltration

and evaporation, the residue was puriﬁed by column chromatography

(ethyl acetate/hexanes = 1:3) to give an oil (340 mg), which was

dissolved in DCM (10 mL), and DIPEA (0.18 mL, 1.42 mmol) and

dimethyl sulfate (0.13 mL, 1.42 mmol) were added and stirred at

room temperature for 7 h. Distilled water (20 mL) was added, and the

mixture was extracted with EtOAc (30 mL × 3), washed with brine,

and dried over Na₂SO₄. After ﬁltration and evaporation, the residue

was puriﬁed by chromatography (ethyl acetate/hexanes = 1:7) to give

6-Hydroxy-5-(methoxymethoxy)-1-indanone (2). To a sol-

ution of 5,6-dihydroxy-1-indanone (1, 2.0 g, 13 mmol) in DMF (30

mL) were added K₂CO₃(2.2 g, 16 mmol) and MOMCl (1.23 mL, 15

mmol). The mixture was stirred at room temperature for 12 h. Water

(180 mL) was added, extracted with EtOAc (200 mL × 3), washed

with brine, and dried over Na₂SO₄. After ﬁltration and evaporation,

the residue was puriﬁed by chromatography (ethyl acetate/hexanes =

1

1:3) to give 1.52 g (65%) of 2 as a white solid, mp 129−131 °C. H

NMR (400 MHz, CDCl₃) δ 7.28 (s, 1H), 7.11 (s, 1H), 5.73 (s, 1H),

5.33 (s, 2H), 3.53 (s, 3H), 3.03 (t, J = 5.6 Hz, 2H), 2.65 (t, J = 5.6 Hz,

2H); ¹³C NMR (100 MHz, CDCl₃) δ 205.9, 150.4, 148.9, 146.1,

131.5, 110.8, 108.5, 95.2, 56.7, 36.5, 25.5.

6-((tert-Butyldimethylsilyl)oxy)-5-methoxymethoxy-1-inda-

none (3). To a solution of 6-hydroxy-5-methoxymethoxy-1-indanone

(2, 2.0 g, 10.4 mmol) in DMF (25 mL), TBSCl (2.4 g, 16.0 mmol)

and imidazole (1.1 g, 16.1 mmol) were added and stirred at room

temperature for 4 h. Distilled water (200 mL) was added, and the

mixture was stirred for 1 h. The resulting precipitate was ﬁltered,

washed with hexanes, and dried to give 2.7 g (86%) of 3 as a white

solid, mp 165−167 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.17 (s, 1H),

7.10 (s, 1H), 5.22 (s, 2H), 3.49 (s, 3H), 3.00 (t, J = 5.5 Hz, 1H), 2.71

(t, J = 5.5 Hz, 2H), 0.98 (s, 9H), 0.15 (s, 6H); ¹³C NMR (150 MHz,

CDCl₃) δ 205.8, 154.9, 150.4, 145.5, 131.1, 114.5, 111.8, 94.7, 56.5,

36.7, 25.6, 25.5, −4.6.

1

190 mg (18%) of 4 as a yellow solid, mp 81−82 °C. H NMR (600

MHz, DMSO-d₆) δ 8.38 (s, 1H), 7.30 (s, 1H), 7.13 (s, 1H), 7.05 (t, J

= 8.1 Hz, 1H), 6.97 (t, J = 2.0 Hz, 1H), 6.82 (dd, J = 8.1, 2.0 Hz, 1H),

6.28 (dd, J = 8.1, 2.0 Hz, 1H), 5.19 (s, 2H), 3.97 (q, J = 7.0 Hz, 2H),

3.92 (s, 3H), 3.43 (s, 3H), 3.38 (s, 2H), 1.32 (t, J = 7.0 Hz, 3H), 1.01

(s, 9H), 0.19 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆) δ 158.6,

147.2, 145.9, 144.3, 143.7, 143.3, 141.5, 128.7, 125.3, 114.2, 112.1,

110.7, 107.2, 103.3, 100.7, 94.2, 61.9, 55.2, 36.4, 28.2, 25.0, 17.6, 14.2,

−5.2. MS (ESI) calcd for C₂₇H₃₈N₃O₄Si [M + H]⁺: 496.3, found:

496.2.

3-((3-Ethoxyphenyl)amino)-6-methoxymethoxy-1-methyl-

1,4-dihydroindeno[1,2-c]pyrazol-7-ol (5). To a solution of 4 (190

mg, 0.38 mmol) in THF (8 mL) was added TBAF (200 mg, 0.76

mmol). The mixture was stirred at room temperature for 0.5 h. After

distilled water (40 mL) was added, the resulting precipitate was

7-((tert-Butyldimethylsilyl)oxy)-N-(3-ethoxyphenyl)-6-(me-

thoxymethoxy)-1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-3-

amine (4). After the solution of 6-((tert-butyldimethylsilyl)oxy)-5-

methoxymethoxy-1-indanone (3, 700 mg, 2.17 mmol) in anhydrous

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ﬁltered and dried to give 110 mg (75%) of 5 as a white solid, mp

136−138 °C. H NMR (600 MHz, DMSO-d₆) δ 9.11 (s, 1H), 8.36

8.1, 2.1 Hz, 1H), 6.29 (dd, J = 8.1, 2.1 Hz, 1H), 4.53 (s, 2H), 3.98 (q,

J = 6.9 Hz, 2H), 3.95 (s, 3H), 3.84 (s, 3H), 3.36 (s, 2H), 1.32 (t, J =

6.9 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 170.8, 159.7, 148.6,

148.5, 146.8, 145.3, 144.9, 143.1, 129.8, 124.8, 112.6, 111.5, 108.3,

106.9, 104.3, 101.8, 69.4, 63.0, 56.3, 37.5, 29.5, 15.3. HRMS (ESI)

calcd for C₂₂H₂₅N₄O₄[M + H]⁺: 409.1870, found: 409.1878.

2-((3-((3-Ethoxyphenyl)amino)-1-methyl-6-propoxy-1,4-

dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetamide (ID03). Fol-

lowing similar procedures to those described for preparing ID02,

compound ID03 was prepared from ID01 (40 mg, 0.10 mmol),

K₂CO₃(28 mg, 0.20 mmol), 1-bromopropane (18 μL, 0.20 mmol) in

80% yield as a white solid, HPLC purity: 99.2%, mp: 114−115 °C. ¹H

NMR (600 MHz, DMSO-d₆) δ 8.43 (s, 1H), 7.58 (s, 1H), 7.46 (s,

1H), 7.39 (s, 1H), 7.33 (s, 1H), 7.12 (t, J = 8.1 Hz, 1H), 7.04 (t, J =

2.0 Hz, 1H), 6.89 (dd, J = 8.1, 2.0 Hz, 1H), 6.34 (dd, J = 8.1, 2.0 Hz,

1H), 4.58 (s, 2H), 4.06 (t, J = 7.4 Hz, 2H), 4.02 (q, J = 7.0 Hz, 2H),

3.44 (s, 2H), 1.96−1.70 (m, 2H), 1.37 (t, J = 7.0 Hz, 3H), 1.06 (t, J =

7.4 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 170.9, 159.7, 148.6,

148.2, 147.1, 145.3, 144.9, 143.5, 129.8, 124.9, 112.8, 112.7, 108.3,

107.8, 104.4, 101.8, 70.6, 69.9, 63.0, 37.5, 29.5, 22.7, 15.3, 10.9.

HRMS (ESI) calcd for C₂₄H₂₉N₄O₄[M + H]⁺: 437.2183, found:

437.2188.

1

(s, 1H), 7.23 (s, 1H), 7.14 (s, 1H), 7.06 (t, J = 8.1 Hz, 1H), 6.97 (t, J

= 2.2 Hz, 1H), 6.83 (dd, J = 8.1, 2.2 Hz, 1H), 6.28 (dd, J = 8.1, 2.2

Hz, 1H), 5.17 (s, 2H), 3.97 (q, J = 7.0 Hz, 2H), 3.90 (s, 3H), 3.44 (s,

3H), 3.34 (s, 2H), 1.32 (t, J = 7.0 Hz, 3H); ¹³C NMR (150 MHz,

DMSO-d₆) δ 159.7, 148.6, 147.1, 145.4, 144.8, 143.8, 139.8, 129.8,

126.7, 116.1, 113.2, 108.3, 107.2, 104.3, 101.8, 95.7, 63.0, 56.2, 37.4,

29.1, 15.3. MS (ESI) calcd for C₂₁H₂₄N₃O₄[M + H]⁺: 382.2, found:

382.1.

Methyl 2-((3-((3-Ethoxyphenyl)amino)-6-(methoxyme-

thoxy)-1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)-

acetate (6). To a solution of 5 (100 mg, 0.26 mmol) in acetone (8

mL) were added methyl chloroacetate (50 μL, 0.52 mmol) and

K₂CO₃(64 mg, 0.52 mmol). The mixture was heated to 65 °C and

stirred for 6 h. After distilled water (20 mL) was added, the resulting

precipitate was ﬁltered and dried to give 100 mg (75%) of 6 as a

1

yellow solid, mp 130−131 °C. H NMR (600 MHz, DMSO-d₆) δ

8.39 (s, 1H), 7.33 (s, 1H), 7.32 (s, 1H), 7.06 (t, J = 8.1 Hz, 1H), 6.99

(t, J = 2.0 Hz, 1H), 6.83 (dd, J = 8.1, 2.0 Hz, 1H), 6.29 (dd, J = 8.1,

2.0 Hz, 1H), 5.21 (s, 2H), 4.93 (s, 2H), 3.98 (q, J = 7.0 Hz, 2H), 3.95

(s, 2H), 3.72 (s, 3H), 3.44 (s, 3H), 3.38 (s, 2H), 1.32 (t, J = 7.0 Hz,

3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 168.7, 158.7, 147.3, 146.8,

144.2, 144.2, 143.8, 141.3, 128.7, 125.5, 115.6, 112.1, 107.2, 104.8,

103.3, 100.8, 94.7, 65.1, 61.9, 55.2, 51.2, 36.5, 28.1, 14.2. MS (ESI)

calcd for C₂₄H₂₈N₃O₆[M + H]⁺: 454.2, found: 454.1.

2-((6-Butoxy-3-((3-ethoxyphenyl)amino)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetamide (ID04). Fol-

lowing the procedures described for preparing ID02, compound ID04

was prepared from ID01 (45 mg, 0.11 mmol), K₂CO₃(30 mg, 0.22

mmol), n-butyl bromide (19 μL, 0.22 mmol) in 82% yield as a white

2-((3-((3-Ethoxyphenyl)amino)-6-(methoxymethoxy)-1-

methyl-1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetamide

(7). To a solution of 6 (100 mg, 0.24 mmol) in MeOH (5 mL) was

added aqueous ammonia (25%, 2 mL). The reaction mixture was

reﬂuxed for 1.5 h. After that, the mixture was stirred at 0 °C for 1 h,

and a white solid was precipitated out. It was ﬁltered, washed with

water, and dried to give 80 mg (83%) of 7 as a white solid, mp 188−

190 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 8.40 (s, 1H), 7.50 (s, 1H),

7.40 (s, 1H), 7.37 (s, 1H), 7.33 (s, 1H), 7.06 (t, J = 8.1 Hz, 1H), 6.99

(t, J = 1.9 Hz, 1H), 6.84 (dd, J = 8.1, 1.9 Hz, 1H), 6.29 (dd, J = 8.1,

1.9 Hz, 1H), 5.23 (s, 2H), 4.57 (s, 2H), 3.98 (q, J = 7.0 Hz, 2H), 3.96

(s, 3H), 3.45 (s, 3H), 3.39 (s, 2H), 1.32 (t, J = 7.0 Hz, 3H); ¹³C

NMR (150 MHz, DMSO-d₆) δ 170.6, 159.7, 148.3, 148.0, 145.6,

145.3, 144.9, 142.7, 129.8, 126.6, 116.4, 113.2, 108.3, 107.0, 104.4,

101.8, 95.9, 69.3, 63.0, 56.4, 37.5, 29.3, 15.3. MS (ESI) calcd for

C₂₃H₂₇N₄O₅[M + H]⁺: 439.2, found: 439.1.

1

solid, HPLC purity: 98.9%, mp: 169−170 °C. H NMR (600 MHz,

DMSO-d₆) δ 8.41 (s, 1H), 7.55 (s, 1H), 7.41 (s, 1H), 7.34 (s, 1H),

7.28 (s, 1H), 7.06 (t, J = 8.1 Hz, 1H), 6.99 (t, J = 2.0 Hz, 1H), 6.83

(dd, J = 8.1, 2.0 Hz, 1H), 6.29 (dd, J = 8.1, 2.0 Hz, 1H), 4.52 (s, 2H),

4.03 (t, J = 6.5 Hz, 2H), 3.97 (q, J = 7.1 Hz, 2H), 3.94 (s, 3H), 3.39

(s, 2H), 1.79−1.68 (m, 2H), 1.55−1.41 (m, 2H), 1.32 (t, J = 7.1 Hz,

3H), 0.95 (t, J = 7.4 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ

170.9, 159.7, 148.6, 148.2, 147.0, 145.3, 144.9, 143.4, 129.8, 124.9,

112.7, 112.6, 108.3, 107.8, 104.3, 101.8, 69.8, 68.7, 63.0, 37.5, 31.4,

29.5, 19.3, 15.3, 14.2. HRMS (ESI) calcd for C₂₅H₃₁N₄O₄[M + H]⁺:

451.2343, found: 451.2340.

2-((3-((3-Ethoxyphenyl)amino)-6-((4-methoxybenzyl)oxy)-

1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)-

acetamide (ID05). Following the procedures described for preparing

ID02, compound ID05 was prepared from ID01 (50 mg, 0.13 mmol),

K₂CO₃(28 mg, 0.26 mmol), p-methoxybenzyl chloride (35 μL, 0.26

mmol) in 66% yield as a white solid, HPLC purity: 99.8%, mp: 201−

202 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 8.41 (s, 1H), 7.50 (s, 1H),

7.43−7.40 (m, 3H), 7.38 (s, 1H), 7.33 (s, 1H), 7.06 (t, J = 8.1 Hz,

1H), 6.99 (t, J = 1.8 Hz, 1H), 6.97 (s, 1H), 6.95 (s, 1H), 6.83 (dd, J =

8.1, 1.8 Hz, 1H), 6.29 (dd, J = 8.1, 1.8 Hz, 1H), 5.07 (s, 2H), 4.54 (s,

2H), 3.98 (q, J = 6.9 Hz, 2H), 3.95 (s, 3H), 3.76 (s, 3H), 3.39 (s,

2H), 1.32 (t, J = 6.9 Hz, 3H);¹³C NMR (150 MHz, DMSO-d₆) δ

169.7, 158.6, 158.4, 147.4, 146.7, 146.2, 144.2, 143.8, 142.1, 129.1,

128.9, 128.7, 128.2, 124.1, 113.2, 113.1, 112.4, 111.6, 107.2, 106.3,

103.2, 100.7, 69.6, 68.5, 61.9, 54.4, 36.3, 28.3, 14.1. HRMS (ESI)

calcd for C₂₉H₃₁N₄O₅[M + H]⁺: 515.2289, found: 515.2295.

N-(3-Ethoxyphenyl)-6-(methoxymethoxy)-1-methyl-7-pro-

poxy-1,4-dihydroindeno[1,2-c]pyrazol-3-amine (8). Following

the procedures described for preparing 6, compound 8 was prepared

from 5 (500 mg, 1.31 mmol), K₂CO₃(362 mg, 2.62 mmol), 1-

bromopropane (248 μL, 2.62 mmol) in 90% yield as a white solid,

mp: 133−135 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.13 (t, J = 7.8 Hz,

1H), 7.07 (s, 1H), 6.62 (s, 1H), 6.59 (d, J = 7.8 Hz, 1H), 6.42 (d, J =

7.8 Hz, 1H), 5.97 (s, 1H), 5.22 (s, 2H), 4.05 (t, J = 7.2 Hz, 4H), 4.00

(s, 3H), 3.55 (s, 3H), 3.38 (s, 2H), 1.83−1.95 (m, 2H), 1.39 (t, J =

7.2 Hz, 3H), 1.08 (t, J = 7.2 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ

160.0, 149.6, 148.9, 145.8, 144.7, 144.2, 141.7, 129.8, 126.8, 116.2,

114.2, 108.6, 105.7, 104.9, 102.4, 96.1, 71.3, 63.3, 56.3, 37.4, 29.7,

22.7, 14.9, 10.6. MS (ESI) calcd for C₂₄H₃₀N₃O₄[M + H]⁺: 424.2,

found: 424.4.

2-((3-((3-Ethoxyphenyl)amino)-6-hydroxy-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetamide (ID01). To a

solution of 7 (80 mg, 0.18 mmol) in MeOH (5 mL) was added CSA

(170 mg, 0.73 mmol). The reaction mixture was heated to 40 °C and

stirred for 2 h. Distilled water (25 mL) was then added, and the

resulting precipitate was ﬁltered and dried to give 65 mg (90%) of

1

ID01 as a yellow solid, HPLC purity: 99.9%, mp: 150−151 °C. H

NMR (600 MHz, DMSO-d₆) δ 9.10 (s, 1H), 8.36 (s, 1H), 8.01 (s,

1H), 7.58 (s, 1H), 7.35 (s, 1H), 7.06 (t, J = 8.1 Hz, 1H), 7.02 (s, 1H),

6.97 (t, J = 2.1 Hz, 1H), 6.81 (dd, J = 8.1, 2.1 Hz, 1H), 6.28 (dd, J =

8.1, 2.1 Hz, 1H), 4.54 (s, 2H), 3.98 (q, J = 6.9 Hz, 2H), 3.96 (s, 3H),

3.33 (s, 2H), 1.32 (t, J = 6.9 Hz, 3H); ¹³C NMR (150 MHz, DMSO-

d₆) δ 170.7, 159.7, 148.9, 145.6, 145.4, 145.0, 144.8, 143.1, 129.8,

123.8, 114.8, 112.3, 108.2, 105.7, 104.3, 101.8, 68.7, 63.0, 37.5, 29.1,

15.3. HRMS (ESI) calcd for C₂₁H₂₃N₄O₄[M + H]⁺: 395.1714,

found: 395.1723.

2-((3-((3-Ethoxyphenyl)amino)-6-methoxy-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetamide (ID02). To a

solution of ID01 (40 mg, 0.10 mmol) in DMF (3 mL) was added

dimethyl sulfate (20 μL, 0.20 mmol) and K₂CO₃(28 mg, 0.20 mmol).

