A critical review of both the synthesis approach and the receptor profile of the 8-chloro-1-(2′,4′-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-Tetrahydrobenzo[6,7]cyclohepta[1,2-c ]pyrazole-3-carboxamide and analogue derivatives

Article abstract of DOI:10.1016/j.ejmech.2016.05.011

8-Chloro-1-(2′,4′-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-Tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide 9a was discovered as potent and selective CB₁ antagonist by part of our group few years ago. In particular it was reported to

Full text of DOI:10.1016/j.ejmech.2016.05.011

Accepted Manuscript
A critical review of both the synthesis approach and the receptor
profile of the 8-chloro-1-(2′,4′-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-
tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide and analogue
derivatives
Paolo Lazzari, Rita Distinto, Ilaria Manca, Gemma Baillie, Gabriele Murineddu,
Marilena Pira, Matteo Falzoi, Monica Sani, Paula Morales, Ruth Ross, Matteo Zanda,
Nadine Jagerovic, Gérard Aimè Pinna
PII:
S0223-5234(16)30388-9
10.1016/j.ejmech.2016.05.011
EJMECH 8600
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 September 2015
Revised Date: 17 April 2016
Accepted Date: 5 May 2016
Please cite this article as: P. Lazzari, R. Distinto, I. Manca, G. Baillie, G. Murineddu, M. Pira, M. Falzoi,
M. Sani, P. Morales, R. Ross, M. Zanda, N. Jagerovic, G.A. Pinna, A critical review of both the synthesis
approach and the receptor profile of the 8-chloro-1-(2′,4′-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-
tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide and analogue derivatives, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.05.011.
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ACCEPTED MANUSCRIPT
R
Q
R1
N
R2
N
9a-i, 10a,b, 12, 13
Compounds
9a-h
R
R1
R2
Q
Cl
H
Carboxamides
H
Cl
H
Cl
H
9i
10a
10b
Cl
O
Cl
Cl
H
H
12
13

ACCEPTED MANUSCRIPT
A critical review of both the synthesis approach and the receptor profile of the 8-chloro-1-
(2’,4’-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-
c]pyrazole-3-carboxamide and analogue derivatives.
e
Paolo Lazzari a,b,c,*, Rita Distinto a,b, Ilaria Manca a,b, Gemma Baillie b,d, Gabriele Murineddu ,
Marilena Pira a,b, Matteo Falzoi f, Monica Sani c,g, Paula Morales h, Ruth Ross b,d, Matteo Zanda b,g
Nadine Jagerovic h, and Gérard Aimè Pinna e
,
a Neuroscienze PharmaNess S.c.a r.l., Edificio 5, Loc. Piscinamanna, 09010 Pula (CA), Italy;
b
Kosterlitz Centre for Therapeutics, Institute of Medical Sciences, College of Life Sciences and
Medicine, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, Scotland, UK;
c KemoTech Srl, Edificio 3, Loc. Piscinamanna, 09010 Pula (CA), Italy.
d
Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto,
Medical Sciences Building, 1 King’s College Circle, Toronto, M5S 1A8, Ontario, Canada;
e
Dipartimento di Chimica e Farmacia, Università di Sassari, Via F. Muroni 23/A, 07100 Sassari,
Italy;
f
Dipartimento di Scienze della Vita e dell’Ambiente, Lab. Genetica, Università di Cagliari, Via
T.Fiorelli 1, 09126 Cagliari (CA), Italy;
g C.N.R. Istituto di Chimica del Riconoscimento Molecolare, Via Mancinelli 7, 20131 Milano, Italy;
h Instituto de Química Médica, CSIC, C/Juan de la Cierva 3, 28006 Madrid, Spain.
* Corresponding
author.
paolo.lazzari@kemotech.it;
Phone:
+3907092433118;
Fax:
+3907092433119 (P. Lazzari).
Abstract
8-Chloro-1-(2’,4’-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-
c]pyrazole-3-carboxamide 9a was discovered as potent and selective CB1 antagonist by part of our
group few years ago. In particular it was reported to have an affinity towards the CB1 cannabinoid
receptor (CB1R), expressed as Ki, of 0.00035 nM. Nevertheless significantly divergent data were
1

ACCEPTED MANUSCRIPT
reported for the same compound from other laboratories. To unequivocally define the receptor
profile of 9a, we have critically reviewed both its synthesis approach and binding data. Here we
report that, in contrast to our previously reported data, 9a showed a Ki value for CB1R in the order
of nanomolar rather than of fentomolar range. The new determined receptor profile of 9a was also
ascertained for analogue derivatives 9b-i, as well as for 12. Moreover, the structural features of the
synthesized compounds necessary for CB1R were investigated. Amongst the novel series, effects on
CB1R intrinsic activity was highlighted due to the substituents at the position 3 of the pyrazole ring
of the 1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole scaffold.
Although the cannabinoid receptor profile of 9a was reviewed in this work, the relevance of this
compound in CB1R antagonist based drug discovery is confirmed.
Keywords: Cannabinoids; endocannabinoid system (ECS); 8-chloro-1-(2’,4’-dichlorophenyl)-N-
piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide; cannabinoid
receptor binding assays; CB1 receptor ligands.
1. Introduction
The endocannabinoid system (ECS) consists of CB1 and CB2 cannabinoid receptors (CB1R
and CB2R, respectively), endogenous ligands such as anandamide (N-arachidonoylethanolamine,
AN) and 2-arachidonoylglycerol (2-AG), and several proteins responsible for their synthesis and
degradation [1-3]. The physiological role of cannabinoid receptors seems to be related to certain
pathophysiological processes such as appetite modulation, pain, and immune system modulation
[4]. In particular, selective CB1R agents might produce beneficial therapeutic effects for the
prevention and/or the treatment of various disorders and diseases as anorexia, emesis, pain,
neurodegenerative diseases, glaucoma, inflammation, sexual dysfunctions, multiple sclerosis,
metabolic syndrome, obesity, and immune system disorders [5-9]. CB1R antagonists have been
2

