Tricyclic pyrazoles. Part 1: synthesis and biological evaluation of novel 1,4-dihydroindeno[1,2-c]pyrazol-based ligands for CB1and CB2 cannabinoid receptors.

Bioorganic & Medicinal Chemistry 11 (2003) 251–263

Tricyclic Pyrazoles. Part 1: Synthesis and Biological Evaluation of

Novel 1,4-Dihydroindeno[1,2-c]pyrazol-based Ligands for CB₁

and CB₂Cannabinoid Receptors

Jean-Mario Mussinu,^aStefania Ruiu,^bAntonio C. Mule,^cAmedeo Pau,^a

Mauro A. M. Carai,^bGiovanni Loriga,^a,b

Gabriele Murineddu^a,band Gerard A. Pinna^a,*

a

`

Dipartimento Farmaco Chimico Tossicologico, Universita di Sassari, via F. Muroni 23/A, 07100 Sassari, Italy

^bNeuroscienze S.c.a.r.l., Zona Industriale Macchiareddu, 09010 Uta, Cagliari, Italy

`

Dipartimento di Scienze del Farmaco, Universita di Sassari, via F. Muroni 23/A, 07100 Sassari, Italy

c

Received 7 May 2002; accepted 18 July 2002

Abstract—Cannabinoids receptors, cellular elements of the endocannabinoid system, have been the focus of extensive studies

because of their potential functional role in several important physiological and pathological processes. To further evaluate the

properties of CB receptors, especially CB₁and CB₂subtypes, we have designed, using SR141716A as a benchmark, a new series of

rigid 1-aryl-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamides. Compounds 1 were synthesized from substituted 1-aryl-1,4-dihydro-

indeno[1,2-c]pyrazole-3-carboxylic acids and requisite amines. The various analogues were assayed for binding both to the brain and

peripheral cannabinoid receptors (CB₁and CB₂). Seven of the new compounds displayed very high in vitro CB₂binding aﬃnities,

especially 1a, 1b, 1c, 1e, 1g, 1h and 1j which showed K_ivalues of 0.34, 0.225, 0.27, 0.23, 0.385, 0.037 and 0.9 nM, respectively.

Compounds 1a, 1b, 1c and 1h showed the highest selectivity for CB₂receptor with K_i(CB₁) to K_i(CB₂) ratios of 6029, 5635, 5814

and 9810, respectively. Noticeably, 1h exhibited the highest aﬃnity and selectivity for CB₂receptors.

Introduction

endocannabinoid system¹⁰suggesting that it may play a

role in antinociception, brain development, retrograde

neuronal communication, memory, appetite, psycho-

motor control, cardiovascular and immune regulation

and cellular proliferation. Recent evidences have indi-

cated that ECS may be a potential therapeutic target for

the treatment of diverse patologies¹¹including asthma,

pain, multiple sclerosis, neurodegenerative, immune and

inﬂammatory diseases.

The endogenous cannabinoid system (ECS) in mammals

incorporates a variety of cellular elements as potential

recognition sites at which a broad group of compounds,

termed cannabinoids, interacts. Biological organization

of this system includes: (a) two subtypes of G-protein

1

coupled membrane receptors, termed the CB₁receptor

2

(primarily present in the nervous system) and the CB₂

receptor (mainly present in the immune system), (b) the

endogenous ligands for these receptors anandamide

(N-arachidonoylethanolamine, AN)³and 2-arachido-

noylglycerol (2-Ara-Gl)⁴named endocannabinoids and

(c) their multiple metabolic pathways for the synthesis

degradation⁵and reuptake (only in the case of ana-

ndamide)^6ꢀ9(Fig. 1).

At present diﬀerent cannabinoid binders have been

identiﬁed and can be classiﬁed into at least ﬁve diverse

chemical families (exocannabinoids). These compounds

include: tricyclic cannabinols¹²(classical cannabinoids

whose structure is based on the dibenzopyran template

of Á⁹-THC), bicyclic cannabinols^13,14(non classical

cannabinoids typiﬁed by CP-55,940), indoles, pirroles

and indenes¹⁵(typiﬁed by WIN-55,212-2), anandamide

analogues¹⁶and diarylpyrazoles¹⁷(SR 141716A is the

prototypical example) (Fig. 2).

Over the past several years a tremendous eﬀort has been

focused on studying the physiological functions of

These various types of binders, to a diﬀerent extent,

interact with both receptors and the development of

*Corresponding author. Tel.: +39-079-228721; fax: +39-079-228720;

e-mail: pinger@ssmain.uniss.it

PII: S0968-0896(02)00319-X

252

J.-M. Mussinu et al. / Bioorg. Med. Chem. 11 (2003) 251–263

Figure 1. Endogenous cannabinoid receptor ligands and numbering system.

Figure 2. Cannabinoid receptor binder structures and their receptor aﬃnities.

potent and selective ligands for CB₁and/or CB₂recep-

tors is of great importance to investigate the involve-

ment of the CB₁and CB₂receptors in some

(physiological) actions of endocannabinoids (a) in the

central nervous system, such as cognition and memory,

control of motor function, perception of pain, and (b)

some peripheral nerves, where they exert an action in

the urogenital, gastrointestinal and cardiovascular sys-

tems. The CB₂receptors are highly expressed in immune

cells, B cells and natural killer cells; availability of

potent and selective CB₂receptor ligands would

enhance our understanding on the role of this receptor

on some eﬀects of cannabinoids such as immunosup-

pressant and anti-inﬂammatory.^11d,18

appropriate structural constraints could restrict a phar-

macophoric structural element to a suﬃciently small

region of conformational space thereby permitting the

ligand to bind, with high aﬃnity and selectivity, to its

designated receptor.²¹In this line, we have designed

(Fig. 3) and synthesized (Table 1) an extensive series of

rigid SR141716A analogues of general structure 1 and

determined in vitro their binding aﬃnities for CB₁and

CB₂receptors.

Chemistry

Synthesis of title compounds 1 is outlined in Scheme 1.

There has been considerable eﬀort in recent years to

determine the relationship of three-dimensional and/or

conformational structure with CB₁and CB₂cannabi-

noid aﬃnity, for some selected molecules, belonging to

the above mentioned groups.¹⁹Understanding the pre-

ferred structure would provide new leads for cannabi-

noid ligands with improved biological properties. Our

approach to this was to minimize the ﬂexibility of the

lead compound through the use of conformationally

restricted analogues.²⁰It has been proposed that

The 1,3-diketoesters 3, as a tautomeric equilibrium shif-

ted towards the alkenylidene structure (3⁰), were prepared

from the indanones 2 and diethyl oxalate in the presence

of sodium ethylate. Compounds 3 and the appropriate

hydrazines were heated in EtOH to aﬀord the desired

1-arylpyrazoles 4. The esters 4 were hydrolized and

resulting acids 5 treated with SOCl₂to aﬀord the acid

chlorides which were allowed to react with the requisite

amines to give the desired amides 1a–o,q,r. Treatment of

1o with acetone in ethyl acetate gave the ketimine 1p.

J.-M. Mussinu et al. / Bioorg. Med. Chem. 11 (2003) 251–263

253

Figure 3. Rigid analogue approach via arrow a to obtain 1,4-dihydroindeno[1,2-c]pirazole derivatives 1.

Table 1. Structures and binding data of compounds 1

Receptor aﬃnity

R

Selectivity ratio

K_iCB₁(nM)^b

Compound 1

R^I

Q

K_iCB₁(nM)^a

K_iCB₁/K_iCB₂(nM)

6029:1

a

6Cl

2^I,4^ICl₂

2050ꢁ90

0.34ꢁ0.06

b

c

d

e

f

6F

6Br

6I

2^I,4^ICl₂

4^ICl

1268ꢁ51

1570ꢁ15

333ꢁ0.5

8.25ꢁ74

723ꢁ53

1152ꢁ65

363ꢁ30

399ꢁ24

1787ꢁ85

>5000

0.225ꢁ0.02

0.27ꢁ0.02

5.5ꢁ0.5

5635:1

5814:1

60:1

5Cl

0.23ꢁ0.036

6.788ꢁ0.47

0.385ꢁ0.04

0.037ꢁ0.003

12.3ꢁ1

3587:1

105:1

2992:1

9810:1

32:1

7Cl

g

h

I

H

6CH₃

6OCH₃

6Cl

j

0.9ꢁ0.09

48ꢁ5

1985:1

104:1

25;1

k

l

6Cl

H

6Cl

4^IOCH₃

2^I,4^ICl₂

3035ꢁ13.5

798ꢁ48

120ꢁ15

m

6Cl

9.9ꢁ0.52

81:1

n

o

p

6Cl

2^I,4^ICl₂

2_I,4^ICl₂

-N(CH₃)₂

-NH₂

1881ꢁ119

2183ꢁ123

2789ꢁ19

144ꢁ20

455ꢁ44

978ꢁ35

13:1

5:1

7:1

q

r

6Cl

2_I,4_ICl₂

2^I,4^ICl₂

>5000

1:1

>5000

SR141716A

SR144528

1.8ꢁ0.075

70ꢁ10

514ꢁ30

0.0035:1

250:1

0.28ꢁ0.04

^aAﬃnnity of compounds for the CB₁receptor was evaluated using mouse cerebellum membrane and [³H]-CP 55,940.

^bAﬃnity of compunds for the CB₂receptor was assayed using mouse spleen omogenate and [³H]-CP 55,940. K_ivaules were obtained from ﬁve

indipendent experiments carry out in triplicate are expressed as the meanꢁstandard error.

254

J.-M. Mussinu et al. / Bioorg. Med. Chem. 11 (2003) 251–263

.

Scheme 1. (a) Na, dry EtOH, (COOEt)₂; (b) ArNHNH₂HCl, EtOH; (c) KOH, MeOH; (d) SOCl₂, C₆H₅–CH₃, CH₂Cl₂, TEA, Q–NH₂; (e)

(CH₃)₂CO, EtOAc.

Biology

had K_ivalues of 363 nM for CB₁receptor, 0.037 nM for

CB₂receptor and a CB₂/CB₁selectivity ratio of 9810.

As a result, 1h had both the highest potency and selec-

tivity among all of the new ligand tested in this study.

The 6-OCH₃substituted analogue 1i possessed a 36-fold

lower CB₂receptor aﬃnity than 1a whereas the CB₁

receptor aﬃnity was increased. When the 2^I,4^I-dichloro

substitution pattern on the N₁-phenyl ring of 1a was

modiﬁed, some impact on CB₂receptor aﬃnity was

observed. While the removal of the C₂,-Cl atom from

the 2^I,4^I-Cl₂phenyl moiety of 1a, leading to 1j, had a

minimal eﬀect on CB₂receptor aﬃnity (1j: K_i=0.9 nM),

the C₂, C₄removal of both chlorine atoms, as illustrated

by compound 1k (K_i=48 nM), led to a marked decrease

in aﬃnity at CB₂receptors. Next, a methoxy in position

C₄, as for compound 1l conferred only modest CB₂

aﬃnity (K_i=120 nM). The importance of the piperidine

ring of the lead compound 1a for binding and selectivity

was conﬁrmed testing compounds 1m–r. Of the piper-

idine replacements made, only the pyrrolydinyl deriva-

tive 1m maintained a CB₂receptor aﬃnity even if

29-fold lower potent than 1a.

Aﬃnities at CB₁and CB₂receptor for compounds 1

were assessed by competition of [³H]-CP 55,940 in

mouse cerebellum membranes and in mouse spleen

omogenate, respectively. For comparison purposes and

as reference values, we have also included results

obtained for the two prototypical cannabinoid ligands

SR141716A and SR144528.