The reaction mixture was stirred at 40 °C for 2.5 h. Distilled water

(180 mL) was added, and the mixture was extracted with EtOAc (100

mL), washed with brine, and dried over Na₂SO₄. After ﬁltration and

evaporation, the residue was puriﬁed by chromatography (ethyl

acetate/hexanes = 4:1) to give 31 mg (75%) of ID02 as a white solid,

HPLC purity: 97.7%, mp: 197−198 °C. ¹H NMR (600 MHz, DMSO-

d₆) δ 8.41 (s, 1H), 7.50 (s, 1H), 7.38 (s, 1H), 7.37 (s, 1H), 7.29 (s,

1H), 7.06 (t, J = 8.1 Hz, 1H), 7.00 (t, J = 2.1 Hz, 1H), 6.84 (dd, J =

3-((3-Ethoxyphenyl)amino)-1-methyl-7-propoxy-1,4-

dihydroindeno[1,2-c]pyrazol-6-ol (9). Following the procedures

O

https://dx.doi.org/10.1021/acs.jmedchem.0c01345

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

described for preparing ID01, compound 9 was prepared from 8 (0.5

64.7, 63.0, 37.3, 29.3, 15.3, 15.2. MS (ESI) calcd for C₂₁H₂₃N₃O₃[M

+ H]⁺: 366.2, found: 366.3.

g, 1.18 mmol), CSA methanol solution (0.5 M, 10 mL) in 80% yield

1

Methyl 2-((6-Ethoxy-3-((3-ethoxyphenyl)amino)-1-methyl-

1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetate (14a). Fol-

lowing the procedures described for preparing 6, compound 14a

was prepared from 13a (1.10 g, 3.01 mmol), K₂CO₃(832 mg, 6.02

mmol), and methyl chloroacetate (0.55 mL, 6.26 mmol) in 74% yield

as a white solid, mp: 139−141 °C. ¹H NMR (600 MHz, DMSO-d₆) δ

8.38 (s, 1H), 7.27 (s, 1H), 7.24 (s, 1H), 7.06 (t, J = 12.1 Hz, 1H),

6.98 (s, 1H), 6.83 (d, J = 12.1 Hz, 1H), 6.29 (d, J = 10.2 Hz, 1H),

4.88 (s, 2H), 4.09 (q, J = 10.2 Hz, 2H), 3.98 (q, J = 10.2 Hz, 2H),

3.93 (s, 3H), 3.72 (s, 3H), 3.38 (s, 2H), 1.35(t, J = 10.2 Hz, 3H), 1.32

(t, J = 10.2 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 169.9, 159.7,

148.6, 147.6, 146.9, 145,3, 144.8, 142.9, 129.7, 124.8, 113.1, 112.7,

108.3, 106.2, 104.4, 101.9, 66.4, 64.7, 63.1, 52.2, 37.5, 29.4, 15.3, 15.2.

MS (ESI) calcd for C₂₄H₂₈N₃O₅[M + H]⁺: 438.2, found: 438.5.

2-((6-Ethoxy-3-((3-ethoxyphenyl)amino)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetic acid (ID07). To a

solution of 14a (40 mg, 0.09 mmol) in MeOH (3 mL) was added

LiOH aqueous solution (1 M, 1.5 mL). The mixture was stirred at 30

°C for 2 h. The resulting solution was adjusted to pH 4 by the

addition of 2 M HCl aqueous solution. After distilled water was

added, a white solid was precipitated out. It was ﬁltered and dried to

give 34 mg (85%) of ID07 as a white solid, HPLC purity: 98.9%, mp:

as a yellow solid, mp: 186−189 °C. H NMR (600 MHz, CDCl₃) δ

7.14 (t, J = 7.8 Hz, 1H), 7.06 (s 1H), 7.01 (s, 1H), 6.58−6.61 (m,

2H), 6.43 (d, J = 7.8 Hz, 1H), 6.05 (s, 1H), 5.82 (s, 1H), 4.08 (t, J =

6.6 Hz, 2H), 4.00 (q, J = 7.2 Hz, 2H), 4.00 (s, 3H), 3.37 (s, 2H),

1.87−1.93 (m, 2H), 1.39 (t, J = 7.2 Hz, 3H), 1.09 (t, J = 7.2 Hz, 3H);

¹³C NMR (150 MHz, CDCl₃) δ 144.9, 144.6, 144.1, 142.3, 129.8,

124.1, 113.3, 112.8, 108.6, 105.7, 102.6, 102.4, 71.0, 63.3, 37.2, 29.8,

22.6, 14.9, 10.5. MS (ESI) calcd for C₂₅H₂₆N₃O₃[M + H]⁺: 380.2,

found: 380.4.

Methyl 2-((3-((3-Ethoxyphenyl)amino)-1-methyl-7-pro-

poxy-1,4-dihydroindeno[1,2-c]pyrazol-6-yl)oxy)acetate (10).

Following the procedures described for preparing 6, compound 10

was prepared from 9 (150 mg, 0.40 mmol), methyl chloroacetate (69

μL, 0.79 mmol), K₂CO₃(109 mg, 0.79 mmol) in 87% yield as a white

solid, mp: 154−156 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.38 (s, 1H),

7.87 (d, J = 1.8 Hz, 1H), 7.34 (s, 1H), 7.23 (s, 1H), 7.06 (t, J = 7.8

Hz, 1H), 6.98 (s, 1H), 6.83 (dd, J = 7.8, 1.8 Hz, 1H), 6.29 (dd, J =

7.8, 1.8 Hz, 1H), 4.48 (s, 2H), 4.07 (t, J = 6.6 Hz, 2H), 3.97 (q, J =

7.2 Hz, 2H), 3.96 (s, 3H), 3.69 (s, 3H), 3.37 (s, 2H), 1.75−1.82 (m,

2H), 1.32 (t, J = 7.2 Hz, 3H), 1.04 (t, J = 7.2 Hz, 3H); ¹³C NMR

(150 MHz, CDCl₃) δ 168.8, 159.7, 148.7, 146.8, 145.3, 144.8, 141.4,

129.8, 126.7, 115.3, 113.1, 108.3, 105.5, 104.4, 101.8, 70.9, 70.1, 63.0,

40.5, 37.6, 29.2, 25.8, 22.7, 15.3, 10.9. MS (ESI) calcd for

C₂₅H₃₀N₃O₅[M + H]⁺: 452.1, found: 452.4.

1

199−200 °C, H NMR (600 MHz, DMSO-d₆) δ 12.97 (s, 1H), 8.39

(s, 1H), 7.25 (s, 1H), 7.23 (s, 1H), 7.06 (t, J = 8.1 Hz, 1H), 6.99 (t, J

= 2.1 Hz, 1H), 6.83 (dd, J = 8.1, 2.1 Hz, 1H), 6.28 (dd, J = 8.1, 2.1

Hz, 1H), 4.78 (s, 2H), 4.08 (q, J = 7.0 Hz, 2H), 3.97 (q, J = 7.0 Hz,

2H), 3.93 (s, 3H), 3.35 (s, 2H), 1.35 (t, J = 7.0 Hz, 3H), 1.32 (t, J =

7.0 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 170.9, 159.7, 148.6,

147.4, 147.0, 145.3, 144.8, 142.4, 129.8, 124.7, 112.8, 112.6, 108.2,

105.5, 104.3, 101.7, 66.0, 64.6, 63.0, 37.4, 29.4, 15.3, 15.2. HRMS

(ESI) calcd for C₂₃H₂₆N₃O₅[M + H]⁺: 424.1867, found: 424.1873.

5-Ethoxy-6-(3-hydroxypropoxy)-1-indanone (16). To a mix-

ture of 5-ethoxy-6-hydroxyl-1-indanone 15 (0.57 g, 2.95 mmol) and

K₂CO₃(0.48 g, 3.54 mmol) in DMF (8 mL), 1-chloro-3-

hydroxypropane (0.21 mL, 3.54 mmol) was added at 75 °C and

stirred for 10 h. Distilled water (40 mL) was added, and the mixture

was extracted with EtOAc (50 mL × 3), washed with brine, and dried

over Na₂SO₄. After ﬁltration and evaporation, the residue was puriﬁed

by chromatography (ethyl acetate/hexanes = 1:1) to give 580 mg

2-((3-((3-Ethoxyphenyl)amino)-1-methyl-7-propoxy-1,4-

dihydroindeno[1,2-c]pyrazol-6-yl)oxy)acetamide (ID06). Fol-

lowing the procedures described for preparing 7, compound ID06 was

prepared from 10 (30 mg, 0.07 mmol), aqueous ammonia (25%, 0.6

mL) in 76% yield as a white solid, HPLC purity: 98.5%, mp: 162−164

1

°C. H NMR (600 MHz, DMSO-d₆) δ 8.38 (s, 1H), 7.48 (s, 1H),

7.34 (s 1H), 7.32 (s, 1H), 7.22 (s, 1H), 7.06 (t, J = 8.4 Hz, 1H), 6.92

(t, J = 2.4 Hz, 1H), 6.83 (dd, J = 8.4, 2.4 Hz, 1H), 6.29 (dd, J = 8.4,

2.4 Hz, 1H), 4.46 (s, 2H), 4.07 (t, J = 6.6 Hz, 2H), 3.97 (q, J = 7.2

Hz, 2H), 3.97 (s, 3H), 3.37 (s, 2H), 1.77−1.84 (m, 2H), 1.32 (t, J =

7.2 Hz, 3H), 1.03 (t, J = 7.2 Hz, 3H); ¹³C NMR (150 MHz, DMSO-

d₆) δ 170.7, 159.7, 148.5, 146.8, 145.3, 144.8, 141.5, 129.8, 126.4,

114.7, 112.9, 108.3, 105.4, 104.4, 101.8, 70.9, 69.4, 63.0, 40.5, 37.6,

29.3, 22.7, 15.3, 11.0. HRMS (ESI) calcd for C₂₄H₂₉N₄O₄[M + H]⁺:

437.2183, found: 437.2191.

1

(79%) of 16 as a white solid, mp: 98−99 °C. H NMR (400 MHz,

7-((tert-Butyldimethylsilyl)oxy)-6-ethoxy-N-(3-ethoxyphen-

yl)-1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-3-amine (12a).

Following similar procedures to those described for preparing 4,

compound 12a was prepared from 11 (1.0 g, 3.2 mmol), LiHMDS

(3.9 mL, 3.9 mmol), 1-ethoxy-3-isothiocyanatobenzene (698 mg, 3.9

mmol), hydrazine hydrate (4.9 mL, 31.2 mmol), DIPEA (401 mL, 2.4

mmol), and dimethyl sulfate (232 mL, 2.4 mmol) in 18% yield as a

DMSO-d₆) δ 7.10 (s, 1H), 7.06 (s, 1H), 4.14 (q, J = 6.9 Hz, 2H), 4.06

(t, J = 6.5 Hz, 2H), 3.61−3.52 (m, 2H), 3.03−2.93 (m, 2H), 2.60−

2.52 (m, 2H), 1.92−1.81 (m, 2H), 1.37 (t, J = 7.0 Hz, 3H); ¹³C NMR

(100 MHz, DMSO-d₆) δ 204.9, 154.8, 150.8, 148.6, 129.3, 109.5,

105.4, 65.9, 64.4, 57.6, 36.2, 32.4, 25.5.

5-Ethoxy-6-(3-(methoxymethoxy)propoxy)-2,3-dihydro-1H-

inden-1-one (17). To a solution of 5-ethoxy-6-hydroxyl-1-indanone

16 (0.52 g, 2.08 mmol) in DCM (8 mL), DIPEA (0.83 mL, 4.16

mmol) and MOMCl (0.31 mL, 4.16 mmol) were added, and the

mixture was stirred for 8 h at room temperature. Distilled water (30

mL) was added, and the mixture was extracted with EtOAc (40 mL ×

3), washed with brine and dried over Na₂SO₄. After ﬁltration and

evaporation, the residue was puriﬁed by chromatography (ethyl

acetate/hexanes = 1:3) to give 420 mg (69%) of 17 as a white solid,

mp: 87−89 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.11 (s, 1H), 7.07

(s, 1H), 4.56 (s, 2H), 4.14 (q, J = 7.0 Hz, 2H), 4.07 (t, J = 6.5 Hz,

2H), 3.62 (t, J = 6.3 Hz, 2H), 3.23 (s, 3H), 3.01−2.95 (m, 2H), 2.56

(dd, J = 6.5, 4.6 Hz, 2H), 1.97 (t, J = 6.3 Hz, 2H), 1.37 (t, J = 7.0 Hz,

3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 205.2, 154.8, 150.2, 148.3,

129.6, 110.1, 106.1, 95.8, 65.9, 64.4, 63.8, 55.1, 36.5, 29.6, 25.3.

6-Ethoxy-N-(3-ethoxyphenyl)-7-(3-(methoxymethoxy)-

propoxy)-1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-3-amine

(18a). Following the procedures described for preparing 4, compound

18a was prepared from 17 (300 mg, 1.02 mmol), LiHMDS (1.22 mL,

1.22 mmol), 1-ethoxy-3-isothiocyanatobenzene (219 mg, 1.22 mmol),

hydrazine hydrate (0.51 mL, 8.16 mmol), DIPEA (70 mL, 0.42

mmol), and dimethyl sulfate (40 mL, 0.42 mmol) in 16% yield as a

1

white solid, mp: 110−112 °C. H NMR (600 MHz, DMSO-d₆) δ

8.36 (s, 1H), 7.22 (s, 1H), 7.12 (s, 1H), 7.06 (t, J = 7.8 Hz, 1H), 6.96

(t, J = 1.8 Hz, 1H), 6.82 (dd, J = 7.8, 1.8 Hz, 1H), 6.29 (dd, J = 7.8,

1.8 Hz, 1H), 4.06 (q, J = 7.2 Hz, 2H), 3.97 (q, J = 7.2 Hz, 2H), 3.91

(s, 3H), 3.37 (s, 2H), 1.37 (t, J = 7.2 Hz, 3H), 1.32 (t, J = 7.2 Hz,

3H), 1.00 (s, 9H), 0.17 (s, 6H); ¹³C NMR (150 MHz, CDCl₃) δ

160.1, 149.9, 149.5, 144.7, 144.1, 142.6, 129.8, 124.6, 113.3, 111.5,

111.3, 108.7, 105.8, 102.5, 64.2, 63.3, 37.1, 30.0, 25.7, 18.4, 14.9, 14.9,

−4.5. MS (ESI) calcd for C₂₇H₃₈N₃O₃Si [M + H]⁺: 480.3, found:

480.5.

6-Ethoxy-3-((3-ethoxyphenyl)amino)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-ol (13a). Following the proce-

dures described for preparing 5, compound 13a was prepared from

12a (1.0 g, 2.3 mmol) and TBAF (1.2 g, 4.6 mmol) in 90% yield as a

1

white solid, mp: 153−155 °C. H NMR (600 MHz, DMSO-d₆) δ

8.83 (s, 1H), 8.34 (s, 1H), 7.15 (s, 1H), 7.11 (s, 1H), 7.05 (t, J = 7.8

Hz, 1H), 6.96 (s, 1H), 6.83 (d, J = 7.8 Hz, 1H), 6.27 (d, J = 7.8 Hz,

1H), 4.06 (q, J = 7.2 Hz, 2H), 3.97 (q, J = 7.2 Hz, 2H), 3.89 (s, 3H),

3.32 (s, 2H), 1.35(t, J = 7.2 Hz, 3H), 1.32 (t, J = 7.2 Hz, 3H); ¹³C

NMR (150 MHz, DMSO-d₆) δ 159.73, 148.8, 146.3, 145.9, 145.4,

144.7, 140.1, 129.8, 125.1, 112.7, 112.6, 108.3, 106.7, 104.3, 101.8,

1

yellow solid, mp: 118−119 °C. H NMR (600 MHz, DMSO-d₆) δ

P

https://dx.doi.org/10.1021/acs.jmedchem.0c01345

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

8.82 (s, 1H), 7.88 (d, J = 7.5 Hz, 1H), 7.58 (s, 1H), 7.41 (t, J = 7.5

Hz, 1H), 7.26 (s, 1H), 6.92 (s, 1H), 6.75 (d, J = 7.5 Hz, 1H), 4.50 (s,

2H), 4.32 (t, J = 6.3 Hz, 2H), 4.29 (q, J = 6.9 Hz, 2H), 4.07 (q, J = 6.9

Hz, 2H), 3.95 (s, 3H), 3.68 (t, J = 6.3 Hz, 2H), 3.36 (s, 2H), 3.27 (s,

3H), 1.97 (p, J = 6.3 Hz, 2H), 1.42 (t, J = 6.9 Hz, 3H), 1.34 (t, J = 6.9

Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 161.1, 158.9, 148.4,

145.2, 144.0, 138.4, 131.1, 130.8, 130.5, 110.4, 109.7, 109.5, 104.4,

102.8, 99.5, 96.3, 65.3, 65.0, 64.9, 64.6, 55.6, 37.9, 29.4, 24.9, 14.8,

14.7. MS (ESI) calcd for C₂₆H₃₃N₃O₅[M + H]⁺: 523.2, found: 523.3.

3-((6-Ethoxy-3-((3-ethoxyphenyl)amino)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-yl)oxy)propan-1-ol (ID08).

Compound 18a (50 mg, 0.11 mmol) was dissolved in a CSA

methanol solution (1 M, 2 mL), and the mixture was heated to 40 °C

and stirred for 2 h. Distilled water (20 mL) was added, and the

mixture was extracted with EtOAc (25 mL × 3), washed with brine,

and dried over Na₂SO₄. After ﬁltration and evaporation, the residue

was puriﬁed by chromatography (ethyl acetate/hexanes = 5:1) to give

32 mg (75%) of ID08 as a white solid, HPLC purity: 99.9%, mp:

Hz, 1H), 6.99 (s, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.29 (d, J = 8.0 Hz,

1H), 4.15−4.03 (m, 4H), 4.00−3.98 (m, 5H), 3.38 (s, 2H), 3.24 (q, J

= 6.1 Hz, 2H), 1.94−1.85 (m, 2H), 1.82 (s, 3H), 1.36 (t, J = 7.4 Hz,

3H), 1.32 (t, J = 7.1 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ

169.6, 159.7, 148.8, 148.0, 147.9, 145.4, 144.8, 142.1, 129.8, 125.1,

113.1, 112.6, 108.3, 106.1, 104.3, 101.8, 67.5, 64.7, 63.0, 37.5, 36.2,

29.5, 29.3, 23.1, 15.4, 15.2. MS (ESI) calcd for C₂₆H₃₂N₄O₄[M +

H]⁺: 465.2, found: 465.4.