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proposed as drugs for the prevention of weight gain and the treatment of obesity with improvement
of cardiovascular and metabolic risk factors, even if the first candidate within this class, Acomplia®
(rimonabant, Figure 1) by Sanofi Aventis [10], has been withdrawn from European market due to
its adverse effects [11].
In previous communications [12,13], the synthesis and structure-affinity relationships (SAR)
regarding CB1R and CB2R of 1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazoles of general
structure I (Figure 1) were described. These compounds were also reported as general classes in a
patent application by Sanofi-Synthélabo (now Sanofi Aventis) [14], and partially reviewed by
Lange et al. and Padgett [15]. Moreover, further SAR studies were reported on various series of
condensed tricyclic pyrazole based cannabinoids (series II-VI) by evidencing key structural
parameters in the modulation of CB1 rather than CB2 affinity and selectivity (Figure 2) [16].
Fig. 1. Rimonabant and corresponding rigid analogues I.
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O
O
O
R
Q
Q
Q
O
N
N
N
H
H
H
R
N
N
N
N
N
N
R1
R1
Cl
R2
R2
Cl
II
III
IV
O
O
Q
Q
S
S
N
N
R
R
H
H
N
N
N
N
R1
R1
Cl
Cl
V
VI
Fig. 2. Series of conformationally constrained tricyclic pyrazole based cannabinoid ligands having
significant activity to CB1 and/or CB2 receptors [16].
In particular it was reported by part of our group that one compound belongs to series I, the
8-chloro-1-(2’,4’-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-
c]pyrazole-3-carboxamide, namely NESS0327 (Fig. 3), showed remarkable binding at the CB1R
and selectivity vs the CB2R. In fact, KiCB1 = 0.00035±0.000005 nM and KiCB2 = 21±0.5 nM were
previously reported as affinity values of NESS0327 as determined using [3H]CP-55,940 and mouse
brain membrane and mouse spleen homogenates, respectively [12]. The compound behaved as a
CB1R antagonist in both in vivo tests (mouse WIN 55,212-2 induced antinociception) and in ex vivo
assays (isolated organs: mouse vas deferens) [12]. Moreover CB1R neutral antagonism instead of
inverse agonism of lead compound rimonabant was highlighted for NESS0327 according to
[35S]GTPγS binding assay results [12].
4

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Fig. 3. Structure of NESS0327 [12].
Independently, two other groups [17-18] described the synthesis and biological evaluation of
conformationally constrained analogues of rimonabant. Amongst these cannabinoid derivatives,
they prepared one compound with the same structure of NESS0327 which showed an affinity for
the CB1R markedly different from that previously reported by our group. Values of KiCB1 equal to
126 nM and 18.4 nM were in fact determined, using [3H]CP-55,940 and hCB1 receptor cloned in
Chinese hamster ovary (CHO) cells, or membranes isolated from a HEK-293 expression system,
respectively [18]. The compound was synthesized both according to the same procedure adopted by
our group [12] and through a different protocol [17].
Based on the discrepancy of literature CB1R binding data regarding the tricyclic derivative
8-chloro-1-(2’,4’-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-
c]pyrazole-3-carboxamide, this work aims to critically review both the synthesis approach of the
compound and its cannabinoid receptor profile. In addition, this paper is focused on the evaluation
of:
(1) the effects of the substituents at both the position 1 and 3 of the pyrazole ring of the 1,4,5,6-
tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole scaffold on CB1R affinity, by structure
relationship activity (SAR) study;
5

ACCEPTED MANUSCRIPT
(2) the conformational and electrostatic properties of the new synthesized compounds in
comparison with those of 9a, taking into account also rimonabant;
(3) the intrinsic activity of lead compounds through preliminary in vitro assays.
2. Chemistry
Two different methods were adopted to re-synthesize the tricyclic derivative 8-chloro-1-
(2’,4’-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7] cyclohepta[1,2-c]pyrazole-3-
carboxamide: our previously developed route (Schemes 1 and 2) [12-13], namely “Procedure A”,
based on the obtainment of the chlorine-substituted 1,3-diketoester 2a as intermediates, and the
modification reported by Stoit et al. (Scheme 4) [17], namely “Procedure B”. The last was
characterized by the synthesis of the corresponding nitro-compound of NESS0327, and the
following steps involving the nitro group reduction and the amino group replacement by the
chlorine atom.
Procedure A was also adopted for the synthesis of novel 9a analogue carboxamides: 9b-i, 10a, and
10b. Moreover, the intermediate ester 5a of the Procedure A was employed as starting material for
the synthesis of derivatives 12 and 13.
2.1. Procedure A: Synthesis of NESS0327 and analogue carboxamides (9a-i, 10a, and 10b)
7-Chlorobenzosuberone 1a and 8-Chlorobenzosuberone 1b, the key intermediates in the first
synthetic route, were synthesized according to previously reported procedures [13].
The 1,3-diketoesters 2a-b were prepared from the known chloro-benzosuberones 1a-b and
diethyl oxalate in the presence of sodium ethylate [13] (Scheme 1). Compounds 2a-b were then
reacted with 2,4-dichlorophenylhydrazine hydrochloride (3) or 4-methylbenzylhydrazine
hydrochloride (4) in ethanol at reflux to afford the key intermediates 5a-b or 6a-b [13,19],
respectively. The esters 5 and 6 were hydrolyzed with KOH in methanol to give the corresponding
carboxylic acids 7a-b and 8a-b [13,19] (Scheme 1).
6

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R
R1
O
1a, R = Cl, R1 = H
1b, R = H, R1 = Cl
(a)
R
O
OEt
R1
O
O
2a, R = Cl, R1 = H
2b, R = H, R1 = Cl
.
.
NHNH2 HCl
NHNH2 HCl
Cl
(b)
(b)
Cl
3
4
O
R
O
OR2
N
R1
OR2
N
R
N
N
R1
Cl
Cl
5a, R = Cl, R1 = H, R2 = Et
5b, R = H, R1 = Cl, R2 = Et
6a, R = Cl, R1 = H, R2 = Et
6b, R = H, R1 = Cl, R2 = Et
(c)
(c)
7a, R = Cl, R1 = H, R2 = H
7b, R = H, R1 = Cl, R2 = H
8a, R = Cl, R1 = H, R2 = H
8b, R = H, R1 = Cl, R2 = H
Scheme 1. Reagents and conditions: (a) Na, (COOEt)2,
dry EtOH; (b) EtOH; (c) KOH, MeOH.
As shown in Scheme 2, the acids 7a-b were allowed to react with the required amines or
hydrazines in the presence of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride
(EDC) and 1-hydroxybenzotriazole hydrate (HOBt) to give the desired compounds 9a-i. Similarly,
carboxamides 10a-b were obtained starting from 8a-b (Scheme 2).
7

ACCEPTED MANUSCRIPT
Scheme 2. Reagents and conditions: (a) EDC, HOBt, X-NH2, CH2Cl2.
2.2. Synthesis of derivatives 11 and 12.
Weinreb amide 11 was synthesized from the ester 5a by reaction with N,O-
dimethylhydroxylamine hydrochloride in the presence of trimethylaluminum (Scheme 3). Reaction
of 11 with (cyclohexylmethyl)magnesium bromide in anhydrous tetrahydrofuran tricyclic ketone
12. Finally, the carbonyl group of 12 was reduced by sodium borohydride at room temperature to
obtain the racemic alcohol (±)-13.
8