Results and Discussion

Examination of Table 1 reveals that several N-piperidin

carboxamides containing the planar 1,4-dihy-

droindeno[1,2-c]pyrazole template displayed very high

in vitro binding aﬃnity for CB₂receptors comparable

to, or exceeding, that of SR144528 claimed as ﬁrst

highly potent and selective ligand for the CB₂recep-

tor.²²Compound 1a, the ground term of this series of

derivatives, possessed high CB₂receptor aﬃnity and low

CB₁receptor aﬃnity exhibiting higher CB₂to CB₁

selectivity (6029-fold) than did SR144528 (250-fold). To

throw light on the signiﬁcance of 6-Cl atom in 1a, the

substitution with a variety of substituents, such as F, Br,

I, CH₃and OCH₃groups, was made. Compounds 1b–d

have a F, Br and I atom, respectively, in place of the

chlorine. The ﬂuoro- and bromo-substituted com-

pounds displayed similar CB₂aﬃnities and selectivities

to those of parent compound while the iodo-derivate was

16-fold less potent. Compounds 1e,f contained a C₅and

a C₇chlorine atom, respectively. Their CB₂receptor

aﬃnities were either slightly increased with C₅substitu-

tion (1e) or decreased with C₇substitution (1f) suggesting

that the shifting of the Cl atom from C₆to C₅was well

tolerated. The C₆unsubstituted compound 1g main-

tained a CB₂receptor aﬃnity that is comparable to that

of 1a. Noticeably the C₆methyl substituted analogue 1h

In conclusion, the present study investigated a series of

1,4-dihydroindeno[1,2-c]pyrazol-derivatives 1 for their

binding aﬃnities for CB₁and CB₂receptors. Several

compounds in this series exhibited a very high degree of

potency and selectivity for CB₂compared to CB₁.

Therefore the synthesis of conformationally restricted

analogues of the lead compound SR141716A resulted in

either markedly improvement of the CB₂binding aﬃ-

nity and selectivity. Hence, restriction of the ﬂexible

5-aryl-4-methyl-pyrazole backbone into the rigid and

planar 1,4-dihydroindeno[1,2-c]pyrazole architecture

suggests that this spatial arrangement is a determining

factor in the potency of these derivatives as CB₂recep-

tor ligands. Moreover, substituents such as F, Cl, Br

and CH₃group, introduced at C₆on the phenyl ring of

J.-M. Mussinu et al. / Bioorg. Med. Chem. 11 (2003) 251–263

255

the tricyclic system, give the highest increase in aﬃnity

and selectivity for CB₂receptors. Among them, 1-(2^I,4^I-

dichlorophenyl)-6-methyl-N-piperidin-1-yl-1,4-dihy-

droindeno[1,2-c]pyrazole-3-carboxamide (1h) deserves

special attention as being, to our knowledge, the most

potent and selective CB₂ligand describe to date.

The analytically pure product was isolated by an

appropriate method of puriﬁcation as below indicated.

6-Chloro-1-(2^I,4^I-dichlorophenyl)-N-piperidin-1-yl-1,4-di-

hydroindeno[1,2-c]pyrazole-3-carboxamide 1a. General

procedure I was used to convert 5a and N-aminopiper-

idine hydrochloride into the title product. The mixture

was reﬂuxed for 3 h puriﬁcation by ﬂash chromato-

graphy [petroleum ether/EtOAc, 6:4] aﬀorded 1a (0.73g,

43%) as a yellow solid. R_f=0.41 (petroleum ether/

EtOAc 6:4); mp 189/191 ^ꢂC (triturated with hexane); IR

1665, 3250; UV l(log e) 254.1 (4.42), 269.4 (4.51), 281.2

Experimental

Chemistry

1

General information. Melting points were obtained on a

Koﬂer melting point apparatus and are uncorrected. IR

spectra were recorded as thin ﬁlms (for oils) or Nujol

mulls (for solids) on NaCl plates with a Perkin–Elmer

781 IR spectrophotometer and are expressed in ꢀ

(cm^ꢀ1). UV–Vis spectra were recorded as ethanolic

solution with a Perkin–Elmer l 5 spectrophotometer

and are the absorption wavelength expressed as l_maxin

(4.55), 295.3 (4.38), 303.0 (4.28); H NMR (CDCl₃) d

1.27–1.48 (m, 2H), 1.51–1.80 (m, 4H), 2.82 (t, 4H), 3.82

(s, 2H), 6.83 (d, 1H), 7.13 (d, 1H), 7.43 (dd, 1H), 7.46 (s,

1H), 7.50 (s, 1H), 7.53–7.64 (m, 2H, NH exch. with

D₂O); ¹³C NMR (DEPT, CDCl₃) 23.29 (CH₂), 25.36

(CH₂ꢃ2), 29.73 (CH₂), 57.12 (CH₂ꢃ2), 119.77 (CH),

126.73 (CH), 126.97 (CH), 128.31 (CH), 129.68 (CH),

130.55 (CH), 128.81 (C), 129.62 (C), 131.74 (C), 133.04

(C), 135.67 (C), 136.24 (C), 141.47 (C), 150.78 (C), 151.29

(C), 158.86 (C); API-ES calcd for C₂₂H₁₉Cl₃N₄O 461.8,

found 461.7 and Anal. calcd: C, 57.22; H, 4.15; Cl, 23.03;

N, 12.13. Found: C, 57.18; H, 4.02; Cl, 23.21; N, 12.33.

3

ꢀ1

.

nm followed by log " in dm mol cm^ꢀ1. All NMR

spectra were taken on a Varian XL-200 NMR spectro-

meter with ¹H and ¹³C being observed at 200 and

50 MHz respectively. Chemical shifts for ¹H and ¹³C

NMR spectra were reported in d or ppm downﬁeld from

TMS [(CH₃)₄Si]. Multiplicities are recorded as s (sing-

let), br s (broad singlet), d (doublet), t (triplet), q

(quartet), dd (doublet of doublets), m (multiplet).

Atmospheric Pressure Ionization Electrospray (API-ES)

mass spectra, when reported, were obtained on a Agi-

lent 1100 series LC/MSD spectrometer. Elemental ana-

lyses were performed by Laboratorio di Microanalisi,

Dipartimento di Chimica, Universita di Sassari, Italy

and are within ꢁ0.4% of the calculated values. All

reactions involving air or moisture-sensitive compounds

were performed under argon atmosphere. Unless other-

wise speciﬁed, all materials, solvents, reagents and pre-

cursors 2a–c,g,i were obtained from commercial

suppliers. Flash chromatography (FC) was performed

using Merck silica gel 60 (230–400 mesh ASTM). Thin

layer chromatography (TLC) was performed with Poly-

gram¹SIL N-HR/HV₂₅₄precoated plastic sheets (0.2

mm). The starting indanone (2h)²³and diketoester

(3g)²⁴were prepared according to the previously descri-

bed literature.

.

1-(2^I,4^I-Dichlorophenyl)-6-ﬂuoro-N-piperidin-1-yl-1,4-di-

hydroindeno[1,2-c]pyrazole-3-carboxamide 1b. General

procedure I was used to convert 5b and N-aminopiper-

idine hydrochloride into the title product. The mixture

was reﬂuxed for 4 h puriﬁcation by crystallization from

ethyl acetate/petroleum ether aﬀorded 1b (0.71g, 40%)

as a white solid. R_f=0.52 (petroleum ether/EtOAc 6:4);

mp 221/222^ꢂC; IR 1590, 1690, 3260; UV l(log e) 245.0

1

(4.46), 277.6 (4.30), 292.6 (4.16), 300.0 (4.09); H NMR

(CDCl₃) d 1.38–1.55 (m, 2H), 1.68–1.88 (m, 4H), 2.88(t,

4H), 3.70 (s, 2H), 6.93 (d,1H), 7.28 (d,1H), 7.43–7.61 (m,

3H), 7.63 (br s, 1H, NH exch. with D₂O), 7.66 (d, 1H);

API-ES calcd for C₂₂H₁₉Cl₂FN₄O 445.3, found 445.2, and

Anal calcd: C, 59.34; H, 4.30; Cl, 15.92; F, 4.27; N, 12.58.

Found: C, 59.06; H, 4.25; Cl, 15.81; F, 4.01; N, 12.77.

6-Bromo-1-(2^I,4^I-dichlorophenyl)-N-piperidin-1-yl-1,4-di-

hydroindeno[1,2-c]pyrazole-3-carboxamide 1c. General

procedure I was used to convert 5c and N-aminopiper-

idine hydrochloride into the title product. The mixture

was reﬂuxed for 5 h puriﬁcation by ﬂash chromato-

graphy (petroleum ether/EtOAc, 1:1) aﬀorded 1c (1.40g,

65%) as a bright yellow solid. R_f=0.67 (petroleum

ether/EtOAc 1:1); mp 181/183 ^ꢂC (methanol); IR 1570,

1665, 3360; UV l(log e) 242.4 (4.22), 254.6 (4.27), 267.6

General procedure I: synthesis of carboxamides and

hydrazides

A mixture of the appropriate 1,4-dihydroindeno[1,2-

c]pyrazole-3-carboxylic acid 5 (1 equiv, 4.0 mmol) and

thionyl chloride (3.0 equiv) in toluene (30 mL) was

reﬂuxed for 2.5–10 h. The solvent and the excess of

SOCl₂were removed under reduced pressure and the

resulting dark solid in CH₂Cl₂(15 mL) was dropwise

added to a solution of requisite amine or hydrazine (1.5

equiv) and Et₃N (1.5 equiv) in CH₂Cl₂(15 mL) at 0 ^ꢂC.

The mixture was warmed to room temperature and

stirred for 3 h. The mixture was then poured into a

separatory funnel and brine was added. The aqueous

layer was separated and extracted with CH₂Cl₂. The

combined organic layer were washed with water, dried

over Na₂SO₄and concentrated under reduced pressure.

1

(4.28), 281.2 (4.28), 295.0 (4.18), 303.4 (4.09); H NMR

(CDCl₃) d 1.38–1.54 (m, 2H), 1.69–1.92 (m, 4H), 2.88 (t,

4H), 3.89 (s, 2H), 6.84 (d, 1H), 7.37 (dd, 1H), 7.48 (dd,

1H), 7.55 (d, 1H), 7.65 (br s, 1H, NH exch. with D₂O),

7.68 (m, 2H); ¹³C NMR (DEPT, CDCl₃) 23.26 (CH₂),

25.34 (CH₂ꢃ2), 29.69 (CH₂), 57.11 (CH₂ꢃ2), 120.13

(CH), 128.32 (CH), 129.62 (CH), 129.67 (CH), 129.83

(CH), 130.55 (CH), 121.09 (C), 128.72 (C), 130.01 (C),

131.72 (C), 135.64 (C), 136.26 (C), 141.42 (C), 150.81

(C), 151.48 (C), 158.86 (C); API-ES calcd for

C₂₂H₁₉BrCl₂N₄O 506.2, found 506.1 and Anal. calcd: C,

52.20; H, 3.78; Br, 15.78; Cl, 14.01; N, 11.07. Found: C,

52.13; H, 3.90; Br, 15.66; Cl, 14.24; N, 11.01.

256

J.-M. Mussinu et al. / Bioorg. Med. Chem. 11 (2003) 251–263

1-(2^I,4^I-Dichlorophenyl)-6-iodo-N-piperidin-1-yl-1,4-dihy-

droindeno[1,2-c]pyrazole-3-carboxamide 1d. General

procedure I was used to convert 5d and N-aminopiper-

idine hydrochloride into the title product. The mixture

was reﬂuxed for 4 h puriﬁcation by crystallization from

ethyl acetate/petroleum ether aﬀorded 1d (0.56g, 25%)

as a yellow solid. R_f=0.57 (petroleum ether/EtOAc 1:1);

mp 174/176 ^ꢂC; IR 1570, 1650, 3170; UV l(log e) 247.6

anal. calcd: C, 57.22; H, 4.15; Cl, 23.03; N, 12.13. Found:

C, 57.00; H, 4.35; Cl, 23.05; N, 12.22.

