Exploration of flexible phenylpropylurea scaffold as novel cardiac myosin activators for the treatment of systolic heart failure

Article abstract of DOI:10.1016/j.ejmech.2017.04.005

A series of flexible urea derivatives have been synthesized and demonstrated as selective cardiac myosin ATPase activator. Among them 1-phenethyl-3-(3-phenylpropyl)urea (1, cardiac myosin ATPase activation at 10?μM?=?51.1%; FS?=?18.90; EF?=?12.15) and 1-benzyl-3-(3-phenylpropyl)urea (9, cardiac myosin ATPase activation?=?53.3%; FS?=?30.04; EF?=?18.27) showed significant activity in?vitro and in?vivo. The change of phenyl ring with tetrahydropyran-4-yl moiety viz., 1-(3-phenylpropyl)-3-((tetrahydro-2H-pyran-4-yl)methyl)urea (14, cardiac myosin ATPase activation?=?81.4%; FS?=?20.50; EF?=?13.10), and morpholine moiety viz., 1-(2-morpholinoethyl)-3-(3-phenylpropyl)urea (21, cardiac myosin ATPase activation?=?44.0%; FS?=?24.79; EF?=?15.65), proved to be efficient to activate the cardiac myosin. The potent compounds 1, 9, 14 and 21 were found to be selective for cardiac myosin over skeletal and smooth myosins. Thus, these urea derivatives are potent scaffold to develop as a newer cardiac myosin activator for the treatment of systolic heart failure.

Full text of DOI:10.1016/j.ejmech.2017.04.005

Accepted Manuscript
Exploration of flexible phenylpropylurea scaffold as novel cardiac myosin activators
for the treatment of systolic heart failure
Manoj Manickam, Hitesh B. Jalani, Thanigaimalai Pillaiyar, Niti Sharma, PullaReddy
Boggu, Eeda Venkateswararao, You-Jung Lee, Eun-Seok Jeon, Sang-Hun Jung
PII:
S0223-5234(17)30260-X
10.1016/j.ejmech.2017.04.005
EJMECH 9352
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 February 2017
Revised Date: 22 March 2017
Accepted Date: 3 April 2017
Please cite this article as: M. Manickam, H.B. Jalani, T. Pillaiyar, N. Sharma, P. Boggu, E.
Venkateswararao, Y.-J. Lee, E.-S. Jeon, S.-H. Jung, Exploration of flexible phenylpropylurea scaffold as
novel cardiac myosin activators for the treatment of systolic heart failure, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.005.
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ACCEPTED MANUSCRIPT
Exploration of flexible phenylpropylurea scaffold as novel cardiac myosin activators for
the treatment of systolic heart failure
GRAPHICAL ABSTRACT

ACCEPTED MANUSCRIPT
Exploration of flexible phenylpropylurea scaffold as novel cardiac myosin activators for
the treatment of systolic heart failure
Manoj Manickama, Hitesh B. Jalania, Thanigaimalai Pillaiyara, Niti Sharmaa, PullaReddy
Boggua, Eeda Venkateswararaoa, You-Jung Leeb, Eun-Seok Jeonb, Sang-Hun Junga*
a College of Pharmacy and Institute of Drug Research and Development, Chungnam National
University, Daejeon 34134, Korea
b Division of Cardiology, Samsung Medical Center, Samsung Biomedical Research Institute, School
of Medicine, Sungkyunkwan University, 81 Irwon-Ro, Gangnam-gu, Seoul 06351, Korea
Abstract
A series of flexible urea derivatives have been synthesized and demonstrated as selective cardiac
myosin ATPase activator. Among them 1-phenethyl-3-(3-phenylpropyl)urea (1, cardiac myosin
ATPase activation at 10 µM = 51.1 %; FS = 18.90; EF = 12.15) and 1-benzyl-3-(3-
phenylpropyl)urea (9, cardiac myosin ATPase activation = 53.3 %; FS = 30.04; EF = 18.27)
showed significant activity in vitro and in vivo. The change of phenyl ring with tetrahydropyran-
4-yl moiety viz., 1-(3-phenylpropyl)-3-((tetrahydro-2H-pyran-4-yl)methyl)urea (14, cardiac
myosin ATPase activation = 81.4 %; FS = 20.50; EF = 13.10), and morpholine moiety viz., 1-(2-
morpholinoethyl)-3-(3-phenylpropyl)urea (21, cardiac myosin ATPase activation = 44.0 %; FS =
24.79; EF = 15.65), proved to be efficient to activate the cardiac myosin. The potent compounds
1, 9, 14 and 21 were found to be selective for cardiac myosin over skeletal and smooth myosins.
Thus, these urea derivatives are potent scaffold to develop as a newer cardiac myosin activator
for the treatment of systolic heart failure.
Key words: Flexible urea, cardiac myosin activator, inotrope, systolic heart failure.