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Scheme 3. Reagents and conditions: (a) AlMe3, MeNHOMe · HCl,
CH2Cl2; (b) (cyclohexylmethyl)magnesium bromide, THF; (c)
NaBH4, MeOH.
2.3 Procedure B: Synthesis of NESS0327
As shown in Scheme 4, the second route to obtain 9a was defined according to the
previously reported procedure developed by Stoit et al. (Scheme 4) [17]. Nitration of the tricyclic
acid
1-(2’,4’-dichlorohenyl)-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxylic
acid 14 [13], achieved by fuming nitric acid in acetic acid, gave a mixture of the 8- and 9-nitro-
isomers 15a and 15b, respectively, in nearly quantitative yield. This mixture of regioisomers was
reacted with 1-aminopiperidine in the presence of O-(Benzotriazol-1-yl)-N,N,N’,N’-
tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA),
followed by chromatographic separation of the resulting regioisomers, to give the 8-nitrohydrazide
16a and the 9-nitrohydrazide 16b in 46% overall yield, as well as a residue of the unsubstituted
analogue 16c.
9

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Nitro group reduction of 16a using iron powder and 1N HCl afforded the arylamine 17 in
84% yield. Finally, the chloro-substituted hydrazide 9a was formed in 27% yield from 17 via a
Sandmeyer reaction.
R1
R2
R1
R2
O
O
O
16a, R1 = NO2, R2 = H
16b, R1 = H, R2 = NO2
16c, R1 = H, R2 = H
(a)
(b)
N
OH
OH
N
N
N
N
N
N
N
H
Cl
Cl
Cl
Cl
Cl
Cl
14
15a, R1 = NO2, R2 = H
15b, R1 = H, R2 = NO2
(c)
for 16a
Cl
R1
O
O
H2N
O
(e)
(d)
N
N
N
N
N
N
N
N
N
N
H
H
N
N
H
Cl
Cl
Cl
Cl
Cl
Cl
9a
9a, R1 = Cl
16c, R1 = H
17
Scheme 4. Reagents and conditions: (a) HNO3, CH3COOH; (b) HBTU, DIPEA, 1-aminopiperidine,
CH2Cl2; (c) Fe, 1N HCl, EtOH/H2O; (d) NaNO2, CuCl, 37% HCl, H2O; (e) chromatography on
silica gel (CH2Cl2/acetone 9/1).
From this reaction we also obtained low yields of the non-chlorinated corresponding
derivative 16c, which was hardly separable by column chromatography from the chloro analogue
9a. According to Stoit et al. [17], the low yield of 9a and the formation of 16c at the end of the
synthetic process were due to partial over reduction of 9a to 16c in the Sandmeyer reaction (step d
of Scheme 4). However, we rather believe that the small amount of 16c in the final product of this
synthetic procedure might result from an incomplete nitration of the acid 14 (step a, Scheme 3),
which produced a mixture of nitro regioisomers 15a and 15b containing a small quantity of residual
starting material 14, which is difficult to detect and separate by chromatography. Indeed, in our
hands the byproduct 16c was inseparable from 16a under the chromatographic condition used by
Stoit et al. [17]. Thus, 16c was recovered unchanged together with 9a after steps c and d of Scheme
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4. The product derived from the Sandmeyer reaction was purified by chromatography on silica gel
to separate 9a from the by-product 16c.
Comparison of the physicochemical properties and spectral data (see Experimental Section)
for the new two samples of NESS0327 obtained as illustrated above confirms their structure, which
is consistent with that of the hydrazide 9a.
3. Biology
3.1. Radioreceptor binding assays
Affinities at CB1 and CB2 receptors for 9a, the corresponding closest analogues 9b-i, and the
derivatives 10a, 10b, 12, and 13 were assessed by competition for [3H]CP-55,940 binding in mouse
whole brain membranes and Chinese hamster ovary (CHO) cell membranes transfected with hCB2,
respectively [20]. The experimental data (IC50 values) were converted into Ki values [21]. The
results from the in vitro binding assays were compared with the Ki values of the lead cannabinoid
ligands CP-55,940 and rimonabant.
3.2. Intrinsic activity by in vitro assays
According to previous papers [22-23], CB1R intrinsic activity of selected synthesized
derivatives was evaluated through in vitro tests based on the determination of phosphorylated ERK
1/2 (P-ERK 1/2) expression in Mouse Neuroblastoma N1E-115 cell line. In fact, it was reported that
this cell line expresses CB1 but not CB2 receptors, and its exposure to the cannabinoid agonists
ACEA and WIN55-212,2 induces a rapid phosphorylation and activation of the ERK 1/2 [22]. This
effect is counteracted by a pre-treatment of N1E-115 cells with CB1 antagonist derivatives [22-23].
4. Results and discussion
4.1. Cannabinoid receptor affinities
11

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The results from binding assays of 9a obtained by both the synthetic procedures were compared
with those of the two reference compounds CP-55,940 and rimonabant, as well as with the
previously reported Ki data of the corresponding compounds (Table 1) [12,17-18].
Table 1
Cannabinoid Receptor Affinities of 9a, CP-55,940, and rimonabant.
Compound
9a from Procedure A
9a from Procedure B
9a [12]
KiCB1 (nM)a
4.20 (0.3-56)c
8.18 (0.9-60)c
0.00035±0.000005d
126
KiCB2 (nM)b
55.7 (18-176)c
85.2 (35-208)c
21±0.5d
9a [17]
not reported
9a [18]
18.4±1.62d
758
0.62 (0.2-1.9)c
2.82 (0.9-9.8)c
1.80±0.075d
9.73 (1.1-18.0)c
1400 (750-2350)c
514±30d
CP-55,940
rimonabant
rimonabant [12]
a Affinity of compounds for the CB1 receptor was evaluated using [3H]-CP 55,940 and mouse whole brain
membranes. Ki values were obtained from five independent experiments carried out in triplicate.
b Affinity of compounds for the CB2 receptor was evaluated using [3H]-CP 55,940 and Chinese hamster ovary
(CHO) cell membranes transfected with hCB2 receptors. Ki values were obtained from five independent
experiments carried out in triplicate.
c 95% confidence limits.
d literature data expressed as ±SEM.
The CB1R affinity value of the novel sample of 9a obtained through Procedure A (KiCB1 = 4.20
nM) disagrees with that previously reported by part of our group [12-13], and it is more in
accordance with the data reported by Zhang et al. [18]. The new nanomolar KiCB1 value for 9a was
confirmed also for the compound obtained from Procedure B after elimination of the by-product 1-
(2’,4’-dichlorophenyl)-N-piperidin-1-yl-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-
carboxamide 16c, although a slightly difference was highlighted for the two KiCB1 values: KiCB1 =
4.20 and 8.18 nM for 9a obtained through Procedure A and B, respectively. However, it is
important to note that the new KiCB1 values of 9a were at least one order of magnitude lower than
that previously measured by Stoit et al. (KiCB1 126 nM) [17-18].
12