1-(2^I,4^I-Dichlorophenyl)-N-piperidin-1-yl-1,4-dihydroin-

deno[1,2-c]pyrazole-3-carboxamide 1g. General proce-

dure I was used to convert 5g and N-aminopiperidine

hydrochloride into the title product. The mixture was

reﬂuxed for 3 h puriﬁcation by ﬂash chromatography

(petroleum ether/EtOAc, 1:1) aﬀorded 1g (0.71 g, 83%)

as a colourless solid. R_f=0.65 (petroleum ether/EtOAc

1:1); mp 228 ^ꢂC (EtOAc/petroleum ether); IR 1610,

1665, 3290; UV l(log e) 245.6 (4.20), 278.0 (4.09), 287.4

1

(4.45), 250.8 (4.37), 278.6 (4.30), 304.2 (4.20); H NMR

(CDCl₃) d 1.35–1.55 (m, 2H), 1.73–1.88 (m, 4H), 2.88 (t,

4H), 3.87 (s, 2H), 6.73 (d,1H), 7.43–7.72 (m, 5H, NH

exch. with D₂O), 7.91 (s, 1H); ¹³C NMR (DEPT,

CDCl₃) 23.28 (CH₂), 25.36 (CH₂ꢃ2), 29.52 (CH₂), 57.11

(CH₂ꢃ2), 120.45 (CH), 128.30 (CH), 129.65 (CH),

130.54 (CH), 135.42 (CH), 135.72 (CH), 92.43 (C),

128.55 (C), 130.52 (C), 131.71 (C), 135.65 (C), 136.24

(C), 141.41 (C), 150.90 (C), 151.58 (C), 158.84 (C); API-

ES calcd for C₂₂H₁₉Cl₂IN₄O 553.2, found 553.1 and

Anal. calcd: C, 47.76; H, 3.46; Cl, 12.82; I, 22.94; N,

10.13. Found: C, 47.74; H, 3.22; Cl, 12.91; I, 22.80; N,

10.35.

1

(4.02), 296.0 (3.98); H NMR (CDCl₃) d 1.34–1.56 (m,

2H), 1.65–1.87 (m, 4H), 2.89 (t, 4H), 3.90 (s, 2H), 6.99

(d, 1H), 7.19–7.39 (m, 2H), 7.44–7.63 (m, 3H), 7.62–7.71

(m, 2H, NH exch. with D₂O); ¹³C NMR (DEPT,

CDCl₃) 23.26 (CH₂), 25.33 (CH₂ꢃ2), 29.71 (CH₂), 57.06

(CH₂ꢃ2), 118.97 (CH), 126.26 (CH), 126.58 (CH),

127.00 (CH), 128.14 (CH), 129.66 (CH), 130.44 (CH),

128.67 (C), 131.03 (C), 131.87 (C), 135.83 (C), 136.01

(C), 141.38 (C), 149.60 (C), 151.69 (C), 159.01 (C); API-

ES calcd for C₂₂H₂₀Cl₂IN₄O 427.3, found 427.2 and

anal. calcd: C, 61.83; H, 4.72; Cl, 16.59; N, 13.11.

Found: C, 61.96; H, 4.52; Cl, 16.44; N, 13.26.

5-Chloro-1-(2^I,4^I-dichlorophenyl)-N-piperidin-1-yl-1,4-di-

hydroindeno[1,2-c]pyrazole-3-carboxamide 1e. General

procedure I was used to convert 5e and N-aminopiper-

idine hydrochloride into the title product. The mixture

was reﬂuxed for 4 h puriﬁcation by ﬂash chromato-

graphy (petroleum ether/EtOAc, 6:4) aﬀorded 1e (1.13

g, 63%) as a colourless solid. R_f=0.76 (petroleum ether/

EtOAc 1:1); mp 160/162 ^ꢂC (EtOAc/petroleum ether);

IR 1570, 1655, 3200; UV l(log e) 246.2 (4.24), 264.4

1-(2^I,4^I-Dichlorophenyl)-6-methyl-N-piperidin-1-yl-1,4-di-

hydroindeno[1,2-c]pyrazole-3-carboxamide 1 h. General

procedure I was used to convert 5h and N-aminopiper-

idine hydrochloride into the title product. The mixture

was reﬂuxed for 5 h puriﬁcation by ﬂash chromato-

graphy (petroleum ether/EtOAc, 1:1) aﬀorded 1h (1.2 g,

67%) as a colourless solid. R_f=0.60 (petroleum ether/

EtOAc 1:1); mp 165 ^ꢂC (EtOAc/petroleum ether); IR

1670, 3250; UV l(log e) 250.6 (4.37), 282.2 (4.22), 293.2

1

(4.26), 276.6 (4.25), 289.6 (4.12), 297.0 (3.90); H NMR

(CDCl₃) d 1.37–1.56 (m, 2H), 1.67–1.90 (m, 4H), 2.90 (t,

4H), 3.92 (s, 2H), 7.69 (d,1H), 7.15- 7.35 (m, 2H), 7.40–

7.58 (m, 2H), 7.65 (s, 1H), 7.67 (br s, 1H, NH exch. with

D₂O); ¹³C NMR (DEPT, CDCl₃) 23.29 (CH₂), 25.35

(CH₂ꢃ2), 29.46 (CH₂), 57.17 (CH₂ꢃ2), 117.41 (CH),

127.27 (CH), 128.29 (CH), 128.37 (CH), 129.70 (CH),

130.53 (CH), 128.44 (C), 131.71 (C), 131.85 (C), 132.58

(C), 135.59 (C), 136.27 (C), 141.53 (C), 147.07 (C), 150.98

(C), 158.77 (C); API-ES calcd for C₂₂H₁₉Cl₃N₄O 461.8,

found 461.7 and anal. calcd: C, 57.22; H, 4.15; Cl, 23.03;

N, 12.13. Found: C, 57.11; H, 4.00; Cl, 23.25 N, 12.34.

1

(4.14), 301.6 (4.13); H NMR (CDCl₃) d 1.37–1.53 (m,

2H), 1.69–1.88 (m, 4H), 2.40(s, 3H), 2.89 (t, 4H), 3.66 (s,

2H), 6.89 (d,1H), 7.04 (d, 1H), 7.38 (s, 1H), 7.47 (dd,

1H), 7.55 (d, 1H), 7.66 (br s, 1H, NH exch. with D₂O),

7.67 (d, 1H); ¹³C NMR (DEPT, CDCl₃) 21.61 (CH₃),

23.31 (CH₂), 25.37 (CH₂ꢃ2), 29.61 (CH₂), 57.10

(CH₂ꢃ2), 118.70 (CH), 127.13 (CH), 127.29 (CH),

128.16 (CH), 129.69 (CH), 130.47 (CH), 128.26 (C),

128.52 (C), 131.87 (C), 135.95 (C), 137.23 (C), 141.38

(C), 144.74 (C), 150.03 (C), 151.81 (C), 159.15 (C); API-

ES calcd for C₂₃H₂₂Cl₂N₄O 441.3, found 441.2 and

anal. calcd: C, 62.59; H, 5.02; Cl, 16.07; N, 12.69.

Found: C, 62.36; H, 5.22; Cl, 16.27; N, 12.61.

7-Chloro-1-(2^I,4^I-dichlorophenyl)-N-piperidin-1-yl-1,4-di-

hydroindeno[1,2-c]pyrazole-3-carboxamide 1f. General

procedure I was used to convert 5f and N-aminopiper-

idine hydrochloride into the title product. The mixture

was reﬂuxed for 4 h puriﬁcation by ﬂash chromato-

graphy (petroleum ether/EtOAc, 4:6) aﬀorded 1f (1.20

g, 64%) as a yellow product. R_f=0.42 (petroleum ether/

EtOAc 1:1); mp 237/238 ^ꢂC (acetone); IR 1610, 1670,

3280; UV l(log e) 248.4 (4.27), 260.6 (4.25), 293.4

1-(2^I,4^I-Dichlorophenyl)-6-methoxy-N-piperidin-1-yl-1,4-

dihydroindeno[1,2-c]pyrazole-3-carboxamide 1i. General

procedure I was used to convert 5i and N-aminopiper-

idine hydrochloride into the title product. The mixture

was reﬂuxed for 2.5 h puriﬁcation by ﬂash chromato-

graphy (petroleum ether/EtOAc, 3:7) aﬀorded 1i (0.84

g, 46%) as a colourless solid. R_f=0.31 (petroleum ether/

EtOAc 1:1); mp 238 ^ꢂC (EtOAc/petroleum ether); IR

1610, 1680, 3330; UV l(log e) 254.2 (4.32), 287. (4.10),

301.2 (4.08), 311.4 (4.06); ¹H NMR (CDCl₃) d 1.37–1.54

(m, 2H), 1.64–1.88 (m, 4H), 2.88 (t, 4H), 3.84 (s, 3H),

3.87 (s, 2H), 6.77 (dd, 1H), 6.90 (d, 1H), 7.12 (s, 1H),

7.47 (dd, 1H), 7.55 (d, 1H), 7.65 (br s, 1H, NH exch.

with D₂O), 7.66 (d, 1H); ¹³C NMR (DEPT, CDCl₃)

55.52 (CH₃), 23.32 (CH₂), 25.39 (CH₂x 2), 29.93 (CH₂),

1

(4.14), 300.2 (3.96); H NMR (CDCl₃) d 1.52–1.62 (m,

2H), 1.69–1.87 (m, 4H), 2.88 (t, 4H), 3.88 (s, 2H), 6.93

(d,1H), 7.26 (dd, 1H), 7.46 (s, 1H), 7.49–7.60 (m, 2H),

7.62 (br s, 1H, NH exch. with D₂O), 7.69 (d, 1H); ¹³C

NMR (DEPT, CDCl₃) 23.29 (CH₂), 25.37 (CH₂ꢃ2),

29.55 (CH₂), 57.13 (CH₂ꢃ2), 118.64 (CH), 126.97 (CH),

127.28 (CH), 128.33 (CH), 128.70 (CH), 129.05 (CH),

128.94 (C), 131.76 (C), 132.65 (C), 132.96 (C), 135.60 (C),

136.25 (C), 141.50 (C), 148.07 (C), 151.73 (C), 158.80 (C);

API-ES calcd for C₂₂H₁₉Cl₃N₄O 461.8, found 461.7 and

J.-M. Mussinu et al. / Bioorg. Med. Chem. 11 (2003) 251–263

257

57.11 (CH₂ꢃ2), 112.13 (CH), 112.55 (CH), 119.69 (CH),

128.18 (CH), 129.71 (CH), 130.48 (CH), 124.28 (C),

127.56 (C), 131.80 (C), 135.91 (C), 135.96 (C), 141.33

(C), 151.64 (C), 151.95 (C), 159.16 (C), 159.27 (C); API-

ES calcd for C₂₃H₂₂Cl₂N₄O₂457.3, found 457.2 and

anal. calcd: C, 60.40; H, 4.85; Cl, 15.50; N, 12.25.

Found: C, 60.59; H, 4.95; Cl, 15.44; N, 12.11.

¹³C NMR (DEPT, CDCl₃) 55.67 (CH₃), 23.30 (CH₂),

25.38 (CH₂ꢃ2), 29.59 (CH₂), 57.15 (CH₂ꢃ2), 114.65

(CHꢃ2), 119.76 (CH), 124.94 (CHꢃ2), 126.79 (CHꢃ2),

129.41 (C), 129.99 (C), 132.59 (C), 132.68 (C), 140.40

(C), 148.67 (C), 151.40 (C), 159.20 (C), 159.65 (C); API-

ES calcd for C₂₃H₂₃ClN₄O₂422.9, found 422.8 and

anal. calcd: C, 65.32; H, 5.48; Cl, 8.38; N, 13.25. Found:

C, 65.39; H, 5.44; Cl, 8.25; N, 13.14.