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*Corresponding author: jungshh@cnu.ac.kr. Tel.; +82 42 821 5939; Fax +82 42 823 6566;
Present address: College of Pharmacy and Institute of Drug Research and development,
Chungnam National University, Daejeon 34134, Korea
1. Introduction
Heart failure is one of the major health and economic burdens worldwide, and its prevalence is
continuously increasing. The Global Burden of Disease 2010 study reported that this disease had
been the most common cause of death worldwide from 1990 to 2010 [1-3]. Heart failure was
responsible for 8.1 million deaths in 2013, which was 15 % of deaths worldwide [4]. Acutely
decompensated chronic heart failure concerns about 70 % of patients with acute heart failure
syndromes [5], and at least half the patients with heart failure have a low ejection fraction (40 %
or less) [6], thus leading to systolic heart failure [7]. Systolic dysfunction is characterized by a
decrease in myocardial contractility resulting in a reduction in the left ventricular ejection
fraction [8]. There are no safe medical therapies to improve cardiac function of heart failure
patients with reduced ejection fraction [9]. Most current therapies are focused on blockade of
neurohormonal activation using inhibitors of the renin–angiotensin pathway, β-adrenergic
blockers, and aldosterone antagonists and do not improve the contractile function of the heart
[10,11]. Currently available inotropes such as adrenergic receptor agonists (i.e., dobutamine),
phosphodiesterase inhibitors (i.e., milrinone), increase cardiac contractility at the expense of
increased intracellular concentrations of calcium and cAMP, contributing to increased heart rate,
hypotension, arrhythmias,and mortality [12,13].
On the other hand, calcium-sensitizing inotropic agents such as levosimendan have been
developed to increase the calcium sensitivity of myofibrils without increasing calcium transients
for treating systolic heart failure [14]. However, it was found that levosimendan also has

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significant phosphodiesterase inhibition activity, this mixed mechanism may be the reason for
levosimendan failure in improvement on mortality [15].
To address these limitations, new agents targeting novel mechanisms are being developed: (i)
istaroxime, a non-glycoside inhibitor of the sodium-potassium-ATPase with additional
stimulatory effects on the sarcoplasmic reticulum calcium pump (SERCA), has shown lusitropic
and inotropic properties in experimental and early clinical studies [16]; (ii) gene therapy
approaches have also been effectively employed to increase myocardial SERCA2a [17]; (iii)
nitroxyl donors have shown positive lusitropic and inotropic as well as potent vasodilatory
effects [18]; (iv) the ryanodine receptor stabilizers reduce pathological leak of calcium from the
sarcoplasmic reticulum with initial promising pre-clinical results [19] and (v) finally the most
attractive class comprises of the cardiac myosin activators, which directly work at the level of the
cardiac sarcomere activating the actin-myosin cross-bridges, the smallest force-producing unit
involved in the contraction mechanism [20].
The selective cardiac myosin activator, omecamtiv mecarbil, which is currently completing late
phase II trials on the verge of phase III trials [21], binds to the myosin catalytic domain and
increases the transition rate of myosin into the strongly actin-bound state that generates force,
while inhibiting ATP turnover in the absence of actin. This mechanism results in more myosin
heads generating force on actin with each beat [22-24]. Studies of healthy volunteers and of
patients with chronic stable heart failure have confirmed that omecamtiv mecarbil prolongs the
duration of systole, which results in increased stroke volume and fractional shortening with
decreased ventricular volumes. Patients with ischemic cardiomyopathy and angina showed no
adverse effects of omecamtiv mecarbil [25]. Unlike prior agents that increased intracellular
cAMP and calcium and decrease ejection time, omecamtiv mecarbil’s unique mechanism of

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action prolongs ejection time, allowing for increased stroke volume without significantly
increasing myocardial oxygen consumption [26,27].
Although omecamtiv mecarbil is currently undergoing clinical trials, a broader range of drugs
that can improve cardiac function is urgently required. Taking advantage of the urea
functionality in the omecamtiv mecarbil (Figure 1) and flexible chain in dobutamine (Figure 1),
we have rationally designed the target molecule to be a flexible urea derivative as selective
cardiac myosin activators for the treatment of systolic heart failure (Figure. 1). Indeed the
presence of molecular flexibility or flexible spacers in molecules has a definite role in the field of
drug discovery [28-30].
Fig. 1. Background for the design of flexible urea with spacer as novel cardiac myosin activators
2. Chemistry
Scheme 1 represents the synthesis of compounds denoted in Table 1. The typical urea 1 was
prepared by the reaction of phenethylamine (34) and 3-phenylpropylisocyanate (35). Urea 1 was

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subsequently converted to its thiourea analog 2 by refluxing with Lawesson’s reagent in toluene.
The sulfonyldiamide 3 was synthesized from the reaction of the 34 and 1,1-sulfonyldiimidazole
to afford the intermediate 36 followed by the substitution with 3-phenylpropylamine. The
preparation of amide 4 was achieved by transformation of 5-phenylvaleric acid (37) to its acid
chloride and then reacting with 34. The amine derivative 5 and 6 were respectively derived from
the substitution reaction of phenylalkylamines 34, 38 and phenylalkylbromides 39, 40 using KI
as catalyst. In order to prepare the ketone 7, the epoxide intermediate 43 was prepared from the
reaction of epichlorohydrin (41) and 2-phenylethanol (42) using NaH as base. Epoxide ring
opening reaction of 43 with benzyl alcohol and KOH yielded the intermediate 44. The secondary
alcohol 44 was then oxidized to the corresponding ketone 7 by Dess-Martin periodinane.