6-Ethoxy-N-(3-ethoxyphenyl)-1-methyl-7-propoxy-1,4-

dihydroindeno[1,2-c]pyrazol-3-amine (ID11). Following the

procedures described for preparing 6, compound ID11 was prepared

from 13a (70 mg, 0.19 mmol), 1-bromopropane (50 μL, 0.38 mmol),

K₂CO₃(60 mg, 0.38 mmol) in 64% yield as a white solid, HPLC

1

purity: 98.7%, mp: 142−143 °C. H NMR (600 MHz, DMSO-d₆) δ

8.38 (s, 1H), 7.28 (s, 1H), 7.20 (s, 1H), 7.06 (t, J = 8.1 Hz, 1H), 6.99

(t, J = 2.0 Hz, 1H), 6.83 (dd, J = 8.1, 2.0 Hz, 1H), 6.28 (dd, J = 8.1,

2.0 Hz, 1H), 4.05 (q, J = 7.0 Hz, 2H), 4.02 (t, J = 6.6 Hz, 2H), 3.98

(q, J = 6.9 Hz, 2H), 3.96 (s, 3H), 3.36 (s, 2H), 1.83−1.71 (m, 2H),

1.34 (t, J = 6.9 Hz, 3H), 1.32 (t, J = 7.0 Hz, 3H), 1.02 (t, J = 7.4 Hz,

3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 158.6, 147.7, 147.1, 146.6,

144.2, 143.7, 140.7, 128.7, 123.9, 111.8, 111.5, 107.1, 104.6, 103.2,

100.7, 69.8, 63.5, 61.9, 36.4, 28.2, 21.6, 14.3, 14.2, 9.9. HRMS (ESI)

calcd for C₂₄H₃₀N₃O₃[M + H]⁺: 408.2282, found: 408.2288.

7-((tert-Butyldimethylsilyl)oxy)-6-ethoxy-N-(4-ethoxyphen-

yl)-1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-3-amine (12b).

Following the procedures described for preparing 4, compound 12b

was prepared from 11 (500 mg, 1.63 mmol), LiHMDS (2.45 mL, 2.45

mmol), 1-ethoxy-4-isothiocyanatobenzene (436 mg, 2.45 mmol),

hydrazine hydrate (0.82 mL, 13.04 mmol), DIPEA (182 μL, 1.10

mmol), and dimethyl sulfate (104 μL, 1.10 mmol) in 17% yield as a

1

162−163 °C. H NMR (400 MHz, DMSO-d₆) δ 8.37 (s, 1H), 7.30

(s, 1H), 7.20 (s, 1H), 7.11−7.04 (t, J = 7.0 Hz, 1H), 6.99 (s, 1H),

6.84 (d, J = 7.0 Hz, 1H), 6.29 (d, J = 7.0 Hz, 1H), 4.55 (s, 1H), 4.14

(t, J = 4.6 Hz, 2H), 4.05 (q, J = 6.4 Hz, 2H), 3.99−3.90 (m, 5H), 3.61

(t, J = 4.6 Hz, 2H), 3.37 (s, 2H), 1.95−1.83 (m, 2H), 1.36 (t, J = 6.4

Hz, 3H), 1.36 (t, J = 6.5 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ

159.7, 148.8, 148.2, 147.7, 145.4, 144.8, 141.8, 129.8, 125.1, 112.9,

112.6, 108.3, 105.5, 104.3, 101.8, 66.7, 64.7, 63.0, 58.0, 37.5, 32.8,

29.4, 15.4, 15.2. MS (ESI) calcd for C₂₄H₂₉N₃O₄[M + Na]⁺: 446.2,

found: 446.3.

tert-Butyl (3-((6-Ethoxy-3-((3-ethoxyphenyl)amino)-1-meth-

yl-1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)propyl)-

carbamate (19a). Following the procedures described for preparing

6, compound 19a was prepared from 13a (40 mg, 0.11 mmol),

K₂CO₃(30 mg, 0.22 mmol), and N-Boc-3-bromopropylamine (50

1

white solid, mp: 168−169 °C. H NMR (600 MHz, DMSO-d₆) δ

8.07 (s, 1H), 7.25 (d, J = 9.0 Hz, 2H), 7.19 (s, 1H), 7.09 (s, 1H), 6.79

(d, J = 9.0 Hz, 2H), 4.04 (q, J = 7.0 Hz, 2H), 3.93 (q, J = 7.0 Hz, 2H),

3.88 (s, 3H), 3.34 (s, 2H), 1.36 (t, J = 7.0 Hz, 3H), 1.29 (t, J = 7.0 Hz,

3H), 1.00 (s, 9H), 0.16 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆) δ

150.6, 147.9, 147.4, 144.6, 142.5, 141.9, 136.8, 123.8, 115.9, 114.2,

111.0, 110.7, 110.5, 63.0, 62.5, 36.3, 28.4, 25.0, 17.6, 14.3, 14.2, −5.3.

MS (ESI) calcd for C₂₇H₃₈N₃O₃Si [M + H]⁺: 480.3, found: 480.3.

3-((7-((tert-Butyldimethylsilyl)oxy)-6-ethoxy-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzonitrile (12c).

Following the procedures described for preparing 4, compound 12c

was prepared from 11 (350 mg, 1.14 mmol), LiHMDS (2.06 mL, 2.06

mmol), 3-isothiocyanatobenzonitrile (329 mg, 2.06 mmol), hydrazine

hydrate (0.57 mL, 9.12 mmol), DIPEA (144 μL, 0.86 mmol), and

dimethyl sulfate (88 μL, 0.86 mmol) in 21% yield as a white solid,

1

mg, 0.22 mmol) in 87% yield as a white solid, mp: 137−138 °C. H

NMR (400 MHz, DMSO-d₆) δ 8.36 (s, 1H), 7.29 (s, 1H), 7.21 (s,

1H), 7.06 (t, J = 8.0 Hz, 1H), 6.98 (s, 1H), 6.83 (s, 1H), 6.82 (d, J =

8.0 Hz, 2H), 6.28 (d, J = 8.0 Hz, 1H), 4.19−4.02 (m, 4H), 4.02−3.83

(m, 5H), 3.37 (s, 2H), 3.14 (q, J = 6.0 Hz, 2H), 1.86 (p, J = 6.1 Hz,

1H), 1.38 (s, 9H), 1.36−1.24 (m, 6H); ¹³C NMR (100 MHz,

DMSO-d₆) δ 159.7, 156.1, 148.8, 148.0, 147.8, 145.3, 144.8, 142.1,

129.8, 125.0, 112.9, 112.6, 108.2, 106.1, 104.3, 101.8, 77.9, 67.7, 64.7,

63.0, 37.7, 37.5, 29.9, 29.3, 28.7, 15.3, 15.2. MS (ESI) calcd for

C₂₉H₃₉N₄O₅[M + H]⁺: 523.3, found: 523.2.

7-(3-Aminopropoxy)-6-ethoxy-N-(3-ethoxyphenyl)-1-meth-

yl-1,4-dihydroindeno[1,2-c]pyrazol-3-amine hydrochloride

(ID09). Acetyl chloride (2.5 mL) was added to ethanol (5 mL) and

stirred for 15 min. To the resulting solution (2 mL), 19a (45 mg, 0.09

mmol) was added, and the mixture was stirred for 20 min. The

resulting precipitate was ﬁltered and dried to give 35 mg (88%) of

1

mp: 110−112 °C. H NMR (400 MHz, DMSO-d₆) δ 8.93 (s, 1H),

7.83 (d, J = 1.6 Hz, 1H), 7.57 (dd, J = 8.0, 1.6 Hz, 1H), 7.45 (dd, J =

8.0, 1.6 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.24 (s, 1H), 7.14 (s, 1H),

4.06 (q, J = 6.9 Hz, 2H), 3.95 (s, 3H), 3.42 (s, 2H), 1.38 (t, J = 6.9

Hz, 3H), 1.00 (s, 9H), 0.17 (s, 6H);¹³C NMR (100 MHz, DMSO-d₆)

δ 149.2, 148.8, 144.7, 143.9, 143.6, 142.9, 130.5, 124.6, 121.7, 120.1,

119.9, 117.4, 112.7, 112.1, 111.9, 111.8, 64.1, 40.6, 40.4, 40.2, 39.9,

39.8, 39.6, 39.4, 37.6, 29.2, 26.1, 18.7, 15.3, −4.2. MS (ESI) calcd for

C₂₆H₃₃N₄O₂Si [M + H]⁺: 461.2, found: 461.6.

1

ID09 as a white solid, HPLC purity: 99.7%, mp: 156−158 °C. H

NMR (600 MHz, DMSO-d₆) δ 8.09 (s, 3H), 7.39 (s, 1H), 7.26 (s,

1H), 7.10 (t, J = 8.1 Hz, 1H), 6.96 (s, 1H), 6.83 (d, J = 8.1 Hz, 1H),

6.36 (d, J = 8.1 Hz, 1H), 4.19 (t, J = 6.2 Hz, 2H), 4.08 (q, J = 6.9 Hz,

2H), 4.00 (s, 2H), 3.97 (d, J = 7.0 Hz, 1H), 3.05−2.98 (m, 2H), 2.13

(p, J = 6.2 Hz, 2H), 1.36 (t, J = 6.9 Hz, 3H), 1.33 (t, J = 7.0 Hz, 3H);

¹³C NMR (150 MHz, DMSO-d₆) δ 159.7, 150.7, 148.8, 147.7, 144.0,

Methyl 3-((7-((tert-Butyldimethylsilyl)oxy)-6-ethoxy-1-

methyl-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate

(12d). Following the procedures described for preparing 4,

compound 12d was prepared from 11 (3 g, 9.8 mmol), LiHMDS

(14.6 mL, 14.6 mmol), methyl 3-isothiocyanatobenzoate (2.84 g, 14.6

mmol), hydrazine hydrate (4.9 mL, 78.4 mmol), DIPEA (1.21 mL,

7.0 mmol), and dimethyl sulfate (0.68 mL, 7.0 mmol) in 18% yield as

a white solid, mp: 122−123 °C. ¹H NMR (600 MHz, CDCl₃) δ 7.69

(s, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.23 (d, J =

7.6 Hz, 1H), 7.02 (s, 1H), 6.99 (s, 1H), 6.22 (s, 1H), 4.05 (q, J = 7.0

Hz, 2H), 3.98 (s, 3H), 3.89 (s, 3H), 3.38 (s, 2H), 1.46 (t, J = 7.0 Hz,

3H), 1.04 (s, 9H), 0.20 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆) δ

162.5, 144.9, 139.4, 138.7, 138.0, 126.3, 124.4, 119.7, 116.1, 115.6,

111.7, 106.8, 106.5, 59.4, 47.4, 32.4, 25.2, 21.1, 21.0, 13.7, 10.2, −9.3.

MS (ESI) calcd for C₂₇H₃₆N₃O₄Si [M + H]⁺: 495.2, found: 495.4.

143.7, 143.1, 130.1, 123.5, 112.2, 111.5, 109.3, 106.6, 106.0, 103.0,

67.3, 64.6, 63.1, 37.3, 36.9, 29.9, 27.3, 15.2, 15.1. HRMS (ESI) calcd

for C₂₄H₃₁N₄O₃[M + H]⁺: 423.2391, found: 423.2394; anal. calcd for

C₂₄H₃₁ClN₄O₃: C, 62.80; H, 6.81; Cl, 7.72; N, 12.21; found: C,

62.92; H, 6.64; Cl, 7.86; N, 12.32.

N-(3-((6-Ethoxy-3-((3-ethoxyphenyl)amino)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-yl)oxy)propyl)acetamide

(ID10). To a solution of ID09 (50 mg, 0.14 mmol) in THF (6 mL)

were added Et₃N (58 μL, 0.42 mmol) and acetic anhydride (22 μL,

0.21 mmol). The mixture was stirred at room temperature for 4 h.

Distilled water was added, and the resulting precipitate was ﬁltered

and dried to give 46 mg (92%) of ID10 as a white solid, HPLC

1

purity: 99.5%, mp: 173−174 °C. H NMR (400 MHz, DMSO-d₆) δ

8.36 (s, 1H), 7.90 (s, 1H), 7.31 (s, 1H), 7.22 (s, 1H), 7.07 (t, J = 8.0

Q

https://dx.doi.org/10.1021/acs.jmedchem.0c01345

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Ethyl 3-((7-((tert-Butyldimethylsilyl)oxy)-6-ethoxy-1-meth-

yl-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate

(12e). Following the procedures described for preparing 4, compound

12e was prepared from 11 (500 mg, 1.63 mmol), LiHMDS (5 mL,

4.89 mmol), ethyl 3-isothiocyanatobenzoate (970 mg, 4.89 mmol),

hydrazine hydrate (0.82 mL, 13.04 mmol), DIPEA (200 μL, 1.0

mmol), and dimethyl sulfate (110 μL, 1.0 mmol) in 19% yield as a

106.8, 64.6, 37.4, 28.9, 15.2. MS (ESI) calcd for C₂₀H₁₉N₄O₂[M +

H]⁺: 347.1, found: 347.3.

Methyl 3-((6-Ethoxy-7-hydroxy-1-methyl-1, 4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate (13d). Fol-

lowing the procedures described for preparing 5, compound 13d was

prepared from 12d (100 mg, 0.20 mmol) and TBAF (106 mg, 0.40

mmol) in 79% yield as a white solid, mp: 153−155 °C. ¹H NMR (600

MHz, DMSO-d₆) δ 8.89 (s, 1H), 8.69 (s, 1H), 7.89 (s, 1H), 7.61 (d,

J= 8.2 Hz, 1H), 7.32 (t, J = 8.2 Hz, 1H), 7.16 (s, 1H), 7.12 (d, J = 8.2

Hz, 1H), 4.06 (q, J = 7.0 Hz, 2H), 3.92 (s, 3H), 3.84 (s, 3H), 3.35 (s,

2H), 1.36 (t, J = 7.0 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ

167.2, 149.1, 146.3, 146.1, 144.5, 144.3, 140.1, 130.6, 129.6, 125.0,

119.8, 119.0, 115.8, 112.8, 112.6, 106.8, 64.7, 52.5, 37.4, 29.2, 15.3.

MS (ESI) calcd for C₂₁H₂₂N₃O₄[M + H]⁺: 380.2, found: 380.1.

Ethyl 3-((6-Ethoxy-7-hydroxy-1-methyl-1,4-dihydroindeno-

[1,2-c]pyrazol-3-yl)amino)benzoate (13e). Following the proce-

dures described for preparing 5, compound 13e was prepared from

12e (120 mg, 0.24 mmol) and TBAF (126 mg, 0.48 mmol) in 86%

yield as a white solid, mp: 184−185 °C. ¹H NMR (600 MHz, DMSO-

d₆) δ 8.86 (s, 1H), 8.70 (s, 1H), 7.90 (s, 1H), 7.64 (d, J = 7.3 Hz,

1H), 7.48−7.28 (m, 2H), 7.16 (s, 1H), 7.14 (s, 1H), 4.30 (q, J = 7.1

Hz, 2H), 4.07 (q, J = 7.0 Hz, 2H), 3.92 (s, 3H), 3.36 (s, 2H), 1.36 (t,

J = 7.0 Hz, 3H), 1.32 (t, J = 7.1 Hz, 3H); ¹³C NMR (150 MHz,

DMSO-d₆) δ 166.6, 149.0, 146.2, 146.0, 144.4, 144.3, 140.1, 130.8,

129.5, 125.0, 119.7, 118.9, 115.7, 112.7, 112.5, 106.7, 64.6, 60.9, 37.3,

29.2, 15.2, 14.6. MS (ESI) calcd for C₂₂H₂₄N₃O₄[M + H]⁺: 394.2,

found: 394.2.

Isopropyl 3-((6-Ethoxy-7-hydroxy-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate (13f). Fol-

lowing the procedures described for preparing 5, compound 13f was

prepared from 12f (100 mg, 0.19 mmol) and TBAF (100 mg, 0.38

mmol) in 80% yield as a white solid, mp: 151−152 °C. ¹H NMR (600

MHz, DMSO-d₆) δ 8.85 (s, 1H), 8.69 (s, 1H), 7.88 (s, 1H), 7.67−

7.60 (m, 1H), 7.39−7.26 (m, 2H), 7.16 (s, 1H), 7.13 (s, 1H), 5.12

(hept, J = 6.2 Hz, 1H), 4.06 (q, J = 7.0 Hz, 2H), 3.96 (s, 3H), 3.36 (s,

2H), 1.35 (t, J = 7.0 Hz, 3H), 1.31 (d, J = 6.2 Hz, 6H); ¹³C NMR

(150 MHz, DMSO-d₆) δ 166.1, 149.0, 146.2, 146.1, 144.4, 144.3,

140.1, 131.1, 129.5, 125.0, 119.6, 118.9, 115.7, 112.6, 112.5, 106.8,

68.3, 64.6, 37.4, 29.2, 22.2, 15.3. MS (ESI) calcd for C₂₃H₂₆N₃O₄[M

+ H]⁺: 408.2, found: 408.3.

1

yellow solid, mp: 160−161 °C. H NMR (600 MHz, DMSO-d₆) δ

8.71 (s, 1H), 7.89 (s, 1H), 7.63 (d, J = 6.8 Hz, 1H), 7.43−7.27 (m,

2H), 7.22 (s, 1H), 7.12 (s, 1H), 4.29 (q, J = 7.0 Hz, 2H), 4.05 (q, J =

6.9 Hz, 2H), 3.92 (s, 3H), 3.39 (s, 2H), 1.37 (t, J = 6.9 Hz, 3H), 1.31

(t, J = 7.0 Hz, 3H), 1.00 (s, 9H), 0.17 (s, 6H); ¹³C NMR (150 MHz,

DMSO-d₆) δ 166.1, 148.6, 148.2, 143.9, 143.8, 143.1, 142.4, 130.3,

129.1, 124.2, 119.2, 118.4, 115.3, 112.2, 111.6, 111.2, 63.6, 60.5, 37.0,

28.9, 25.6, 18.2, 14.7, 14.2, −4.6. MS (ESI) calcd for C₂₈H₃₈N₃O₄Si

[M + H]⁺: 508.3, found: 508.2.

