Identification of dual role of piperazine-linked phenyl cyclopropyl methanone as positive allosteric modulator of 5-HT2C and negative allosteric modulator of 5-HT2B receptors

Article abstract of DOI:10.1016/j.ejmech.2018.12.070

Allosteric modulators of G-protein-coupled receptors have lately gained significant traction in drug discovery. Recent studies have shown that allosteric modulation of serotonin 2C receptor (5-HT_2C) as a viable strategy for the treatment of various central nervous system (CNS) disorders. Considering the critical role of 5-HT_2C in the modulation of appetite, a selective positive allosteric modulator (PAM) of 5-HT_2C offers a new opportunity for anti-obesity therapeutic development. In this study, phenyl cyclopropyl-linked N-heterocycles were synthesized and evaluated at 5-HT_2C for agonist and PAM activity. Our study shows that imidazole linked phenyl cyclopropyl methanones has PAM activity on both 5-HT_2C and serotonin 2B receptor (5-HT_2B). Interestingly, piperazine linked phenyl cyclopropyl methanones (58) was active as PAM of 5-HT_2C (increased the E_max of 5-HT to 139%), and as negative allosteric modulator (NAM) of 5-HT_2B (decreases EC₅₀ of 5-HT 10 times without affecting E_max). Similar effect of compound 58 was observed with synthetic orthosteric agonist lorcaserin on 5-HT_2B. Molecular docking study revealed that all active compounds were binding to the predicted allosteric site on 5-HT_2C and shared a common interacting residues. Finally, compound 58 suppressed food intake in Sprague Dawley (SD) rats similar to lorcaserin after i.c.v. administration. Therefore, these results suggest that piperazine moiety is essential for dual activity (PAM & NAM) of compounds 58, and supports the hypothesis of 5-HT_2C PAM for the treatment of obesity similar to the full agonist.

Full text of DOI:10.1016/j.ejmech.2018.12.070

Accepted Manuscript
Identification of dual role of piperazine-linked phenyl cyclopropyl methanone as
positive allosteric modulator of 5-HT
receptors
and negative allosteric modulator of 5-HT
2B
2C
Kartikey Singh, Chandan Sona, Vikash Ojha, Maninder Singh, Ankita Mishra, Ajeet
Kumar, Mohammad Imran Siddiqi, Rama P. Tripathi, Prem N. Yadav
PII:
S0223-5234(18)31116-4
DOI:
https://doi.org/10.1016/j.ejmech.2018.12.070
EJMECH 11004
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 October 2018
Revised Date: 5 December 2018
Accepted Date: 26 December 2018
Please cite this article as: K. Singh, C. Sona, V. Ojha, M. Singh, A. Mishra, A. Kumar, M.I. Siddiqi,
R.P. Tripathi, P.N. Yadav, Identification of dual role of piperazine-linked phenyl cyclopropyl methanone
as positive allosteric modulator of 5-HT
2C
and negative allosteric modulator of 5-HT receptors,
2B
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2018.12.070.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Identification of Dual Role of Piperazine-Linked Phenyl Cyclopropyl Methanone
as Positive Allosteric Modulator of 5-HT2C and Negative Allosteric Modulator of
5-HT2B Receptors
Kartikey Singh,#[a] Chandan Sona,#[b,c] Vikash Ojha,[a] Maninder Singh,[d] Ankita Mishra,[b] Ajeet
Kumar,[b] Mohammad Imran Siddiqi,[d] Rama P. Tripathi*[a,c,e] and Prem N. Yadav,*[b,c]
[a]
[b]
Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute (CSIR-
CDRI),Sector 10, Jankipuram Extension, Sitapur Road, Lucknow-226031, India.
Pharmacology Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Sector 10,
Jankipuram Extension, Sitapur Road, Lucknow-226031, India.
[c]
[d]
Academy of Scientific and Innovative Research (AcSIR), New Delhi-110001, India.
Computational Biology and Bioinformatics Unit, Molecular and Structural Biology
Division, CSIR-Central Drug Research Institute (CSIR-CDRI), Sector 10, Jankipuram
Extension, Sitapur Road, Lucknow-226031, India.
[e]
Dean, National Institute of Pharmaceutical Education and Research Raebarely, New
Transit Campus, Bijnor Road, Sarojani Nagar Near CRPF Base Camp, Lucknow 226002,
U.P., India
*Address for Correspondence:
Prem N. Yadav
Sr. Scientist & Ramanujan Fellow
Division of Pharmacology
CSIR-Central Drug Research Institute
Sector-10, Jankipuram Extension
Sitapur Road, Lucknow-226031, U.P., INDIA
Email: pn.yadav@cdri.res.in
Tel: +91 5222772450 X-4614; FAX: +91 5222771941
Rama P. Tripathi
Consultant/Advisor National Institute of
Pharmaceutical Education and Research,
Raebareli-229010, U.P., INDIA
Ex. Chief Scientist & Professor AcSIR
Medicinal and Process Chemistry Division
CSIR-Central Drug Research Institute
Lucknow-226031, U.P., INDIA
Email: rpt.cdri@gmail.com
Tel: +91 9415004443
Keywords: Serotonin, 5-HT2C receptor, 5-HT2B receptor, positive allosteric modulator,
negative allosteric modulator, food intake, anti-obesity
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Abstract
Allosteric modulators of G protein-coupled receptors have lately gained significant traction in
drug discovery. Recent studies have shown that allosteric modulation of serotonin 2C receptor
(5-HT2C) as a viable strategy for the treatment of various central nervous system (CNS)
disorders. Considering the critical role of 5-HT2C in the modulation of appetite, a selective
positive allosteric modulator (PAM) of 5-HT2C offers a new opportunity for anti-obesity
therapeutic development. In this study, phenyl cyclopropyl-linked N-heterocycles were
synthesized and evaluated at 5-HT2C for agonist and PAM activity. Our study shows that
imidazole linked phenyl cyclopropyl methanones has PAM activity on both 5-HT2C and serotonin
2B receptor (5-HT2B). Interestingly, piperazine linked phenyl cyclopropyl methanones (58) was
active as PAM of 5-HT2C (increased the Emax of 5-HT to 139 %), and as negative allosteric
modulator (NAM) of 5-HT2B (decreases EC50 of 5-HT 10 times without affecting Emax). Similar
effect of compound 58 was observed with synthetic orthosteric agonist lorcaserin on 5-HT2B.
Molecular docking study revealed that all active compounds were binding to the predicted
allosteric site on 5-HT2C and shared a common interacting residues. Finally, compound 58
suppressed food intake in Sprague Dawley (SD) rats similar to lorcaserin after i.c.v.
administration. Therefore, these results suggest that piperazine moiety is essential for dual
activity (PAM & NAM) of compounds 58, and supports the hypothesis of 5-HT2C PAM for the
treatment of obesity similar to the full agonist.