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Scheme 1. Synthesis of 1-7 denoted in Table 1.
Reagents and conditions: (a) acetonitrile, RT, 8 h; (b) Lawesson’s reagent, toluene, reflux, 5 h;
(c) 1,1'-sulfonyldiimidazole, TEA, THF, 60 °C, 2 h; (d) 3-phenylpropylamine, TEA, THF, 60 °C,
3 h; (e) (i) SOCl2, CH2Cl2, reflux 3 h, (ii) TEA, CH2Cl2, 3 h, RT (f) K2CO3, KI, DMF, 60 °C, 8
h; (g) NaH, THF, reflux, 15 h; (h) benzylalcohol, KOH, neat condition, 60 °C 3 h; (i) Dess-
Martin periodinane, CH2Cl2, RT, 1 h.
The reaction of various amine (‘L’ group and ‘n’ are in Table 2) with 3-phenylpropylisocyanate
(35) afforded the corresponding urea derivatives 8-21 (Scheme 2). The preparation of compound

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9 was previously reported by our group [31]. To prepare the urea 22, 2-morpholinoethylamine
was reacted with 4-phenylbutyl isocyanate. Similarly the reaction of phenethylisocyanate (45)
and amine (‘R’ group is in Table 2) yielded the urea derivatives 23-25. Urea 26 was prepared by
the reaction of 2-cyclohexylethanamine with triphosgene in the presence of N,N-
diisopropylethylamine for 0.5 h stirring at 0 °C followed by the addition of 3-cyclohexylpropan-
1-amine (47). The synthesis of 3-cyclohexylpropan-1-amine (47) was in turn achieved from 3-
cyclohexylpropan-1-ol (46) by bromination reaction with HBr and subsequent treatment with
phthalimide followed by reaction with hydrazine hydrate.
Scheme 2. Synthesis of 8-26 denoted in Table 2.
Reagents and conditions: (a) Acetonitrile, RT, 8 h, (b) (i) HBr, reflux, 5 h (ii) phthalimide, THF,
reflux, 2 h (iii) NH2NH2.H2O, ethanol, 60 °C, 30 min, (c) 2-cyclohexylethanamine, triphosgene,
DIPEA, THF, 0 °C to RT, 8 h. The ‘L, R’ group of the amine and ‘n,m’ are denoted in Table 2.

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To analyze the effect of rigidification of the flexible spacer of 1, compound 27-32 were prepared
as denoted in Scheme 3. Chlorination of pyrrolidine 48/piperidine 49 with N-chlorosuccinimide
in diethyl ether afforded 50/51 which was reacted with phenyl lithium at 35 °C in diethyl ether to
obtain 2-phenylpyrrolidine (52)/2-phenylpiperidine (53). The reaction of 52 and 53 with 3-
phenylpropylisocyanate (35) in acetonitrile afforded the respective ureas 27 and 28. The urea 29
was synthesized from the reaction of 35 with 3-phenylpiperidine (55), which was in turn derived
from the Pt/C assisted hydrogenation of 3-phenylpyridine (54) in presence of acetic acid at 55
psi. Reaction of 2,3-dihydro-1H-inden-2-amine with 35 gave the urea 30. Similarly the reaction
of phenethylisocyanate (45) with 4-phenylpiperidine (57)/4-phenylpiperazine (58) yielded the
respective ureas 31 and 32. To mask the hydrogen bonding donor (NH) group of the urea 1, it
was methylated using methyliodide in presence of sodium hydride as base to give the urea 33.

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Scheme 3. Synthesis of 27-33 denoted in Table 2.
Reagents and conditions: (a) N-chlorosuccinimide, diethyl ether, RT, 1 h; (b) PhLi diethyl ether,
35°C, 1 h (c) 35, acetonitrile, RT, 8 h; (d) Pt/C/H2, AcOH, 55psi RT, 20 h; (e) acetonitrile, RT, 8
h, (f) CH3I, NaH, DMF, RT, 5 h.
3. Pharmacology
In the sarcomere, force generation is directly coupled to ATP hydrolysis by myosin ATPase.
Actin stimulated ATPase activity was assayed spectrophotometrically using sarcomere assay
[32] with modifications. Compounds that activate the sarcomere were identified by measuring %

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increase in myosin ATPase activity at 10 µM. The results are shown in Table 1 and 2.
Compound specificity with respect to muscle type was evaluated by comparing the effect of the
compound on actin stimulated ATPase activity of a panel of myosin isoforms including cardiac
(bovine (10 µM)) [32], skeletal (rabbit (100 µM)) [33,34,35] and smooth muscle (chicken
gizzard (100 µM)) [33,36,37] at a single dose of the compound. Omecamtiv mecarbil was used
as positive control for cardiac myosin ATPase activity and (-)-blebbistatin was used as a negative
control for measurement of skeletal or smooth muscle myosin ATPase activity [38]. The results
are shown in Table 3. Positive inotropic effect was tested by measuring % increase of left
ventricle fractional shortening (FS) and ejection fraction (EF) with/without samples using
echocardiography [39] in seven-week-old Sprague-Dawley male rats. The results are shown in
Table 2.
4. Results and Discussion
The importance of the urea functionality is demonstrated from the comparison between similar
scaffolds as shown in Table 1. Initially the compound 1 holding a urea functional group showed
comparable cardiac myosin ATPase activation (51.1 % at 10 µM) to omecamtiv mecarbil (OM,
58.0 % at 10 µM) and proved potent when compared to its thiourea 2 (cardiac myosin ATPase
activation = 10.3 %) and sulfonyldiamide 3 (cardiac myosin ATPase activation = 32.1 %). This
demonstrates the importance of enhanced hydrogen bonding acceptor (HBA) property of the
carbonyl functional group. Proving once again the same above statement, amide 4 retained the
activity (cardiac myosin ATPase activation = 39.1 %), while removing the carbonyl function as
shown in the amines 5 and 6 completely abolished the activity (cardiac myosin ATPase
activation = 1.6 % and 11.9 % respectively). The importance of C=O functionality in the NH-