Isopropyl 3-((7-((tert-Butyldimethylsilyl)oxy)-6-ethoxy-1-

methyl-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate

(12f). Following the procedures described for preparing 4, compound

12f was prepared from 11 (300 mg, 0.98 mmol), LiHMDS (1.96 mL,

1.96 mmol), isopropyl 3-isothiocyanatobenzoate (430 mg, 1.96

mmol), hydrazine hydrate (0.49 mL, 7.84 mmol), DIPEA (76 μL,

0.46 mmol), and dimethyl sulfate (44 μL, 0.46 mmol) in 13% yield as

1

a yellow solid, mp: 114−116 °C. H NMR (600 MHz, DMSO-d₆) δ

8.70 (s, 1H), 7.87 (d, J = 1.6 Hz, 1H), 7.66−7.59 (m, 1H), 7.37−7.28

(m, 2H), 7.21 (s, 1H), 7.12 (s, 1H), 5.12 (hept, J = 6.3 Hz, 1H), 4.04

(q, J = 7.0 Hz, 2H), 3.92 (s, 3H), 3.39 (s, 2H), 1.36 (t, J = 7.0 Hz,

3H), 1.31 (d, J = 6.3 Hz, 6H), 1.00 (s, 9H), 0.16 (s, 6H); ¹³C NMR

(150 MHz, DMSO-d₆) δ 165.0, 148.0, 147.6, 143.3, 142.5, 141.8,

130.1, 128.4, 123.6, 118.6, 117.9, 114.7, 111.6, 110.9, 110.6, 67.2,

63.0, 36.4, 28.3, 25.0, 21.1, 17.6, 14.1, −5.2. MS (ESI) calcd for

C₂₉H₄₀N₃O₄Si [M + H]⁺: 522.3, found: 522.2.

3-((7-((tert-Butyldimethylsilyl)oxy)-6-ethoxy-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)-N-methylbenza-

mide (12g). Following the procedures described for preparing 4,

compound 12g was prepared from 11 (1.5 g, 4.90 mmol), LiHMDS

(5.64 mL, 5.64 mmol), 3-isothiocyanato-N-methylbenzamide (1.08 g,

5.64 mmol), hydrazine hydrate (2.45 mL, 39.2 mmol), DIPEA (0.45

mL, 2.72 mmol), and dimethyl sulfate (0.26 mL, 2.72 mmol) in 18%

yield as a yellow solid, mp: 106−108 °C. ¹H NMR (600 MHz,

DMSO-d₆) δ 8.59 (s, 1H), 8.31 (q, J = 4.4 Hz, 1H), 7.62 (s, 1H), 7.44

(d, J = 8.1 Hz, 1H), 7.25 (t, J = 8.1 Hz, 1H), 7.21 (s, 1H), 7.14 (d, J =

8.1 Hz, 1H), 7.12 (s, 1H), 4.07−3.99 (m, 2H), 3.91 (s, 3H), 3.36 (s,

2H), 2.74 (d, J = 4.4 Hz, 3H), 1.36 (t, J = 6.8 Hz, 3H), 0.99 (s, 9H),

0.16 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆) δ 167.7, 149.1, 148.7,

144.4, 144.3, 143.5, 142.9, 135.7, 129.1, 124.6, 117.8, 116.6, 114.3,

112.8, 111.9, 111.7, 64.0, 37.5, 29.5, 26.7, 26.1, 18.7, 15.3, −4.2. MS

(ESI) calcd for C₂₇H₃₇N₄O₃Si [M + H]⁺: 493.3, found: 493.7.

6-Ethoxy-3-((4-ethoxyphenyl)amino)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-ol (13b). Following the proce-

dures described for preparing 5, compound 13b was prepared from

12b (140 mg, 0.29 mmol) and TBAF (150 mg, 0.58 mmol) in 76%

yield as a white solid, mp: 183−184 °C. ¹H NMR (600 MHz, DMSO-

d₆) δ 8.81 (s, 1H), 8.06 (s, 1H), 7.25 (d, J = 9.0 Hz, 2H), 7.14 (s,

1H), 7.10 (s, 1H), 6.79 (d, J = 9.0 Hz, 2H), 4.05 (q, J = 7.0 Hz, 2H),

3.93 (q, J = 7.0 Hz, 2H), 3.87 (s, 3H), 3.30 (s, 2H), 1.35 (t, J = 7.0

Hz, 3H), 1.29 (t, J = 7.0 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ

150.6, 147.7, 145.1, 144.8, 144.6, 139.0, 136.8, 124.1, 115.9, 114.2,

111.5, 110.8, 105.6, 63.6, 62.5, 36.2, 28.1, 14.3, 14.2. MS (ESI) calcd

for C₂₁H₂₄N₃O₃[M + H]⁺: 366.2, found: 366.2.

3-((6-Ethoxy-7-hydroxy-1-methyl-1,4-dihydroindeno[1,2-c]-

pyrazol-3-yl)amino)-N-methylbenzamide (13g). Following the

procedures described for preparing 5, compound 13g was prepared

from 12g (90 mg, 0.18 mmol) and TBAF (95.5 mg, 0.36 mmol) in

1

89% yield as a white solid, mp: 123−124 °C. H NMR (600 MHz,

DMSO-d₆) δ 8.90 (s, 1H), 8.58 (s, 1H), 8.31 (q, J = 4.3 Hz, 1H), 7.62

(d, J = 8.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H),

7.15 (s, 1H), 7.13 (s, 1H), 4.07−4.03 (m, 2H), 3.91 (s, 3H), 3.33 (s,

2H), 2.75 (d, J = 4.3 Hz, 3H), 1.35 (t, J = 7.0 Hz, 3H); ¹³C NMR

(150 MHz, DMSO-d₆) δ 167.7, 149.0, 146.2, 144.4, 144.3, 140.1,

135.8, 124.9, 117.7, 116.5, 112.8, 112.4, 106.7, 64.5, 60.2, 37.36, 29.3,

26.7, 21.2, 15.3, 14.5. MS (ESI) calcd for C₂₁H₂₃N₄O₃[M + H]⁺:

379.2, found: 379.6.

Methyl 2-((6-Ethoxy-3-((4-ethoxyphenyl)amino)-1-methyl-

1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetate (14b). Fol-

lowing the procedures described for preparing 6, compound 14b

was prepared from 13b (100 mg, 0.27 mmol), methyl chloroacetate

(48 μL, 0.54 mmol), K₂CO₃(76 mg, 0.54 mmol) in 71% yield as a

1

white solid, mp: 155−157 °C. H NMR (600 MHz, DMSO-d₆) δ

8.09 (s, 1H), 7.30−7.24 (m, 3H), 7.22 (s, 1H), 6.79 (d, J = 9.0 Hz,

2H), 4.88 (s, 2H), 4.07 (q, J = 7.0 Hz, 2H), 3.93 (q, J = 7.0 Hz, 2H),

3.91 (s, 3H), 3.71 (s, 3H), 3.34 (s, 2H), 1.34 (t, J = 7.0 Hz, 3H), 1.29

(t, J = 7.0 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 168.8, 150.6,

147.4, 146.4, 145.8, 144.7, 141.8, 136.7, 123.8, 115.9, 114.1, 111.9,

110.8, 105.0, 65.3, 63.6, 62.5, 51.1, 36.3, 28.3, 14.3, 14.2. MS (ESI)

calcd for C₂₄H₂₈N₃O₅[M + H]⁺: 438.2, found: 438.1.

3-((6-Ethoxy-7-hydroxy-1-methyl-1,4-dihydroindeno[1,2-c]-

pyrazol-3-yl)amino)benzonitrile (13c). Following the procedures

described for preparing 5, compound 13c was prepared from 12c

(180 mg, 0.39 mmol) and TBAF (110 mg, 0.78 mmol) in 81% yield

as a white solid, mp: 130−132 °C. ¹H NMR (400 MHz, DMSO-d₆) δ

8.90 (s, 1H), 8.87 (s, 1H), 7.79 (d, J = 8.1 Hz, 1H), 7.57 (d, J = 8.1

Hz, 1H), 7.32 (t, J = 8.1 Hz 1H), 7.17 (s, 1H), 7.14 (s, 1H), 4.07 (q, J

= 7.0 Hz, 2H), 3.93 (s, 3H), 3.38 (s, 2H), 1.36 (t, J = 7.0 Hz, 3H);

¹³C NMR (100 MHz, DMSO-d₆) δ 149.1, 146.3, 146.1, 144.73 143.9,

Methyl 2-((3-((3-Cyanophenyl)amino)-6-ethoxy-1-methyl-

1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetate (14c). Fol-

lowing the procedures described for preparing 6, compound 14c

was prepared from 13c (110 mg, 0.32 mmol), methyl chloroacetate

140.1, 130.4, 124.9, 121.6, 120.0, 119.9, 117.4, 112.7, 112.5, 111.9,

R

https://dx.doi.org/10.1021/acs.jmedchem.0c01345

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

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(60 μL, 0.64 mmol), K₂CO₃(87 mg, 0.64 mmol) in 91% yield as a

white solid, mp: 151−152 °C. H NMR (400 MHz, DMSO-d₆) δ

°C. H NMR (600 MHz, DMSO-d₆) δ 8.13 (s, 1H), 7.52 (s, 1H),

7.38 (s, 2H), 7.27 (d, J = 9.0 Hz, 2H), 7.25 (s, 1H), 6.80 (d, J = 9.0

Hz, 2H), 4.53 (s, 2H), 4.09 (q, J = 7.0 Hz, 2H), 3.94 (q, J = 7.0 Hz,

1H), 3.91 (s, 3H), 1.37 (t, J = 7.0 Hz, 3H), 1.30 (t, J = 7.0 Hz, 3H);

¹³C NMR (150 MHz, DMSO-d₆) δ 170.9, 151.6, 148.5, 147.8, 147.0,

145.7, 143.3, 137.7, 124.9, 116.9, 115.1, 112.6, 111.7, 107.4, 69.7,

64.5, 63.5, 37.3, 29.4, 15.3, 15.3. HRMS (ESI) calcd for C₂₃H₂₇N₄O₄

[M + H]⁺: 423.2027, found: 423.2034.

1

8.93 (s, 1H), 7.85 (s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.39 (t, J = 7.9

Hz, 1H), 7.30 (s, 1H), 7.26 (s, 1H), 7.14 (d, J = 7.9 Hz, 1H), 4.89 (s,

2H), 4.09 (q, J = 6.9 Hz, 2H), 3.98 (s, 3H), 3.72 (s, 3H), 3.42 (s,

3H), 1.36 (t, J = 6.9 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ

169.8, 148.9, 147.8, 147.0, 144.7, 144.0, 142.9, 130.4, 124.7, 121.7,

120.11, 119.9, 117.5, 113.1, 112.8, 111.9, 106.3, 66.5, 64.7, 52.1, 37.6,

29.1, 15.2. MS (ESI) calcd for C₂₃H₂₃N₄O₄[M + H]⁺: 419.2, found:

419.4.

2-((3-((3-Cyanophenyl)amino)-6-ethoxy-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetamide (ID13). Fol-

lowing the procedures described for preparing 7, compound ID13 was

prepared from 14c (30 mg, 0.07 mmol), aqueous ammonia (25%, 2

mL) in 95% yield as a white solid, HPLC purity: 96.0%, mp: 133−135

°C. 1H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H), 8.93 (s, 1H),

7.86 (s, 1H), 7.86 (s, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.51 (s, 1H),

7.41−7.25 (m, 3H), 7.14 (d, J = 7.2 Hz, 1H), 4.54 (s, 2H), 4.10 (q, J

= 6.9 Hz, 2H), 3.98 (s, 3H), 3.43 (s, 2H), 1.38 (t, J = 6.9 Hz, 3H);

13C NMR (100 MHz, DMSO-d6) δ 170.9, 148.8, 148.1, 147.2, 144.7,

144.0, 143.4, 130.5, 124.7, 121.7, 120.1, 119.9, 117.5, 112.7, 111.9,

107.6, 69.7, 64.7, 37.6, 29.2, 15.2. HRMS (ESI) calcd for C₂₂H₂₂N₅O₃

[M + H] +: 404.1717, found: 404.1723.

Methyl 3-((7-(2-Amino-2-oxoethoxy)-6-ethoxy-1-methyl-

1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate (ID14).

Following the procedures described for preparing 7, compound ID14

was prepared from 14d (1.1 g, 2.43 mmol), aqueous ammonia (25%,

10 mL) in 92% yield as a white solid, HPLC purity: 99.7%, mp: 216−

217 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.72 (s, 1H), 7.94 (s, 1H),

7.62−7.59 (m, 1H), 7.51 (s, 1H), 7.40 (s, 1H), 7.32−7.28 (m, 3H),

7.27 (s, 1H), 4.53 (s, 2H), 4.17−4.03 (q, J = 7.0 Hz, 2H), 3.94 (s,

3H), 3.81 (s, 3H), 1.37 (t, J = 7.0 Hz, 3H); ¹³C NMR (100 MHz,

DMSO-d₆) δ 171.0, 167.2, 148.8, 148.0, 147.1, 144.4, 143.4, 130.6,

129.6, 124.8, 119.9, 119.1, 115.8, 112.8, 112.7, 110.2, 107.6, 69.7,

64.7, 52.5, 37.5, 29.4, 15.2. HRMS (ESI) calcd for C₂₃H₂₅N₄O₅[M +

H]⁺: 437.1819, found: 437.1823.

Methyl 3-((6-Ethoxy-7-(2-methoxy-2-oxoethoxy)-1-methyl-

1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate (14d).

Following the procedures described for preparing 6, compound 14d

was prepared from 13d (60 mg, 0.16 mmol), methyl chloroacetate

(28 μL, 0.32 mmol), K₂CO₃(44 mg, 0.32 mmol) in 71% yield as a

1

white solid, mp: 136−137 °C. H NMR (600 MHz, DMSO-d₆) δ

8.71 (s, 1H), 7.89−7.86 (m, 2H), 7.62 (s, 1H), 7.33 (s, 1H), 7.29 (s,

1H), 7.26 (s, 1H), 4.89 (s, 2H), 4.09 (q, J = 6.9 Hz, 2H), 3.96 (s,

3H), 3.84 (s, 3H), 3.72 (s, 3H), 1.35 (t, J = 6.9 Hz, 3H); ¹³C NMR

(150 MHz, DMSO-d₆) δ 169.9, 167.2, 148.8, 147.8, 147.0, 144.5,

144.4, 143.0, 130.6, 129.6, 124.8, 119.9, 119.1, 115.8, 113.1, 112.9,

106.3, 66.5, 64.8, 52.5, 52.2, 37.5, 29.3, 15.2. MS (ESI) calcd for

C₂₄H₂₆N₃O₆[M + H]⁺: 452.2, found: 452.5.

Ethyl 3-((6-Ethoxy-7-(2-methoxy-2-oxoethoxy)-1-methyl-

1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate (14e).

Following the procedures described for preparing 6, compound 14e

was prepared from 13e (80 mg, 0.20 mmol), methyl chloroacetate (50

μL, 0.40 mmol), K₂CO₃(70 mg, 0.40 mmol) in 77% yield as a white

1

solid, mp: 155−156 °C. H NMR (600 MHz, DMSO-d₆) δ 8.73 (s,

1H), 7.91 (s, 1H), 7.64 (d J = 7.3 Hz, 1H), 7.35−7.30 (m, 2H), 7.29

(s, 1H), 7.25 (s, 1H), 4.89 (s, 2H), 4.30 (q, J = 7.1 Hz, 2H), 4.08 (q, J

= 7.0 Hz, 2H), 3.96 (s, 3H), 3.72 (s, 3H), 3.40 (s, 2H), 1.35 (t, J = 7.0

Hz, 3H), 1.32 (t, J = 7.1 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ

169.9, 166.6, 148.7, 147.7, 146.9, 144.4, 144.3, 142.8, 130.8, 129.5,

124.7, 119.7, 119.0, 115.8, 112.9, 112.7, 106.1, 66.3, 64.6, 60.8, 52.2,

37.5, 29.3, 15.2, 14.6. MS (ESI) calcd for C₂₅H₂₇N₃O₆Na [M + Na]⁺:

480.2, found: 480.5.

Ethyl 3-((7-(2-Amino-2-oxoethoxy)-6-ethoxy-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate (ID15). Fol-

lowing the procedures described for preparing 7, compound ID15 was

prepared from 14e (40 mg, 0.08 mmol), aqueous ammonia (25%, 2

mL) in 84% yield as a white solid, HPLC purity: 99.2%, mp: 177−179

Isopropyl 3-((6-Ethoxy-7-(2-methoxy-2-oxoethoxy)-1-meth-

yl-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate

(14f). Following the procedures described for preparing 6, compound

14f was prepared from 13f (62 mg, 0.16 mmol), methyl chloroacetate

(35 μL, 0.32 mmol), K₂CO₃(44 mg, 0.32 mmol) in 78% yield as a

1

°C. H NMR (600 MHz, DMSO-d₆) δ 8.73 (s, 1H), 7.90 (s, 1H),

7.65−7.62 (m, 1H), 7.50 (s, 1H), 7.41 (s, 1H), 7.38−7.29 (m, 3H),

7.27 (s, 1H), 4.53 (s, 2H), 4.30 (q, J = 7.1 Hz, 2H), 4.10 (q, J = 6.9

Hz, 2H), 3.96 (s, 3H), 3.41 (s, 2H), 1.37 (t, J = 6.9 Hz, 3H), 1.32 (t, J

= 7.1 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 170.9, 166.6,

148.7, 147.9, 147.1, 144.5, 144.3, 143.3, 130.8, 129.5, 124.7, 119.8,

119.0, 115.8, 112.7, 112.6, 107.5, 69.7, 64.5, 61.0, 37.5, 29.4, 15.2,

14.6. HRMS (ESI) calcd for C₂₄H₂₇N₄O₅[M + H]⁺: 451.1976,

found: 451.1984.

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white solid, mp: 123−124 °C. H NMR (600 MHz, DMSO-d₆) δ

8.72 (s, 1H), 7.89 (s, 1H), 7.68−7.59 (m, 1H), 7.38−7.28 (m, 3H),

7.25 (s, 1H), 5.12 (hept, J = 6.2 Hz, 1H), 4.89 (s, 2H), 4.08 (q, J =

7.0 Hz, 2H), 3.96 (s, 3H), 3.72 (s, 3H), 3.40 (s, 2H), 1.35 (t, J = 7.0

Hz, 3H), 1.31 (d, J = 6.2 Hz, 6H); ¹³C NMR (150 MHz, DMSO-d₆)

δ 169.9, 166.1, 148.7, 147.7, 146.9, 144.5, 144.3, 142.8, 131.2, 129.5,

124.7, 119.7, 119.0, 115.8, 112.9, 112.7, 106.2, 68.3, 66.4, 64.7, 52.2,

37.5, 29.4, 22.2, 15.3. MS (ESI) calcd for C₂₆H₃₀N₃O₆[M + H]⁺:

480.2, found: 480.2.

Isopropyl 3-((7-(2-Amino-2-oxoethoxy)-6-ethoxy-1-methyl-

1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate (ID16).