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Introduction
5-HT2C is a Gq coupled G protein-coupled receptor (GPCR) widely known for its important role in
central nervous system (CNS) disorders, including obesity, anxiety, Parkinson’s disease,
schizophrenia, and drug addiction [1-3]. Compounds like meta-chlorophenylpiperazine (m-CPP)
(1) and related are known to be the earliest tool compounds used in pharmacological studies
for understanding the pharmacology and biology of the 5-HT2C (Fig. 1) [4, 5]. Apart from these
compounds, recent finding suggests 2-phenylcyclopropylmethylamine (2) scaffold as 5-HT2C
agonist with drug like properties in rodent models of schizophrenia [6, 7]. Vabicaserin (3), which
is a high-affinity full agonist of 5-HT2C and 5-HT2B was tested in clinical trials for the treatment of
schizophrenia and depression but failed to meet the primary endpoints (ClinicalTrials.gov
Identifier: NCT00563706) [8]. Fenfluramine which was approved as an appetite suppressant,
but later on discontinued due to 5HT2B mediated cardiac valvulopathy, which underlines the
importance of 5-HT2C selectivity over 5-HT2B [9]. Lorcaserin (Belviq®, 4) is the first FDA approved
drug targeting 5-HT2C receptor with 100-fold selectivity for 5-HT2C over 5-HT2B [10-13]. So the
major challenge in designing 5-HT2C specific agonist is to develop selectivity over 5-HT2A and 5-
HT2B, which result in significant CNS (5-HT2A) and cardiovascular (5-HT2B) adverse effects.
Given the highly conserved orthosteric site across all 5-HT receptors, designing of receptor-
specific positive allosteric modulators (enhances the binding affinity or efficacy, PAMs) or
negative allosteric modulators (decreases the binding affinity and/or efficacy, NAMs) appears to
be more appropriate approach to develop drug candidates targeting 5-HT receptor. The first
known PAM of 5-HT2C is PNU-69176E 5a with high receptor subtype selectivity profile
(Pharmacia, Pfizer), but exhibit response in high micro molar range [14]. Furthermore, a recent
report showed that N-[(1-benzyl-1H-indol-3-yl) methyl]pyridin-3-amine (VA012, 5b) exhibit
selective PAM activity at 5-HT2C, induces significant anorexia and weight loss without causing
CNS-related malaise [15]. Thus, PAMs and NAMs provide a unique and advantageous way of
inducing selectivity and reducing side effects of not so selective 5-HT2C agonist [16]. In this
study, we synthesized compounds containing piperazine[17] and N-heteroaryl-phenyl
cyclopropyl methanone with an objective of designing selective agonist for 5-HT2C.
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Fig. 1. 5-HT2C agonist (1-4) and PAM (5a&b)
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Result and Discussion
N-linked heteroaryl, arylpiperazine (e.g., m-CPP 1) and 2-Phenylcyclopropylmethylamine 2
containing scaffolds are known to act as an agonist on the 5-HT2C [1, 18-23]. Moreover,
cyclopropyl ring containing scaffold in drugs have shown enhanced potency, pharmacokinetic
properties, aqueous solubility and increase in blood-brain barrier (BBB) permeability [24-28] In
continuation of our endeavor to develop new chemical entities (NCE) as CNS active agents [29],
we embarked upon a new series of compounds consisting of piperazine, N-heteroaryl, and
cyclopropyl scaffolds were evaluated for agonist and PAM activity on 5-HT2C (Fig. 2).
Fig. 2 Screening of N-heterocycle linked phenyl cyclopropyl derivatives allowed to identify hit compound
40a and 41a, which entered in the further modification on heterocycle unit for the search of PAM of the
5-HT2C Receptor.
Chemistry
The synthesis of cyclopropyl methane/methanone derivatives were achieved by employing
similar methods reported by us previously, starting from the commercially available 4-chloro-4-
fluorobutyrophenone 7 [30, 31]. Two series of phenyl cyclopropyl-linked N-heterocycle
derivatives were synthesized. The first series of compounds 19-26 (Scheme 1) were synthesized
by CuAAC reactions with phenyl cyclopropyl methyl azide 10. Azide intermediate 10 was
synthesized from pre-synthesized phenylcyclopropylmethanol 9 using sodium azide in acidic
condition [32]. Different alkyne derivatives were used during synthesis that are commercially
available (19, 23, 24 and 25), or pre-synthesized sulfonamide (20, 21), ester (22) and sugar
derivative (26). Having the azide derivative 1-[azido(cyclopropyl)methyl]-4-(benzyloxy)benzene
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10 and alkynes derivatives, the CuAAC reactions were performed in t-BuOH/H2O (1:1) using
equimolar quantities of the reactants, CuSO4.5H2O (10 mol%) and sodium ascorbate (20 mol%)
at ambient temperature that lead to synthesis of 1-(cyclopropyl(phenyl)methyl)-1H-1,2,3-
triazole derivatives in excellent yield 88-93 % (Scheme 1).
Scheme 1 Synthesis of phenyl cyclopropyl triazolyl methanes (19-26). Reagents and conditions: (a) NaH,
THF, TBAB, 0 °C- rt, 6 h (b) NaBH4, MeOH, 0 °C - rt, 4 h (c) NaN3, 56% H2SO4-CHCl3 (1:1), 0 °C- rt, 5 h (d)
CuSO4 (10 mol%), sodium ascorbate (20 mol%), tert-BuOH-H2O (1:1), 30 °C, 3-4h.
In scheme 2, 4-chloro-4-fluorobutyrophenone 7 was used to synthesize phenyl cyclopropyl
methanone-linked N-heterocyclic compounds (39a-50a and 58-62). Different N-heteroaryls
were fused with 4-chloro-4-fluorobutyrophenone in the presence of K2CO3 in DMSO at 140 °C.
The reaction proceeded through intramolecular cyclization, followed by SNAr substitution
reactions (39a-50a, Scheme 2). N-heterocycles linked cyclopropyl phenyl methanols (39b-47b)
were synthesized by carrying out the reduction of the cyclopropyl phenyl methanones with
NaBH4 at ambient temperature (Scheme 2).
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Scheme 2 Synthesis of 4-heterocycle substituted phenyl cyclopropyl methanones and methanols
compounds. Reagents and conditions: (a) K2CO3, DMSO, 140 °C, 14-16 h (b) NaBH4, MeOH, 0 °C - r.t., 4-5
h.
Further, we use different piperazines to synthesized piperazine-linked phenyl cyclopropyl
methanones (58-62) in same reaction conditions as described above (Scheme 3).
Scheme 3 Synthesis of 4-piperazinyl phenyl cyclopropyl methanones compounds 58-62. Reagents and
conditions: (a) K2CO3, DMSO, 140 °C, 12-14h.
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Pharmacology
Identification of novel 5-HT2C PAM
The phenyl cyclopropyl methane/methanone derivates (8, 19-26, 39a-45a, 44a' 39b-45b) were
evaluated on human 5-HT2C for agonist and PAM activity using NFAT-RE luciferase assay in
HEK293-T cells. For a compound to be active as an agonist, it should have more than 2-fold
stimulation above the baseline. None of the triazole-linked phenyl cyclopropyl methane
derivatives (19-26) and phenyl cyclopropyl methanone and methanol derivatives (8, 39a-45a,
39b-45b) were active as an agonist on the 5-HT2C (Fig. 3A). We further evaluated these
compounds for PAM activity on the 5-HT2C. For a compound to be active as a PAM, the
compound should either increase maximum efficacy (Emax) of serotonin by 2 fold or decrease
the EC50 value of serotonin on 5-HT2C. Interestingly, we identified three active molecules (40a,
41a and 22), out of these two imidazoles derived phenyl cyclopropyl methanones (40a, 41a)
were found consistently active as PAM on the 5-HT2C (Fig. 3B, C). Compound 22 was discarded
due to inconsistency in the PAM activity. We also evaluated these two active compounds on 5-
HT2B to determine selectivity (Fig. 4A-D), which shows that 40a and 41a were non-selective and
exhibited 5-HT2B PAM response similar to 5-HT2C (Fig 4A-D). Further, reduction of the active
cyclopropyl methanone derivatives to respective methanol derivatives (40b, 41b), which did
not exhibit any PAM activity at 5-HT2C.