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Although a different receptor matrix has been adopted for CB2 affinity evaluation in this work, the
new determined values of KiCB2 were comparable to that previously reported for NESS0327 [12],
with KiCB2 in the order of 50 nM.
CB1R and CB2R affinities of 9a were compared to those determined for derivatives 9b-i, as
well as for compounds 10a-b, and 12-13 (Table 2). As for 9a, also the re-synthesized derivatives
9b-d, evidenced higher KiCB1 values relative to previously reported data, which were in the order
of 0.001-0.3 nM [13]. All the investigated 9a closest analogues 9b-h, evidenced KiCB1 data in the
range of 3.6-24.8 nM, in line with the novel value determined for the lead compound. Thus,
amongst the synthesized derivatives, CB1R affinity was not substantially affected by the
replacement of hydrazide groups of 9a-c with carboxamide groups containing cycloalkyl or
methylene-cycloalkyl moieties (compounds 9d-h). The introduction of a methylene spacer between
cyclohexyl ring and carboxamide group at 3 position of the pyrazole ring (from 9d to 9e)
determined a doubling of KiCB1, although this modification maintained good CB1R affinity (9e:
KiCB1 = 19.4 nM). No significant effect was recorded due to the replacement of cyclohexyl ring in
9e to give derivatives 9f-h. However, the compound bearing the bulky myrtanil substituent (9h)
showed the best CB1R affinity, with a KiCB1 = 3.6 nM, which was equivalent to that determined for
the lead 9a.
As highlighted from comparison of compounds 9h and 9i, the shifting of chlorine atom from 8 to 9
position of the myrtanil based tricyclic derivatives induced a decrease of CB1R affinity. However,
also derivative 9i evidenced good CB1R affinity (9i: KiCB1 = 17.3 nM). Although the comparison
was limited to these two derivatives, the shifting of chlorine atom from 8 to 9 position didn’t
significantly affect the CB1R affinity as instead previously showed for other analogues compounds
taking into account the original KiCB1 value of 9a [13].
13

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Table 2
Structure and binding data of the synthesized compounds according to Procedure A.
Receptor affinity (nM)a
KiCB2/
KiCB1
Compd
9a
R
R1
R2
Q
b
c
KiCB1
KiCB2
Cl
H
4.2 (0.3-56)
17.2 (1-244)
4.5 (1-24)
55.7 (18-176)
182 (95-348)
28.1 (11-74)
17.9 (10-31)
31.8 (15-68)
13.3 (6-28)
13.26
10.58
6.24
1.75
1.64
0.69
2.10
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
9b
9c
9d
9e
9f
10.2 (1-97)
19.4 (7-58)
19.1 (7-49)
24.8 (3-226)
O
N
H
O
N
O
52.1 (21-131)
9g
H
Cl
H
H
Cl
H
3.6 (2-7)
19.5 (13-30)
13.6 (4-48)
9.5 (7-14)
5.42
0.79
0.03
0.30
9h
9i
17.3 (3-121)
313 (28-3536)
169 (48-587)
Cl
H
10a
10b
Cl
51.1 (18-95)
14

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Cl
Cl
H
H
8.5 (6-13)
325 (147-722)
24 (7-88)
38.24
0.16
12
13
154 (97-245)
a 95% confidence limits.
b
Affinity of compounds for the CB1 receptor was evaluated using [3H]-CP 55,940 and mouse whole brain
membranes. Ki values were obtained from five independent experiments carried out in triplicate.
c
Affinity of compounds for the CB2 receptor was evaluated using [3H]-CP 55,940 and Chinese hamster ovary
(CHO) cell membranes transfected with hCB2 receptors. Ki values were obtained from five independent
experiments carried out in triplicate.
The replacement of 2,4-dichloro-phenyl with 4-methyl-benzyl substituent at 1 position of
the pyrazole ring of 9h and 9i determined compounds 10a and 10b, respectively. In both cases, the
adopted modification elicited a significant reduction of CB1R affinity, with 2-3 orders of magnitude
increasing of KiCB1 values.
No significant effect on CB1R affinity was instead observed by the introduction of a ketone group in
3 position of the pyrazole ring. Compound 12 showed in fact KiCB1 = 8.5 nM, which is equivalent
to that of the closest analogues 9d. It is important to note that a reduction of the ketone group of 12
to get derivative 13 determined a 18-fold increasing of KiCB1 value by evidencing a more
favourable availability of the electrons of ketone oxygen to form hydrogen bonds with the CB1R
rather than those of the hydroxyl moiety.
Amongst the synthesized compounds, 12 evidenced the best CB1R selectivity, with
KiCB2/KiCB1 = 38.24 (Table 2). In contrast to previously reported data [12-13] and according to the
novel determined KiCB1 values and confirmation of KiCB2 values, all the other compounds didn’t
appear as marked selective CB1R derivatives. Acceptable CB1R selectivity was recorded only for 9a
and 9b (KiCB2/KiCB1 of 13.26 and 10.58, respectively), while equivalent or lower KiCB2 relative to
15

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KiCB1 values were highlighted for the other compounds. The new determined CB1R selectivity
value of 9a is in accordance with that reported by Zhang et al. [18].
4,2 Conformational analysis and electrostatic potential maps
In an effort to further study the conformational and electrostatic properties of the new synthesized
pyrazoles, it was first interesting to compare 9a (NESS0327) with rimonabant. Comparative
conformational studies have been already reported for both compounds [13,17,18,24]. No
significant conformational difference was reported by Stoit et al. [17] using the MOPAC/PM3
module within SYBYL and by some of us at B3LYP/6-31G* level [13]. Zhang et al. [18] and
Thomas et al. [24] focussed their conformational comparative analysis on the constrained rotation
of the diaryl rings using the MMFF94 force field within SYBYL concluding to a suboptimal
orientation of the conformational restricted analogues for receptor interaction. In the present study,
we first compare the electrostatic potential maps of 9a and rimonabant calculated from the
minimum energy conformers of each molecule using the level of theory HF 6-31G*. Then, we
selected three of the new analogues (9d, 12 and 13) for conformational and electrostatic
calculations in order to define some structural features in relation with the C3-pyrazole substituent.
Initial conformational analysis of rimonabant (see Supporting Information) are in agreement with
the data reported by Hurst et al. [25]. In the global minimum energy conformer of 9a the piperidine
ring is in chair conformation, with the nitrogen lone pair of electrons and the carboxamide oxygen
group pointing in the opposite direction. We have also considered the conformer that displays the
piperidine nitrogen lone pair and the carboxamide oxygen in the same direction as proposed by
Hurst et al. [25] for the bioactive conformer of rimonabant. The energy differences between both
conformers was ∆E = 0.84 kcal/mol for 9a and 0.91 kcal/mol for rimonabant. In the electrostatic
potential maps of rimonabant and 9a (see Supporting Information) significant difference concerning
the orientation of the 4-chlorophenyl ring is observed. The heptacyclic ring constraint may
disfavour the coulombic interactions as well as the aromatic stacking compared to rimonabant.
16