Following the procedures described for preparing 7, compound ID16

was prepared from 14f (50 mg, 0.11 mmol), aqueous ammonia (25%,

2 mL) in 73% yield as a white solid, HPLC purity: 97.2%, mp: 179−

181 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 8.76 (s, 1H), 7.89 (s, 1H),

7.68−7.61 (m, 1H), 7.53 (s, 1H), 7.41 (s, 1H), 7.39 (s, 1H), 7.36−

7.29 (m, 2H), 7.27 (s, 1H), 5.12 (hept, J = 6.2 Hz, 1H), 4.54 (s, 2H),

4.10 (q, J = 7.0 Hz, 2H), 3.96 (s, 3H), 3.41 (s, 2H), 1.37 (t, J = 7.0

Hz, 3H), 1.32 (d, J = 6.2 Hz, 6H); ¹³C NMR (150 MHz, DMSO-d₆)

δ 170.9, 166.1, 148.7, 147.8, 147.1, 144.5, 144.3, 143.3, 131.2, 129.5,

124.8, 119.6, 119.0, 115.8, 112.6, 112.6, 107.5, 69.6, 68.2, 64.6, 37.5,

29.4, 22.1, 15.2. HRMS (ESI) calcd for C₂₅H₂₉N₄O₅[M + H]⁺:

465.2132, found: 465.2140.

Methyl 2-((6-Ethoxy-1-methyl-3-((3-(methylcarbamoyl)-

phenyl)amino)-1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)-

acetate (14g). Following the procedures described for preparing 6,

compound 14g was prepared from 13g (30 mg, 0.08 mmol), methyl

chloroacetate (0.01 mL, 0.16 mmol), K₂CO₃(21.9 mg, 0.16 mmol) in

1

86% yield as a white solid, mp: 136−137 °C. H NMR (600 MHz,

DMSO-d₆) δ 8.60 (s, 1H), 8.32−8.27 (m, 2H), 7.64 (s, 1H), 7.47 (d,

J = 7.6 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.24 (s, 1H), 7.14 (d, J = 7.6

Hz, 1H), 4.89 (s, 2H), 4.07 (q, J = 7.0 Hz, 2H), 3.94 (s, 3H), 3.71 (s,

3H), 3.36 (s, 2H), 2.74 (d, J = 4.4 Hz, 3H), 1.34 (t, J = 7.0 Hz, 3H);

¹³C NMR (150 MHz, DMSO-d₆) δ 169.9, 167.6, 148.7, 147.6, 146.8,

144.5, 142.8, 135.7, 132.1, 132.0, 129.4, 124.6, 117.7, 116.6, 114.4,

112.7, 105.9, 66.2, 65.5, 52.2, 37.5, 30.4, 19.3, 14.0. MS (ESI) calcd

for C₂₄H₂₇N₄O₅[M + H]⁺: 451.2, found: 451.5.

2-((6-Ethoxy-3-((4-ethoxyphenyl)amino)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetamide (ID12). Fol-

lowing the procedures described for preparing 7, compound ID12 was

prepared from 14b (86 mg, 0.13 mmol), aqueous ammonia (25%, 2

mL) in 91% yield as a white solid, HPLC purity: 99.4%, mp: 198−200

3-((7-(2-Amino-2-oxoethoxy)-6-ethoxy-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)-N-methylbenza-

mide (ID17). Following the procedures described for preparing 7,

compound ID17 was prepared from 14g (20 mg, 0.04 mmol),

aqueous ammonia (25%, 2 mL) in 79% yield as a white solid, HPLC

purity: 99.7%, mp: 196−197 °C. H NMR (600 MHz, DMSO-d₆) δ

8.61 (s, 1H), 8.34 (s, 1H), 7.65 (s, 1H), 7.54 (s, 1H), 7.49 (dd, J =8.0,

1.7, 1H), 7.40 (s, 2H), 7.28−7.23 (m, 2H), 7.15 (d, J = 8.0 Hz, 1H),

1

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https://dx.doi.org/10.1021/acs.jmedchem.0c01345

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

4.54 (s, 2H), 4.13−4.05 (m, 2H), 3.96 (s, 3H), 2.76 (s, 3H), 1.37 (t, J

= 6.9, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 171.0, 167.7, 148.7,

148.0, 147.1, 144.6, 144.3, 143.4, 135.8, 129.1, 124.8, 117.81, 116.7,

114.5, 112.9, 112.7, 107.6, 69.7, 64.6, 37.5, 29.5, 26.7, 15.2. HRMS

(ESI) calcd for C₂₃H₃₀N₅O₃[M + H]⁺: 436.1979, found: 436.1983.

tert-Butyl (3-((7-((tert-Butyldimethylsilyl)oxy)-6-ethoxy-1-

methyl-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)phenyl)-

(ethyl)carbamate (12h). Following the procedures described for

preparing 4, compound 12h was prepared from 11 (400 mg, 1.31

mmol), LiHMDS (1.70 mL, 1.70 mmol), tert-butyl ethyl(3-

isothiocyanatophenyl)carbamate (454 mg, 1.70 mmol), hydrazine

hydrate (0.65 mL, 10.48 mmol), DIPEA (111 μL, 0.68 mmol), and

dimethyl sulfate (63 μL, 0.68 mmol) in 13% yield as a white solid,

144.4, 140.1, 137.7, 129.5, 125.0, 119.7, 118.1, 114.2, 112.7, 112.6,

106.8, 64.7, 37.4, 31.7, 29.2, 15.3, 8.8. MS (ESI) calcd for

C₂₂H₂₄N₃O₃[M + H]⁺: 378.2, found: 378.3.

Methyl 2-((6-Ethoxy-3-((3-(ethylamino)phenyl)amino)-1-

methyl-1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetate

(14h). Following the procedures described for preparing 6,

compound 14h was prepared from 13h (55 mg, 0.13 mmol), methyl

chloroacetate (31 μL, 0.26 mmol), and K₂CO₃(48 mg, 0.26 mmol) in

1

81% yield as a white solid, mp: 147−148 °C. H NMR (600 MHz,

DMSO-d₆) δ 8.03 (s, 1H), 7.26 (s, 1H), 7.23 (s, 1H), 6.87 (t, J = 8.2

Hz, 1H), 6.45−6.44 (m, 2H), 5.97 (d, J = 8.2 Hz, 1H), 5.28 (t, J = 5.3

Hz, 1H), 4.88 (s, 2H), 4.07 (q, J = 7.0 Hz, 2H), 3.92 (s, 3H), 3.72 (s,

3H), 3.36 (s, 2H), 3.18−2.91 (m, 2H), 1.34 (t, J = 7.0 Hz, 3H), 1.15

(t, J = 7.1 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 168.8, 149.0,

147.5, 146.5, 145.8, 144.0, 143.9, 141.8, 128.4, 123.8, 111.9, 111.8,

105.0, 103.2, 102.3, 98.3, 65.3, 63.6, 51.1, 36.8, 36.3, 28.7, 14.2, 13.9.

MS (ESI) calcd for C₂₄H₂₉N₄O₄[M + H]⁺: 437.2, found: 437.2.

Methyl 2-((6-Ethoxy-1-methyl-3-((3-propionylphenyl)-

amino)-1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetate

(14i). Following the procedures described for preparing 6, compound

14i was prepared from 13i (110 mg, 0.29 mmol), methyl

chloroacetate (60 μL, 0.58 mmol), and K₂CO₃(80 mg, 0.58 mmol)

in 85% yield as a white solid, mp: 133−134 °C. ¹H NMR (600 MHz,

DMSO-d₆) δ 8.68 (s, 1H), 7.86 (s, 1H), 7.65−7.59 (m, 1H), 7.39−

7.31 (m, 2H), 7.29 (s, 1H), 7.25 (s, 1H), 4.89 (s, 2H), 4.08 (q, J = 7.0

Hz, 2H), 3.96 (s, 3H), 3.72 (s, 3H), 3.39 (s, 2H), 3.00 (q, J = 7.2 Hz,

2H), 1.35 (t, J = 7.0 Hz, 3H), 1.09 (t, J = 7.2 Hz, 3H); ¹³C NMR

(150 MHz, DMSO-d₆) δ 201.1, 169.9, 148.7, 147.7, 146.9, 144.5,

144.5, 142.9, 137.7, 129.5, 124.7, 119.8, 118.2, 114.4, 112.9, 112.7,

106.2, 66.4, 64.7, 52.2, 37.5, 31.7, 29.3, 15.3, 8.7. MS (ESI) calcd for

C₂₅H₂₈N₃O₅[M + H]⁺: 450.2, found: 450.2.

1

mp: 172−173 °C. H NMR (600 MHz, DMSO-d₆) δ 8.45 (s, 1H),

7.24 (s, 1H), 7.20 (s, 1H), 7.17−7.13 (m, 2H), 7.11 (s, 1H), 6.64−

6.49 (m, 1H), 4.05 (q, J = 7.0 Hz, 2H), 3.90 (s, 3H), 3.56 (q, J = 7.0

Hz, 2H), 3.39 (s, 2H), 1.39 (s, 9H), 1.36 (q, J = 7.0 Hz, 3H), 1.08 (t,

J = 7.0 Hz, 3H), 1.00 (s, 9H), 0.17 (s, 6H); ¹³C NMR (150 MHz,

DMSO-d₆) δ 154.0, 149.0, 148.6, 144.7, 144.5, 143.6, 143.1, 142.9,

129.1, 124.8, 116.6, 113.9, 113.0, 112.6, 112.0, 111.6, 79.4, 64.1, 37.5,

29.3, 28.4, 26.1, 18.7, 15.3, 14.2, −4.2. MS (ESI) calcd for

C₃₂H₄₇N₄O₄Si [M + H]⁺: 579.3, found: 579.0.

7-((tert-Butyldimethylsilyl)oxy)-6-ethoxy-N-(3-(2-ethyl-1,3-

dioxolan-2-yl)phenyl)-1-methyl-1,4-dihydroindeno[1,2-c]-

pyrazol-3-amine (12i). Following the procedures described for

preparing 4, compound 12i was prepared from 11 (100 mg, 0.33

mmol), LiHMDS (0.59 mL, 0.59 mmol), 2-ethyl-2-(3-isothiocyana-

tophenyl)-1,3-dioxolane (138 mg, 0.59 mmol), hydrazine hydrate

(0.17 mL, 2.64 mmol), DIPEA (40 μL, 0.24 mmol), and dimethyl

sulfate (23 μL, 0.24 mmol) in 22% yield as a white solid, mp: 141−

142 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 8.40 (s, 1H), 7.30 (d, J =

7.8, Hz, 1H), 7.20 (s, 1H), 7.20 (s, 1H), 7.15 (t, J = 7.8 Hz, 1H), 7.11

(s, 1H), 6.73 (d, J = 7.8 Hz, 1H), 4.05 (q, J = 7.0 Hz, 2H), 3.95 (t, J =

7.7 Hz, 2H), 3.90 (s, 3H), 3.71 (t, J = 7.7 Hz, 2H), 3.37 (s, 2H), 1.81

(q, J = 7.4 Hz, 2H), 1.37 (t, J = 7.0 Hz, 3H), 1.00 (s, 9H), 0.81 (t, J =

7.4 Hz, 4H), 0.17 (s, 6H); ¹³C NMR (150 MHz, DMSO-d₆) δ 149.1,

148.6, 144.7, 144.0, 143.6, 143.3, 142.8, 128.9, 124.8, 115.7, 114.6,

112.7, 112.6, 112.0, 111.6, 110.6, 64.6, 64.1, 37.4, 33.3, 29.5, 26.2,

26.1, 18.6, 15.2, 8.5, −4.1. MS (ESI) calcd for C₃₀H₄₂N₃O₄Si [M +

H]⁺: 536.3, found: 536.2.

2-((6-Ethoxy-3-((3-(ethylamino)phenyl)amino)-1-methyl-

1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetamide (ID18).

Following the procedures described for preparing 7, compound

ID18 was prepared from 14h (40 mg, 0.09 mmol), aqueous ammonia

(2 mL) in 92% yield as a white solid, HPLC purity: 99.5%, mp: 195−

1

196 °C. H NMR (400 MHz, CDCl₃) δ 8.03 (s, 1H), 7.50 (s, 1H),

7.38 (s, 1H), 7.36 (s, 1H), 7.25 (s, 1H), 6.87 (t, J = 7.9 Hz, 1H),

6.48−6.40 (m, 2H), 5.98 (d, J = 7.9 Hz, 1H), 5.30 (s, 1H), 4.53 (s,

2H), 4.09 (q, J = 6.9 Hz, 2H), 3.92 (s, 3H), 3.37 (s, 3H), 2.99 (q, J =

6.5 Hz, 2H), 1.37 (t, J = 6.9 Hz, 3H), 1.15 (t, J = 6.5 Hz, 3H); ¹³C

NMR (100 MHz, CDCl₃) δ 175.7, 154.8, 153.2, 152.6, 151.9, 149.9,

149.7, 148.1, 134.2, 129.7, 117.7, 117.5, 112.3, 109.0, 108.2, 104.1,

74.5, 69.4, 42.6, 42.1, 34.5, 20.0, 19.8. MS (ESI) calcd for

C₂₃H₂₇N₅O₃[M + H]⁺: 422.2, found: 422.4.

6-Ethoxy-3-((3-(ethylamino)phenyl)amino)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-ol (13h). Acetyl chloride (2.5

mL) was added to ethanol (5 mL) and stirred for 15 min. To the

resulting solution (2 mL), 12h (45 mg, 0.09 mmol) was added, and

the mixture was stirred for 2 h. After concentration, the resulting

mixture was adjusted to pH 7 by the addition of a saturated NaHCO₃

aqueous solution. The resulting precipitate was ﬁltered, washed with

hexane, and dried to give 55 mg (98%) of 13h as a white solid, mp:

2-((6-Ethoxy-1-methyl-3-((3-propionylphenyl)amino)-1,4-

dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetamide (ID19). Fol-

lowing the procedures described for preparing 7, compound ID19 was

prepared from 14i (86 mg, 0.19 mmol), aqueous ammonia (2 mL) in

72% yield as a white solid, HPLC purity: 95.9%, mp: 218−219 °C. ¹H

NMR (600 MHz, DMSO-d₆) δ 8.71 (s, 1H), 7.87 (s, 1H), 7.67−7.59

(m, 1H), 7.53 (s, 1H), 7.41 (s, 1H), 7.38 (s, 1H), 7.36−7.31 (m, 2H),

7.27 (s, 1H), 4.54 (s, 2H), 4.10 (q, J = 7.0 Hz, 2H), 3.96 (s, 3H), 3.40

(s, 2H), 3.00 (q, J = 7.2 Hz, 2H), 1.37 (t, J = 7.0 Hz, 3H), 1.09 (t, J =

7.2 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 200.0, 169.8, 147.6,

146.9, 146.0, 143.4, 143.3, 142.2, 136.6, 128.4, 123.7, 118.6, 117.1,

113.1, 111.6, 111.5, 106.4, 68.6, 63.5, 36.4, 30.6, 28.3, 14.1, 7.6.

HRMS (ESI) calcd for C₂₄H₂₇N₄O₄[M + H]⁺: 435.2027, found:

435.2027.

1

143−145 °C. H NMR (600 MHz, DMSO-d₆) δ 8.81 (s, 1H), 7.99

(s, 1H), 7.14 (s, 1H), 7.10 (s, 1H), 6.86 (t, J = 7.8 Hz, 1H), 6.59−

6.35 (m, 2H), 5.97 (d, J = 7.8 Hz, 1H), 5.28 (t, J = 5.3 Hz, 1H), 4.05

(q, J = 7.0 Hz, 2H), 3.87 (s, 3H), 3.32 (s, 2H), 3.10−2.91 (m, 2H),

1.35 (t, J = 7.0 Hz, 3H), 1.15 (t, J = 7.1 Hz, 3H); ¹³C NMR (150

MHz, DMSO-d₆) δ 149.0, 147.7, 145.1, 144.8, 143.9, 139.0, 128.4,

124.1, 114.2, 111.9, 111.5, 105.6, 103.2, 102.3, 98.2, 63.6, 36.8, 36.2,

28.5, 14.2, 13.9. MS (ESI) calcd for C₂₁H₂₅N₄O₂[M + H]⁺: 365.2,

found: 365.2.

1-(3-((6-Ethoxy-7-hydroxy-1-methyl-1,4-dihydroindeno-

[1,2-c]pyrazol-3-yl)amino)phenyl)propan-1-one (13i). Acetyl

chloride (2.5 mL) was added to ethanol (5 mL) and stirred for 15

min. To the resulting solution (2 mL), 12i (175 mg, 0.33 mmol) was

added, and the mixture stirred for 2 h. After concentration, the

resulting mixture was adjusted to pH 7 by the addition of a saturated

NaHCO₃aqueous solution. The resulting precipitate ﬁltered, washed

with hexane, and dried to give 110 mg (89%) of 13i as a white solid,

2-((3-((3-Cyanophenyl)amino)-6-ethoxy-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-yl)oxy)-N-methylacetamide

(ID20). Following the procedures described for preparing 7,

compound ID20 was prepared from 14c (30 mg, 0.07 mmol),

aqueous methylamine solution (25%, 1 mL) in 83% yield as a white

1

solid, HPLC purity: 96.5%, mp: 149−151 °C. H NMR (400 MHz,

DMSO-d₆) δ 8.95 (s, 1H), 7.91 (s, 1H), 7.85 (s, 1H), 7.58 (d, J = 8.1

Hz, 1H), 7.41 (s, 1H), 7.39 (t, J = 8.1 Hz, 1H), 7.29 (s, 1H), 7.14 (d,

J = 8.1 Hz, 1H), 4.56 (s, 2H), 4.11 (q, J = 7.0 Hz, 2H), 3.98 (s, 3H),

3.43 (s, 2H), 3.35 (s, 3H), 1.38 (t, J = 7.0 Hz, 3H); ¹³C NMR (100

MHz, DMSO-d₆) δ 168.9, 148.8, 148.2, 147.1, 144.6, 144.0, 143.6,

130.5, 124.7, 121.7, 120.1, 119.9, 117.4, 114.3, 112.7, 111.9, 108.0,

1

mp: 181−182 °C. H NMR (400 MHz, DMSO-d₆) δ 8.85 (s, 1H),

8.64 (s, 1H), 7.84 (s, 1H), 7.61 (s, 1H), 7.34 (s, 2H), 7.16 (s, 1H),

7.13 (s, 1H), 4.06 (q, J = 7.0 Hz, 2H), 3.92 (s, 3H), 3.35 (s, 2H), 3.00

(q, J = 6.8 Hz, 2H), 1.35 (t, J = 6.8 Hz, 3H), 1.09 (t, J = 7.0 Hz, 3H);

¹³C NMR (100 MHz, DMSO-d₆) δ 201.1, 149.1, 146.3, 146.1, 144.5,

T

https://dx.doi.org/10.1021/acs.jmedchem.0c01345

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

70.2, 64.6, 37.6, 29.2, 25.8, 15.3. HRMS (ESI) calcd for C₂₃H₂₄N₅O₃

[M + H]⁺: 418.1874, found: 418.1883.