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Fig. 3A) Representative bar graph shows 5-HT2C dependent increase in relative luminescence units (RLU)
after treatment with test compounds (8, 19-26, 39a-45a, 39b-45b) at10 µM for 6 hrs for agonist activity
at 5-HT2C. Serotonin (5-HT, 1µM) was used as positive control. Dashed lines represent half of the Emax. B)
Representative bar graph shows stimulation of 5-HT2C as measured of increase of RLU after treatment
with test compounds (8, 19-26, 39a-45a, 39b-45b) at 10 µM along with 5-HT (1µM for PAM activity at 5-
HT2C. Dashed lines represents Emax of 5-HT. C) Representative bar graph shows RLU of active compounds
(40a, 41a and 22) at 10 µM + serotonin (1µM for PAM activity at 5-HT2C).
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Fig. 4. Concentration response curve of 40a + 5-HT (A and B) and 41a + 5-HT (C and D) with at 5-HT2C (A
and C) and 5-HT2B (B and D) for PAM activity in transiently transfected HEK293T cells with NFAT-RE
luciferase along with either 5-HT2C or 5-HT2B.
After identification of the imidazole linked phenyl cyclopropyl methanone scaffold important
for PAM activity, we synthesized more azole-linked phenyl cyclopropyl methanones (46a-50a)
and also replaced the imidazole unit with piperazine unit (58-62). These compounds were also
evaluated for agonist activity and PAM activity on 5-HT2C (Fig. 5A).Surprisingly, none of these
compounds were active as agonist on 5-HT2C. Interestingly however, compound 58 (piperazine-
linked phenyl cyclopropyl methanone) was found active as PAM of 5-HT2C and increased the
maximum efficacy (Emax) of 5-HT from 100% to 149 % at 10 µM and 139 % at 1µM but was
inactive at other concentrations (Fig. 5B, D). PAM activity of compound 58 was also confirmed
by using another functional assay in which we measured intracellular calcium flux (iCa++) in
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HEK293T cells transiently expressing 5-HT2C in response to 5-HT either alone or combination of
compound 58. Indeed, we observed the increase of 5-HT activity by compound 58 at 1 µM (Emax
from 100 to 123.94, Fig. 5E).Compound 58 on Upon further evaluation of receptor subtype
selectivity of compound 58, we found that this compound exhibit NAM activity at 5-HT2B as it
decreased serotonin EC50 by 10 fold. Moreover, the NAM effect of compound 58 was found to
be concentration dependent as shown in Fig. 5C. Further change of 58 to allylpiperazine
derivative 59 resulted in the loss of PAM activity. The activity of these compounds was also
sensitive to steric bulk, as piperazine derivative with biphenyl (60) and piperonyl unit (61) were
inactive at this receptor. In this series, none of the phenyl cyclopropyl methyl triazole
derivatives were active as PAM or agonist on 5-HT2C. In case of the phenyl cyclopropyl
methanone-linked azoles, only imidazoles derivatives (40a, 41a) were active as PAM at 5-HT2C
and 5-HT2B, while the bicyclic imidazoles and other azoles (39a, 42a-50a) derivatives were
inactive. Since 40a were active as 5-HT2C PAM, we sought to evaluate the activity of imidazole
linked phenyl cyclopropyl methanone with propyl linker 52, and observed that this modification
lead to loss of the PAM activity in 52. These results suggest that imidazole should be directly
linked to the phenyl cyclopropyl methanone scaffold for PAM activity at 5-HT2C (Fig. 6).
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Fig. 5. Evaluation of phenyl cyclopropyl methanone derivatives on 5-HT2C for PAM activity using NFAT-
RE luciferase assay. A) Representative bar graph showing change in relative luminescence units after
addition of vehicle or compounds (8, 19-26, 39a-45a, 3b-45b) at 10 µM followed by serotonin (5-HT,
1µM). The compound 58 showed about 2 fold stimulation over 5-HT alone. B) Representative
concentration response curve of 5-HT (Emax =100% and log EC50 -8.01± 0.09) shows increased efficacy
with 10 µM (Emax =149%, log EC50 = -8.1± 0.16), and 1 µM of compound 58 (Emax =139% and log EC50 = -
8.21± 0.09). Data were plotted as mean ± SEM (n=3-4). C) Representative concentration response curve
of 5-HT at 5-HT2B showing log EC50 -8.75 ± 0.17 were as 5-HT + 58 (1µM) shows affinity of log EC50 -7.28 ±
0.10, 5-HT+ 58 (100 nM) shows affinity of log EC50 -8.12 ± 0.15 and 5-HT+ 58 (10 nM) shows affinity of
log EC50 -7.89 ± 0.13 did on 5-HT2B receptor. Data were plotted as mean ± SEM (n=3). D) Bar graph shows
increase in Emax of 5-HT (100 nM) in the presence of compound 58 at various concentrations Data were
plotted as mean ± SEM (n=3-4). **p<0.001, one way ANOVA Newman Keuls multiple comparisons post
hoc test. E) Representative bar graph showing significant increase in Emax of intracellular calcium flux
[iCa++] in HEK293T cells transfected with 5-HT2C upon prior stimulation with compound 58 (1 µM). The
Emax were plotted as mean ± SEM. *p<0.05, unpaired t-test. (n=4).
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Fig. 6. SAR analysis of all synthesized N-heterocycle-linked phenyl cyclopropyl methanones.
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Selectivity of phenyl cyclopropyl methanones on 5-HT2A and 5-HT2B receptors
Since activation of 5-HT2C analogous receptors 5-HT2A and 5-HT2B are widely known to induce
severe adverse effects such as hallucination/psychosis (5-HT2A) and cardiac valvulopathy (5-
HT2B), we determined the activity of compound 58 at 5-HT2A and 5-HT2B receptors. Our result
showed that PAM activity of piperazine-linked phenyl cyclopropyl methanone derivative 58 (1
µM) and imidazole-linked phenyl cyclopropyl methanones 40a and 41a (1 µM) showed equal
increase in the efficacy (100% to 140%) of 5-HT without affecting EC50 (Table 1) at 5-HT2C (Fig.
7C). As the compound 58 was found active at 1 µM on the 5-HT2C, we used this concentration
for selectivity profile for all active compounds. All three active compounds (58, 40a and 41a)
were inactive at 5-HT2A receptor as an agonist (Supp. Fig. 1) or as PAM (Fig. 7A, Table 1).
However, our initial screening showed that phenyl cyclopropyl methanone-linked imidazoles
(40a, 41a) exhibited non-selective PAM activity at 5-HT2B receptor without affecting EC50 of 5-
HT (Fig. 7B, Fig. 4C, D, Table 1). Interestingly, phenyl cyclopropyl methanone-linked piperazine
derivative 58 showed NAM activity and decreased the EC50 of 5-HT 10 times at 5-HT2B (Fig7B,
and Table 1). These data reveal that the presence of benzylpiperazine moiety on phenyl
cyclopropyl methanone scaffold is essential for dual activity of negative allosterism on 5-HT2B
and positive allosterism at 5-HT2C. We also wanted to determine if compound 58 via NAM
activity at 5-HT2B receptor can increase selectivity of another orthosteric agonist such as
lorcaserin. Lorcaserin has been reported to be 100 fold selective compared to 5-HT on 5-HT2B
[10]. We observed that compound 58 (1 µM) shifted EC50 of lorcaserin about 10 times at 5-HT2B
receptor (LogEC50 from -6.33 to -5.1, Fig. 8) which overall increases 1000 fold selectivity with
respect to 5-HT (5-HT log EC50 -8.54 and compound 58 + lorcaserin log EC50 -5.1, Fig. 8). Thus,
compound 58 might potentially be used to enhance the therapeutic window by decreasing the
chances of 5-HT2B-mediated adverse effect profile.