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C=O linkage was established by evaluating the ketone 7. Though it retained the activity (cardiac
myosin ATPase activation = 42.9 %), its unstability [40] made it unsuitable to develop further as
a drug scaffold.
Table 1. Cardiac myosin activation of urea scaffold and its isosteric replacements
ATPase
activity
Comp.
No
CLogP
Functional Group (FG)
% at 10 µM
1
51.1
10.3
32.1
39.1
4.025
4.305
4.430
4.024
2
3
4
1.6
5.113
4.963
5
6
11.9
42.9
58.0
3.263
3.266
7
Omecamtiv
mecarbil
-
The present work focuses the establishment of urea functionality as selective cardiac myosin
activator and therefore we synthesized the compounds listed in Table 2 by varying the spacer and

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groups on both sides. Moreover, the importance of the flexible spacer and HBD of urea NH were
also established.
Table 2. Optimization of spacer and groups in the urea scaffold
ATPase
Fractional
Shoretning
% increase
Ejection
Fraction
% increase
activity
% at 10
µM
Comp.
No
CLogP
L
R
n
m
1
8
Phenyl
Phenyl
Phenyl
Phenyl
2
0
1
1
51.1
46.2
18.90
---
12.15
---
4.025
3.577
9
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
1
3
0
1
1
1
53.3
45.5
20.3
30.04
---
18.27
---
3.696
4.404
3.965
10
11
Cyclohexyl
---
---
Precipitated during
12
13
14
Cyclohexyl
Cyclohexyl
Phenyl
Phenyl
Phenyl
1
2
1
1
1
1
60.2
61.4
81.4
4.578
5.107
2.179
formulation
Precipitated during
formulation
20.50
---
13.10
---
15
16
17
Phenyl
Phenyl
Phenyl
1
1
1
1
1
1
47.6
36.7
37.2
3.342
2.872
3.686
---
---
---
---

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18
19
20
21
Phenyl
Phenyl
Phenyl
Phenyl
2
2
2
2
1
1
1
1
30.0
29.6
30.6
44.0
2.528
3.671
3.201
2.408
---
---
---
---
---
---
24.79
15.65
22
23
Phenyl
2
2
2
1
42.7
37.9
---
---
---
---
2.608
5.257
Phenyl
Cyclohexyl
24
25
Phenyl
Phenyl
2
2
1
1
32.6
32.9
---
---
---
---
1.862
2.208
26
27
Cyclohexyl Cyclohexyl
2
1
44.4
32.4
---
---
---
---
6.339
4.216
28
73.0
13.32
---
9.26
---
4.775
29
30
35.2
61.9
4.575
3.910
precipitated during
formulation
precipitated during
formulation
31
46.6
4.126
32
33
36.6
72.4
---
---
3.676
4.307
5.74
3.41

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Omeca
mtiv
mecarb
il
---
58.0
20.32
13.35
3.266
---, activity not checked
Based on urea scaffold 1 with good cardiac myosin activation (51.1 % at 10 µM), compounds 8
(n = 0, cardiac myosin ATPase activation = 46.2 %), 9 (n = 1, cardiac myosin ATPase activation
= 53.3 %) and 10 (n = 3, cardiac myosin ATPase activation = 45.5 %) were synthesized varying
the length of the carbon chain between urea functional group and the phenyl ring (L). The results
proved that compound 1 with one carbon spacer or 9 with two carbon spacer retained the cardiac
myosin ATPase activity compared to OM (cardiac myosin ATPase activation = 58.0 %). Thus in
vivo fractional shortening (FS) and ejection fraction (EF) of compounds 1 and 9 were measured.
Compound 1 (FS = 18.90; EF = 12.15) showed good activity and compound 9 (FS = 30.04; EF =
18.27) exhibited highly potent activity, which was more potent than the standard omecamtiv
mecarbil (FS = 20.32; EF = 13.35).
The replacement of the phenyl ring with cyclohexyl moiety as shown in 11 (n = 0, ATPase
activation = 20.3 %), 12 (n = 1, ATPase activation = 60.2 %) and 13 (n = 2, ATPase activation =
61.4 %) confirmed once again that one or two carbon chain is preferred. However, the
hydrophobic cyclohexyl compounds 12 (CLog P = 4.578) and 13 (CLog P = 5.107) were not
suitable for in vivo studies since they were precipitated during formulation.
Introducing an oxygen atom in the cyclohexyl ring of 12 as shown in tetrahydropyran-4-ylurea
derivative 14 improved not only the in vitro ATPase activity (81.4 %) but also water solubility
during formulation and therefore demonstrated good in vivo activity (FS = 20.50; EF = 13.10).
Hence we thought to introduce other heterocycles on the left side (L) with the preferred carbon
chain length as one or two as shown in 15-22. The thiophen-2-yl (15, ATPase activation = 47.6
%), furan-2-yl (16, ATPase activation = 36.7 %), indol-5-yl (17, ATPase activation = 37.2 %)