15.3, 15.2. HRMS (ESI) calcd for C₂₂H₂₆N₅O₄[M + H]⁺: 424.1979,

found: 424.1984.

Methyl 3-((6-Ethoxy-1-methyl-7-(2-(methylamino)-2-oxoe-

thoxy)-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate

(ID21). Following the procedures described for preparing 7,

compound ID21 was prepared from 14d (52 mg, 0.12 mmol),

aqueous methylamine solution (25%, 2 mL) in 92% yield as a white

3-((6-Ethoxy-7-(3-(methoxymethoxy)propoxy)-1-methyl-

1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzonitrile

(18b). Following the procedures described for preparing 4,

compound 18b was prepared from 17 (0.40 g, 1.36 mmol), NaH

(220 mg, 5.44 mmol), 3-isothiocyanatobenzonitrile (0.327 g, 2.04

mmol), hydrazine hydrate (0.47 mL, 8.0 mmol), DIPEA (104 μL,

0.63 mmol), and dimethyl sulfate (61 μL, 0.63 mmol) in 14% yield as

1

solid, HPLC purity: 96.3%, mp: 184−186 °C. H NMR (600 MHz,

DMSO-d₆) δ 8.72 (s, 1H), 7.90 (s, 2H), 7.63 (d, J = 7.3 Hz, 1H), 7.40

(t, J = 7.3 Hz, 1H), 7.32 (s, 1H), 7.26 (d, J = 7.3 Hz, 1H), 4.55 (s,

2H), 4.10 (q, J = 6.8 Hz, 2H), 3.95 (s, 3H), 3.83 (s, 3H), 3.40 (s,

2H), 2.71 (s, 3H), 1.37 (t, J = 6.8 Hz, 3H); ¹³C NMR (150 MHz,

DMSO-d₆) δ 169.0, 167.1, 148.72, 148.2, 147.1, 145.3, 144.4, 143.62,

130.6, 129.6, 124.8, 119.8, 119.1, 115.85, 112.8, 112.2, 108.1, 70.3,

64.6, 52.5, 37.5, 29.4, 25.8, 15.3. HRMS (ESI) calcd for C₂₄H₂₇N₄O₅

[M + H]⁺: 451.1976, found: 451.1985.

1

a yellow solid, mp: 122−124 °C. H NMR (400 MHz, DMSO-d₆) δ

8.92 (s, 1H), 7.88 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0

Hz, 1H), 7.33 (s, 1H), 7.23 (s, 1H), 7.14 (d, J = 8.0 Hz, 1H), 4.59 (s,

2H), 4.15 (t, J = 6.3 Hz, 2H), 4.06 (q, J = 7.0 Hz, 2H), 3.99 (s, 3H),

3.67 (t, J = 6.3 Hz, 2H), 3.41 (s, 2H), 3.25 (s, 3H), 2.05−1.95 (m,

2H), 1.35 (t, J = 7.0 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ

149.1, 148.1, 148.0, 144.7, 143.9, 142.1, 130.5, 124.9, 121.7, 120.1,

119.9, 117.4, 113.0, 112.7, 111.9, 106.1, 96.2, 66.7, 64.70, 64.2, 55.0,

37.6, 29.8, 29.1, 15.3. MS (ESI) calcd for C₂₅H₂₉N₄O₄[M + H]⁺:

449.2, found: 449.5.

7-((tert-Butyldimethylsilyl)oxy)-6-ethoxy-N-(6-ethoxypyri-

din-2-yl)-1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-3-amine

(12j). Following the procedures described for preparing 4, compound

12j was prepared from 11 (220 mg, 0.72 mmol), LiHMDS (1.44 mL,

1.44 mmol), 2-ethoxy-6-isothiocyanatopyridine (274 mg, 1.44 mmol),

hydrazine hydrate (0.36 mL, 5.76 mmol), DIPEA (76 μL, 0.46

mmol), and dimethyl sulfate (45 μL, 0.46 mmol) in 20% yield as a

Methyl 3-((6-Ethoxy-7-(3-(methoxymethoxy)propoxy)-1-

methyl-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate

(18c). Following the procedures described for preparing 4, compound

18c was prepared from 17 (500 mg, 1.70 mmol), LiHMDS (2.50 mL,

2.50 mmol), methyl 3-isothiocyanatobenzoate (490 mg, 2.5 mmol),

hydrazine hydrate (0.85 mL, 13.6 mmol), DIPEA (212 μL, 1.28

mmol), and dimethyl sulfate (124 μL, 1.28 mmol) in 19% yield as a

1

white solid, mp: 147−148 °C. H NMR (600 MHz, DMSO-d₆) δ

8.88 (s, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.20 (s, 1H), 7.11 (s, 1H), 6.90

(d, J = 7.9 Hz, 1H), 6.09 (d, J = 7.9 Hz, 1H), 4.22 (q, J = 7.0 Hz, 2H),

4.04 (q, J = 7.0 Hz, 2H), 3.91 (s, 3H), 3.46 (s, 2H), 1.36 (t, J = 7.0

Hz, 3H), 1.29 (t, J = 7.0 Hz, 3H), 1.00 (s, 9H), 0.16 (s, 6H); ¹³C

NMR (150 MHz, DMSO-d₆) δ 162.9, 154.4, 149.0, 148.6, 143.5,

142.9, 142.9, 140.6, 124.7, 114.4, 112.0, 111.6, 101.1, 99.0, 64.1, 61.2,

37.4, 30.0, 26.1, 18.6, 15.2, 15.2, −4.1. MS (ESI) calcd for

C₂₆H₃₇N₄O₃Si [M + H]⁺: 481.2, found: 481.5.

1

yellow solid, mp: 117−119 °C. H NMR (600 MHz, DMSO-d₆) δ

8.71 (s, 1H), 7.90 (s, 1H), 7.68−7.57 (dt, J = 7.5, 2.0 Hz, 1H), 7.41−

7.28 (m, 3H), 7.21 (s, 1H), 4.59 (s, 2H), 4.15 (t, J = 6.3 Hz, 2H),

4.06 (q, J = 7.0 Hz, 2H), 3.98 (s, 3H), 3.84 (s, 3H), 3.66 (t, J = 6.3

Hz, 2H), 3.38 (s, 2H), 3.25 (s, 3H), 2.00 (p, J = 6.3 Hz, 2H), 1.34 (t,

J = 7.0 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 167.1, 148.9,

148.1, 147.9, 144.5, 144.3, 142.1, 130.6, 129.6, 125.0, 119.8, 118.9,

115.8, 113.0, 112.7, 106.1, 96.1, 66.6, 64.7, 64.2, 55.0, 52.4, 37.6, 29.8,

29.2, 15.3. MS (ESI) calcd for C₂₆H₃₂N₃O₆[M + H]⁺: 482.2, found:

482.1.

6-Ethoxy-3-((6-ethoxypyridin-2-yl)amino)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-ol (13j). Following the procedures

described for preparing 5, compound 13j was prepared from 12j (120

mg, 0.25 mmol) and TBAF (110 mg, 0.50 mmol) in 81% yield as a

1

white solid, mp: 201−202 °C. H NMR (600 MHz, DMSO-d₆) δ

Ethyl 3-((6-Ethoxy-7-(3-(methoxymethoxy)propoxy)-1-

methyl-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate

(18d). Following the procedures described for preparing 4,

compound 18d was prepared from 17 (500 mg, 1.70 mmol),

LiHMDS (5.1 mL, 5.10 mmol), ethyl 3-isothiocyanatobenzoate (1.0

g, 5.10 mmol), hydrazine hydrate (0.85 mL, 13.6. mmol), DIPEA

(185 μL, 1.12 mmol), and dimethyl sulfate (108 μL, 1.12 mmol) in

8.86 (s, 1H), 8.82 (s, 1H), 7.46 (t, J = 7.9 Hz, 1H), 7.14 (s, 1H), 7.12

(s, 1H), 6.92 (d, J = 7.9 Hz, 1H), 6.09 (d, J = 7.9 Hz, 1H), 4.23 (q, J =

7.0 Hz, 2H), 4.06 (q, J = 7.0 Hz, 2H), 3.91 (s, 3H), 3.42 (s, 2H), 1.35

(t, J = 7.0 Hz, 3H), 1.29 (t, J = 7.0 Hz, 3H); ¹³C NMR (150 MHz,

DMSO-d₆) δ 162.9, 154.5, 148.8, 146.2, 146.0, 142.9, 140.6, 140.1,

125.1, 114.5, 112.5, 106.7, 101.0, 99.0, 64.6, 61.2, 37.3, 29.8, 15.3,

15.2. MS (ESI) calcd for C₂₀H₂₃N₄O₃[M + H]⁺: 367.2, found: 367.1.

Methyl 2-((6-Ethoxy-3-((6-ethoxypyridin-2-yl)amino)-1-

methyl-1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetate

(14j). Following the procedures described for preparing 6, compound

14j was prepared from 13j (74 mg, 0.20 mmol), methyl chloroacetate

(60 μL, 0.40 mmol), and K₂CO₃(55 mg, 0.40 mmol) in 81% yield as

1

13% yield as a yellow solid, mp: 138−139 °C. H NMR (600 MHz,

DMSO-d₆) δ 8.72 (s, 1H), 7.91 (s, 1H), 7.65 (dt, J = 7.3, 2.2 Hz, 1H),

7.45−7.27 (m, 3H), 7.21 (s, 1H), 4.59 (s, 2H), 4.30 (q, J = 7.1 Hz,

2H), 4.15 (t, J = 6.3 Hz, 2H), 4.06 (q, J = 6.9 Hz, 2H), 3.98 (s, 3H),

3.67 (t, J = 6.3 Hz, 2H), 3.39 (s, 2H), 3.26 (s, 3H), 2.00 (p, J = 6.3

Hz, 2H), 1.35 (t, J = 6.9 Hz, 3H), 1.32 (t, J = 7.1 Hz, 3H); ¹³C NMR

(150 MHz, DMSO-d₆) δ 166.6, 148.9, 148.0, 147.9, 144.4, 144.3,

142.1, 130.8, 129.5, 125.0, 119.7, 118.9, 115.8, 112.9, 112.6, 106.1,

96.1, 66.6, 64.7, 64.1, 60.9, 55.0, 37.5, 29.8, 29.3, 15.3, 14.6. MS (ESI)

calcd for C₂₇H₃₄N₃O₆[M + H]⁺: 496.2, found: 496.2.

1

a white solid, mp: 111−112 °C. H NMR (600 MHz, DMSO-d₆) δ

8.89 (s, 1H), 7.47 (t, J = 7.9 Hz, 1H), 7.27 (s, 1H), 7.23 (s, 1H), 6.92

(d, J = 7.9 Hz, 1H), 6.09 (d, J = 7.9 Hz, 1H), 4.88 (s, 2H), 4.23 (q, J =

7.0 Hz, 2H), 4.08 (q, J = 7.0 Hz, 2H), 3.95 (s, 3H), 3.72 (s, 3H), 3.46

(s, 2H), 1.35 (t, J = 7.0 Hz, 3H), 1.29 (t, J = 7.0 Hz, 3H); ¹³C NMR

(150 MHz, DMSO-d₆) δ 169.9, 162.9, 154.4, 148.6, 147.6, 146.8,

143.0, 142.9, 140.6, 124.7, 114.5, 112.8, 106.1, 101.1, 99.0, 66.4, 64.6,

61.2, 52.2, 37.5, 30.0, 15.3, 15.2. MS (ESI) calcd for C₂₃H₂₇N₄O₅[M

+ H]⁺: 439.2, found: 439.3.

Isopropyl 3-((6-Ethoxy-7-(3-(methoxymethoxy)propoxy)-1-

methyl-1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate

(18e). Following the procedures described for preparing 4, compound

18e was prepared from 17 (500 mg, 1.70 mmol), LiHMDS (4.25 mL,

4.25 mmol), isopropyl 3-isothiocyanatobenzoate (930 mg, 5.10

mmol), hydrazine hydrate (0.85 mL, 13.6. mmol), DIPEA (234 μL,

1.42 mmol), and dimethyl sulfate (138 μL, 1.42 mmol) in 23% yield

as a yellow solid, mp: 120−121 °C. ¹H NMR (600 MHz, DMSO-d₆)

δ 8.72 (s, 1H), 7.89 (s, 1H), 7.69 (dd, J = 7.6, 1.8 Hz, 1H), 7.40−7.27

(m, 3H), 7.21 (s, 1H), 5.13 (hept, J = 6.3 Hz, 1H), 4.59 (s, 2H), 4.15

(t, J = 6.3 Hz, 2H), 4.06 (q, J = 7.0 Hz, 2H), 3.98 (s, 3H), 3.67 (t, J =

6.3 Hz, 2H), 3.39 (s, 2H), 3.34 (s, 3H), 2.00 (p, J = 6.3 Hz, 2H), 1.34

(t, J = 7.0 Hz, 3H), 1.32 (d, J = 6.3 Hz, 6H); ¹³C NMR (150 MHz,

DMSO-d₆) δ 166.1, 148.9, 148.1, 147.9, 144.4, 144.4, 142.1, 131.2,

129.4, 125.0, 119.6, 118.9, 115.7, 112.9, 112.6, 106.0, 96.1, 68.2, 66.6,

64.7, 64.2, 55.0, 37.5, 29.8, 29.3, 22.1, 15.3. MS (ESI) calcd for

C₂₈H₃₆N₃O₆[M + H]⁺: 510.3, found: 510.1.

2-((6-Ethoxy-3-((6-ethoxypyridin-2-yl)amino)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-yl)oxy)acetamide (ID22). Fol-

lowing the procedures described for preparing 7, compound ID22 was

prepared from 14j (70 mg, 0.16 mmol), aqueous ammonia (2 mL) in

82% yield as a white solid, HPLC purity: 98.1%, mp: 148−149 °C. ¹H

NMR (600 MHz, DMSO-d₆) δ 8.91 (s, 1H), 7.50 (s, 1H), 7.47 (t, J =

7.9 Hz, 1H), 7.39 (s, 1H), 7.36 (s, 1H), 7.25 (s, 1H), 6.92 (d, J = 7.9

Hz, 1H), 6.09 (d, J = 7.9 Hz, 1H), 4.53 (s, 2H), 4.22 (q, J = 7.0 Hz,

2H), 4.10 (q, J = 7.0 Hz, 2H), 3.95 (s, 3H), 3.47 (s, 2H), 1.37 (t, J =

7.0 Hz, 3H), 1.29 (t, J = 7.0 Hz, 3H); ¹³C NMR (150 MHz, DMSO-

d₆) δ 170.9, 162.9, 154.4, 148.5, 147.9, 147.0, 143.4, 143.0, 140.6,

124.8, 114.4, 112.5, 107.5, 101.1, 99.1, 69.7, 64.6, 61.2, 37.4, 30.1,

U

https://dx.doi.org/10.1021/acs.jmedchem.0c01345

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

3-((6-Ethoxy-7-(3-(methoxymethoxy)propoxy)-1-methyl-

1,4-dihydroindeno[1,2-c]pyrazol-3-yl)amino)-N-methylbenza-

mide (18f). Following the procedures described for preparing 4,

compound 18f was prepared from 17 (250 mg, 0.85 mmol), LiHMDS

(1.28 mL, 1.28 mmol), 3-isothiocyanato-N-methylbenzamide (245

mg, 1.28 mmol), hydrazine hydrate (0.43 mL, 6.80 mmol), DIPEA

(89 μL, 0.54 mmol), and dimethyl sulfate (52 μL, 0.54 mmol) in 15%

yield as a yellow solid, mp: 162−164 °C. ¹H NMR (600 MHz,

DMSO-d₆) δ 8.56 (s, 1H), 8.27 (dd, J = 8.0, 1.7 Hz, 1H), 7.65 (t, J =

1.7 Hz, 1H), 7.47 (dd, J = 8.0, 1.7 Hz, 1H), 7.32 (s, 1H), 7.25 (t, J =

8.0 Hz, 1H), 7.20 (s, 1H), 7.15 (d, J = 8.0 Hz, 1H), 4.58 (s, 2H), 4.14

(t, J = 6.3 Hz, 2H), 4.05 (q, J = 7.0 Hz, 2H), 3.97 (s, 3H), 3.66 (t, J =

6.3 Hz, 2H), 3.36 (s, 3H), 2.76 (d, J = 4.5 Hz, 3H), 2.00 (p, J = 6.2

Hz, 2H), 1.34 (t, J = 7.0 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ

166.6, 147.8, 146.9, 146.8, 143.4, 143.2, 141.0, 134.7, 127.9, 123.9,

116.7, 115.5, 113.4, 111.9, 111.7, 105.0, 95.1, 65.6, 63.6, 63.1, 53.9,

36.4, 28.7, 28.3, 25.6, 14.2. MS (ESI) calcd for C₂₆H₃₃N₄O₅[M +

H]⁺: 481.2, found: 481.2.

95.2%, mp: 169−170 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 8.74 (s,

1H), 7.90 (s, 1H), 7.65 (dt, J = 7.5, 1.8 Hz, 1H), 7.40−7.25 (m, 3H),

7.21 (s, 1H), 4.56 (t, J = 6.1 Hz, 1H), 4.30 (q, J = 7.1 Hz, 2H), 4.14

(t, J = 6.4 Hz, 2H), 4.05 (q, J = 6.9 Hz, 2H), 3.98 (s, 3H), 3.61 (q, J =

6.1 Hz, 2H), 3.38 (s, 2H), 1.90 (p, J = 6.1 Hz, 2H), 1.34 (t, J = 6.9

Hz, 3H), 1.32 (t, J = 7.1 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ

166.6, 149.0, 148.1, 147.7, 144.4, 144.3, 141.7, 130.8, 129.5, 124.9,

119.7, 118.9, 115.7, 112.7, 112.6, 105.5, 66.5, 64.6, 60.9, 58.0, 37.5,

32.8, 29.3, 15.3, 14.6. HRMS (ESI) calcd for C₂₅H₃₀N₃O₅[M + H]⁺:

452.2180, found: 452.2187.