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Fig. 7. Selectivity of phenyl cyclopropyl methanones derivates (58, 40a and 41a) on 5-HT receptors for
allosteric activity using NFAT- RE luciferase assay. A) Representative dose response curve of 5-HT
(Emax100% and log EC50 -7.5± 0.13) does not show any change in efficacy (Emax) or affinity (EC50) of 5-HT
upon prior addition of 1µM of compound 58, 40a and 41a in 5-HT2A expressing HEK293T cells. Data were
plotted as mean ± sem (n=3). Emax was normalized in all the experiments by 5-HT alone response. B)
Representative dose response curve of 5-HT (Emax102% and log EC50 -8.75± 0.11) shows very little
increase in EC50 upon prior addition of 1µM of compound 58, 40a and 41a, whereas compound 58 shows
a decrease in EC50 of 5-HT on 5-HT2B receptor but no effect by compound 40a and 41a. Data were
plotted as mean ± SEM (n=3). C) Representative dose response curve of 5-HT (Emax100% and log EC50 -
8.1± 0.17) shows increased efficacy (Emax) and EC50 of 5-HT upon prior addition of 1µM of compound 58,
40a and 41a on the 5-HT2C receptor. Data were plotted as mean ± SEM (n=3-4).
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Fig. 8. Compound 58 increases selectivity of lorcaserin compared to 5-HT on 5-HT2B receptor.
Representative dose response curve of 5-HT (Emax100% and log EC50 -8.54 ± 0.11) shows no
increase in efficacy upon prior addition of 1µM of compound 58, whereas compound 58 shows
decrease in EC50 of lorcaserin from log EC50 -6.331 ± 0.08 (100 fold selective compared to 5-HT)
to log EC50 -5.1 ± 0.22 (> 1000 fold selective compared to 5-HT) on 5-HT2B receptor. Data were
plotted as mean ± SEM (n=4).
Table 1. Mean Emax(%) and log EC50 values obtained after normalizing response with 5-HT on different 5-
HT receptors. Emax was normalized by the 5-HT individual response.
Compound
5-HT
Structure
5-HT2C
5-HT2B
5-HT2A
Log EC50
Emax
100
Log EC50
Emax
102
Log EC50
Emax
100
-8.1 ± 0.09
-8.75± 0.11
7.45± 0.13
5-HT + 58
146*
143*
-8.30± 0.11ns
-8.37± 0.09ns
118*
119*
-7.93± 0.1*
-8.9± 0.09ns
99ns
7.42± 0.16ns
7.30± 0.15ns
5-HT + 40a
96ns
5-HT + 41a
140*
-8.38± 0.1ns
123*
-8.9±.13ns
100ns
7.34± 0.14ns
*p<0.01 compared with 5-HT in all experiments; ns= non significant. Analysis was done by in built
statistical analysis of top and EC50 comparison using GraphPad Prism 5 software.
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Molecular Docking studies at 5-HT2C allosteric site.
To further support our claim regarding allosteric activity of the active compounds, we
performed molecular docking studies on predicted allosteric site of 5-HT2C as reported recently
by Christopher et.al, [33]. For docking experiments, crystal structure of human serotonin
receptor protein 5-HT2C was retrieved from protein data bank (PDB) in its active form co-
crystallized with agonist ligand ergotamine (PDB ID: 6BQG, 3Ao resolution)[34]. Schrodinger
suite was used for all the protein-ligand preparation and docking experiments. Binding site and
receptor grid information for the docking of PAMs and serotonin were taken from Christopher
et al., [33] as described in the experimental section. Molecular docking studies were carried out
on all the three active compounds (40a, 41a and 58) to identify possible binding to allosteric
site for each identified PAM compounds in the extracellular region of the receptor (PDB ID:
6BQG). Fig. 9 depicted the binding mode of compound 40a (A), 41a (B) and 58 (C) in the
proposed allosteric site of the 5-HT2C protein. Top scoring poses measured by binding energy
(as shown in table 2) were selected for the interaction analysis of PAM compounds and 5-HT2C
protein.
Table 2: Binding energies of identified PAM compounds of the 5-HT2C protein
Compound ID Binding energy (kcal/mol)
40a
41a
58
-6.091
-6.276
-6.182
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Fig. 9. Binding mode of PAM compounds (green). Binding mode of compound 40a (A), compound 41a
(B), and compound 58 (C) in the allosteric site with respect to the position of 5-HT (pink) in the
orthosteric site of the 5-HT2C protein.
In compound 40a (Fig. 10A, B), the N-3 atom of the imidazole ring bonded to the side chain
amide group of ASN331 (TMH6) through conventional hydrogen bond while imidazole core was
involved in the π-alkyl interactions with VAL208 and LEU209 in the extracellular loop (EL2).
VAL354 (TMH7) hold aromatic ring through π-sigma interaction. Bridging carbonyl oxygen atom
bonded to SER110 (TMH2) with conventional hydrogen bond. Terminal cyclopropane formed
alkyl bonds with ILE114, TYR118, TRP355 and VAL354. These bonding and non-bonding
interactions are crucial for the higher affinity of compound 40a in the allosteric site of the 5-
HT2C protein.
Compound 41a (Fig. 10C, D) contain 4-nitroimidazole instead of imidazole forming an additional
hydrogen bond with the ASN331. Moreover, compound 41a formed same type of interactions
as exhibited by compound 40a, with an additional hydrogen bond between 4-nitroimidazole
and ASN331 as depicted in Fig. 10C & 10D directly contributing to binding energy (Table 2).
Compound 58 (Fig. 10E, F) is relatively more complex than compound 40a and 41a. It has
piperazine ring in place of imidazole which form alkyl-alkyl hydrophobic interactions with
VAL208 and LEU209. LEU209 involved in the Carbon-Hydrogen Bond with the methylene
bridging piperazine and benzene ring. An extra aromatic ring in the terminal involved in π-alkyl
hydrophobic interactions with VAL215. Both the interactions were directly involved in
increasing the binding energy of compound 58. Although crystal structures of 5-HT2B and 5-HT1B
are available, nothing is known about allosteric sites in these receptors, and therefore we could
not perform docking studies with our active molecules at these receptors. Understandably, this
is one of the limitations of this study.
18

ACCEPTED MANUSCRIPT
Figure 10: Interaction of PAM compounds (light green/gray) with the 5-HT2C protein. Amino acid
residues mediating interaction between 5-HT2C protein and compound 40a (A, B), compound 41a (C, D),
and compound 58 (E, F). Hydrogen bonds represented in green and cyan. Alkyl/ π-alkyl and π-sigma
interactions were represented in pink and purple respectively. Superscripts showed GPCR db labeling
[35] of amino acid residues.
19

ACCEPTED MANUSCRIPT
Effect of compound 58 on food intake in Sprague Dawley (SD) rats
Since 5-HT2C activation in brain induces anorexia [36-38], we evaluated the effect of compound
58 in vivo to determine if this class of molecules can exhibit drug like properties in comparison
to full agonist lorcaserin and ultimately induce anorexia. We administered compound 58 (2
pmols/rat/2 µL) via intracerebroventricular (i.c.v.) route and measured suppression of food
intake (anorexia) in SD rats. As expected, compound 58 suppressed food intake after 3 and 6
hours of administration and is comparable to reference anti-obesity drug lorcaserin
administered at same dose. Since the effect of compound 58 as well as reference drug did not
exhibit any effect on cumulative food intake after 24 hrs, most likely both compounds are
getting metabolized after 6 hrs (Fig. 11). These results suggest that both allosteric modulator
(compound 58) and full agonist (lorcaserin) had similar in vivo effect at least in terms of
suppression of food intake.