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with one carbon chain as well as pyridin-2-yl (18, ATPase activation = 30.0 %), thiophen-2-yl
(19, ATPase activation = 29.6 %), furan-2-yl (20, ATPase activation = 30.6 %) with two carbon
chain did not improve the activity when compared to the phenyl compounds 1 and 9 and
tetrahydropyran-4-yl derivative 14.
However, the morpholin-1-yl (21, ATPase activation = 44.0 %; FS = 20.79; EF = 15.65) showed
good activity both in vitro and in vivo. Further increment in the carbon chain between urea and
phenyl ring (R) as demonstrated in the morpholin-1-yl derivative 22 (m = 4) did not improve the
activity which clearly say that n = 1 or 2 and m = 3 should be the optimum chain length of the
urea pharmacophore for potent cardiac myosin activation.
Replacement of the phenyl ring at the right side (R) of 1 with cyclohexyl ring as shown in
compound 23 (ATPase activation = 37.9 %) did not improve the activity.
On the other hand, replacement with other heterocycles such as imidazol-1-yl (24, ATPase
activation = 32.6 %) and morpholin-1-yl (25, ATPase activation = 32.9 %) showed decrement in
the activity.
Finally to study the importance of either of the phenyl ring, compound 26 was prepared where
both the phenyl rings were replaced with cyclohexyl rings which also did not increase the
activity (ATPase activation = 44.4 %) when compared to the compound 1 or 9.
In order to find the importance of the flexible carbon chain on either side, we synthesized the
rigid compounds 27-32. The rigidification of the flexible spacer of compound 9 (ATPase
activation = 53.3 %) with five membered ring as shown in 27 (ATPase activation = 32.4 %)
caused the decrement in activity whereas rigidification with six membered ring (28, ATPase
activation = 73.0 %) showed good in vitro activity. However it failed to prove the effectiveness
in the animal model contributing low FS and EF (13.32 and 9.26). Similarly rigidification of

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compound 1 as demonstrated in compound 29 (ATPase activation = 35.2 %) was not fruitful.
However 30 (ATPase activation = 61.9 %) successfully showed good in vitro activity but
exhibited poor water solubility. On the other hand, rigidification on the other side as shown in
compound 31 (ATPase activation = 46.6 %, CLogP = 4.126) retained the cardiac myosin
activation. Its poor water solubility obstructed it to formulate for in vivo studies. To improve the
water solubility additional nitrogen atom was introduced in compound 31 as shown in compound
32. Unfortunately, the cardiac myosin ATPase activity was decreased to 36.6 %.
Finally to find the importance of the hydrogen bonding donor (HBD) of urea functional group,
both the NH were masked with methyl group. Surprisingly the ATPase activity improved to 72.4
%. However, the in vivo activity was very weak (FS = 5.74; EF = 3.41).
The potent compounds 1, 9, 14 and 21 were analyzed for selectivity in cardiac myosins over
skeletal and smooth myosins (Table 3). None of the compound showed significant activity for
myosin ATPase using skeletal and smooth myosin S1. Thus, from these results it is evident that
these urea derivatives are selective for cardiac myosin S1.
Table 3. Selectivity study in myosins
ATPase % activity
Compound No.
Cardiac myosin Skeletal myosin Smooth myosin
(at 10 µM)
(at 100 µM)
(at 100 µM)
1
51.1
˗ 2.1
˗ 4.7
53.3
1.4
˗ 3.7
9
81.4
4.4
5.1
14
21
44.0
˗ 5.3
3.1
Omecamtiv mecarbil
58.0
2.8
˗ 5.5

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----
˗ 50.7
˗ 35.7
(˗)Blebbistatin
Fig. 2. SAR of urea as a selective cardiac myosin activator
5. Conclusion
Novel urea scaffold with flexible spacers as selective cardiac myosin activator for the treatment
of systolic heart failure was explored. The structure activity relationship study (Figure 2)
demonstrated that phenyl ring with three carbon spacer is favorable for potent cardiac myosin
activation on one side whereas on the other side phenyl ring with one carbon spacer viz., 1-
benzyl-3-(3-phenylpropyl)urea (9 cardiac myosin ATPase activation at 10 µM = 53.3 %; FS =
30.04; EF = 18.27) or 1-(3-phenylpropyl)-3-((tetrahydro-2H-pyran-4-yl)methyl)urea (14 cardiac
myosin ATPase activation = 81.4 %; FS = 20.50; EF = 13.10) and two carbon spacer viz., 1-
phenethyl-3-(3-phenylpropyl)urea (1 cardiac myosin ATPase activation = 51.1 %; FS = 18.90;
EF = 12.15) or 1-(2-morpholinoethyl)-3-(3-phenylpropyl)urea (21 cardiac myosin ATPase
activation = 44.0 %; FS = 24.79; EF = 15.65) are favorable. The flexibility of the carbon spacer
is well tolerated. Rigidification of the carbon chain on either side has adverse effect on the
ATPase activity. Masking the hydrogen bonding donor property of the NH group of the urea is
not favorable for cardiac myosin ATPase activity. The potent compounds 1, 9, 14, 21 were
highly selective for cardiac myosin over skeletal and smooth myosins and thus proving them