6-Chloro-1-(4^I-chlorophenyl)-N-piperidin-1-yl-1,4-dihy-

droindeno[1,2-c]pyrazole-3-carboxamide 1j. General pro-

cedure I was used to convert 5j and N-aminopiperidine

hydrochloride into the title product. The mixture was

reﬂuxed for 10 h puriﬁcation by ﬂash chromatography

(petroleum ether/EtOAc, 1:1) aﬀorded 1j (0.95g, 55%)

as a colourless solid. R_f=0.63 (petroleum ether/EtOAc

1:1); mp 233–234 ^ꢂC (EtOAc/petroleum ether); IR 1595,

1690, 3290; UV l(log e) 255.0 (4.32), 279.8 (4.40), 295.4

(4.27), 304.4 (4.17); ¹H NMR (CDCl₃) 1.39–1.58 (m,

2H), 1.70–1.89 (m, 4H), 2.90 (t, 4H), 3.88 (s, 2H), 7.22–

7.39 (m, 2H), 7.53–7.77 (m, 6H, NH exch. with D₂O);

¹³C NMR (DEPT, CDCl₃) 23.30 (CH₂), 25.39 (CH₂ꢃ2),

29.60 (CH₂), 57.14 (CH₂ꢃ2), 119.88 (CH), 124.55

(CHꢃ2), 126.94 (CHꢃ2), 129.78 (CHꢃ2), 129.71 (C),

130.16 (C), 133.01 (C), 134.23 (C), 138.03 (C), 141.14

(C), 148.60 (C), 151.48 (C), 158.93 (C); API-ES calcd

for C₂₂H₂₀Cl₂N₄O 427.3, found 427.2 and anal. calcd:

C, 61.83; H, 4.72; Cl, 16.59; N, 13.11. Found: C, 61.94;

H, 4.59; Cl, 16.45; N, 13.16.

6-Chloro-1-(2^I,4^I-dichlorophenyl)-N-pyrrolidin-1-yl-1,4-

dihydroindeno[1,2-c]pyrazole-3-carboxamide 1m. Gen-

eral procedure I was used to convert 5a and N-amino-

pyrrolidine hydrochloride into the title product.

Because of an excess of the hydrochloride salt, 3 equiv

of TEA were used in this reaction. The mixture was

reﬂuxed for 3 h puriﬁcation by crystallization from

EtOAc aﬀorded 1m (1.55 g, 86%) as a colourless solid.

R_f=0.38 (petroleum ether/EtOAc 1:1); mp 213/215 ^ꢂC;

IR 1665, 3200; UV l(log e) 255.0 (4.30), 266.8 (4.29),

281.6 (4.27), 294.6 (4.16), 302.8 (4.99); ¹H NMR

(CDCl₃) d 1.84–2.02 (m, 4H), 2.95–3.12 (m, 4H), 3.91 (s,

2H), 6.90 (d, 1H), 7.22 (dd, 1H), 7.43–7.60 (m, 3H), 7.61

(br s, 1H, NH exch. with D₂O), 7.67 (d, 1H); ¹³C NMR

(DEPT, CDCl₃) 22.24 (CH₂ꢃ2), 29.76 (CH₂), 55.54

(CH₂ꢃ2), 119.75 (CH), 126.72 (CH), 126.96 (CH),

128.29 (CH), 129.66 (CH), 130.54 (CH), 128.64 (C),

129.58 (C), 131.76 (C), 133.03 (C), 135.63 (C), 136.25

(C), 141.35 (C), 150.78 (C), 151.26 (C), 159.74 (C); API-

ES calcd for C₂₁H₁₇Cl₃N₄O 447.7, found 447.6 and

anal. calcd: C, 56.33; H, 3.83; Cl, 23.75; N, 12.51.

Found: C, 56.39; H, 3.73; Cl, 23.56; N, 12.77.

6 - Chloro - 1 - phenyl - N - piperidin - 1 - yl - 1,4 - dihydroin-

deno[1,2-c]pyrazole-3-carboxamide 1k. General proce-

dure I was used to convert 5k and N-aminopiperidine

hydrochloride into the title product. The mixture was

reﬂuxed for 7 h puriﬁcation by ﬂash chromatography

(petroleum ether/EtOAc, 1:1) aﬀorded 1k (1.06 g, 67%)

as a colourless solid. R_f=0.53 (petroleum ether/EtOAc

1:1); mp 191–193 ^ꢂC (EtOAc/petroleum ether); IR 1595,

1690, 3330; UV l(log e) 253.6 (4.30), 270.8 (4.37), 278.6

6-Chloro-1-(2^I,4^I-dichlorophenyl)-N’,N’-dimethyl-1,4-di-

hydroindeno[1,2-c]pyrazole-3-carbohydrazide 1n. Gen-

eral procedure I was used to convert 5a and dimethyl

hydrazine into the title product. The mixture was

reﬂuxed for 3 h puriﬁcation by crystallization from

EtOAc aﬀorded 1n (0.95 g, 67%) as a colourless solid.

R_f=0.43 (petroleum ether/EtOAc 2.5:7.5); mp 214/

215 ^ꢂC; IR 1680, 3200; UV l(log e) 255.6 (4.30), 266.4

1

(4.38), 295.4 (4.23), 304.4 (4.14); H NMR (CDCl₃) d

1.39–1.53 (m, 2H), 1.69–1.87 (m, 4H), 2.89 (t, 4H), 3.88 (s,

2H), 7.23 (d, 1H), 7.35 (d, 1H), 7.43–7.78 (m, 7H, NH

exch. with D₂O); ¹³C NMR (DEPT, CDCl₃) 23.31 (CH₂),

25.39 (CH₂ꢃ2), 29.59 (CH₂), 57.14 (CH₂ꢃ2), 119.96

(CH), 123.35 (CHꢃ2), 126.82 (CHꢃ2), 128.51 (CHꢃ2),

129.60 (CH), 129.92 (C), 129.95 (C), 132.79 (C), 139.50

(C), 140.84 (C), 148.56 (C), 151.46 (C), 159.11 (C); API-

ES calcd for C₂₂H₂₁ClN₄O 392.9, found 392.8 and anal.

calcd: C, 67.26; H, 5.39; Cl, 9.02 N, 14.26. Found: C,

67.40; H, 5.31; Cl, 9.21 N, 14.06.

1

(4.29), 279.6 (4.28), 294.8 (4.15), 302.8 (4.06); H NMR

(CDCl₃) d 2.73 (s, 6H), 3.92 (s, 2H), 6.90 (d, 1H), 7.23

(dd, 1H), 7.43–7.60 (m, 3H), 7.62 (br s, 1H, NH exch.

with D₂O), 7.67 (d, 1H); ¹³C NMR (DEPT, CDCl₃)

29.76 (CH₂), 47.72 (CH₃ꢃ2), 119.74 (CH), 126.71 (CH),

126.97 (CH), 128.30 (CH), 129.65 (CH), 130.54 (CH),

128.61 (C), 129.53 (C), 131.75 (C), 133.05 (C), 135.59

(C), 136.28 (C), 141.24 (C), 150.81 (C), 151.23 (C),

159.21 (C); API-ES calcd for C₁₉H₁₅Cl₃N₄O 421.7,

found 421.6 and anal. calcd: C, 54.11; H, 3.59; Cl, 25.22;

N, 13.29. Found: C, 54.30; H, 3.38; Cl, 25.33; N, 13.20.

6-Chloro-1-(4^I-methoxyphenyl)-N-piperidin-1-yl-1,4-dihy-

droindeno[1,2-c]pyrazole-3-carboxamide 1l. General pro-

cedure I was used to convert 5l and N-aminopiperidine

hydrochloride into the title product. The mixture was

reﬂuxed for 3 h puriﬁcation by trituration with petro-

leum ether containing a few drops of EtOAc aﬀorded 1l

(1.28 g, 75%) as a colourless solid. R_f=0.40 (petroleum

ether/EtOAc 1:1); mp 166–167 ^ꢂC; IR 1595, 1680, 3330;

UV l(log e) 254.0 (4.24), 271.8 (4.36), 279.6 (4.39),

295.4 (4.24), 303.8 (4.13); ¹H NMR (CDCl₃) d 1.38–1.53

(m, 2H), 1.69–1.91 (m, 4H), 2.89 (t, 4H), 3.87 (s, 2H),

3.91 (s, 3H), 7.05–7.13 (m, 2H), 7.18–7.35 (m, 2H),

7.52–7.64 (m, 3H), 7.71 (br s, 1H, NH exch. with D₂O);

6-Chloro-1-(2^I,4^I-dichlorophenyl)-1,4-dihydroindeno[1,2-

c]pyrazole-3-carbohydrazide 1o. General procedure I

was used to convert 5a and hydrazine hydrate into the

title product. The mixture was reﬂuxed for 3 h puriﬁ-

cation by trituration with from petroleum ether aﬀor-

ded 1o (1.40 g, 90%) as a pink solid. R_f=0.29 (CHCl₃/

MeOH 8.5:1.5); mp 201/202 ^ꢂC; IR 1660, 3160, 3340;

UV l(log e) 253.4 (4.12), 281.6 (4.10), 294.4 (3.98),

1

302.8 (3.88); H NMR (CDCl₃) 3.89 (s, 2H), 4.06 (br s,

2H, NH₂exch. with D₂O), 6.91 (d, 1H), 7.24 (dd, 1H),

7.44–7.54 (m, 2H), 7.55 (s, 1H), 7.66 (d, 1H), 8.03 (br s,

258

J.-M. Mussinu et al. / Bioorg. Med. Chem. 11 (2003) 251–263

1H, NH exch. with D₂O); ¹³C NMR (DEPT, CDCl₃/

DMSO) 28.52 (CH₂), 118.87 (CH), 125.56 (CH), 125.96

(CH), 127.43 (CH), 128.91 (CH), 129.31 (CH), 126.92

(C), 128.57 (C), 130.33 (C), 131.59 (C), 134.56 (C), 135.00

(C), 139.85 (C), 149.38 (C), 149.94 (C), 160.68 (C); API-

ES calcd for C₁₇H₁₁Cl₃N₄O 393.6, found 393.5 and anal.

calcd: C, 51.87; H, 2.82; Cl, 27.02; N, 14.23. Found: C,

51.83; H, 2.70; Cl, 27.21; N, 14.29.

(petroleum ether/EtOAc 1:1); mp 201/202 ^ꢂC; IR 1730,

3360; UV l(log e) 263.6 (4.36), 282.2 (4.27), 294.4

1

(4.12), 303.0 (4.01); H NMR (CDCl₃) d 1.97 (s, 3H),

2.15 (s, 3H), 3.94 (s, 2H), 6.92 (d, 1H), 7.23 (dd, 1H), 7.51

(dd, 1H), 7.55 (s, 1H), 7.59 (s, 1H), 7.68 (d, 1H), 9.51 (br s,

1H, NH exch. with D₂O); ¹³C NMR (DEPT, CDCl₃) 16.62

(CH₃), 25.55 (CH₃), 29.80 (CH₂), 119.75 (CH), 126.73

(CH), 126.99 (CH), 128.26 (CH), 129.63 (CH), 130.56

(CH), 128.79 (C), 129.53 (C), 131.78 (C), 133.11 (C), 135.62

(C), 136.27 (C), 141.09 (C), 150.97 (C), 151.25 (C), 154.93

(C), 157.29 (C); API-ES calcd for C₂₀H₁₅Cl₃N₄O 433.7,

found 433.6 and anal. calcd: C, 55.38; H, 3.49; Cl, 24.52; N,

12.92. Found: C, 55.60; H, 3.32; Cl, 24.39; N, 12.87.

6-Chloro-1-(2^I,4^I-dichlorophenyl)-N-(4-methylpiperazin-1-

yl)-1,4-dihydroindeno[1,2-c]pyrazole-3-carboxamide 1q.

General procedure I was used to convert 5a and

N-amino-N-methyl piperazine into the title product.

The mixture was reﬂuxed for 3 h puriﬁcation by crys-

tallization from acetone containing a few drop of

EtOAc aﬀorded 1q (1.42 g, 60%) as a colourless solid.