Fig. 11. Acute food intake in SD rats after central administration (i.c.v.) of compound 58. Each
histogram represents mean ± SEM (n=4). ***p<0.001 and *p<0.05, one way ANOVA Newman
Keuls multiple comparisons posthoc analysis, ns (non significant).
20

ACCEPTED MANUSCRIPT
Conclusions
In this study we evaluated allosteric activity of phenyl-cyclopropyl-linked N-heterocycles
at 5-HT2C receptor. Our result suggests that imidazole-linked phenyl cyclopropyl
methanones (40a and 41a) and benzylpiperazine linked phenyl cyclopropyl methanones
(
58) acts as 5-HT2C PAM activity. Notably, compound 58 showed unique dual
characteristics by exhibiting PAM response at 5-HT2C and NAM at 5-HT2B receptors, which
is a unique property that is rarely reported for allosteric modulators. Such molecules
could be further developed as adjuvant therapy to enhance selectivity over receptor
subtype. Molecular docking studies of compound 58, 40a and 41a further supports our
observation of allosteric binding on 5-HT2C. Furthermore, compound 58 exhibited in vivo
efficacy and the effect was found to be comparable to FDA approved drug lorcaserin,
suggesting the strong potential of compound 58 for further development as therapeutics
for obesity. However, further molecular docking studies on allosteric site of serotonin
receptor subtype family and experimental pharmaco-kinetic studies are needed to
completely understand the dual mechanism exhibited by compound 58
.
21

ACCEPTED MANUSCRIPT
Experimental Section
General methods
Commercially available reagent grade chemicals were used as received. All reactions
were monitored by TLC on E. Merck Kieselgel 60 F254, with detection by UV light,
spraying 20% aq. KMnO4 solution or spraying 4% ethanolic H2SO4. Column
chromatography was performed on Silica Gel (60–120 mesh, E. Merck). IR spectra were
recorded as thin films or in KBr pellet with a Perkin-Elmer Spectrum RX-1 (4000–450 cm-
1
1) spectrophotometer. H NMR spectra were recorded on 500, 400 and 300 MHz Bruker
NMR spectrometers in CDCl3 or DMSO-d6. Chemical shift values are reported in δ ppm
relative to the residual signals of TMS in CDCl3 or deuterated solvent DMSO-d6. 13C NMR
spectra were recorded on 100 and 75 MHz Bruker NMR spectrometers. Unless
otherwise stated; J in Hertz. ESI mass spectra were recorded using a Quattro II
(Micromass) instrument. HRMS spectra were recorded using a mass spectrometer Q-
TOF. The purity of all tested compounds was characterized by HPLC analysis (Discovery
HS C-18 HPLC) system. The HPLC system consisted of a pump (LC-10AT VP with FCV-10AL
VP), degasser (DGU-14A) and auto-injector (SIL-HTc, fixed with a 100 μl loop) (Shimadzu,
Japan). Eluents were monitored at 260 nm with a UV-Vis multiple wavelength detector
and chromatograms were integrated using Class-VP (version 6.12 SP5) software
(Shimadzu, Japan). Individual compounds with a purity of >95% were used for
subsequent experiments.
1-{Azido(cyclopropyl)methyl}-4-(benzyloxy)benzene (10). The reaction of
9 (0.5 g,
1.96 mmol) in an ice cold solution (34 ml) of 56% H2SO4 and chloroform (1:1) in which
NaN3 (0.38 g, 5.90 mmol) was added slowly in portion at 0 °C and the reaction was
stirred at r.t. for about 5 h. After the completion of reaction, the workup was done with
ethylacetate and water, then the organic layer was separated, dried over anhydrous
Na2SO4 and concentrated under reduced pressure passed through column to obtain the
compound 10 as white solid in 65% yield (0.36 g); mp 62-64 °C; IR (νmax, cm-1): 3010 (C-
H), 2095 (N3 asymmetric), 1610 (C=C), 1510 (C=C), 1457 (C=C), 1240 (N3 symmetric), 824
1
(C-H bend); H NMR (400 MHz, CDCl3) 7.40 (m, 4H, Ar-H), 7.34 (m, 1H, Ar-H), 7.28 (d, J =
22

ACCEPTED MANUSCRIPT
8.66 Hz, 2H, Ar-H), 6.97 (d, J = 8.66 Hz, 2H, Ar-H), 5.06 (s, 2H, CH2), 3.81 (d, J = 8.60 Hz,
1H, CH), 1.28 (m, 1H, CH), 0.73 (m, 1H, CH), 0.56 (m, 2H, CH), 0.30 (m, 1H, CH); 13C NMR
(100 MHz, CDCl3) δ 158.6 (Ar-C), 136.8 (Ar-C), 132.1 (Ar-C), 128.6 (Ar-C), 128.1 (Ar-C),
128.0 (Ar-C), 127.4 (Ar-C), 114.8 (Ar-C), 70.0 (CH2), 69.4 (CH), 16.3 (CH), 4.3 (CH2).
General procedure for the synthesis of compounds 19-26
Substituted propargyl alcohol and selected alkynes 11-18 (1 mmol) and synthesized
azide 10 (1 mmol) were suspended in a mixture of 1:1 t-butanol-water (20 mL) and kept
for stirring at room temperature. To this stirring reaction mixture freshly prepared 0.5
mL solution of sodium ascorbate (20 mol %) and 0.2 mL solution of CuSO4.5H2O (10 mol
%) in were sequentially added. The reaction mixture was stirred at ambient temperature
for 12-16h. After completion of reaction (TLC), the workup was done with EtOAc and
water. The organic layer was dried (anhd. Na2SO4) and evaporated under reduced
pressure to give a crude mass. The latter was purified by silica gel (60-120 mesh) column
chromatography using ethylacetate: hexane (1:1) as eluent to give the cycloaddition
products 19-26 in excellent yields.
1-{(4-Benzyloxyphenyl)(cyclopropyl)methyl}-1H-1,2,3-triazol-4-yl-methanol (19). The
reaction of 1-(azido(cyclopropyl)methyl)-4-(benzyloxy)benzene 10 (0.1 g, 0.367 mmol)
.
and propargyl alcohol 11 (0.02 ml, 0.40 mmol), CuSO4 5H2O (0.01 g, 0.04 mmol) and
sodium ascorbate (0.02 g, 0.08 mmol) in 1:1 t-butanol-water (20 ml) in 88 % yield (0.1 g)
as white solid; mp 112-114 °C; IR (νmax, cm-1): 3367 (O-H), 1610 (C=C), 1512 (C=C
1
triazole), 1455 (N=N), 1241 (C-O), 755 (C-H bend); H NMR (400 MHz, CDCl3) δ 7.59 (s,
1H, triazole-H), 7.44-7.38 (m, 4H, Ar-H), 7.34 (m, 1H, Ar-H), 7.26 (d, J= 8.64 Hz ,2H, Ar-H),
6.97 (d, J = 8.64 Hz, 2H, Ar-H), 5.075 (s, 2H, CH2), 4.93 (d, J = 9.64 Hz, 1H, CH), 4.81 (s, 2H,
CH2), 2.38 (s, 1H, OH), 1.65 (m, 1H, CH), 0.90-0.72 (m, 2H, CH), 0.53 (m, 2H, CH); 13C NMR
(100 MHz, CDCl3) δ 158.8 (Ar-C), 136.7 (Ar-C), 130.9 (Ar-C), 128.6 (Ar-C), 128.2 (Ar-C),
128.0 (Ar-C), 127.4 (Ar-C), 115.0 (Ar-C), 70.0 (CH2), 69.0 (CH), 56.64 (CH2), 15.9 (CH), 5.3
(CH2); Anal. Found: C, 71.61; H, 6.29; N, 12.51. Calcd: C, 71.62; H, 6.31; N, 12.53; HRMS:
Calcd. Accurate mass for (C20H22N3O2): 336.1707. Found 336.1700 [M+H]+, (C17H17O):
23

ACCEPTED MANUSCRIPT
Calcd. 237.1274. Found 237.1273 [M-98], (C7H7): Calcd. 91.0542. Found 91.0545 [M-
244].