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these new urea derivatives is a novel scaffold for discovery of cardiac myosin activators for the
treatment of systolic heart failure.
6. Experimental section
6.1 Chemistry
Melting points were determined on Electro thermal 1A 9100 MK2 apparatus and are
uncorrected. All commercial chemicals were used as obtained and all solvents were purified by
the standard procedures [41] prior to use. Thin layer chromatography was performed on E Merck
silica gel GF-254 pre-coated plates and the identification was done with UV light and
colorization with spray 10 % phosphomolybdic acid followed by heating. Flash column
chromatography was performed with E Merck silica gel (230-400 mesh). Infrared spectrum was
recorded by using sample as such on FT-IR spectrum with Nicolet - 380 models. NMR spectra
were measured against the peak of tetramethylsilane by JEOL, JNM-AL-400 (Alice) 400 FT-
NMR spectrometer. High resolution mass spectra (HRMS) were measured in ESI ionization
using AB Sciex TripleTOF 5600 LCMS instrument.
6.1.1. General synthetic procedure for the preparation of Urea derivatives:
To the corresponding amine (1.10 mmol) in acetonitrile (5 mL), isocyanate (1.00 mmol) was
added and allowed to stir for 8 h at ambient temperature. The resulting mixture was portioned
between water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and
evaporated under reduced pressure. The crude mixture was subjected to column chromatography
to obtain the pure compounds.
6.1.1.1.
1-Phenethyl-3-(3-phenylpropyl)urea
(1);
Phenethylamine
34
and
3-
phenylpropylisocyanate 35 were reacted according to the general procedure. Yield 92 %. White
°
solid. Rf 0.32 (1 : 1 Ethylacetate : Hexane) mp 68-70 C. IR (neat): 3340, 2939, 2862, 1620,

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1570, 1523, 1454 cm-1. 1H-NMR (CDCl3) δ 1.76-1.83 (m, 2H), 2.63 (t, J = 7.20 Hz, 2H), 2.79 (t,
J = 6.80 Hz, 2H), 3.15 (t, J = 7.20 Hz, 2H), 3.41 (t, J = 6.80 Hz, 2H), 4.25 (bs, 2H), 7.15-7.32
(m, 10H). 13C-NMR (CDCl3) δ 31.70, 33.09, 36.31, 40.00, 41.55, 125.96, 126.43, 128.39,
128.46, 128.61, 128.85, 139.23, 141.61, 158.21. HRMS Calcd for C18H22N2O m/z [M+H]
283.1811, found 283.1835.
6.1.1.2. 1-Phenethyl-3-(3-phenylpropyl)thiourea (2); To a solution of the urea (1, 0.5 mmol) in
toluene (10 mL) Lawesson’s reagent (1.00 mmol) was added and the mixture was heated at 100
°C for 5 h. After the completion of the reaction, solvent was evaporated and the crude mixture
was portioned between water and ethyl acetate. The organic layer was dried over anhydrous
sodium sulfate and evaporated under reduced pressure. The crude mixture was subjected to
column chromatography to obtain the pure compounds. Yield 78 %, Pale yellow solid. Rf 0.46 (1
: 1 Ethylacetate : Hexane) mp 104-105 °C. IR (neat): 3352, 2968, 2897, 1565, 1519, 1453, 1160
cm-1. 1H-NMR (CDCl3) δ 1.83-1.90 (m, 2H), 2.65 (t, J = 7.60 Hz, 2H), 2.87 (t, J = 7.20 Hz, 2H),
3.25-3.33 (m, 2H), 3.58-3.67 (m, 2H), 5.56 (bs, 1H), 5.67 (bs, 1H), 7.14-7.19 (m, 5H), 7.25-7.32
(m, 5H); 13C-NMR (CDCl3) δ 30.17, 33.15, 35.12, 35.14, 45.54, 126.41, 126.96, 128.51,
128.77, 128.91, 128.97, 138.46, 141.00, 181.69. HRMS Calcd for C18H22N2S m/z [M+H]
299.1583, found 299.1606.
6.1.1.3. 1-Phenethyl-3-(3-phenylpropyl)sulfuricdiamide (3); To a solution of phenethylamine
(34, 0.15 mmol) in THF (5 mL), triethylamine (0.15 mmol) was added. To this mixture, 1,1'-
sulfonyldiimidazole (0.15 mmol) was added and the reaction mixture was stirred at 60 °C for 2
h. After the completion of the reaction monitored by TLC, water was added and the reaction
mixture was extracted using ethyl acetate. The organic layer was dried over anhydrous sodium
sulfate and concentrated under vacuum. The obtained intermediate 36 was used for next step

ACCEPTED MANUSCRIPT
without further purification. In the next step, 3-phenylpropylamine (0.15 mmol) was dissolved in
THF and triethylamine (0.15 mmol) was added. To this mixture, 36 was added. The reaction was
stirred at 60 °C for 3 h. The reaction was monitored by TLC. After the completion of the
reaction, the reaction mixture was quenched with water and the whole mixture was extracted
using ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and evaporated
under reduced pressure. The crude mixture was purified by column chromatography to afford the
pure product 3. Yield 72 %, Pale yellow solid. Rf 0.30 (2 : 1 Ethylacetate : Hexane), mp. 135-137
°C; IR (neat): 3362, 2950, 2822, 1560, 1523, 1345, 1170, 945 cm-1. 1H-NMR (CDCl3) δ 1.79-
1.87 (m, 2H), 2.64 (t, J = 8.00 Hz, 2H), 2.85 (t, J = 7.20 Hz, 2H), 2.92-2.97 (m, 2H), 3.28-3.33
(m, 2H), 4.07-4.13 (m, 2H), 7.15-7.33 (m, 10H); 13C-NMR (CDCl3) δ 31.13, 32.94, 35.81,
42.68, 44.31, 126.35, 127.04, 128.50, 128.71, 128.97, 128.98, 138.10, 141.04. HRMS Calcd for
C17H22N2O2S m/z [M+H] 319.1481, found 319.1507.
6.1.1.4. N-Phenethyl-5-phenylpentanamide (4); To a solution of 5-phenylvaleric acid 37 (0.30
mol) in methylene chloride (50 mL), thionyl chloride (0.60 mol) was added and the mixture was
refluxed for 3 h. After the completion of the reaction, the solvent was removed under reduced
pressure to obtain the 5-phenylvaleroyl chloride. To a solution of phenethylamine 34 (0.30 mol)
in methylene chloride (50 mL), triethylamine (0.60 mol) was added and cooled to 0 °C. To this
reaction mixture the obtained 5-phenylvaleroyl chloride was added and allowed to stir for 3 h at
ambient temperature. After the completion of the reaction, the mixture was washed with water.
The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced
pressure. The crude product was purified by column chromatography. Yield 82 %, White solid,
Rf 0.40 (1 : 1 Ethylacetate : Hexane), mp. 73-75 °C; IR (neat): 3323, 2966, 2946, 1650, 1538,
1425, 1211 cm-11H-NMR (CDCl3) δ 1.60-1.68 (m, 4H), 2.13 (t, J = 6.80 Hz, 2H), 2.61 (t, J =