R_f=0.61 (CHCl₃/MeOH 8.5:1.5); mp 172/173 ^ꢂC; IR

1695, 3300; UV l(log e) 255.8 (4.36), 265.6 (4.35), 280.4

General procedure II: synthesis of carboxylic acids

To a mixture of ester 4 (1.0 equiv, 5 mmol) in methanol

(25 mL) was added a solution of potassium hydroxide

(2.0 equiv) in methanol (18 mL). The resulting mixture

was heated under reﬂux overnight. The mixture was

allowed to cool to room temperature and then poured

into water and acidiﬁed with 1N hydrochloric acid. The

precipitate was ﬁltered, washed with water and air-dried

to yield the analytically pure acid.

1

(4.33), 282.2 (4.32), 295.0 (4.20), 303.0 (4.11); H NMR

(CDCl₃) d 2.33 (s, 3H), 2.60–2.74 (m, 4H), 2.90–3.09 (m,

4H), 3.89 (s, 2H), 6.90 (d, 1H), 7.22 (dd, 1H), 7.45–7.58

(m, 3H), 7.60 (br s, 1H, NH exch. with D₂O), 7.67 (d,

1H); ¹³C NMR (DEPT, CDCl₃) 45.76 (CH₃), 29.74

(CH₂), 54.31 (CH₂ꢃ2), 55.58 (CH₂ꢃ2), 119.71 (CH),

126.66 (CH), 126.93 (CH), 128.26 (CH), 129.63 (CH),

130.48 (CH), 128.69 (C), 129.48 (C), 131.69 (C), 133.00

(C), 135.48 (C), 136.24 (C), 141.13 (C), 151.10 (C),

151.14 (C), 158.87 (C); API-ES calcd for C₂₂H₂₀Cl₃N₅O

476.8, found 476.7 and anal. calcd: C, 55.42; H, 4.23; Cl,

22.31; N, 14.69. Found: C, 55.26; H, 4.50; Cl, 22.20; N,

14.67.

6-Chloro-1-(2^I,4^I-dichlorophenyl)-1,4-dihydroindeno[1,2-

c]pyrazole-3-carboxylic acid 5a. General procedure II

was used to convert 4a into the title product 5a (1.84 g,

97%) as a bright yellow solid. R_f=0.60 (CHCl₃/MeOH

6:4); mp 263/265 ^ꢂC; IR 1590, 1710, 3440; ¹H NMR

(CDCl₃/DMSO) d 3.43 (br s, 1H, OH exch. with D₂O),

3.86 (s, 2H), 6.94 (d, 1H), 7.24 (d, 1H), 7.47 (dd, 1H),

7.55 (s, 1H), 7.59 (s, 1H), 7.64 (dd, 1H). Anal. calcd for

C₁₇H₉Cl₃N₂O₂: C, 53.79; H, 2.39; Cl, 28.02; N,7.38.

Found: C, 53.65; H, 2.59; Cl, 28.33; N,7.22.

6-Chloro-1-(2^I,4^I-dichlorophenyl)-N-[(1-ethylpyrrolidin-

2-yl)methyl]-1,4-dihydroindeno[1,2-c] pyrazole-3-carbox-

amide 1r. General procedure I was used to convert 5a

and N-ethyl-2-aminomethylpirrolidine into the title

product. The mixture was reﬂuxed for 3 h puriﬁcation

by crystallization from ethyl acetate aﬀorded 1r (1.54g,

79%) as a colourless solid. R_f=0.64 (CHCl₃/MeOH

8.5:1.5); mp 213–215 ^ꢂC; IR 1655, 3390; UV l(log e)

253.6 (4.46), 265.4 (4.43), 279.4 (4.41), 294.6 (4.28),

303.0 (4.19); ¹H NMR (CDCl₃) d 1.12 (t, 3H), 1.60–2.01

(m, 4H), 2.10–2.40 (m, 2H), 2.63–3.00 (m, 2H), 3.13–

3.35 (m, 2H), 3.65–3.80 (m, 1H), 3.89 (s, 2H), 6.91 (d,

1H), 7.18–7.38 (m, 2H, NH exch. with D₂O), 7.48 (dd,

1H), 7.51–7.61 (m, 2H), 7.66 (d, 1H); ¹³C NMR (DEPT,

CDCl₃) 13.98 (CH₃), 22.87 (CH₂), 28.58 (CH₂), 29.71

(CH₂), 41.30 (CH₂), 48.54 (CH₂), 53.69 (CH₂), 62.78

(CH), 119.74 (CH), 126.71 (CH), 126.94 (CH), 128.24

(CH), 129.63 (CH), 130.54 (CH), 128.22 (C), 129.77 (C),

131.69 (C), 132.88 (C), 135.80 (C), 136.06 (C), 142.11

(C), 150.74 (C), 151.20 (C), 161.97 (C); API-ES calcd

for C₂₄H₂₃Cl₃N₄O 489.8, found 489.7 and anal. calcd:

C, 55.85; H, 4.73; Cl, 21.71; N, 11.44. Found: C, 58.68;

H, 4.88; Cl, 21.61; N, 11.49.

1-(2^I,4^I-Dichlorophenyl)-6-ﬂuoro-1,4-dihydroindeno[1,2-

c]pyrazole-3-carboxylic acid 5b. General procedure II

was used to convert 4b into the title product 5b (1.78 g,

98%) as a colourless solid. R_f=0.25 (petroleum ether/

EtOAc 6:4); mp 274/275 ^ꢂC; IR 1600, 1700, 3435; H

1

NMR (CDCl₃/DMSO) d 2.68 (br s, 1H, OH exch. with

D₂O), 3.86 (s, 2H), 6.95 (d, 1H), 7.27 (d, 1H), 7.47 (d,

1H), 7.56 (s, 1H), 7.64 (m, 2H). Anal. calcd for

C₁₇H₉Cl₂FN₂O₂: C, 56.22; H, 2.50; Cl, 19.52F, 5.23; N,

7.71. Found: C, 56.10; H, 2.32; Cl, 19.72; F, 5. 32; N,

7.66.

6-Bromo-1-(2^I,4^I-dichlorophenyl)-1,4-dihydroindeno[1,2-

c]pyrazole-3-carboxylic acid 5c. General procedure II

was used to convert 4c into the title product 5c (1.80 g,

96%) as a lighter brown solid. R_f=0.49 (CHCl₃/MeOH,

9:1); mp 253/254 ^ꢂC; IR 1590, 1720, 3400; ¹H NMR

(DMSO/TFA) d 3.58 (br s, 1H, OH exch. with D₂O),

3.85 (s, 2H), 6.90 (d, 1H), 7.25 (s, 1H), 7.43 (dd, 1H),

7.62 (dd, 1H), 7.70–7.87 (m, 2H). Anal. calcd for

C₁₇H₉BrCl₂N₂O₂: C, 48.15; H, 2.14; Br, 18.84; Cl,

16.72 N, 6.61. Found: C, 48.33; H, 2.21; Br, 18.77; Cl,

16.70 N, 6.43.

6-Chloro-1-(2^I,4^I-dichlorophenyl)-N’-(1-methylethyli-

dene)-1,4-dihydroindeno[1,2-c]pyrazole-3-carbohydrazide

1p. Compound 1o (0.5 g, 1.27 mmol) was dissolved in

ethyl acetate (5 mL) and acetone (2 mL). The reaction

mixture was stirred at reﬂux for 1 h and then cooled to

room temperature. The crystallized product was ﬁltered

oﬀ to give the title compound 1p (0.46g, 84%). R_f=0.23

1-(2^I,4^I-Dichlorophenyl)-6-iodo-1,4-dihydroindeno[1,2-

c]pyrazole-3-carboxylic acid 5d. General procedure II

was used to convert 4d into the title product. Because of

J.-M. Mussinu et al. / Bioorg. Med. Chem. 11 (2003) 251–263

259

the low solubility of 4d in methanol, a mixture of tetra-

hydrofuran/dioxane/ethanol (28/28/18.5 mL) was used.

Compound 5d (2.17 g, 92%) was isolated as a yellow

solid. R_f=0.38 (petroleum ether/EtOAc, 1:1); mp

calcd for C₁₈H₁₂Cl₂N₂O₃: C, 57.62; H, 3.22; Cl, 18.90;

N, 7.47. Found: C, 57.53; H, 3.17; Cl, 18.80; N, 7.66.

6-Chloro-1-(4^I-chlorophenyl)-1,4-dihydroindeno[1,2-c]pyr-

azole-3-carboxylic acid 5j. General procedure II was

used to convert 4j into the title product 5j (1.69 g,

98%) as a white solid. R_f=0.76 (CHCl₃/MeOH,

292 ^ꢂC; IR 1590, 1710, 3380; H NMR (DMSO) d 3.67

1

(br s, 1H, OH exch. with D₂O), 3.83 (s, 2H), 6.77 (d,

1H), 7.60–7.94 (m, 4H), 8.03 (d, 1H). Anal. calcd for

C₁₇H₉Cl₂IN₂O₂: C, 43.34; H, 1.93; Cl, 15.05; I, 26.94,

16.72 N, 5.95. Found: C, 43.38; H, 1.76; Cl, 15.35; I,

27.03, N, 5.75.

8.5/1.5); mp 258 ^ꢂC; IR 1720, 3440; H NMR (CDCl₃/

1

DMSO) d 3.83 (s, 2H), 3.99 (br s, 1H, OH exch. with

D₂O), 7.23–7.44 (m, 3H), 7.52–7.66 (m, 3H), 7.71

(d,1H). Anal. calcd for C₁₇H₁₀Cl₂N₂O₂: C, 59.15; H,

2.92; Cl, 20.54; N, 8.12. Found: C, 59.39; H, 3.04; Cl,

20.33; N, 8.26.

5-Chloro-1-(2^I,4^I-dichlorophenyl)-1,4-dihydroindeno[1,2-

c]pyrazole-3-carboxylic acid 5e. General procedure II

was used to convert 4e into the title product 5e (1.83 g,

96%) as a cream solid. R_f=0.61 (CHCl₃/MeOH, 9:1);

mp 247/249 ^ꢂC; IR 1610, 1720, 3420; ¹H NMR (CDCl₃/

DMSO) d 3.87 (s, 2H), 4.25 (br s, 1H, OH exch. with

D₂O ), 6.93 (d, 1H), 7.18–7.34 (m, 2H), 7.49 (s, 1H),

7,57–7.72 (m, 2H). Anal. calcd for C₁₇H₉Cl₃N₂O₂: C,

53.79; H, 2.39; Cl, 28.02; N,7.38. Found: C, 53.68; H,

2.52; Cl, 28.35; N,7.20.

6-Chloro-1-phenyl-1,4-dihydroindeno[1,2-c]pyrazole-3-

carboxylic acid 5k. General procedure II was used to

convert 4k into the title product 5k (1.50 g, 97%) as a

colourless solid. R_f=0.73 (CHCl₃/MeOH, 8.5/1.5); mp

247–248 ^ꢂC; IR 1720, 3400; ¹H NMR (CDCl₃/DMSO) d

3.84 (s, 2H), 7.21–7.47 (m, 2H), 7.48–7.63 (m, 6H, OH

exch. with D₂O), 7.75 (d, 1H). Anal. calcd for

C₁₇H₁₁ClN₂O₂: C, 65.71; H, 3.57; Cl, 11.41; N, 9.02.

Found: C, 65.80; H, 3.41; Cl, 11.27; N, 9.22.

7-Chloro-1-(2^I,4^I-dichlorophenyl)-1,4-dihydroindeno[1,2-

c]pyrazole-3-carboxylic acid 5f. General procedure II

was used to convert 4f into the title product 5f (1.74 g,

92%) as a cream solid. R_f=0.38 (CHCl₃/MeOH, 9:1);

mp >300 ^ꢂC dec.; IR 1610, 1700, 3410; ¹H NMR

(CDCl₃/DMSO) d 3.68 (br s, 1H, OH exch. with D₂O ),

3.83 (s, 2H), 6.93 (d, 1H), 7.27 (dd, 1H), 7.46–7.73 (m,

4H). Anal. calcd for C₁₇H₉Cl₃N₂O₂: C, 53.79; H, 2.39;

Cl, 28.02; N,7.38. Found: C, 53.78; H, 2.33; Cl, 28.25;

N,7.22.