N-1-{(4-benzyloxyphenyl)(cyclopropyl)methyl}-1H-1,2,3-triazol-4-yl-methyl
4-
fluorobenzenesulfonamide (20). The reaction of 1-(azido(cyclopropyl)methyl)-4-
(benzyloxy)benzene 10 (0.1 g, 0.367 mmol) and N-propargyl-4-fluoro-
.
benzenesulfonamide 12 (0.076 g, 0.40 mmol), CuSO4 5H2O (0.01 g, 0.04 mmol) and
sodium ascorbate (0.02 g, 0.08 mmol) in 1:1 t-butanol-water (20 ml) in 92% yield (0.16 g)
as white solid; mp 170-172 °C; IR (νmax, cm-1): 3583 (N-H), 1612 (C=C), 1531 (C=C
1
triazole), 1216 (C-O), 1148 (S=O), 756 (C-H bend); H NMR (400 MHz, CDCl3) δ 7.83 (m,
2H, Ar-H), 7.43 (s, 1H, triazole-H), 7.41-7.35 (m, 4H, Ar-H), 7.33 (m, 1H, Ar-H), 7.18 (d, J =
8.64 Hz, 2H, Ar-H), 7.09 (m, 2H, Ar-H), 6.94 (d, J = 8.64 Hz, 2H, Ar-H), 5.37 (t, J = 5.88 Hz,
1H, NH), 5.06 (s, 2H, CH2), 4.82 (d, J = 9.68 Hz, 1H, CH), 4.27 (d, J = 6.08 Hz, 2H, CH2), 1.58
(m, 1H, CH), 0.86-0.68 (m, 2H, CH), 0.46 (m, 2H, CH); 13C NMR (100 MHz, CDCl3) δ 165.04
(d, 1JCF = 253.1 Hz), 158.93 (Ar-C), 143.06 (Ar-C), 136.66 (Ar-C), 135.92 (Ar-C), 130.65 (Ar-
3
C), 129.85 (d, JCF = 9.1 Hz), 128.65 (Ar-C), 128.21(Ar-C), 128.10 (Ar-C), 127.45 (Ar-C),
2
120.60 (Ar-C), 116.28 (d, JCF = 22.43 Hz), 115.15 (Ar-C), 77.22 (CH), 70.10 (CH2), 69.20
(CH), 38.74 (CH2), 15.85 (CH), 5.29 (CH2); Anal. Found: C, 63.17; H, 5.12; N, 11.35. Calcd:
C, 63.40; H, 5.12; N, 11.37; HRMS: Calcd. Accurate mass for (C26H26FN4O3S): 493.1704.
Found 493.1694 [M+H]+, (C17H17O): Calcd. 237.1274. Found 237.1280 [M-255], (C7H7):
Calcd. 91.0542. Found 91.0542 [M-401].
N-1-{(4-benzyloxyphenyl)(cyclopropyl)methyl}-1H-1,2,3-triazol-4-yl-methyl
4-
methylbenzenesulfonamide (21). The reaction of 1-(azido(cyclopropyl)methyl)-4-
(benzyloxy)benzene 10 (0.1 g, 0.367 mmol) and N-propargyl-4-methyl-
.
benzenesulfonamide 13 (0.074 g, 0.40 mmol), CuSO4 5H2O (0.01 g, 0.04 mmol) and
sodium ascorbate (0.02 g, 0.08 mmol) in 1:1 t-butanol-water (20 ml) in 90% yield (0.16 g)
as a white solid; mp 156-158 °C; IR (νmax, cm-1): 3583 (N-H), 1610 (C=C), 1454 (N=N),
1324 (C-N), 1243 (C-O), 1158 (S=O), 753 (C-H bend); 1H NMR (400 MHz, CDCl3) δ 7.72 (d,
J = 8.20 Hz, 2H, Ar-H), 7.44 (s, 1H, triazole-H), 7.42-7.36 (m, 4H, Ar-H), 7.32 (m, 1H, Ar-H),
7.26 (d, J = 7.76 Hz, 2H, Ar-H), 7.18 (d, J = 8.64 Hz, 2H, Ar-H), 6.94 (d, J = 8.64 Hz, 2H, Ar-
24

ACCEPTED MANUSCRIPT
H), 5.07 (s, 1H, NH), 5.05 (s, 2H, CH2), 4.83 (d, J = 9.60 Hz, 1H, CH), 4.24 (d, J = 6.08, 2H,
13
CH2), 2.40 (s, 3H, CH3), 1.57 (m, 1H, CH), 0.88-0.66 (m, 2H, CH), 0.46 (m, 2H, CH); C
NMR (100 MHz, CDCl3) δ 158.8 (Ar-C), 143.6 (Ar-C), 136.6 (Ar-C), 130.7 (Ar-C), 129.7 (Ar-
C), 128.6 (Ar-C), 128.1 (Ar-C), 128.0 (Ar-C), 127.4 (Ar-C), 127.1 (Ar-C), 120.7 (Ar-C), 115.1
(Ar-C), 70.1 (CH2), 69.1 (CH), 38.9 (CH2), 21.5 (CH3), 15.8 (CH), 5.3 (CH2); Anal. Found: C,
66.36; H, 5.77; N, 11.45. Calcd: C, 66.37; H, 5.78; N, 11.47; HRMS: Calcd. Accurate mass
for (C27H29N4O3S): 489.1955. Found 489.1945 [M+H]+, (C17H17O): Calcd. 237.1274. Found
237.1274 [M-251], (C7H7): Calcd. 91.0542. Found 91.0541 [M-397].
1-{(4-Benzyloxyphenyl)(cyclopropyl)methyl}-1H-1,2,3-triazol-4-yl-methyl
3,4,5-
trimethoxybenzoate (22). The reaction of 1-(azido(cyclopropyl)methyl)-4-
(benzyloxy)benzene 10 (0.1 g, 0.36 mmol) and propargyl-3,4,5-trimethoxybenzoate 14
.