ACCEPTED MANUSCRIPT
6.80 Hz, 2H), 2.80 (d, J = 7.20 Hz, 2H), 3.49-3.53 (m, 2H), 5.37 (bs, 1H), 7.15-7.32 (m, 10H);
13C-NMR (CDCl3) δ 25.38, 31.07, 35.72, 35.74, 36.72, 40.52, 125.92, 126.67, 128.47, 128.56,
128.80, 128.92, 139.07, 142.36, 172.99. HRMS Calcd for C19H23NO m/z [M+H] 282.1859,
found 282.1884.
6.1.1.5. N-Phenethyl-5-phenylpentan-1-amine (5); To a solution of phenethylamine 34 (0.8
mmol) in DMF (5 mL), K2CO3 (1.00 mmol) was added. To this reaction mixture, (5-
bromopentyl)benzene 39 (0.8 mmol) and KI (0.2 mmol) was added and heated at 60 °C for 8 h.
After the completion of the reaction, water was added to the mixture and extracted using ethyl
acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under
vacuum. The crude product was purified by column chromatography. Yield 75 %, Colorless oil,
Rf 0.25 (1 : 9 MeOH : CH2Cl2), IR (neat): 3373, 2945, 1515, 1211, 967, 868 cm-1; 1H-NMR
(CDCl3) δ 1.29-1.37 (m, 2H), 1.49-1.56 (m, 2H), 1.57-1.65 (m, 2H), 2.02 (bs, 1H), 2.57-2.65 (m,
4H), 2.79-2.91 (m, 4H), 7.15-7.31 (m, 10H); 13C-NMR (CDCl3) δ 26.98, 29.70, 31.36, 35.89,
36.16, 49.72, 51.15, 125.79, 126.34, 128.41, 128.55, 128.64, 128.87, 140.07, 142.79. HRMS
Calcd for C19H25N m/z [M+H] 268.2066, found 268.2095.
6.1.1.6. 4-Phenyl-N-(3-phenylpropyl)butan-1-amine (6); To a solution of 4-phenylbutylamine 38
(0.8 mmol) in DMF (5 mL), K2CO3 (1.00 mmol) was added. Then, (3-bromopropyl)benzene 40
(0.8 mmol) and KI (0.2 mmol) were added and heated at 60 °C for 8 h. After the completion of
the reaction, water was added to the mixture and extracted using ethyl acetate. The organic layer
was dried over anhydrous sodium sulfate and concentrated under vacuum. The crude product
was purified by column chromatography. Yield 72 %, Colorless oil, Rf 0.25 (1 : 9 MeOH :
CH2Cl2), IR (neat): 3380, 2950, 1510, 1130, 969, 870 cm-1; 1H-NMR (CDCl3) δ 1.60-1.65 (m,
4H), 1.87-1.94 (m, 2H), 2.85-2.70 (m, 8H), 7.15-7.19 (m, 6H), 7.25-7.29 (m, 4H); 13C-NMR

ACCEPTED MANUSCRIPT
(CDCl3) δ 28.79, 29.10, 30.68, 33.53, 35.73, 49.03, 49.36, 125.92, 126.08, 128.47, 128.52,
128.54, 128.55, 141.76, 142.39. HRMS Calcd for C19H25N m/z [M+H] 268.2066, found
268.2083.
6.1.1.7. 1-(Benzyloxy)-3-phenethoxypropan-2-one (7)
6.1.1.7.1. Preparation of 2-(phenethoxymethyl)oxirane (43): To a cooled suspension of NaH (60
mmol) in THF (30 mL), 2-phenylethanol (42, 50 mmol) was added and stirred at ambient
temperature for 45 minutes. Then the reaction mixture was cooled and epichlorohydrin (41, 60
mmol) was added. The reaction mixture was stirred at reflux for 15 h. Saturated NH4Cl solution
was added to neutralize excess NaH and extracted using ethyl acetate. The organic layer was
dried over anhydrous sodium sulfate and concentrated under vacuum. The crude product was
purified by column chromatography. Yield 70 %
6.1.1.7.2. Preparation of 1-(benzyloxy)-3-phenethoxypropan-2-ol (44): Potassium hydroxide
(4.76 mmol) was added to benzyl alcohol (4.33 mmol) and heated at 60 °C for 30 minutes to
form a white viscous liquid. To that 2-(phenethoxymethyl)oxirane (43, 4.33 mmol) was added
and heated at 60 °C for 3 h. After the completion of the reaction, water was added to the reaction
mixture and the extracted using ethyl acetate. The organic layer was dried over anhydrous
sodium sulfate. After the evaporation of the solvent, the crude product was taken for the next
step. Yield 65 % Colorless oil; IR (neat): 3330, 2870, 1496, 1268, 1084, 780, 697 cm-1; 1H-
NMR (CDCl3) δ 2.40 (d, J = 4.40 Hz, 1H), 2.88 (t, J = 7.60 Hz, 2H), 3.45-3.55 (m, 4H), 3.66-
3.71 (m, 2H), 3.93-3.99 (m, 1H), 4.53 (s, 2H), 7.20-7.22 (m, 3H), 7.27-7.37 (m, 7H).
6.1.1.7.3. Preparation of (7). To a solution of 1-(benzyloxy)-3-phenethoxypropan-2-ol (44, 1.30
mmol) in methylene chloride (10 mL) Dess-Martin Periodinane (1.56 mmol) was added and
stirred for 1 h at ambient temperature. The reaction was monitored by TLC. After the completion