6-Chloro-1-(4^I-methoxyphenyl)-1,4-dihydroindeno[1,2-

c]pyrazole-3-carboxylic acid 5l. General procedure II

was used to convert 4l into the title product. Because of

the low solubility of 4l in methanol, a mixture of

methanol/tetrahydrofuran (45/12 mL) was used. Com-

pound 5l (1.56 g, 92%) was isolated as a yellow solid.

R_f=0.52 (CHCl₃/MeOH, 8.5/1.5); mp 245–247 ^ꢂC; IR

1

1710, 3410; H NMR (CDCl₃/DMSO) d 3.82 (s, 2H),

3.91 (s, 3H), 6.51 (br s, 1H, OH exch. with D₂O), 7.07

(d, 2H), 7.22–7.40 (m, 2H), 7.53 (s, 1H), 7.63 (d, 2H).

Anal. calcd for C₁₈H₁₃ClN₂O₂: 63.44; H, 3.85; Cl, 10.40;

N, 8.22. Found: 63.54; H, 3.66; Cl, 10.61; N, 8.11.

1-(2^I,4^I-Dichlorophenyl)-1,4-dihydroindeno[1,2-c]pyra-

zole-3-carboxylic acid 5g. General procedure II was

used to convert 4g into the title product 5g (1.32 g,

75%) as a cream solid. R_f=0.48 (CHCl₃/MeOH, 8/2);

mp 271–272 ^ꢂC; IR 1705, 3350; ¹H NMR (CDCl₃/

DMSO) d 3.49(br s, 1H, OH exch. with D₂O), 3.82 (s,

2H), 6.99 (d, 1H), 7.20–7.39 (m, 3H), 7.58 (d, 1H), 7.66

(s, 1H), 7.73 (d, 1H). Anal. calcd for C₁₇H₁₀Cl₂N₂O₂: C,

59.15; H, 2.92; Cl, 20.54; N, 8.12. Found: C, 59.33; H,

2.81; Cl, 20.42; N, 8.23.

General procedure III: synthesis of tricyclic esters

To a stirred mixture of diketoester 3 (1.0 equiv, 4 mmol)

and the requisite phenyl hydrazine hydrochloride (1.15

equiv) in EtOH (28 mL) was heated under reﬂux for 2–5

h. The reaction was allowed to cool to room tempera-

ture and the insoluble material was collected by ﬁltra-

tion and washed with a small volume of ice-cool

ethanol. Puriﬁcation by ﬂash chromatography aﬀorded

the analytically pure product.

1-(2^I,4^I-Dichlorophenyl)-6-methyl-1,4-dihydroindeno[1,2-

c]pyrazole-3-carboxylic acid 5h. General procedure II

was used to convert 4h into the title product 5h (1.78 g,

98%) as a cream solid. R_f=0.49 (CHCl₃/MeOH, 9/1);

ꢂ

1

mp 267 C; IR 1710, 3440; H NMR (CDCl₃/DMSO) d

2.40 (s, 3H), 2.74 (br s, 1H, OH exch. with D₂O), 3.81

(s, 2H), 6.91 (d, 1H), 7.07 (d, 1H), 7.38 (s, 1H), 7.45 (dd,

1H), 7.58 (s, 1H), 7.63 (d, 1H). Anal. calcd for

C₁₈H₁₂Cl₂N₂O₂: C, 60.19; H, 3.37; Cl, 19.74; N, 7.80.

Found: C, 60.02; H, 3.17; Cl, 19.60; N, 7.96.

Ethyl 6-chloro-1-(2^I,4^I-dichlorophenyl)-1,4-dihydroin-

deno[1,2-c]pyrazole-3-carboxylate 4a. General proce-

dure

III

was

used

to

convert

3a

and

2,4-dichlorophenylhydrazine hydrochloride into the

title product. The mixture was heated at reﬂux for 5 h.

Puriﬁcation by ﬂash chromatography (petroleum

ether/EtOAc, 8.5/1.5) aﬀorded 4a (1.12 g, 80%) as a

oﬀ-white solid. R_f=0.54 (petroleum ether/EtOAc, 8:2);

mp 210–212 ^ꢂC (triturated with petroleum ether); IR

1-(2^I,4^I-Dichlorophenyl)-6-methoxy-1,4-dihydroindeno[1,2-

c]pyrazole-3-carboxylic acid 5i. General procedure II

was used to convert 4i into the title product 5i (1.61 g,

86%) as a white solid. R_f=0.45 (CHCl₃/MeOH, 9/1);

mp 274–275 ^ꢂC; IR 1710, 3400; ¹H NMR (CDCl₃/

DMSO) d 3.00 (br s, 1H, OH exch. with D₂O), 3.82 (s,

2H), 3.85 (s, 3H), 6.79 (dd, 1H), 6.92 (d, 1H), 7.12 (s,

1H), 7.45 (dd, 1H), 7.59 (s, 1H), 7.64 (d, 1H). Anal.

1

1710; H NMR (CDCl₃) d 1.45 (t, 3H), 3.85 (s, 2H),

4.41–4.54 (q, 2H), 6.92 (d, 1H), 7.24 (d, 1H), 7.46

(dd, 1H), 7.55 (s, 1H), 7.57 (s, 1H), 7.63 (dd, 1H).

Anal. calcd for C₁₉H₁₃Cl₃N₂O₂: C, 55.98; H, 3.21; Cl,

26.09; N, 6.87. Found: C, 55.77; H, 3.20; Cl, 26.39;

N, 6.55.

260

J.-M. Mussinu et al. / Bioorg. Med. Chem. 11 (2003) 251–263

Ethyl 1-(2^I,4^I-dichlorophenyl)-6-ﬂuoro-1,4-dihydroin-

deno[1,2-c]pyrazole-3-carboxylate 4b. General proce-

mixture was heated at reﬂux for 3 h. Puriﬁcation by

ﬂash chromatography (petroleum ether/EtOAc, 8.5/1.5)

aﬀorded 4f (0.80 g, 58%) as a yellowish solid. R_f=0.40

(petroleum ether/EtOAc, 8/2); mp 193–195 ^ꢂC (tritu-

rated with petroleum ether); IR 1730; ¹H NMR (CDCl₃)

d 1.45 (t, 3H), 3.84 (s, 2H), 4.40–4.55 (q, 2H), 6.95 (d,

1H), 7.28 (dd, 1H), 7.43–7.54 (m, 2H), 7.58 (d, 1H), 7.67

(d, 1H). Anal. calcd for C₁₉H₁₃Cl₃N₂O₂: C, 55.98; H,

3.21; Cl, 26.09; N, 6.87. Found: C, 55.71; H, 3.22; Cl,

26.22; N, 6.74.

dure

III

was

used

to

convert

3b

and

2,4-dichlorophenylhydrazine hydrochloride into the title

product. The mixture was heated at reﬂux for 3 h. Pur-

iﬁcation by ﬂash chromatography (petroleum ether/

EtOAc, 8/2) aﬀorded 4b (1.15 g, 85%) as a colourless

solid. R_f=0.59 (petroleum ether/EtOAc, 8/2); mp 197 ^ꢂC

(triturated with petroleum ether); IR 1725; ¹H NMR

(CDCl₃) d 1.45 (t, 3H), 3.86 (s, 2H), 4.40–4.55 (q, 2H),

6.95 (d, 1H), 7.28 (d, 1H), 7.45 (dd, 1H), 7.57 (s, 1H), 7.61

(s, 1H), 7.64 (dd, 1H). Anal. calcd for C₁₉H₁₃FCl₂N₂O₂:

C, 58.33; H, 3.35; Cl, 18.12; F, 4.86; N, 7.16. Found: C,

58.61; H, 3.25; Cl, 18.15; F, 4.77; N, 7.38.

Ethyl 1 - (2^I,4^I- dichlorophenyl) - 1,4 - dihydroindeno[1,2 -

c]pyrazole-3-carboxylate 4g. General procedure III was

used to convert 3g and 2,4-dichlorophenylhydrazine

hydrochloride into the title product. The mixture was

heated at reﬂux for 3 h. Puriﬁcation by ﬂash chromato-

graphy (petroleum ether/EtOAc, 8.5/1.5) aﬀorded 4g

(0.90 g, 70%) as a oﬀ-white solid. R_f=0.63 (petroleum

ether/EtOAc, 8/2); mp 165 ^ꢂC (triturated with petro-

Ethyl 6-bromo-1-(2^I,4^I-dichlorophenyl)-1,4-dihydroin-

deno[1,2-c]pyrazole-3-carboxylate 4c. General procedure

III was used to convert 3c and 2,4-dichlorophenylhy-

drazine hydrochloride into the title product. The mix-

ture was heated at reﬂux for 5 h. Puriﬁcation by ﬂash

chromatography (petroleum ether/EtOAc, 8.5/1.5)

aﬀorded 4c (1.27 g, 80%) as a yellow solid. R_f=0.54

(petroleum ether/EtOAc, 8/2); mp 208–209 ^ꢂC (tritu-

rated with petroleum ether); IR 1715; ¹H NMR (CDCl₃)

d 1.45 (t, 3H), 3.85 (s, 2H), 4.41–4.55 (q, 2H), 6.86 (d,

1H), 7.39 (dd, 1H), 7.49 (dd, 1H), 7.56 (s, 1H), 7.61 (s,

1H), 7.67 (dd, 1H). Anal. calcd for C₁₉H₁₃BrCl₂N₂O₂C,

50.47; H, 2.90; Br, 17.67; Cl, 15.68; N, 6.20. Found C,

50.57; H, 3.03; Br, 17.55; Cl, 15.49; N,6.55.

1

leum ether); IR 1710; H NMR (CDCl₃) d 1.45 (t, 3H),

3.88 (s, 2H), 4.41–4.55 (q, 2H), 7.01 (d, 1H), 7.31 (m,

3H), 7.44 (dd, 1H), 7.56 (d, 1H), 7.62 (d, 1H). Anal.

calcd for C₁₉H₁₄Cl₂N₂O₂: C, 61.14; H, 3.78; Cl, 19.00;

N, 7.51. Found: C, 61.22; H, 3.68; Cl, 19.21; N, 7.45.

Ethyl 1-(2^I,4^I-dichlorophenyl)-6-methyl-1,4-dihydroin-

deno[1,2-c]pyrazole-3-carboxylate 4h. General proce-

dure

III

was

used

to

convert

3h

and

2,4-dichlorophenylhydrazine hydrochloride into the title

product. The mixture was heated at reﬂux for 3 h. Pur-

iﬁcation by ﬂash chromatography (petroleum ether/

EtOAc, 9.5/0.5) aﬀorded 4h (1.25 g, 93%) as a colour-

less solid. R_f=0.55 (petroleum ether/EtOAc, 9/1); mp

Ethyl 1 - (2^I,4^I- dichlorophenyl) - 6 - iodo - 1,4 - dihydroin-

deno[1,2-c]pyrazole-3-carboxylate 4d. General proce-

dure

III

was

used

to

convert

3d

and

199 ^ꢂC (triturated with petroleum ether); IR 1715; H

1

2,4-dichlorophenylhydrazine hydrochloride into the title

product. The mixture was heated at reﬂux for 4 h. Pur-

iﬁcation by ﬂash chromatography (petroleum ether/

EtOAc, 8/2) aﬀorded 4d (1.44 g, 81%) as a yellow solid.

R_f=0.42 (petroleum ether/EtOAc, 8/2); mp 236–238 ^ꢂC

NMR (CDCl₃) d 1.45 (t, 3H), 2.41 (s, 3H), 3.82 (s, 2H),

4.40–4.54 (q, 2H), 6.90 (d, 1H), 7.06 (d, 1H), 7.39 (s,

1H), 7.44 (dd, 1H), 7,61 (s, 1H), 7.63 (d, 1H). Anal.

calcd for C₂₀H₁₆Cl₂N₂O₂: C, 62.03; H, 4.16; Cl, 18.31;

N, 7.23. Found: C, 62.00; H, 4.01; Cl, 18.20; N, 7.44.