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Extra rotational bonds in the C3-pyrazole substituent of 9d, 12 and 13 compared to 9a generated a
higher number of conformers in the initial conformational search (9a: 33 conformers; 9d: 59
conformers; 12: 78 conformers; 13: 171 conformers). The global minimum energy conformers of
9d, 12 and 13 as initially identified by molecular mechanics and then optimized by ab initio
Hartree-Fock 6-31G* calculations are represented in Fig 4A. Electrostatic potential maps from the
corresponding minimum energy conformers are illustrated in Fig 4B. The structural modifications
realized on the C3-substituent of 9a preserve the geometry of the phenyl rings and the pyrazole
core. However, there is a crucial difference in the electrostatic potential map of 13 compared to 9a,
9d, and 12. This difference concerns the orientation of the most electronegative region of the
molecule. An intramolecular hydrogen bond between the hydroxyl group and the pyrazole nitrogen
may stabilize the minimum energy conformer. This conformation hinders the availability of the
oxygen lone pair to interact with CB1R explaining the decrease of binding affinity.
It has been previously reported that the carboxamide oxygen of rimonabant is engaged in a
hydrogen bond with the residue K3.28 of CB1R and that the absence of the piperidine nitrogen does
not affect affinity [26]. The same behaviour has been observed in the constrained analogues
described herein.
Furthermore, the carboxamide functional group has also been reported to play a crucial role for
CB2R affinity, for instance, with the contribution of hydrogen bonding with the EC-3 loop residue
D275 [27]. The comparative binding data reported in Table 3 suggest that the conformational
restricted analogues (9a, 9d, 12, and 13) are more favourable to CB2R binding that their
corresponding rimonabant derivatives (rimonabant, CHASR [26,28], VII [23], and VIII [23]).
Table 3
Comparative binding data of conformational restricted derivatives and rimonabant analogues
17

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Compd
Q
KiCB1 (nM)
KiCB2 (nM)
Selectivity
CHASR [26,28]
9d
2.4 ± 0.1
228 ± 2
95
10.2 (1-97)
17.9 (10-31)
1.75
VII [23]
12
46.1 ± 1.0
8.5 (6-13)
2233 ± 390.0
325 (147-722)
48.44
38.24
VIII [23]
13
175.0 ± 14.0
154 (97-245)
4200.0 ± 200.0
24 (7-88)
24
0.16
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Fig. 4. A) Minimum energy conformers of 9a, 9d, 12 and 13. B) Molecular electrostatic potential
maps of the global minimum energy conformations of compounds 9a, 9d, 12 and 13. The
electrostatic potential scale (in kJ/mol) is provided as a color scale. This scale is from blue (most
electropositive) to red (most electronegative).
4.3 Intrinsic activity by in vitro assays
Preliminary in vitro assays were carried out to evaluate CB1R mediated intrinsic activity of
the synthesized compounds, with the exception of derivatives 10a-b and 13 having KiCB1 values
higher than 150 nM.
According to rimonabant [23], compounds 9a-g and 12 didn’t affect P-ERK 1/2 expression
in N1E-115 cells in the concentration range 1 nM - 10 µM (Table 4 for data at 1 µM; dose response
data not shown). Moreover, all these derivatives inhibited the P-ERK 1/2 expression up-regulation
19

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induced by the reference cannabinoid agonist WIN55,212-2 (Table 3). These data thus confirm the
previously reported CB1R antagonism profile of 9a [12], and evidence an analogue behaviour for
derivatives 9b-g and 12.
In contrast to the other compounds, derivatives 9h-i bearing a myrtanil substituent at the
carbamoyl moiety in 3 position of pyrazole ring enhanced P-ERK 1/2 expression in N1E-115 cells.
This effect was statistically recorded starting from 1 nM concentration of the compounds. CB1R
agonism profile of both the derivatives was further ascertained through the rimonabant
counteraction of the elicited P-ERK 1/2 expression up-regulation by 9h-i. Representative results
regarding 9h have been reported in Fig.5. Equivalent behaviour has been detected for 9i (data not
shown).
Table 4
Effect of compounds 9a-g and 12 at the dose of 1 µM on P-ERK 1/2 expression in N1E-115 cells
after 10 min of exposure (second column) and as 5 min of pretreatment before exposure of the same
cell lines to the cannabinoid agonist WIN55,212-2 (25 nM) (third column). Data are expressed as a
*
mean percentage of vehicle ±SEM and are the results of five separate experiments. P<0.05 vs
vehicle; #P<0.05 vs WIN55,212-2 (25 nM) without CB1R antagonist pretreatment.
P-ERK 1/2 (% of vehicle)
Compd
Compd (1 µM)
100±10
111±12
95±9
Compd (1 µM) + WIN55,212-2 (25 nM)
185±12*
115±13#
103±9#
Vehicle
9a
9b
9c
107±10
113±9
102±12#
117±10#
109±12#
108±12#
118±8#
9d
9e
100±14
98±10
9f
9g
12
113±11
115±7
105±15#
108±7#
rimonabant
110±8
20

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Fig. 5. a) Dose response studies of P-ERK 1/2 expression following a 10 min exposure of N1E-115
cells to different concentrations of derivative 9h or the reference cannabinoid agonist WIN 55,212-2
(25 nM). b) Results of competition study of P-ERK 1/2 expression mediated by a 5 min pre-
treatment of N1E-115 cells with reference CB1R antagonist/inverse agonist rimonabant (SR)
followed by a 10 min exposure to WIN 55,212-2 (25 nM) or 9h (10 nM). In a) and b) data are
expressed as a mean percentage of vehicle ± SEM (five independent experiments). A one-way
Anova followed by Dunnet’s multiple comparison post test was performed for statistical analysis.
5. Conclusion
We have re-synthesized ex-novo the 8-chloro-1-(2’,4’-dichlorophenyl)-N-piperidin-1-yl-
1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide 9a (NESS0327), following
21

ACCEPTED MANUSCRIPT
two alternative routes according to previously reported synthetic procedures. Comparison of the
physicochemical and spectral data of the samples unequivocally demonstrates their structural
equivalence. However, the procedure of Schemes 1-2 afforded the final product 9a in higher yield
and purity relative to that of Scheme 4.
The CB1R affinity values of the new samples of 9a disagree with those previously reported
by part of our group [12-13]. In accordance with the data reported by Zhang et al. [18], 9a
evidenced KiCB1 values in the order of nM units, with KiCB2/KiCB1 = 13.26. The reasons for the
discrepancy relative to our previous data are unclear. However, it doesn’t appear to be due to
structural differences in the synthesized compounds but rather may be related to anomalies in the
procedures used in the original pharmacological testing (binding assays) of the compound. The new
reported data indicate that the CB1R affinity of 9a is consistent with that of the lead compound
rimonabant, as well as of those of the novel analogue derivatives 9b-i.
According to intrinsic activity assays, the substituent at carbamoyl group at 3 postion of the
pyrazole ring determines significant differences in CB1R modulation. In contrast to 9a and other
investigated analogues, the myrtanil carboxamides 9h,i show in fact a biological profile consistent
with that of CB1 agonist compounds.
The importance of compound 9a and analogues remains high considering both the new
CB1R affinity value and the confirmation of antagonism profile of the compound re-synthesized for
this work. The relevance of derivative 9a has been furtherly evidenced by the recent published work
by Meye et al. [29]. In fact, it has been reported that 9a induces rimonabant equivalent reduction of
weight gain and food intake. In addition, the neutral CB
instead highlighted in the case of rimonabant administration (i.e. suppression of the constitutive
CB R in ventral tegmental area and basolateral amygdala, which are involved in the regulation of
1
R antagonist does not elicit the side effects
1
motivation and emotions; anxiety; reduction of motivation for reward).
6. Experimental protocols
22