Isopropyl 3-((6-Ethoxy-7-(3-hydroxypropoxy)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate (ID26). Fol-

lowing the procedures described for preparing ID08, compound ID26

was prepared from 18e (60 mg, 0.12 mmol) and CSA methanol

solution (1 M, 2 mL) in 83% yield as a white solid, HPLC purity:

97.9%, mp: 159−160 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 8.74 (s,

1H), 7.88 (s, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.37−7.27 (m, 3H), 7.21

(s, 1H), 5.12 (hept, J = 6.2 Hz, 1H), 4.56 (t, J = 6.3 Hz, 1H), 4.14 (t,

J = 6.3 Hz, 2H), 4.05 (q, J = 7.0 Hz, 2H), 3.97 (s, 3H), 3.61 (q, J =

6.3 Hz, 2H), 3.39 (s, 2H), 1.90 (p, J = 6.3 Hz, 2H), 1.34 (t, J = 7.0

Hz, 3H), 1.31 (d, J = 6.2 Hz, 6H); ¹³C NMR (150 MHz, DMSO-d₆)

δ 166.1, 148.9, 148.1, 147.7, 144.3, 141.7, 131.2, 129.5, 124.9, 119.6,

118.9, 115.7, 112.7, 112.5, 105.5, 68.3, 66.6, 64.6, 58.0, 37.6, 32.8,

29.3, 26.8, 22.2, 15.3. HRMS (ESI) calcd for C₂₆H₃₂N₃O₅[M + H]⁺:

466.2336, found: 466.2342.

6-Ethoxy-N-(3-(2-ethyl-1,3-dioxolan-2-yl)phenyl)-7-(3-

(methoxymethoxy)propoxy)-1-methyl-1,4-dihydroindeno-

[1,2-c]pyrazol-3-amine (18g). Following the procedures described

for preparing 4, compound 18g was prepared from 17 (250 mg, 0.85

mmol), LiHMDS (1.70 mL, 1.70 mmol), 2-ethyl-2-(3-isothiocyana-

tophenyl)-1,3-dioxolane (399 mg, 1.70 mmol), hydrazine hydrate

(0.41 mL, 6.80 mmol), DIPEA (123 μL, 0.74 mmol), and dimethyl

sulfate (72 μL, 0.74 mmol) in 21% yield as a yellow solid, mp: 162−

3-((6-Ethoxy-7-(3-hydroxypropoxy)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)-N-methylbenza-

mide (ID27). Following the procedures described for preparing

ID08, compound ID27 was prepared from 18f (48 mg, 0.10 mmol)

and CSA methanol solution (1 M, 2 mL) in 72% yield as a white

1

164 °C°C. H NMR (600 MHz, DMSO-d₆) δ 8.41 (s, 1H), 7.37−

7.27 (m, 2H), 7.21 (s, 1H), 7.20 (s, 1H), 7.16 (t, J = 7.8 Hz, 1H),

6.73 (d, J = 7.8 Hz, 1H), 4.58 (s, 2H), 4.14 (t, J = 6.3 Hz, 2H), 4.05

(q, J = 7.0 Hz, 2H), 3.99−3.91 (m, 5H), 3.71 (dd, J = 7.6, 6.1 Hz,

2H), 3.66 (t, J = 6.3 Hz, 2H), 3.36 (s, 2H), 3.25 (s, 3H), 1.99 (p, J =

6.3 Hz, 2H), 1.81 (q, J = 7.4 Hz, 2H), 1.34 (t, J = 7.0 Hz, 3H), 0.81

(t, J = 7.4 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 147.7, 146.9,

146.8, 143.6, 142.9, 142.2, 141.0, 127.8, 124.0, 114.6, 113.5, 111.9,

111.6, 109.5, 105.0, 95.1, 65.6, 63.6, 63.5, 63.1, 53.9, 36.4, 32.2, 28.7,

28.3, 14.2, 7.4. MS (ESI) calcd for C₂₉H₃₈N₃O₆[M + H]⁺: 524.3,

found: 524.3.

1

solid, HPLC purity: 95.7%, mp: 219−220 °C. H NMR (400 MHz,

DMSO-d₆) δ 8.56 (s, 1H), 8.28 (s, 1H), 7.65 (s, 1H), 7.48 (d, J = 7.7

Hz, 1H), 7.32 (s, 1H), 7.26 (t, J = 7.7 Hz, 1H), 7.20 (s, 1H), 7.15 (d,

J = 7.7 Hz, 1H), 4.55 (s, 1H), 4.14 (t, J = 6.0 Hz, 2H), 4.05 (q, J = 6.6

Hz, 2H), 3.97 (s, 3H), 3.61 (t, J = 6.0 Hz, 2H), 3.36 (s, 3H), 2.76 (s,

3H), 1.91 (p, J = 6.0 Hz, 1H), 1.34 (t, J = 6.6 Hz, 3H); ¹³C NMR

(100 MHz, DMSO-d₆) δ 167.7, 149.0, 148.1, 147.7, 144.5, 144.4,

141.8, 135.8, 129.0, 125.0, 117.7, 116.6, 114.5, 112.9, 112.8, 105.6,

66.7, 64.6, 58.0, 37.5, 32.8, 29.4, 26.7, 15.3. HRMS (ESI) calcd for

C₂₄H₂₉N₄O₄[M + H]⁺: 437.2183, found: 437.2180.

3-((6-Ethoxy-7-(3-hydroxypropoxy)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzonitrile (ID23).

Following the procedures described for preparing ID08, compound

ID23 was prepared from 18b (70 mg, 0.16 mmol) and CSA methanol

solution (1 M, 4 mL) in 83% yield as a white solid, HPLC purity:

96.6%, mp: 170−172 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.93 (s,

1H), 7.85 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H),

7.33 (s, 1H), 7.22 (s, 1H), 7.14 (d, J = 8.0 Hz, 1H), 4.14 (t, J = 6.4

Hz, 2H), 4.06 (q, J = 7.0 Hz, 2H), 4.00 (s, 3H), 3.40 (s, 2H), 1.90 (p,

J = 6.3 Hz, 2H), 1.35 (t, J = 7.0 Hz, 3H); ¹³C NMR (100 MHz,

DMSO-d₆) δ 149.2, 148.2, 147.9, 144.7, 143.9, 141.8, 130.5, 124.9,

121.7, 120.1, 119.9, 117.5, 112.9, 112.7, 111.9, 105.4, 66.7, 64.7, 58.0,

37.59, 32.8, 29.1, 15.3. HRMS (ESI) calcd for C₂₃H₂₅N₄O₃[M + H]⁺:

405.1921, found: 405.1928.

Methyl 3-((6-Ethoxy-7-(3-hydroxypropoxy)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate (ID24). Fol-

lowing the procedures described for preparing ID08, compound ID24

was prepared from 18c (26 mg, 0.05 mmol) and CSA methanol

solution (1 M, 2 mL) in 63% yield as a white solid, HPLC purity:

99.4%, mp: 164−165 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 8.71 (s,

1H), 7.91 (s, 1H), 7.65 (dd, J = 7.0, 2.0 Hz, 1H), 7.38−7.28 (m, 3H),

7.21 (s, 1H), 4.14 (t, J = 6.4 Hz, 2H), 4.05 (q, J = 7.0 Hz, 2H), 3.98

(s, 3H), 3.84 (s, 3H), 3.61 (t, J = 6.3 Hz, 2H), 3.38 (s, 2H), 1.93−

1.86 (m, 2H), 1.34 (t, J = 7.0 Hz, 3H); ¹³C NMR (150 MHz, DMSO-

d₆) δ 167.1, 149.1, 148.2, 147.8, 144.5, 144.3, 141.8, 130.6, 129.5,

124.9, 119.8, 119.0, 115.8, 112.8, 112.7, 105.6, 66.6, 64.6, 58.0, 52.4,

37.5, 32.8, 29.2, 15.3. HRMS (ESI) calcd for C₂₅H₃₀N₃O₄[M + H]⁺:

438.2038, found: 438.2023.

1-(3-((6-Ethoxy-7-(3-hydroxypropoxy)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)phenyl)propan-1-

one (ID28). Following the procedures described for preparing ID08,

compound ID28 was prepared from 18g (30 mg, 0.06 mmol) and

CSA methanol solution (1 M, 2 mL) in 67% yield as a white solid,

HPLC purity: 95.2%, mp: 218−220 °C. ¹H NMR (600 MHz, DMSO-

d₆) δ 8.75 (s, 1H), 7.85 (s, 1H), 7.62 (dt, J = 7.0, 2.0 Hz, 1H), 7.43−

7.29 (m, 3H), 7.21 (s, 1H), 4.14 (t, J = 6.3 Hz, 2H), 4.05 (q, J = 7.0

Hz, 2H), 3.98 (s, 3H), 3.61 (t, J = 6.3 Hz, 2H), 3.38 (s, 2H), 3.01 (q,

J = 7.2 Hz, 2H), 1.90 (p, J = 6.3 Hz, 2H), 1.34 (t, J = 7.0 Hz, 3H),

1.09 (t, J = 7.2 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 201.0,

149.2, 148.1, 147.8, 144.3, 144.2, 141.8, 137.6, 129.5, 124.7, 119.8,

118.3, 114.3, 112.7, 112.4, 105.5, 66.6, 64.5, 57.9, 37.5, 32.7, 31.6,

29.3, 15.3, 8.7. HRMS (ESI) calcd for C₂₅H₃₀N₃O₄[M + H]⁺:

436.2231, found: 436.2240.

3-((6-Ethoxy-7-(3-hydroxypropoxy)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoic acid (ID29).

Following the procedures described for preparing ID07, compound

ID24 was prepared from ID29 (25 mg, 0.06 mmol) and LiOH

aqueous solution (1 M, 1.5 mL) in 83% yield as a white solid, HPLC

1

purity: 97.8%, mp: 234−235 °C. H NMR (600 MHz, DMSO-d₆) δ

8.69 (s, 1H), 7.86 (s, 1H), 7.61−7.51 (m, 1H), 7.32 (s, 1H), 7.32 (s,

1H), 7.31 (s, 1H), 7.21 (s, 1H), 4.14 (t, J = 6.3 Hz, 2H), 4.05 (q, J =

7.0 Hz, 2H), 3.98 (s, 3H), 3.61 (t, J = 6.3 Hz, 2H), 3.37 (s, 2H), 1.90

(p, J = 6.3 Hz, 2H), 1.34 (t, J = 7.0 Hz, 3H); ¹³C NMR (150 MHz,

DMSO-d₆) δ 168.2, 149.1, 148.1, 147.7, 144.3, 144.2, 141.7, 131.6,

129.4, 124.8, 119.5, 119.3, 116.0, 112.7, 112.6, 105.5, 66.5, 64.5, 57.9,

37.5, 32.7, 29.3, 15.3. HRMS (ESI) calcd for C₂₃H₂₆N₃O₅[M + H]⁺:

424.1867, found: 424.1870.

Ethyl 3-((6-Ethoxy-7-(3-hydroxypropoxy)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzoate (ID25). Fol-

lowing the procedures described for preparing ID08, compound ID25

was prepared from 18d (60 mg, 0.12 mmol) and CSA methanol

solution (1 M, 2 mL) in 85% yield as a white solid, HPLC purity:

V

https://dx.doi.org/10.1021/acs.jmedchem.0c01345

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

6-Ethoxy-N-(6-ethoxypyridin-2-yl)-7-(3-(methoxymethoxy)-

propoxy)-1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-3-amine

(18h). Following the procedures described for preparing 4,

compound 18h was prepared from 17 (400 mg, 1.36 mmol l),

LiHMDS (2.00 mL, 2.00 mmol), 2-ethyl-2-(3-isothiocyanatophenyl)-

1,3-dioxolane (367 mg, 2.00 mmol), hydrazine hydrate (l0.68 mL,

10.88 mmol), DIPEA (174 μL, 0.10 mmol), and dimethyl sulfate (98

191 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 8.99 (s, 1H), 7.87 (s, 1H),

7.59 (d, J = 7.9 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H), 7.33 (s, 1H), 7.22

(s, 1H), 7.14 (d, J = 7.9 Hz, 1H), 4.13 (t, J = 6.6 Hz, 2H), 4.05 (q, J =

7.0 Hz, 2H), 4.00 (s, 3H), 3.41 (s, 2H), 2.76 (t, J = 6.6 Hz, 2H), 1.84

(p, J = 6.6 Hz, 2H), 1.34 (t, J = 7.0 Hz, 3H); ¹³C NMR (150 MHz,

DMSO-d₆) δ 149.1, 148.1, 147.8, 144.6, 143.9, 141.8, 130.5, 124.8,

121.6, 120.1, 119.9, 117.4, 112.6, 112.5, 111.9, 105.5, 67.6, 64.5, 38.9,

37.6, 32.7, 29.0, 15.3. HRMS (ESI) calcd for C₂₃H₂₆N₅O₂[M + H]⁺:

404.2081, found: 404.2087.

1

μL, 0.10 mmol) in 17% yield as a yellow solid, mp: 98−99 °C. H

NMR (600 MHz, DMSO-d₆) δ 8.88 (s, 1H), 7.47 (t, J = 7.9 Hz, 1H),

7.31 (s, 1H), 7.19 (s, 1H), 6.92 (d, J = 7.9 Hz, 1H), 6.09 (d, J = 7.9

Hz, 1H), 4.58 (s, 2H), 4.23 (q, J = 7.0 Hz, 2H), 4.14 (t, J = 6.3 Hz,

2H), 4.05 (q, J = 7.0 Hz, 2H), 3.96 (s, 3H), 3.66 (t, J = 6.3 Hz, 2H),

3.45 (s, 2H), 3.33 (s, 3H), 1.99 (p, J = 6.3 Hz, 2H), 1.34 (t, J = 7.0

Hz, 3H), 1.29 (t, J = 7.0 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ

162.9, 154.4, 148.8, 148.0, 147.9, 142.9, 142.2, 140.6, 125.0, 114.5,

112.9, 106.0, 101.1, 99.1, 96.1, 66.6, 64.7, 64.2, 61.2, 55.0, 37.5, 29.9,

29.8, 15.3, 15.2. MS (ESI) calcd for C₂₅H₃₃N₄O₅[M + H]⁺: 469.2,

found: 469.6.

1-(3-((7-(3-Aminopropoxy)-6-ethoxy-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)phenyl)propan-1-

one hydrochloride (ID32). Following the procedures described for

preparing ID09, compound ID32 was prepared from 19i (50 mg, 0.09

mmol), acetyl chloride (2.5 mL) and ethanol (5 mL) in 88% yield as a

1

white solid, HPLC purity: 99.2%, mp: 170−171 °C. H NMR (600

MHz, DMSO-d₆) δ 8.15 (s, 3H), 7.87 (s, 1H), 7.61 (d, J = 7.8 Hz,

1H), 7.40 (s, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H),

7.25 (s, 1H), 4.19 (t, J = 6.1 Hz, 2H), 4.08 (q, J = 7.0 Hz, 2H), 4.02

(s, 3H), 3.14−2.92 (m, 4H), 2.08 (p, J = 6.1 Hz, 1H), 1.36 (t, J = 7.0

Hz, 3H), 1.09 (t, J = 7.2 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ

201.0, 149.7, 148.3, 147.6, 144.0, 143.9, 142.7, 137.7, 129.6, 124.2,

120.3, 118.8, 114.7, 112.5, 112.2, 106.3, 67.2, 64.6, 37.5, 37.0, 31.7,

29.6, 27.3, 15.2, 8.7. HRMS (ESI) calcd for C₂₅H₃₁N₄O₃[M + H]⁺:

435.2391, found: 435.2394; anal. calcd for C₂₅H₃₁ClN₄O₃: C, 63.75;

H, 6.63; Cl, 7.53; N, 11.90; found: C, 63.62; H, 6.75; Cl, 7.71; N,

11.76.

3-((6-Ethoxy-3-((6-ethoxypyridin-2-yl)amino)-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-7-yl)oxy)propan-1-ol (ID30). Fol-

lowing the procedures described for preparing ID08, compound ID30

was prepared from 18h (60 mg, 0.13 mmol) and CSA methanol

solution (1 M, 2 mL) in 74% yield as a white solid, HPLC purity:

99.0%, mp: 157−159 °C. ¹H NMR (600 MHz, DMSO-d₆) δ 8.83 (s,

1H), 7.38 (t, J = 7.8 Hz, 1H), 7.22 (s, 1H), 7.10 (s, 1H), 6.84 (d, J =

7.8 Hz, 1H), 6.01 (d, J = 7.8 Hz, 1H), 4.47 (s, 1H), 4.14 (q, J = 6.7

Hz, 2H), 4.04 (t, J = 5.3 Hz, 2H), 3.96 (q, J = 6.9 Hz, 2H), 3.88 (s,

3H), 3.52 (q, J = 5.3 Hz, 2H), 3.36 (s, 2H), 1.82 (p, J = 5.3 Hz, 2H),

1.25 (t, J = 6.7 Hz, 3H), 1.20 (t, J = 6.9 Hz, 3H); ¹³C NMR (150

MHz, DMSO-d₆) δ 162.9, 154.4, 148.8, 148.0, 147.7, 142.9, 141.8,

140.6, 124.9, 114.4, 112.7, 105.4, 101.1, 99.0, 66.6, 64.6, 61.2, 58.0,

37.5, 32.8, 29.9, 15.3, 15.2. HRMS (ESI) calcd for C₂₃H₂₉N₄O₄[M +

H]⁺: 425.2183, found: 425.2193.

tert-Butyl (3-((6-Ethoxy-3-((6-ethoxypyridin-2-yl)amino)-1-

methyl-1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)propyl)-

carbamate (19j). Following the procedures described for preparing

6, compound 19j was prepared from 13j (90 mg, 0.25 mmol), K₂CO₃

(60 mg, 0.50 mmol), and N-Boc-3-bromopropylamine (116 mg, 0.50

mmol) in 70% yield as a white solid, mp: 155−156 °C. ¹H NMR (600

MHz, DMSO-d₆) δ 8.89 (s, 1H), 7.47 (t, J = 7.9 Hz, 1H), 7.29 (s,

1H), 7.20 (s, 1H), 6.92 (d, J = 7.9 Hz, 1H), 6.83 (s, 1H), 6.09 (d, J =

7.9 Hz, 1H), 4.23 (q, J = 7.0 Hz, 2H), 4.16−4.02 (m, 4H), 3.97 (s,

3H), 3.45 (s, 2H), 3.14 (q, J = 6.4 Hz, 2H), 1.86 (p, J = 6.4 Hz, 2H),

1.38 (s, 9H), 1.34 (t, J = 7.0 Hz, 3H), 1.29 (t, J = 7.0 Hz, 3H); ¹³C

NMR (150 MHz, DMSO-d₆) δ 162.9, 156.1, 154.4, 148.8, 147.5,

147.8, 142.9, 142.2, 140.6, 124.9, 114.4, 112.8, 106.1, 101.1, 99.0,

77.9, 67.7, 64.6, 61.2, 37.7, 37.5, 30.0, 29.8, 28.7, 15.3, 15.2. MS (ESI)

calcd for C₂₈H₃₈N₅O₅[M + H]⁺: 524.3, found: 524.2.

tert-Butyl (3-((3-((3-Cyanophenyl)amino)-6-ethoxy-1-meth-

yl-1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)propyl)-

carbamate (19c). Following the procedures described for preparing

6, compound 19c was prepared from 13c (100 mg, 0.29 mmol),

K₂CO₃(64 mg, 0.58 mmol), and N-Boc-3-bromopropylamine (109

1

mg, 0.58 mmol) in 76% yield as a white solid, mp: 136−138 °C. H

NMR (600 MHz, DMSO-d₆) δ 8.92 (s, 1H), 7.85 (s, 1H), 7.57 (d, J =

7.8 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.31 (s, 1H), 7.23 (s, 1H), 7.13

(d, J = 7.8 Hz, 1H), 6.84 (s, 1H), 4.15−4.02 (m, 4H), 3.99 (s, 3H),

3.41 (s, 2H), 3.14 (t, J = 6.4 Hz, 2H), 1.86 (p, J = 6.4 Hz, 2H), 1.38

(s, 9H), 1.35 (t, J = 7.0 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ

156.1, 149.1, 148.0, 144.6, 143.9, 142.1, 130.4, 124.8, 121.7, 120.1,

119.9, 117.4, 112.8, 112.6, 111.9, 106.1, 77.9, 67.7, 64.6, 37.7, 37.6,

29.8, 29.0, 28.7, 15.3. MS (ESI) calcd for C₂₈H₃₄N₅O₄[M + H]⁺:

504.3, found: 504.1.