(0.09 g, 0.36 mmol), CuSO4 5H2O (0.01 g, 0.04 mmol) and sodium ascorbate (0.02 g, 0.08
mmol) in 1:1 t-butanol-water (20 ml) in 85 % yield (165 mg) as white solid; mp 100-102
°C; IR (νmax, cm-1): 2938 (C-H), 1713 (C=O), 1589 (C=C), 1416 (N=N), 1345 (C-N), 1224 (C-
O), 1178 (C-O), 863 (C-H bend); 1H NMR (400 MHz, CDCl3) δ 7.72 (s,1 H, triazole-H), 7.44-
7.38 (m, 4H, Ar-H), 7.35 (m, 1H, Ar-H), 7.31 (s, 2H, Ar-H), 7.25 (s, 1H, Ar-H), 6.96 (d, J =
8.60 Hz, 2H, Ar-H), 5.40 (s, 2H, CH2), 5.07 (s, 2H, CH2), 4.92 (d, J = 9.68 Hz, 1H, CH), 3.91
(s, 3H, CH3), 3.90 (s, 6H, CH3), 1.69 (m, 1H, CH), 0.88-0.70 (m, 2H, CH), 0.52 (m, 2H, CH);
13C NMR (100 MHz, CDCl3) δ 166.1 (C=O), 158.8 (Ar-C), 152.9 (Ar-C), 142.7 (Ar-C), 136.6
(Ar-C), 130.8 (Ar-C), 128.6 (Ar-C), 128.2 (Ar-C), 128.0 (Ar-C), 127.4 (Ar-C), 124.7 (Ar-C),
115.1 (Ar-C), 107.0 (Ar-C), 70.1 (CH2), 69.2 (CH), 60.9 (CH3), 58.1 (CH3), 56.2 (CH2), 15.9
(CH), 5.3 (CH2); Anal. Found: C, 68.02; H, 5.89; N, 7.89. Calcd: C, 68.04; H, 5.90; N, 7.93;
HRMS: Calcd. Accurate mass for (C30H32N3O6): 530.2286. Found 530.2272 [M+H]+,
(C17H17O): Calcd. 237.1274. Found 237.1270 [M-292], (C7H7): Calcd. 91.0542. Found
91.0535 [M-438].
4-{1-((4-Benzyloxyphenyl)(cyclopropyl)methyl)-1H-1,2,3-triazol-4-yl}butan-1-ol (23).
The reaction of 1-(azido(cyclopropyl)methyl)-4-(benzyloxy)benzene 10 (0.1 g, 0.367
.
mmol), 5-hexyne-1-ol 15 (0.04 ml 0.367 mmol), CuSO4 5H2O (0.01 g, 0.04 mmol) and
sodium ascorbate (0.02 g, 0.08 mmol) in 1:1 t-butanol-water (20 ml) in 85% yield (0.12 g)
25

ACCEPTED MANUSCRIPT
as white solid; mp 112-114 °C; IR (νmax, cm-1): 3367 (O-H), 2936 (C-H), 1610 (C=C), 1511
1
(C=C triazole), 1455 (N=N), 1381 (C-N), 1242 (C-O), 1110 (C-O), 799 (C-H bend); H NMR
(400 MHz, CDCl3) δ 7.42 (s, 1H, triazole-H), 7.40-7.31 (m, 4H, Ar-H), 7.30 (m, 1H, Ar-H),
7.23 (d, J= 8.66 Hz ,2H, Ar-H), 6.94 (d, J = 8.66 Hz, 2H, Ar-H), 5.05 (s, 2H, CH2), 4.88 (d, J =
9.64 Hz, 1H, CH), 3.66 (t, J= 6.32 Hz, 2H, CH2), 2.73 (t, J= 7.40 Hz, 2H, CH2), 1.76 (m, 2H,
13
CH2), 1.65 (m, 2H, CH2), 1.61 (m, 1H, CH), 0.88-0.68 (m, 2H, CH), 0.49 (m, 2H, CH); C
NMR (100 MHz, CDCl3) δ 158.7 (Ar-C), 147.9 (Ar-C), 136.7 (Ar-C), 131.3 (Ar-C), 128.6 (Ar-
C), 128.1 (Ar-C), 128.0 (Ar-C), 127.4 (Ar-C), 119.2(Ar-C), 115.0(Ar-C), 70.0 (CH2), 68.7
(CH), 62.44 (CH2), 32.24 (CH2), 25.52 (CH2), 25.41 (CH2), 15.9 (CH), 5.3 (CH2); Anal. Found:
C, 73.17; H, 7.20; N, 11.13. Calcd: C, 73.18; H, 7.21; N, 11.13; HRMS: Calcd. Accurate
mass for (C23H28N3O2): 378.2171. Found 378.2167 [M+H]+, (C17H17O): Calcd. 237.1274.
Found 237.1258 [M-100], (C7H7): Calcd. 91.0542. Found 91.0538 [M-246].
1-{(4-Benzyloxyphenyl)(cyclopropyl)methyl}-4-phenyl-1H-1,2,3-triazole (24). The
reaction of 1-(azido(cyclopropyl)methyl)-4-(benzyloxy)benzene 10 (0.1 g, 0.36 mmol),
.
phenylacetylene 16 (0.04 ml 0.36 mmol), CuSO4 5H2O (0.01 g, 0.04 mmol) and sodium
ascorbate (0.02 g, 0.08 mmol) in 1:1 t-butanol-water (20 ml) in 90% (0.13 g) as white
solid; mp 158-160 °C; IR (νmax, cm-1): 2926 (C-H), 1610 (C=C), 1511 (C=C, triazole), 1455
1
(N=N), 1244 (C-O), 764 (C-H bend); H NMR (400 MHz, CDCl3) δ 7.82 (m, 2H, Ar-H), 7.75
(s, 1H, triazole-H), 7.45-7.34 (m, 6H, Ar-H), 7.33-7.26 (m, 4H, Ar-H), 6.96 (d, J = 8.72 Hz,
2H, Ar-H), 5.05 (s, 2H, CH2), 4.97 (d, J = 9.64 Hz, 1H, CH), 1.70 (m, 1H, CH), 0.81 (m, 2H,
CH), 0.55 (m, 2H, CH); 13C NMR (100 MHz, CDCl3) δ 158.8 (Ar-C), 147.7 (Ar-C), 136.7 (Ar-
C), 131.1 (Ar-C), 130.8 (Ar-C), 128.7 (Ar-C), 128.6 (Ar-C), 128.2 (Ar-C), 128.1 (Ar-C), 128.0
(Ar-C), 127.4 (Ar-C), 125.6 (Ar-C), 118.1(Ar-C), 115.1(Ar-C), 70.1 (CH2), 68.9 (CH), 16.0
(CH), 5.4 (CH2); Anal. Found: C, 78.68; H, 6.07; N, 11.02. Calcd: C, 78.71; H, 6.08; N,
11.02; HRMS: Calcd. Accurate mass for (C25H24N3O): 382.1914. Found 382.1908 [M+H]+,
(C17H17O): Calcd. 237.1274. Found 237.1270 [M-144], (C7H7): Calcd. 91.0542. Found
91.0548 [M-290].
1-{(4-Benzyloxyphenyl)(cyclopropyl)methyl}-4-pentyl-1H-1,2,3-triazole (25). The
reaction of 1-(azido(cyclopropyl)methyl)-4-(benzyloxy)benzene 10 (0.1 g, 0.36 mmol),
26

ACCEPTED MANUSCRIPT
.
heptyne-1 17 (0.048 ml, 0.36 mmol), CuSO4 5H2O (0.01 g, 0.04 mmol) and sodium
ascorbate (0.02 g, 0.08 mmol) in 1:1 t-butanol-water (20 ml) in 85% (0.12 g) as a white
solid; mp 86-88 °C; IR (νmax, cm-1): 2927 (C-H), 1610 (C=C), 1531 (C=C, triazole), 1454
1
(N=N), 1243 (C-O), 665 (C-H bend); H NMR (400 MHz, CDCl3) δ 7.42 (s, 1H, triazole-H),
7.40-7.31 (m, 4H, Ar-H), 7.28 (m, 1H, Ar-H), 7.23 (d, J= 8.60 Hz ,2H, Ar-H), 6.94 (d, J =
8.60 Hz, 2H, Ar-H), 5.05 (s, 2H, CH2), 4.89 (d, J = 9.56 Hz, 1H, CH), 2.69 (t, J= 7.72 Hz, 2H,
CH2), 1.64 (m, 2H, CH2), 1.63 (m, 1H, CH), 1.32 (m, 4H, CH2), 0.88 (t, J= 6.68 Hz, 3H, CH3),
0.84-0.68 (m, 2H, CH), 0.50 (m, 2H, CH); 13C NMR (100 MHz, CDCl3) δ 158.7 (Ar-C), 136.7
(Ar-C), 131.4 (Ar-C), 128.6 (Ar-C), 128.1 (Ar-C), 128.0 (Ar-C), 127.4 (Ar-C), 119.1(Ar-C),
114.9(Ar-C), 70.0 (CH2), 68.6 (CH), 31.5 (CH2), 29.0 (CH2), 25.8 (CH2), 22.3 (CH2), 15.9
(CH), 14.0 (CH3), 5.3 (CH2); Anal. Found: C, 76.75; H, 7.77; N, 11.19. Calcd: C, 76.76; H,
7.78; N, 11.19; HRMS: Calcd. Accurate mass for (C24H30N3O): 376.2383 Found 376.2381
[M+H]+, (C17H17O): Calcd. 237.1274. Found 237.1253 [M-138], (C7H7): Calcd. 91.0542.