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6.1. Chemistry
6.1.1. General Methods
Melting points were obtained on a Stuart SMP10 Melting point apparatus and are
uncorrected. IR spectra were recorded on a Bruker Alpha-T FT-IR Spectrophotometer equipped
with an Alpha Eco ATR as sampling module and are expressed in ν (cm-1). All NMR spectra were
taken on a Bruker Ultra Shield 400 Avance III NMR Spectrometer, with 1H and 13C observed at 400
1
and 100 MHz, respectively. Chemical shifts for H and 13C NMR spectra are expressed in δ
downfield from TMS (Me4Si). Multiplicities are reported as s (singlet), bs (broad singlet), d
(doublet), t (triplet), q (quartet), qu (quintuplet), dd (doublet of doublets), m (multiplet). ESI mass
spectra were recorded on a Bruker Esquire 3000 Plus Ion Trap mass spectrometer in the positive
mode. Combustion analyses of all target compounds were performed by Laboratorio di
Microanalisi, Dipartimento di Chimica, Università di Sassari, Italy, and are within ± 0.4% of the
theoretical values, corresponding to ≥95% purity. All reactions involving air or moisture sensitive
compounds were performed under highly pure nitrogen atmosphere. Flash chromatography (FC)
was performed using Merck silica gel 60 (230-400 mesh ASTM). Thin layer chromatography
(TLC) was performed with Polygram® SIL N-HR/HV254 precoated plastic sheets (0.2 mm).
Unless otherwise specified, all materials, solvents, and reagents were obtained from Sigma-Aldrich
(Milan, Italy).
The requisite carboxylic acid compounds to get final derivatives were prepared as previously
reported in literature. Physicochemical properties and spectral data of the synthesized intermediates
were in accordance with those previously recorded [13,17,19]
6.1.2. General procedure A: preparation of carbohydrazides 9a-c and carboxamides 9d-i.
A mixture of the appropriate carboxylic acid 7a-b or 8a-b (1.0 eq, 0.25 mmol), 1-
hydroxybenzotriazole hydrate (HOBt) (1.2 eq, 0.30 mmol) and N-(3-dimethylaminopropyl)-N’-
ethylcarbodiimide hydrochloride (EDC) (1.2 eq, 0.30 mmol) in dichloromethane (2.0 mL) was
23

ACCEPTED MANUSCRIPT
stirred at room temperature for 1 h. A solution of the requisite hydrazine or amine (2.0 eq, 0.50
mmol) in dichloromethane (3.0 mL) was added drop-wise. The resulting mixture was stirred at
room temperature for 22 h. The solvent was removed under vacuum and the residue was purified by
flash-chromatography to afford the desired compound. When the starting hydrazines or amines
were used as hydrochloride salt, triethylamine (TEA) (2.0 eq, 0.50 mmol) was added to the mixture.
6.1.2.1. N-Piperidin-1-yl-8-chloro-1-(2’,4’-dichlorophenyl)-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta
[1,2-c]pyrazole-3-carboxamide (9a) [12].
General procedure A was used to convert 7a and N-aminopiperidine into the title product. White
solid; yield: 79%; mp 199-200°C (acetone), (202 °C [12]); Rf 0.40 (diethyl ether/petroleum ether,
1
6:4); IR 3200, 1650; ESI-MS (m/z): 513.1 (M+Na); H-NMR (CDCl3) δ 1.40-1.48 (m, 2H), 1.72-
1.80 (m, 4H), 2.19-2.30 (m, 2H), 2.66 (t, 2H, J = 6.6 Hz), 2.83-2.90 (m, 4H), 2.90-3.48 (m, 2H),
6.56 (d, 1H, J = 8.3 Hz), 7.00 (dd, 1H, J = 2.2 and 8.3 Hz), 7.30 (d, 1H, J = 2.2 Hz), 7.40 (dd, 1H, J
13
= 2.2 and 8.3 Hz); 7.42-7.48 (m, 2H), 7.66 (bs, 1H); C-NMR (CDCl3) δ 20.2, 23.4, 25.5, 31.4,
32.6, 57.1, 122.7, 126.2, 127.7, 128.0, 128.2, 129.9, 130.4, 130.5, 132.5, 134.2, 135.9, 136.0, 142.2,
143.6, 143.8, 160.0. Anal. C24H23Cl3N4O (C, H, Cl, N).
6.1.2.2.
N-Pyrrolidin-1-yl-8-chloro-1-(2’,4’-dichlorophenyl)-1,4,5,6-tetrahydrobenzo[6,7]
cyclohepta[1,2-c]pyrazole-3-carboxamide (9b) [13].
General procedure A was used to convert 7a and N-aminopyrrolidine hydrochloride into the title
product. Because of an excess of the hydrochloride salt, 2 eq of TEA were used in this reaction.
1
White solid; yield: 60%; mp 127-128°C; Rf 0.33 (petroleum ether/EtOAc 4:6); IR 1651, 3206; H-
NMR (CDCl3) δ 1.82-1.96 (m, 4H), 2.25 (t, 2H, J = 6.4 Hz), 2.66 (t, 2H, J = 6.4 Hz), 2.90-3.10 (m,
6H), 6.57 (d, 1H, J = 8.2 Hz), 6.99 (dd, 1H, Jo = 8.2 Hz, Jm = 2.2 Hz), 7.28-7.31 (m, 1H), 7.37-7.49
13
(m, 3H), 7.66 (br s, 1H, NH exch. with D2O); C-NMR (CDCl3) δ 20.04 (CH2), 22.20 (CH2 x 2),
24