7-(3-Aminopropoxy)-6-ethoxy-N-(6-ethoxypyridin-2-yl)-1-

methyl-1,4-dihydroindeno[1,2-c]pyrazol-3-amine dihydro-

chloride (ID33). Following the procedures described for preparing

ID09, compound ID33 was prepared from 19j (31 mg, 0.06 mmol),

acetyl chloride (2.5 mL), and ethanol (5 mL) in 90% yield as a white

1

solid, HPLC purity: 99.8%, mp: 230−231 °C. H NMR (600 MHz,

DMSO-d₆) δ 9.86 (s, 1H), 8.17 (s, 3H), 7.61 (t, J = 8.0 Hz, 1H), 7.43

(s, 1H), 7.27 (s, 1H), 6.90 (d, J = 8.0 Hz, 1H), 6.25 (d, J = 8.0 Hz,

1H), 4.28 (q, J = 7.0 Hz, 2H), 4.20 (t, J = 6.2 Hz, 2H), 4.09 (q, J = 7.0

Hz, 2H), 4.05 (s, 3H), 3.04−2.98 (td, J = 5.7, 6.2 Hz, 2H), 2.07 (p, J

= 6.2 Hz, 2H), 1.36 (t, J = 7.0 Hz, 3H), 1.32 (t, J = 7.0 Hz, 3H); ¹³C

NMR (150 MHz, DMSO-d₆) δ 162.1, 152.9, 150.1, 148.7, 147.6,

143.0, 141.8, 141.7, 123.8, 113.5, 112.3, 106.5, 102.2, 99.4, 67.3, 64.6,

62.3, 37.5, 37.0, 30.1, 27.3, 15.3, 15.1. HRMS (ESI) calcd for

C₂₃H₃₀N₅O₃[M + 2H]²⁺: 212.6208, found: 212.6216; anal. calcd for

C₂₄H₃₂Cl₂N₄O₃: C, 58.18; H, 6.51; Cl, 14.31; N, 11.31; found: C,

58.36; H, 6.75; Cl, 14.12; N, 11.24.

tert-Butyl (3-((6-Ethoxy-1-methyl-3-((3-propionylphenyl)-

amino)-1,4-dihydroindeno[1,2-c]pyrazol-7-yl)oxy)propyl)-

carbamate (19i). Following the procedures described for preparing

6, compound 19i was prepared from 13i (80 mg, 0.21 mmol), K₂CO₃

(59 mg, 0.42 mmol), and N-Boc-3-bromopropylamine (113 mg, 0.42

mmol) in 74% yield as a white solid, mp: 152−153 °C. ¹H NMR (600

MHz, DMSO-d₆) δ 8.67 (s, 1H), 7.86 (s, 1H), 7.62 (s, 1H), 7.36−

7.31 (m, 3H), 7.22 (s, 1H), 6.84 (s, 1H), 4.10−4.06 (m, 4H), 3.98 (s,

3H), 3.39 (s, 2H), 3.15 (t, J = 5.7 Hz, 2H), 3.01 (q, J = 7.1 Hz, 2H),

1.91−1.81 (p, J = 5.7 Hz 2H), 1.39 (s, 9H), 1.35 (t, J = 7.1 Hz, 3H),

1.10 (t, J = 7.1 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 156.1,

148.9, 148.0, 147.9, 144.5, 142.1, 137.7, 129.5, 124.9, 119.7, 118.1,

114.2, 112.9, 112.6, 106.1, 77.9, 67.7, 64.6, 37.7, 37.5, 31.6, 29.8, 29.3,

28.7, 15.3, 8.7. MS (ESI) calcd for C₂₈H₃₈N₅O₅[M + H]⁺: 535.3,

found: 535.1.

3-((7-(3-Aminopropoxy)-6-ethoxy-1-methyl-1,4-

dihydroindeno[1,2-c]pyrazol-3-yl)amino)benzonitrile (ID31).

To a solution of 19c (90 mg, 0.18 mmol) in DCM (5 mL) was

added triﬂuoroacetic acid (200 μL) and stirred at room temperature

for 2 h. After concentration, the resulting mixture was adjusted to pH

7 by the addition of a saturated NaHCO₃aqueous solution. The

resulting precipitate ﬁltered, washed with hexane, and dried to give 62

mg (90%) of ID31 as a white solid, HPLC purity: 98.9%, mp: 190−

In Vitro Tubulin Polymerization Assay. The tubulin polymer-

ization assay was performed using the Tubulin Polymerization Assay

Kit (BK011P, Cytoskeleton) according to the manual. The tubulin

reaction mix contained 2 mg/mL porcine brain tubulin, 2 mM MgCl₂,

0.5 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic

acid (EGTA), 1 mM guanosine triphosphate (GTP), and 15%

glycerol. A 96-well plate was incubated with 5 μL of the tested

compounds in various concentrations at 37 °C for 1 min. Then, the

tubulin reaction mix (50 μL) was added rapidly. The ﬂuorescence

intensity enhancement was monitored by excitation at 355 nm and

emission at 460 nm every 60 s for 60 min in a multifunction

microplate reader. The IC₅₀values were deﬁned as the compound

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concentration inhibiting the polymerization by 50% after 60 min. The

IC₅₀values were calculated by GraphPad Prism 5 software and

expressed as the mean SD from the dose−response curves of at

least three independent experiments.

Cell Lines and Culture. Human liver carcinoma HepG2 (HB-

8065), human lung adenocarcinoma A549 (CCL-185), human

colorectal carcinoma HCT116 (CCL-247), and normal liver LO2

(CRL-12461) cell lines were purchased from American Type Culture

Collection (ATCC, Manassas, VA). Paclitaxel selected MDR subline

A549/Tax was obtained from the Second Hospital of Shandong

University (Shandong, China). All of these cells were cultured in

RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37

°C in a humidiﬁed atmosphere of 5% CO₂and 95% air.

min in the dark at room temperature. The samples were detected by a

ﬂow cytometer.

Wound-Healing Assay. The exponentially growing HepG2 cells

were seeded into six-well cell plates (3 × 10⁵cells/well) and allowed

to grow to full conﬂuence in six-well plates. Monolayer cells were

wounded by scratching with a pipette tip and washed with PBS. The

cells were incubated in serum-free DMEM medium and treated with

the tested compounds at the diﬀerent concentrations for 24 h. The

cell images of 0 and 24 h were detected by a ﬂuorescence inverted

microscope.

Western Blot Analysis. The exponentially growing HepG2 cells

were seeded into six-well cell plates at 5 × 10⁵cells per well and

allowed to adhere overnight. The HepG2 cells were incubated with

various concentrations of the test compounds for 24 h. The cells were

harvested, and cell extracts were prepared for western blot analysis.

The total protein samples (20 μg) were separated by 10%

polyacrylamide gel under the condition of the ice bath. The protein

was transferred to the PVDF membrane and blocked with 5% nonfat

milk at room temperature for 1 h. Then, the PVDF membrane was

incubated with primary antibodies for 16 h at 4 °C. The primary

antibodies included cleaved caspase-3, PARP, Bcl-2, and Bax. On the

next day, the membranes were incubated with the HRP-conjugated

secondary antibody for 1 h. Immunoreactive bands were visualized

using an enhanced chemiluminescence reagent and quantiﬁed by

densitometry using a G:BOX chemi XR5 molecular imager. The

intensities of the blots were quantiﬁed by Gel-Pro32.

Aqueous Solubility Study. The tested compounds were initially

dissolved in DMSO at a concentration of 10 mg/mL and then diluted

to ﬁve concentration gradients with methanol. The peak area under

various concentrations was determined by HPLC, and the standard

curve was established. The tested compounds were added into 1 mL

of buﬀer solution to prepare a saturated solution. The absorption peak

area of the saturated solution was analyzed by HPLC. The saturated

solubility of the tested compounds was calculated using the standard

curve equation. The absorption peak area was determined using an

Agilent 1200 series equipment (column, Diamonil C18, 4.6 mm ×

150 mm, 5 μm; mobile phase, methanol/H₂O/CF₃COOH

(65:35:0.1%); ﬂow rate, 1.0 mL/min; UV wavelength, 254 nm).

Log P Measurement. The tested compounds were initially

dissolved in DMSO at a concentration of 5 mg/mL; 20 μL of the

resulting solution was added into a mixture solution of n-octanol (1

mL) and distilled water (1 mL). The mixture was stirred at room

temperature for 10 h and left at 4 °C overnight. The aqueous and

organic phases of the mixture were analyzed by HPLC, separately.

The instruments and conditions were the same as those for the

aqueous solubility assay. The Log P was calculated by the peak area

ratio in n-octanol and water.

Antitumor Activity In Vivo. The experimental procedures

conformed to the animal experiment guidelines of the Animal Care

and Welfare Committee of Shandong University. Five-week-old

pathogen-free female Balb/c-nu mice were obtained from the Animal

Centre of China Academy of Medical Sciences (Beijing, China). The

HepG2 xenograft model was established by injecting 200 μL PBS

containing HepG2 cells (1 × 10⁷cells/mL) subcutaneously in the

right armpit of mice. When the tumor volume reached 90 mm³, nude

mice were randomly divided into groups with ﬁve mice per group:

vehicle group (distilled water, po, n = 5), ID09 (25 mg/kg, po, n = 5)

group, ID33 (25 mg/kg, po, n = 5) group, vehicle group (distilled

water, iv, n = 5), ID09 (5 mg/kg, iv, n = 5), and ID09 (10 mg/kg, iv,

n = 5). ID09 and ID33 were dissolved in distilled water to form a 2.5

mg/mL solution for intragastric administration. ID09 was dissolved in

distilled water to form a 2.0 mg/mL solution for intravenous injection.

The drugs and the vehicle were administrated by po once a day or iv

every two days for 21 consecutive days. Tumor volumes and body

weights were recorded every 3 days after drug treatment. At the end

of the experiment, the mice were sacriﬁced, and the tumors were

removed and weighed. Tumor volumes were calculated with a formula

of L (long axis) × W²(short axis)/2. Relative tumor volume = mean

tumor volume at day 21/mean tumor volume at day 0. The

therapeutic eﬀect of drugs was evaluated by the relative tumor

Cell Growth Inhibitory Assay. The tested compounds were

evaluated for their antiproliferative activity by the 3-(4,5-dimethylth-

iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The

exponentially growing cells were seeded into 96-well cell plates (4−

7 × 10³cells/well) and allowed to adhere overnight. The cells were

exposed to various concentrations of the test compounds. After 72 h

of incubation in the incubator, 20 μL of MTT (2.5 mg/mL) was

added to each well and incubated for 4 h. After the suspension was

discarded, DMSO (150 μL) was added to dissolve the crystals. The

absorbance at 570 nm was measured by a microplate spectropho-

tometer. The GI₅₀values of the test compounds were deﬁned as the

molar drug concentrations required to cause 50% tumor cell growth

inhibition, were calculated by GraphPad Prism 5 software and

expressed as the mean SD from the dose−response curves of at

least three independent experiments.

Immunoﬂuorescence Staining. The exponentially growing

HepG2 cells were seeded into 24-well cell plates (5 × 10⁴cells/

well) containing coverslips and allowed to adhere overnight. The

HepG2 cells were exposed to the tested compounds for 24 h. After

ﬁxed in a mixture of ice-cold methanol/acetic acid (3:1) for 10 min,

the HepG2 cells were permeabilized with 0.3% Triton X-100 for 10

min and blocked with 5% bovine serum albumin for 30 min. Then,

they were incubated with mouse antitubulin antibody at 4 °C

overnight. On the next day, the cells were incubated with goat

antimouse IgG-TRITC antibody for 2 h at 37 °C in the dark. Hoechst

33342 was used to stain the nucleus in the dark at room temperature

for 10 min. The coverslips were immediately visualized on a Zeiss

LSM 880 laser scanning confocal microscope (Carl Zeiss, Germany).

EBI Competition Assay. The exponentially growing HepG2 cells

were seeded into six-well cell plates at 5 × 10⁵cells per well. After

overnight adherence, the cells were ﬁrst exposed to the tested

compounds for 2 h and afterward treated with EBI (100 μM) for

another 1.5 h. The cells were harvested, and cell extracts were

prepared for Western blot analysis. The total protein sample (20 μg)

was separated by 10% polyacrylamide gel under the condition of an

ice bath. The protein was transferred to the poly(vinylidene ﬂuoride)

(PVDF) membrane and blocked with 5% nonfat milk at room

temperature for 1 h. Then, the PVDF membrane was incubated with

anti-β-tubulin antibody for 16 h at 4 °C. On the next day, the

membranes were incubated with HRP-conjugated secondary antibody

for 1 h. Immunoreactive proteins were ﬁnally detected by

chemiluminescence.

Cell Cycle Analysis. The exponentially growing HepG2 cells were

seeded into six-well cell plates (3 × 10⁵cells/well) and allowed to

adhere overnight. They were incubated with various concentrations of

ID09 and ID33 for 24 h. Then, cells were harvested and ﬁxed in cold

70% ethanol overnight at 4 °C. After being washed with ice-cold

phosphate-buﬀered saline (PBS), the cells were incubated with

staining buﬀer (485 μL), PI solution (5 μL), and RNaseA solution

(10 μL) at 37 °C for 15 min. Cell cycle distribution was analyzed

using a ﬂow cytometer.

Apoptosis Analysis. The exponentially growing HepG2 cells

were seeded in six-well plates (3 × 10⁵cells/well). After overnight

adherence, they were incubated in the presence or absence of

compounds ID09 and ID33 with diﬀerent concentrations for 12 or 24

h. Then, the cells were harvested, suspended in Annexin V binding

buﬀer, and subsequently stained by Annexin V-PE and 7-AAD for 15

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inhibition rate = (1 − relative tumor volume of treated group/relative

tumor volume of vehicle group) × 100%.

Statistical Analysis. Prism Software 5.0 was used for data

analyses. The statistical signiﬁcance (P < 0.05) was determined by

one-way analysis of variance (ANOVA), followed by Dunnett’s

multiple comparison test, comparing each treated group to the

control.

Author Contributions

^§Y.-J.C. and C.L. contributed equally.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by the National Natural Science

Foundation of China (NSFC, Grant No. 81573275), the Key

Research and Development Program of Shandong Province

(2017CXGC1401), and the Fundamental Research Funds of

Shandong University (Grant Nos. 21310082033116 and

21310072064056).

ASSOCIATED CONTENT

* Supporting Information

■

sı

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01345.

Results of the in vitro tubulin polymerization assay as

well as some bioassays, NMR spectra, and HPLC charts

for all of the screening compounds (PDF)

ABBREVIATIONS USED

■

MTAs, microtubule-targeting agents; ADCs, antibody−drug

conjugates; FDA, Food and Drug Administration; CA-4,

combretastatin A-4; DIPEA, N,N-diisopropylethylamine;

LiHMDS, lithium hexamethyldisilazide; CSA, camphorsulfonic

acid; EBI, N,N′-ethylene-bis(iodoacetamide); VCR, vincris-

tine; GTP, guanosine triphosphate; EGTA, ethylene glycol-

bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; MTT, (3-

(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-

mide); PI, propidium iodide

Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Authors

■

Jing-De Wu − Department of Medicinal Chemistry, Key

Laboratory of Chemical Biology (Ministry of Education),

School of Pharmaceutical Sciences, Cheeloo College of

Medicine, Shandong University, Jinan 250012, P. R. China;

Phone: +86 (0)531 88382006; Email: wujingde70@

sdu.edu.cn

REFERENCES

■

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Authors

Ying-Jie Cui − Department of Medicinal Chemistry, Key

Laboratory of Chemical Biology (Ministry of Education),

School of Pharmaceutical Sciences, Cheeloo College of

Medicine, Shandong University, Jinan 250012, P. R. China

Chao Liu − Department of Medicinal Chemistry, Key

Laboratory of Chemical Biology (Ministry of Education),

School of Pharmaceutical Sciences, Cheeloo College of

Medicine, Shandong University, Jinan 250012, P. R. China

Chen-Chen Ma − Central Laboratory, The Aﬃliated Hospital

of Shandong University of Traditional Chinese Medicine,

Jinan 250012, P. R. China

Ya-Ting Ji − Department of Medicinal Chemistry, Key

Laboratory of Chemical Biology (Ministry of Education),

School of Pharmaceutical Sciences, Cheeloo College of

Medicine, Shandong University, Jinan 250012, P. R. China

Yi-Li Yao − Department of Medicinal Chemistry, Key

Laboratory of Chemical Biology (Ministry of Education),

School of Pharmaceutical Sciences, Cheeloo College of

Medicine, Shandong University, Jinan 250012, P. R. China

Long-Qian Tang − Department of Medicinal Chemistry, Key

Laboratory of Chemical Biology (Ministry of Education),

School of Pharmaceutical Sciences, Cheeloo College of

Medicine, Shandong University, Jinan 250012, P. R. China

Cheng-Mei Zhang − Department of Medicinal Chemistry, Key

Laboratory of Chemical Biology (Ministry of Education),

School of Pharmaceutical Sciences, Cheeloo College of

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J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

SAR Investigation and Discovery of Water-Soluble 1-Methyl-1,4-dihydroindeno[1,2- c]pyrazoles as Potent Tubulin Polymerization Inhibitors

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