Found 91.0539 [M-284].
1-{(4-Benzyloxyphenyl)(cyclopropyl)methyl-4-(1’,2’:5’,6’-di-O-isopropylidene-α-D-
glucofuranos-3’-yloxy)methyl}-1H-1,2,3-triazole
(26).
The
reaction
of
1-
(azido(cyclopropyl)methyl)-4-(benzyloxy)benzene (0.1 g, 0.36 mmol), propargylated
.
diacetonide protected glucose 18 (0.11 g, 0.40 mmol), CuSO4 5H2O (0.01 g, 0.04 mmol)
and sodium ascorbate (0.02 g, 0.08 mmol) in 1:1 t-butanol-water (5 ml), purification by
column chromatography using 60-120 mesh silica gel (50% EtOAc/Hexane), gave the
titled compound 26 (0.18 g, 86%) as a sticky glassy solid; IR (νmax, cm-1): 2988 (C-H), 2929
(C-H), 1611 (C=C), 1512 (C=C, triazole), 1456 (N=N), 1377 (C-N), 1220 (C-O), 848 (C-H
1
bend); H NMR (400 MHz, CDCl3) δ 7.61 (s, 1H, triazole-H), 7.46-7.35 (m, 4H, Ar-H), 7.32
(m, 1H, Ar-H), 7.23 (d, J= 8.60 Hz ,2H, Ar-H), 6.95 (d, J = 8.60 Hz, 2H, Ar-H), 5.85 (d, J = 3.4
Hz, 1H, CH), 5.05 (s, 2H, CH2), 4.91 (d, J = 9.52 Hz, 1H, CH), 4.81 (m, 2H, CH2), 4.58 (d, J =
4.08 Hz, 1H, CH), 4.29 (m, 1H, CH), 4.10 (m, 1H, CH), 4.04 (m, 2H, CH), 3.99 (d, J = 5.46
Hz, 1H, CH), 1.66 (m, 1H, CH), 1.48 (s, 3H, CH3), 1.35 (s, 3H, CH3), 1.30 (s, 3H, CH3), 1.28
13
(s, 3H, CH3), 0.86-0.70 (m, 2H, CH), 0.52 (m, 2H, CH); C NMR (100 MHz, CDCl3) δ 158.8
(Ar-C), 136.6 (Ar-C), 131.0 (Ar-C), 128.6 (Ar-C), 128.2 (Ar-C), 128.1 (Ar-C), 128.0 (Ar-C),
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ACCEPTED MANUSCRIPT
127.4 (Ar-C), 121.0 (Ar-C), 115.1 (C-O), 111.8 (Ar-C), 109.0 (C-O), 105.2 (C-O), 82.6 (C-O),
81.8 (C-O), 81.1 (C-O), 72.4 (C-O), 70.9 (CH2), 68.9 (CH), 67.4 (C-O), 64.3 (CH2), 29.7 (CH3),
26.8 (CH3), 26.2 (CH3), 25.4 (CH3), 15.9 (CH), 5.3 (CH2); HRMS: Calcd. Accurate mass for
(C32H40N3O7): 578.2861. Found 578.2849 [M+H]+, (C17H17O): Calcd. 237.1274. Found
237.1255 [M-340], (C7H7): Calcd. 91.0542. Found 91.0540 [M-485].
Cyclopropyl-{4-(1H-benzo[d]imidazol-1-yl)-phenyl}methanone (39a). Following the
same procedure and the workup used for the synthesis of (40a), the reaction of 4-
chloro-4-fluorobutyrophenone
7 (0.5 mL, 3.04 mmol), benzimidazole 27 (0.36g, 3.04
mmol) and K2CO3 (1 g, 7.23 mmol) in DMSO gave the title compound as white solid in
78% yield (0.62 g); mp 138-140 °C; IR (νmax, cm-1): 3016 (C-H), 1667 (C=O), 1531 (C=C),
1514 (C=N), 1419 (C-N), 1385 (C-C), 843 (C-H bend); 1H NMR (400 MHz, CDCl3) δ 8.16 (d,
J= 8.52 Hz ,2H, Ar-H), 8.10 (s, 1H, Ar-H), 7.82 (m, 1H, Ar-H), 7.58 (d, J = 8.52 Hz, 2H, Ar-H),
7.52 (m, 1H, Ar-H), 7.29 (m, 2H, Ar-H), 2.63 (m, 1H, CH), 1.24 (m, 2H, CH), 1.04 (m, 2H,
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CH); C NMR (100 MHz, CDCl3) δ 199.2 (C=O),144.2 (Ar-C), 141.8 (Ar-C), 139.9 (Ar-C),
137.1 (Ar-C), 133.2 (Ar-C), 130.0 (Ar-C), 124.1 (Ar-C), 123.4 (Ar-C), 123.2 (Ar-C), 120.9
(Ar-C), 110.4 (Ar-C), 17.3 (CH), 12.0 (CH2); Anal. Found: C, 77.81; H, 5.37; N, 10.64. Calcd:
C, 77.84; H, 5.38; N, 10.68; HRMS: Calcd. Accurate mass for (C17H15N2O): 263.1179.
Found 263.1168 [M+H]+, (C13H10N2): Calcd. 194.0838. Found 194.0840 [M-68], (C10H9O):
Calcd. 145.0648. Found 145.0623 [M-117].
Cyclopropyl-{4-(1H-imidazol-1-yl)phenyl}methanone (40a)[39]. The reaction carried
out with 4-chloro-4-fluorobutyrophenone
7 (0.5 ml, 3.04 mmol) with imidazole 28 (0.21
g, 3.04 mmol) and K2CO3 (1 g, 7.23 mmol) in DMSO at 140 °C till the completion of
reaction (TLC). After the completion of reaction, the workup was done with ethylacetate
and water and then the organic layer was separated, dried over anhydrous Na2SO4 and
evaporated under reduced pressure. The latter was purified by silica gel (60-120 mesh)
column chromatography using ethylacetate: hexane (1:1) as eluent to give the title
compound in 80% yield (0.51 g); mp 108-110 °C; IR (νmax, cm-1): 3392 (C-H), 1663 (C=O),
1
1604 (C=C), 1520 (C=N), 1485 (C-N), 1305 (C-N), 840 (C-H bend); H NMR (400 MHz,
CDCl3) δ 8.16 (d, J = 8.70 Hz, 2H, Ar-H), 8.01 (s, 1H, Ar-H), 7.53 (d, J = 8.70 Hz, 2H, Ar-H),
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