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31.32 (CH2), 32.41 (CH2), 55.31 (CH2 x 2), 122.47 (C), 126.10 (CH), 127.50 (C), 127.93 (CH),
128.11 (CH), 129.81 (CH), 130.29 (CH), 130.37 (CH), 132.36 (C), 134.06 (C), 135.84 (C), 142.13
(C), 143.34 (C), 143.67 (C), 160.72 (C), 169.98 (CO); API-ES calcd for 475.8, found 475.10; Anal.
calcd for C23H21Cl3N4O: C, 58.06; H, 4.45; Cl, 22.35; N, 11.78. Found: C, 58.25; H, 4.46; Cl,
22.37; N, 11.81.
6.1.2.3.
N-Homopiperidin-1-yl-8-chloro-1-(2’,4’-dichlorophenyl)-1,4,5,6-tetrahydrobenzo[6,7]
cyclohepta[1,2-c]pyrazole-3-carboxamide (9c) [13].
General procedure A was used to convert 7a and N-aminohomopiperidine into the title product.
1
White solid; yield: 35%; mp 160-161°C; Rf 0.54 (petroleum ether/EtOAc 6:4); IR 1659, 3174; H-
NMR (CDCl3) δ 1.60-1.80 (m, 10H), 2.25 (qu, 2H, J = 6.0 Hz), 2.65 (t, 2H, J = 6.2 Hz), 3.15 (t, 4H,
J = 5.6 Hz), 6.56 (d, 1H, J = 8.4 Hz), 6.99 (dd, 1H, Jo = 8.6 Hz, Jm = 1.8 Hz), 7.30 (s, 1H), 7.37-
7.48 (m, 3H), 8.10 (br s, 1H, NH exch. with D2O); 13C-NMR (CDCl3) δ 20.10 (CH2), 26.36 (CH2),
26.95 (CH2 x 2), 29.67 (CH2), 31.41 (CH2), 32.49 (CH2), 58.32 (CH2 x 2), 122.57 (C), 126.17 (CH),
127.62 (C), 127.10 (CH), 128.14 (CH), 129.89 (CH), 130.32 (CH), 130.46 (CH), 132.42 (C),
134.11 (C x 2), 135.86 (C), 142.17 (C), 143.74 (C x 2), 160.72 (CO); API-ES calcd for 503.9, found
503.01; Anal. calcd for C25H25Cl3N4O: C, 59.59; H, 5.00; Cl, 21.10; N, 11.12. Found: C, 59.76; H,
5.02; Cl, 21.15; N, 11.13.
6.1.2.4. N-Cyclohexyl-8-chloro-1-(2’,4’-dichlorophenyl)-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta
[1,2-c]pyrazole-3-carboxamide (9d) [13].
General procedure A was used to convert 7a and cyclohexylamine into the title product. White
1
solid; yield: 50%; mp 96-98°C; Rf 0.54 (petroleum ether/EtOAc 8:2); IR 1633, 3201; H-NMR
(CDCl3) δ 1.16-1.45 (m, 7H), 1.62-1.82 (m, 2H), 1.96-2.06 (m, 2H), 2.25 (qu, 2H, J = 6.4 Hz), 2.66
(t, 2H, J = 6.6 Hz), 2.95-3.12 (m, 2H), 6.57 (d, 1H, , J = 8.4 Hz), 6.86 (d, 1H, , J = 8.6 Hz), 6.99
25

ACCEPTED MANUSCRIPT
(dd, 1H, Jo = 8.0 Hz, Jm = 2.0 Hz), 7.30 (d, 1H, J = 2.2 Hz), 7.38-7.46 (m, 2H), 7.50 (br s, 1H, NH
13
exch. with D2O); C-NMR (CDCl3) δ 20.18 (CH2), 25.01 (CH2 x 2), 25.59 (CH2), 31.49 (CH2),
32.50 (CH2), 33.19 (CH2 x 2), 47.92 (CH), 122.22 (C), 126.16 (CH), 127.74 (C), 127.99 (CH),
128.17 (CH), 129.88 (CH), 130.38 (CH), 130.45 (CH), 132.46 (C), 134.05 (C), 135.85 (C), 135.98
(C), 142.28 (C), 143.73 (C), 144.25 (C), 161.77 (CO); API-ES calcd for 488.84, found 488.10;
Anal. calcd for C25H24Cl3N3O: C, 61.43; H, 4.95; Cl, 21.76; N, 8.60. Found: C, 61.25; H, 4.94; Cl,
21.69; N, 8.58.
6.1.2.5.
N-Cyclohexylmethyl-8-chloro-1-(2’,4’-dichlorophenyl)-1,4,5,6-tetrahydrobenzo[6,7]
cyclohepta [1,2-c]pyrazole-3-carboxamide (9e).
General procedure A was used to convert 7a and cyclohexanemethylamine into the title product.
1
White solid; yield: 44%; Rf 0.45 (petroleum ether/ethyl acetate 9:1); IR 3410, 1670; H-NMR
(CDCl3) δ 0.92-1.03 (m, 2H); 1.10-1.32 (m, 4H); 1.53-1.84 (m, 5H); 2.20-2.31 (m, 2H); 2.60-2.72
(m, 3H); 2.80-3.15 (m, 1H); 3.20-3.30 (m, 2H); 6.58 (d, 1H, J = 8.0 Hz); 6.97-7.08 (m, 2H); 7.30
13
(d, 1H, J = 1.6 Hz); 7.40 (dd, 1H, J = 1.7 and 8.4 Hz); 7.42-7.48 (m, 2H); C-NMR (CDCl3) δ
20.2, 25.9, 26.4, 31.0, 31.5, 32.5, 38.1, 45.3, 122.2, 126.2, 127.8, 128.1, 128.2, 129.9, 130.4, 130.5,
132.5, 134.1, 135.9, 136.1, 142.3, 143.8, 144.2, 162.7. Anal. calc. for C26H26Cl3N3O: C, 62.10; H,
5.21; Cl, 21.15; N, 8.36. Found: C, 62.03; H, 5.20; Cl, 21.13; N, 8.34.
6.1.2.6.
N-(1-Adamantylmethyl)-8-chloro-1-(2’,4’-dichlorophenyl)-1,4,5,6-tetrahydrobenzo[6,7]
cyclohepta[1,2-c]pyrazole-3-carboxamide (9f).
General procedure A was used to convert 7a and 1-adamantylmethylamine into the title product.
1
White solid; yield: 59%; Rf 0.41 (petroleum ether/ethyl acetate 9:1); IR 3405, 1669; H-NMR
(CDCl3) δ 1.57 (bs, 6H); 1.61-1.75 (m, 6H); 1.95-2.02 (m, 3H); 2.20-2.30 (m, 2H); 2.63-2.73 (m,
3H); 2.82-3.18 (m, 3H); 6.58 (d, 1H, J = 8.4 Hz); 6.97-7.09 (m, 2H); 7.30 (bs, 1H); 7.40 (dd, 1H, J
26