1

(triturated with ethanol); IR 1720; H NMR (CDCl₃) d

1.45 (t, 3H), 3.83 (s, 2H), 4.38–4.53 (q, 2H), 6.75 (d,

1H), 7.25 (s, 1H), 7.45 (dd, 1H), 7.56 (s, 1H), 7,62 (dd,

1H), 7.92 (s, 1H). Anal. calcd for C₁₉H₁₃Cl₂IN₂O₂: C,

45.72; H, 2.63; Cl, 14.21; I, 25.43; N, 5.61. Found: C,

45.59; H, 2.50; Cl, 14.28; I, 25.31; N, 5.79.

Ethyl 1-(2^I,4^I-dichlorophenyl)-6-methoxy-1,4-dihydroin-

deno[1,2-c]pyrazole-3-carboxylate 4i. General procedure

III was used to convert 3i and 2,4-dichlorophenylhy-

drazine hydrochloride into the title product. The mix-

ture was heated at reﬂux for 4 h. Puriﬁcation by ﬂash

chromatography (petroleum ether/EtOAc, 9/1) aﬀorded

4i (1.20 g, 85%) as a colourless solid. R_f=0.62 (petro-

leum ether/EtOAc, 8/2); mp 168–169 ^ꢂC (triturated with

Ethyl 5-chloro-1-(2^I,4^I-dichlorophenyl)-1,4-dihydroin-

deno[1,2-c]pyrazole-3-carboxylate 4e. General procedure

III was used to convert 3e and 2,4-dichlorophenylhy-

drazine hydrochloride into the title product. The mixture

was heated at reﬂux for 2 h. Puriﬁcation by ﬂash chro-

matography (petroleum ether/EtOAc, 9/1) aﬀorded 4e

(0.93 g, 63%) as a oﬀ-white solid. R_f=0.63 (petroleum

ether/EtOAc, 8:2); mp 191–193 ^ꢂC (triturated with pet-

roleum ether); IR 1715; ¹H NMR (CDCl₃) d 1.46 (t, 3H),

3.88 (s, 2H), 4.43–4.56 (q, 2H), 6.92 (d, 1H), 7.17–7.34 (m,

2H), 7.46 (d, 1H), 7.59 (d, 1H), 7.65 (d, 1H). Anal. calcd

for C₁₉H₁₃Cl₃N₂O₂: C, 55.98; H, 3.21; Cl, 26.09; N, 6.87.

Found: C, 55.76; H, 3.42; Cl, 26.31; N, 6.85.

1

petroleum ether); IR 1720; H NMR (CDCl₃) d 1.44 (t,

3H), 3.83 (s, 2H), 3.84 (s, 3H), 4.40–4.54 (q, 2H), 6.78

(dd, 1H), 6.91 (d, 1H), 7.12 (d, 1H), 7.44 (dd, 1H), 7,59

(d, 1H), 7.63 (d, 1H). Anal. calcd for C₂₀H₁₆Cl₂N₂O₃:

C, 59.57; H, 4.00; Cl, 17.58; N, 6.95. Found: C, 59.38;

H, 4.13; Cl, 17.29; N, 7.10.

Ethyl 6-chloro-1-(4^I-chlorophenyl)-1,4-dihydroindeno[1,2-

c]pyrazole-3-carboxylate 4j. General procedure III was

used to convert 3a and 4-chlorophenylhydrazine hydro-

chloride into the title product. The mixture was heated

at reﬂux for 2.5 h. Puriﬁcation by ﬂash chromatography

(petroleum ether/EtOAc, 9.5/0.5) aﬀorded 4j (1.20 g,

91%) as a yellow solid. R_f=0.78 (petroleum ether/EtOAc,

8.5/1.5); mp 197–198 ^ꢂC (triturated with ethanol); IR

Ethyl 7-chloro-1-(2^I,4^I-dichlorophenyl)-1,4-dihydroin-

deno[1,2-c]pyrazole-3-carboxylate 4f. General procedure

III was used to convert 3f and 2,4-dichlorophenyl-

hydrazine hydrochloride into the title product. The

J.-M. Mussinu et al. / Bioorg. Med. Chem. 11 (2003) 251–263

261

1

1590, 1730; H NMR (CDCl₃) d 1.45 (t, 3H), 3.82 (s,

for 2 h at room temperature. Compound 3b (1.26g,

84%) was isolated as a oﬀ-white solid. R_f=0.51 (petro-

leum ether/EtOAc, 8/2); mp 107 ^ꢂC (triturated with

2H), 4.40–4.55 (q, 2H), 7.22–7.38 (m, 3H), 7.50–7.63 (m,

3H), 7.68 (d, 1H). Anal. calcd for C₁₉H₁₄Cl₂N₂O₂: C,

61.14; H, 3.78; Cl, 19.00; N, 7.51. Found: C, 61.02; H,

3.58; Cl, 19.21; N, 7.44.

1

petroleum ether); IR 1590, 1680, 1720, 3430; H NMR

(CDCl₃) d 1.44 (t, 3H), 3.99 (s, 2H), 4.35–4.52 (q, 2H),

7.08 (m, 2H), 7.87 (t, 1H), 13.18 (br s, 1H, OH exch.

with D₂O). Anal. calcd for C₁₃H₁₁FO₄: C, 62.40; H,

4.43; F, 7.59. Found: C, 62.33; H, 4.41; F, 7.69.

Ethyl 6-chloro-1-phenyl-1,4-dihydroindeno[1,2-c]pyra-

zole-3-carboxylate 4k. General procedure III was used

to convert 3a and phenylhydrazine hydrochloride into

the title product. The mixture was heated at reﬂux for

2.5 h. Puriﬁcation by ﬂash chromatography (petroleum

ether/EtOAc, 9.5/0.5) aﬀorded 4k (1.14 g, 97%) as a oﬀ-

white solid. R_f=0.63 (petroleum ether/EtOAc, 8.5/1.5);

mp 155–157 ^ꢂC (triturated with petroleum ether); IR

Ethyl ꢂ-(5-bromo-1-oxo-2,3-dihydro-1H-inden-2-yl)-ꢀ-

oxo-acetate 3c. General procedure IV was used to con-

vert 2c into the title product. The mixture was stirred

for 8 h at room temperature. Compound 3c (1.69 g,

91%) was isolated as a yellowish solid. R_f=0.29 (petro-

leum ether/EtOAc, 8/2); mp 107 ^ꢂC (triturated with

1

1600, 1710; H NMR (CDCl₃) d 1.46 (t, 3H), 3.84 (s,

1

2H), 4.42–4.56 (q, 2H), 7.24 (s, 1H), 7.37 (d, 1H), 7.44–

7.66 (m, 5H), 7.74 (d, 1H). Anal. calcd for

C₁₉H₁₅ClN₂O₂: C, 67.36; H, 4.46; Cl, 10.46; N, 8.27.

Found: C, 67.22; H, 4.55; Cl, 10.25; N, 8.32.

petroleum ether); IR 1600, 1670, 1720, 3400; H NMR

(CDCl₃) d 1.43 (t, 3H), 3.98 (s, 2H), 4.35–4.52 (q, 2H),

7.57 (d, 1H), 7.70–7.82 (m, 2H), 12.95 (br s, 1H, OH

exch. with D₂O). Anal. calcd for C₁₃H₁₁BrO₄: C, 50.18;

H, 3.56; Br, 25.68. Found: C, 50.43; H, 3.61; Br, 25.44.

Ethyl 6 - chloro - 1 - (4^I- methoxyphenyl) - 1,4 - dihydroin-

deno[1,2-c]pyrazole-3-carboxylate 4l. General procedure

III was used to convert 3a and 4-methoxyphenylhy-

drazine hydrochloride into the title product. The mix-

ture was heated at reﬂux for 3 h. Puriﬁcation by ﬂash

chromatography (petroleum ether/EtOAc, 8/2) aﬀorded

4l (1.08 g, 84%) as a yellow product. R_f=0.52 (petro-

leum ether/EtOAc, 8/2); mp 199–200 ^ꢂC (triturated with

Ethyl ꢂ-(5-iodo-1-oxo-2,3-dihydro-1H-inden-2-yl)-ꢀ-

oxo-acetate 3d. General procedure IV was used to con-

vert 2d into the title product. The mixture was stirred

for 6 h at room temperature. Compound 3d (2.0 g,

66%) was isolated as a yellow solid. R_f=0.40 (petro-

leum ether/EtOAc, 8/2); mp 117–118 ^ꢂC (triturated with

1

petroleum ether); IR 1595, 1670, 1720, 3400; H NMR

1

petroleum ether); IR 1700; H NMR (CDCl₃) d 1.45 (t,

(CDCl₃) d 1.43 (t, 3H), 3.97 (s, 2H), 4.36–4.51 (q, 2H),

7.58 (d, 1H), 7.81 (d, 1H), 7.97 (s, 1H), 14.22 (br s, 1H,

OH exch. with D₂O). Anal. calcd for C₁₃H₁₁IO₄: C,

43.60; H, 3.10; I, 35.44. Found: C, 43.65; H, 3.22; I,

35.32.

3H), 3.82 (s, 2H), 3.90 (s, 3H), 4.40–4.55 (q, 2H), 7.06 (d,

2H), 7.22–7.36 (m, 2H), 7.54 (s, 1H), 7.62 (d, 2H,). Anal.

calcd for C₂₀H₁₇ClN₂O₃: C, 65.13; H, 4.65; Cl, 9.61; N,

7.60. Found: C, 65.19; H, 4.52; Cl, 9.55; N, 7.41.

Ethyl ꢂ-(4-Chloro-1-oxo-2,3-dihydro-1H-inden-2-yl)-ꢀ-

oxo-acetate 3e. General procedure IV was used to con-

vert 2e into the title product. The mixture was stirred for

2 h at room temperature. Compound 3e (1.1 g, 69%) was

isolated as a green solid. R_f=0.21 (petroleum ether/

EtOAc, 8/2); mp 86 ^ꢂC (triturated with petroleum ether);

General procedure IV: synthesis of ꢀ,ꢁ-diketoesters

Sodium metal (2.0 equiv) was added in small portion to

dry ethanol (5 mL) and stirred until all the sodium had

reacted. Ethyl oxalate (1.0 equiv) was added, followed

by dropwise addition of a solution of appropriate inda-

none starting material (1.0 equiv, 6 mmol) in dry etha-

nol (30 mL). The solution was stirred at room

temperature for 2–8 h. The mixture was slowly poured

over 2 N hydrochloride acid and the resulting pre-

cipitate was collected by ﬁltration and washed with a

small volume of ice-cold ethanol and water. The air-

dried residue aﬀorded the analytically pure product.

1

IR 1590, 1610, 1670, 1720, 2540; H NMR (CDCl₃) d

1.45 (t, 3H), 4.00 (s, 2H), 4.39–4.56 (q, 2H), 7.43 (t, 1H),

7.64 (d, 1H), 7.78 (d, 1H), 9.19 (br s, 1H, OH exch. with

D₂O). Anal. calcd for C₁₃H₁₁ClO₄: C, 58.55; H, 4.16;

Cl, 13.29. Found: C, 58.30; H, 4.19; Cl, 13.25.