ACCEPTED MANUSCRIPT
13
= 1.9 and 8.4 Hz); 7.44-7.50 (m, 2H); C-NMR (CDCl3) δ 20.3, 28.3, 31.4, 32.5, 34.1, 37.0, 40.3,
50.5, 122.2, 126.2, 127.8, 128.1, 128.2, 129.9, 130.4, 130.5, 132.5, 134.1, 135.9, 136.1, 142.3,
143.8, 144.3, 162.9. Anal. calc. for C30H30Cl3N3O: C, 64.93; H, 5.45; Cl, 19.17; N, 7.57. Found: C,
64.81; H, 5.44; Cl, 19.10; N, 7.55.
6.1.2.7. N-(2,3,4,5-Tetrahydrofurfuryl)-8-chloro-1-(2’,4’-dichlorophenyl)-1,4,5,6-tetrahydrobenzo
[6,7] cyclohepta[1,2-c]pyrazole-3-carboxamide (9g).
General procedure A was used to convert 7a and R-(–)-tetrahydrofurfurylamine into the title
1
product. White solid; yield: 54%; Rf 0.38 (petroleum ether/ethyl acetate 7:3); IR 3409, 1667; H-
NMR (CDCl3) δ 1.58-1.74 (m, 1H); 1.84-1.96 (m, 2H); 1.97-2.08 (m, 1H); 2.19-2.31 (m, 2H); 2.61-
2.72 (m, 2H); 2.75-3.28 (m, 2H); 3.31-3.41 (m, 1H); 3.66-3.81 (m, 2H); 3.84-3.94 (m, 1H); 4.04-
4.12 (m, 1H); 6.57 (d, 1H, J = 8.0 Hz); 7.00 (dd, 1H, J = 1.9 and 8.4 Hz); 7.27-7.32 (m, 2H); 7.39
13
(dd, 1H, J = 1.9 and 8.4 Hz); 7.41-7.48 (m, 2H); C-NMR (CDCl3) δ 19.7, 25.3, 28.2, 30.9, 31.9,
42.2, 67.6, 77.3, 121.6, 125.6, 126.4, 127.2, 127.5, 127.6, 129.3, 129.8, 129.9, 131.8, 133.5, 135.2,
141.6, 143.2, 143.4, 162.3. Anal. calc. for C24H22Cl3N3O2: C, 58.73; H, 4.52; Cl, 21.67; N, 8.56.
Found: C, 58.69; H, 4.51; Cl, 21.65; N, 8.54.
6.1.2.8.
N-(Myrtanyl)-8-chloro-1-(2’,4’-dichlorophenyl)-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta
[1,2-c]pyrazole-3-carboxamide (9h).
General procedure A was used to convert 7a and (–)-cis-myrtanylamine into the title product. White
1
solid; yield: 81%; Rf 0.48 (petroleum ether/ethyl acetate 6:4); IR 3400, 1662; H-NMR (CDCl3) δ
1.07 (s, 3H); 1.21 (s, 3H); 1.52-1.63 (m, 5H); 1.82-2.05 (m, 4H); 2.15-2.29 (m, 4H); 2.64 (t, 2H, J =
6.6 Hz); 3.35-3.45 (m, 2H); 6.59 (d, 1H, J = 8.0 Hz); 7.00 (dd, 1H, J = 2.2 e 8.0 Hz); 7.25-7.32 (m,
13
1H); 7.37-7.43 (m, 3H); 7.51 (bt, 1H, J = 5.4 Hz); C-NMR (CDCl3) δ 19.86 (CH3), 20.20 (CH3),
23.23 (CH2), 26.02 (CH2), 27.96 (CH2), 31.44 (CH2), 33.49 (CH2), 33.28 (CH2), 38.70 (C), 41.32
27

ACCEPTED MANUSCRIPT
(CH), 41.48 (CH), 43.81 (CH), 44.58 (CH2), 122.15 (C), 126.16 (CH), 127.68 (CH), 128.01 (CH),
128.15 (CH), 129.88 (CH), 130.33 (CH), 130.45 (CH), 132.45 (C), 134.05 (C), 135.85 (C), 135.97
(C), 142.27 (C), 143.72 (C), 144.15 (C), 162.61 (C=O). Anal. calc. for C29H30Cl3N3O: C, 64.15; H,
5.57; Cl, 19.59; N, 7.74. Found: C, 64.09; H, 5.55; Cl, 19.56; N, 7.72.
6.1.2.9.
N-(Myrtanyl)-9-chloro-1-(2’,4’-dichlorophenyl)-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta
[1,2-c]pyrazole-3-carboxamide (9i).
General procedure A was used to convert 7b and (–)-cis-myrtanylamine into the title product. White
1
solid; yield: 87%; Rf 0.50 (petroleum ether/ethyl acetate 6:4); IR 3405, 1664; H-NMR (CDCl3) δ
1.08 (s, 3H); 1.20 (s, 3H); 1.53-1.61 (m, 5H); 1.84-2.03 (m, 4H); 2.19-2.30 (m, 4H); 2.63 (t, 2H, J =
6.4 Hz); 3.37-3.46 (m, 2H); 6.61 (s, 1H); 6.93 (bt, 1H, J = 5.5 Hz); 6.98-7.10 (m, 1H); 7.17-7.23
(m, 2H); 7.49-7.55 (m, 2H); 13C-NMR (CDCl3) δ 19.3, 19.6, 22.6, 25.5, 27.4, 31.0, 31.5, 32.7, 38.1,
40.8, 41.0, 43.3, 44.0, 122.0, 126.2, 127.6, 127.8, 129.8, 129.9, 130.2, 130.5, 131.1, 132.0, 135.3,
135.4, 139.7, 141.5, 143.6, 162.0. Anal. calc. for C29H30Cl3N3O: C, 64.15; H, 5.57; Cl, 19.59; N,
7.74. Found: C, 64.03; H, 5.56; Cl, 19.57; N, 7.73.
6.1.2.10. N-(Myrtanyl)-8-chloro-1-(4’-methylbenzyl)-1,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1,2-
c]pyrazole-3-carboxamide (10a).
General procedure A was used to convert 8a and (–)-cis-myrtanylamine into the title product. White
1
solid; yield: 56%; Rf 0.47 (petroleum ether/ethyl acetate 7:3); IR 3405, 1658; H-NMR (CDCl3) δ
1.08 (s, 3H); 1.21 (s, 3H); 1.50-1.70 (m, 3H); 1.90-2.02 (m, 4H); 2.03-2.50 (m, 9H); 2.83-2.95 (m,
13
2H); 3.38-3.50 (m, 2H); 5.37 (s, 2H); 6.85-7.31 (m, 8H); C-NMR (CDCl3) δ 19.90 (CH3), 21.07
(CH3), 21.35 (CH3), 23.24 (CH2), 26.04 (CH2), 28.01 (CH2), 30.75 (CH2), 33.48 (CH2), 33.27
(CH2), 38.73 (C), 41.37 (CH), 41.61 (CH), 43.93 (CH), 44.97 (CH2), 52.09 (CH2), 115.75 (C),
116.40 (C), 125.65 (CH), 126.72 (CH x 2), 127.13 (CH x 2), 127.38 (CH), 129.55 (CH), 132.25
28