Ethyl ꢂ-(6-chloro-1-oxo-2,3-dihydro-1H-inden-2-yl)-ꢀ-

oxo-acetate 3f. General procedure IV was used to con-

vert 2f into the title product. The mixture was stirred for

8 h at room temperature. Compound 3e (1.2 g, 75%)

was isolated as a brownish solid. R_f=0.37 (petroleum

ether/EtOAc, 8/2); mp 128 ^ꢂC (triturated with petro-

Ethyl ꢂ-(5-chloro-1-oxo-2,3-dihydro-1H-inden-2-yl)-ꢀ-

oxo-acetate 3a. General procedure IV was used to con-

vert 2a into the title product. The mixture was stirred

for 5 h at room temperature. Compound 3a (1.36 g, 85%)

was isolated as a yellowish solid. R_f=0.61 (petroleum

ether/EtOAc, 8/2); mp 122–124 ^ꢂC (triturated with petro-

leum ether); IR 1600, 1670, 1710, 3400; ¹H NMR (CDCl₃)

d 1.43 (t, 3H), 3.99 (s, 2H), 4.35–4.49 (q, 2H), 7.43 (d, 1H),

7.55 (s, 1H), 7.80 (s, 1H), 13.28 (br s, 1H, OH exch. with

D₂O). Anal. calcd for C₁₃H₁₁ClO₄: C, 58.55; H, 4.16; Cl,

13.29. Found: C, 58.43; H, 4.23; Cl, 13.22.

1

leum ether); IR 1600, 1730, 3400; H NMR (CDCl₃) d

1.45 (t, 3H), 2.70 (br s, 1H, OH exch. with D₂O), 3.97 (s,

2H), 4.36–4.50 (q, 2H), 7.49 (d, 1H), 7.60 (dd, 1H), 7.83

(s, 1H). Anal. calcd for C₁₃H₁₁ClO₄: C, 58.55; H, 4.16;

Cl, 13.29. Found: C, 58.50; H, 4.09; Cl, 13.33.

Ethyl ꢂ-(1-Oxo-2,3-dihydro-1H-inden-2-yl)-ꢀ-oxo-ace-

tate 3g.²⁴General procedure IV was used to convert 2g

into the title product. The mixture was stirred for 5 h at

room temperature. Compound 3f (1.5 g, 98%) was iso-

lated as a yellow solid. R_f=0.30 (petroleum ether/

Ethyl ꢂ-(5-Fluoro-1-oxo-2,3-dihydro-1H-inden-2-yl)-ꢀ-

oxo-acetate 3b. General procedure IV was used to con-

vert 2b into the title product. The mixture was stirred

262

J.-M. Mussinu et al. / Bioorg. Med. Chem. 11 (2003) 251–263

EtOAc, 9/1); mp 68–70 ^ꢂC (triturated with petroleum

with previously reported spectra.²⁶Anal. calcd for

C₉H₇ClO: C, 64.88; H, 4.23; Cl, 21.28. Found: C, 64.65;

H, 4.11; Cl, 21.12.

ether) (lit.²⁴69–70 ^ꢂC); IR 1610, 1670, 1730, 3420; H

1

NMR (CDCl₃) d 1.44 (t, 3H), 4.00 (s, 2H), 4.36–4.52 (q,

2H), 7.41 (t, 1H), 7.56 (d, 1H), 7.66 (t, 1H), 7.88 (d, 1H),

13.68 (br s, 1H, OH exch. with D₂O). Anal. calcd for

C₁₃H₁₂O₄: C, 67.23; H, 5.21. Found C, 67.13; H, 5.35.

5-Iodoindan-1-one 2d. A solution of NaNO₂(1.2 equiv)

in water (1.1 mL) was added dropwise to a stirred solu-

tion of the 5-amino-indanone²⁷starting material

(1 equiv, 3.1 mmol) in 15% HCl (2.5 mL) at 0 ^ꢂC. To

the resulting solution was added a solution of potassium

iodide (1.2 equiv) in water (2 mL) and the whole was

stirred at room temperature for 1.5 h and then warmed

at 60 ^ꢂC until gas development ceased. The mixture was

allowed to cool to room temperature and then extracted

with ether. The combined organic layers were washed

with 5% solution of sodium tiosulfate, dried over

Na₂SO₄and concentrated under reduced pressure to

aﬀorded a crude oil. Puriﬁcation by ﬂash chromato-

graphy (petroleum ether/EtOAc, 9/1) aﬀorded 2d

(0.75g, 55.5%) as a red oil. R_f=0. 78 (petroleum ether/

EtOAc, 8/2); IR 1590, 1700; ¹H NMR (CDCl₃) d 2.67 (t,

2H), 3.13 (t, 2H), 7.47 (d, 1H), 7.73 (d, 1H), 7.91 (s, 1H).

Anal. calcd for C₉H₇IO: C, 41.89; H, 2.73; I, 49.18.

Found: C, 41.77; H, 2.57; I, 49.31.

Ethyl ꢂ-(5-methyl-1-oxo-2,3-dihydro-1H-inden-2-yl)-ꢀ-

oxo-acetate 3h. General procedure IV was used to con-

vert 2h into the title product. The mixture was stirred for

5 h at room temperature. Compound 3h (1.16 g, 78%)

was isolated as a yellow solid. R_f=0.57 (petroleum ether/

EtOAc, 9:1); mp 110–112 ^ꢂC (triturated with petroleum

1

ether); IR 1620, 1670, 1720, 3420; H NMR (CDCl₃) d

1.43 (t, 3H), 2.48 (s, 3H), 3.94 (s, 2H), 4.43 (q, 2H), 7.25

(d, 1H), 7.34 (s, 1H), 7.75 (d, 1H),13.70 (br s, 1H, OH

exch. with D₂O). Anal. calcd for C₁₄H₁₄O₄: C, 68.28; H,

5.73. Found: C, 68.18; H, 5.52.

Ethyl ꢂ-(5-methoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)-

ꢀ-oxo-acetate 3i. General procedure IV was used to

convert 2i into the title product. The mixture was stirred

for 5 h at room temperature. Compound 3i (1.37 g,

87%) was isolated as a yellow solid. R_f=0.47 (petro-

leum ether/EtOAc, 8/2); mp 110 ^ꢂC (triturated with

Biology

1

petroleum ether); IR 1610, 1660, 1720, 3420; H NMR

(CDCl₃) d 1.43 (t, 3H), 3.92 (s, 3H), 3.94 (s, 2H), 4.34–

4.49 (q, 2H), 6.92–7.04 (m, 2H), 7.79 (d, 1H), 13.19 (br

s, 1H, OH exch. with D₂O). Anal. calcd for C₁₄H₁₄O₅

C, 64.12; H, 5.38. Found: C, 64.02; H, 5.45.

General information. Male CD1 mice weighting 20–25 g,

(Charles River, Calco, LC Italy) were housed in the

animal care quarters maintained at 22ꢁ2 ^ꢂC on a 12 h

light/dark cycle, and food and water were available ad

libitum. All experimental protocols were accepted by

the Ethical Committee at the University of Cagliari and

performed in strict accordance with the E.C. regulation

for care and use of experimental animals (EEC N^ꢂ86/

609).

General procedure V: synthesis of chloroindanones

A solution of NaNO₂(1.2 equiv) in water (2.6 mL) was

added dropwise to a stirred solution of the amino-inda-

none starting material (1 equiv, 7.27 mmol) in 15% HCl

(12 mL) at 0 ^ꢂC. The resulting solution was added to a

stirred solution of CuCl (3.5 equiv) in concd HCl (20

mL) at 0 ^ꢂC. When the addition was complete, the mix-

ture was stirred at room temperature for 2 h, poured

into water and extracted with EtOAc (15 ꢃ 3 mL). The

combined layers were dried over Na₂SO₄and con-

centrated under reduced pressure. Puriﬁcation by ﬂash

chromatography aﬀorded the analytically pure product.

[³H]-CP-55,940 (speciﬁc activity 180 Ci/mmol) was pur-

chased from New England Nuclear (Boston, MA,

USA). CP 55,940, was obtained from Tocris Cookson

Ltd (Bristol UK). For biochemical experiments, drugs

were dissolved in dimethyl-sulfoxide (DMSO). DMSO

concentration in the diﬀerent assays never exceeded 0.1%

(v/v) and was without eﬀect on radioligand binding.

Tissue preparation. Mice were killed by cervical disloca-

tion and the brain (minus cerebellum) for CB₁receptor

and spleen for CB₂receptor were rapidly removed and

placed on an ice-cold plate. After thawing, tissues were

homogenated in 20 vol (w/v) of ice-cold TME buﬀer (50

mM Tris–HCl, 1 mM EDTA and 3.0 mM MgCl₂, pH

7.4). The homogenates were centrifuged at 1086 ꢃ g for

10 min at 4 ^ꢂC, and the resulting supernatants were

centrifuged at 45,000 ꢃ g for 30 min in a Beckman

SW41 swing-out rotor, at 4 ^ꢂC.

4-Chloroindan-1-one 2e. General procedure V was used

to convert 4-amino-indanone²⁵into the title product.

Puriﬁcation by ﬂash chromatography (petroleum ether/

EtOAc, 8.5/1.5) aﬀorded 2e (0.76 g, 63%) as a brown

solid. R_f=0.72 (petroleum ether/EtOAc, 8:2); mp 94–

96 ^ꢂ_ꢂC (triturated with petroleum ether) (lit²⁶mp 93–

with previously reported spectra.²⁶Anal. calcd for

C₉H₇ClO: C, 64.88; H, 4.23; Cl, 21.28. Found: C, 64.59;

H, 4.41; Cl, 21.19.

1

94 C); IR, H NMR, and ¹³C NMR were in agreement

Binding study at CB₁and CB₂receptor. [³H]-CP-55,940

binding was performed by a modiﬁcation of a method

previously described.²⁸Brieﬂy, the membranes (30–80

mg of protein) were incubated with 0.5–1 nM of [³H]-

CP55940 for 1 h at 30 ^ꢂC in a ﬁnal volume of 0.5 mL of

TME buﬀer containing 5 mg/mL of fatty acid-free

bovine serum albumin (BSA). Non-speciﬁc binding was

estimated in the presence of 10 mM of CP55940. All

6-Chloroindan-1-one 2f. General procedure V was used

to convert 6-amino-indanone²⁵into the title product.

Puriﬁcation by ﬂash chromatography (petroleum ether/

EtOAc, 8.5/1.5) aﬀorded 2f (0.98 g, 98%) as a brown

solid. R_f=0.57 (petroleum ether/EtOAc, 8:2); mp 76–

78 ^ꢂC (triturated with petroleum ether) (lit²⁶mp 77–

80 ^ꢂC); IR, H NMR, and ¹³C NMR were in agreement

1

J.-M. Mussinu et al. / Bioorg. Med. Chem. 11 (2003) 251–263

263

binding studies were performed in disposable glass tubes

pre-treated with Sigma-Cote (Sigma Chemical Co. Ltd.,

Poole, UK), in order to reduce non-speciﬁc binding.

The reaction was terminated by rapid ﬁltration through

Whatman GF/C ﬁlters presoaked in 0.5% poly-

ethyleneimine (PEI) using a Brandell 96-sample har-

vester (Gaithersburg, MD, USA). Filters were washed

ﬁve times with 4 mL aliquots of ice cold Tris–HCl buﬀer

(pH 7.4) containing 1 mg/mL BSA The ﬁlter bound

radioactivity was measured in a liquid scintillation

counter (Tricarb 2100, Packard, Meridien, USA) with 4

mL of scintillation ﬂuid (Ultima Gold MV, Packard).

Protein determination was performed by means of

Bradford²⁹protein assay using BSA as a standard

according to the protocol of the supplier (Bio-Rad,

Milan, Italy).

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Data Analysis

All experiments were performed in triplicate and results

were conﬁrmed in at least ﬁve independent experiments.

Data from radioligand inhibition experiments were

analyzed by nonlinear regression analysis of a Sigmoid

Curve using Graph Pad Prism program. IC₅₀values

were derived from the calculated curves and converted

to K_ivalues as described previously.³⁰

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Article Doi

Tricyclic pyrazoles. Part 1: synthesis and biological evaluation of novel 1,4-dihydroindeno[1,2-c]pyrazol-based ligands for CB1and CB2 cannabinoid receptors.

DOI: 10.1016/S0968-0896(02)00319-X

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