ACCEPTED MANUSCRIPT
of the reaction, diethyl ether (5 mL) was added followed by the addition of 1 % sodium
thiosulfate solution. The resulting mixture was stirred for 15 mins. The organic layer was
separated and dried over anhydrous sodium sulfate. After the evaporation of the solvent, the
crude product was purified by column chromatography. Yield 82 %; Pale yellow oil; Rf 0.48 (1 :
3 Ethylacetate : Hexane), IR (neat): 2829, 1747, 1701, 1686, 1201, 1121, 713, 698 cm-1; 1H-
NMR (CDCl3) δ 2.91 (t, J = 7.20 Hz, 2H), 3.69 (t, J = 7.20 Hz, 2H), 4.18 (s, 2H), 4.21 (s, 2H),
4.56 (s, 2H), 7.19-7.37 (m, 10H). 13C-NMR (CDCl3) δ 36.21, 72.85, 73.57, 73.64, 74.94,
126.55, 128.10, 128.24, 128.59, 128.71, 129.07, 137.23, 138.61, 206.24. HRMS Calcd for
C18H20O3 m/z [M+H] 285.1491, found 285.1512.
6.1.1.8. 1-Phenyl-3-(3-phenylpropyl)urea (8); Prepared by general procedure. Yield 80 %; White
solid; Rf 0.64 (1 : 1 Ethylacetate : Hexane), mp 88-90 °C; IR (neat): 3328, 3292, 2937, 2357,
2341, 1624 cm-1; 1H-NMR (CDCl3) δ 1.77-1.85 (m, 2H), 2.61 (t, J = 8.00 Hz, 2H), 3.22-3.27 (m,
13
2H), 5.08 (bs, 1H), 6.74 (bs, 1H), 7.03-7.08 (m, 1H), 7.12-7.19 (m, 3H), 7.23-7.30 (m, 6H); C
NMR (CDCl3) δ 31.62, 33.11, 39.88, 121.17, 123.86, 126.00, 128.38, 128.49, 129.33, 138.63,
141.54, 156.12. HRMS Calcd for C16H18N2O m/z [M+H] 255.1498, found 255.1526.
6.1.1.9. 1-Benzyl-3-(3-phenylpropyl)urea (9); Prepared by general procedure. Yield 89 %;
Colorless solid; mp 96-98 °C; Rf 0.30 (1 : 1 Ethylacetate : Hexane), IR (neat): 3308, 2868, 1620,
1559, 1494, 1453 cm-1; 1H-NMR (CDCl3) δ 1.76-1.84 (m, 2H), 2.61 (t, J = 7.80 Hz, 2H), 3.17-
3.21 (m, 2H), 4.33 (d, J = 6.00 Hz, 2H), 4.39 (bs, 1H), 4.66 (bs, 1H), 7.13-7.19 (m, 3H), 7.24-
7.34 (m, 7H). 13C-NMR (CDCl3) δ 31.70, 33.02, 39.97, 44.36, 125.94, 127.28, 127.38, 128.38,
128.44, 128.84, 139.32, 141.62, 158.44. HRMS Calcd for C17H20N2O m/z [M+H] 269.1654,
found 269.1682.

ACCEPTED MANUSCRIPT
6.1.1.10. 1,3-Bis(3-phenylpropyl)urea (10); Prepared by general procedure. Yield 85 %;
Colorless solid; mp 94-96 °C; Rf 0.38 (1 : 1 Ethylacetate : Hexane), IR (neat): 3313, 3060, 2862,
1621, 1560, 1493, 1452 cm-1; 1H-NMR (CDCl3) δ 1.76-183 (m, 4H), 2.63 (t, J = 8.00 Hz, 4H),
3.13-3.18 (m, 4H), 4.46 (bs, 2H), 7.15-7.28 (m, 10H). 13C-NMR (CDCl3) δ 31.41, 32.80, 39.72,
125.66, 128.08, 128.16, 141.32, 158.05. HRMS Calcd for C19H24N2O m/z [M+H] 297.1968,
found 297.1992.
6.1.1.11. 1-Cyclohexyl-3-(3-phenylpropyl)urea (11); Prepared by general procedure. Yield 78 %;
White solid; mp 103-105°C; Rf 0.32 (1 : 2 Ethylacetate : Hexane), IR (neat): 3327, 2927, 2850,
1644, 1618, 1560, 1494 cm-1; 1H-NMR (CDCl3) δ 1.02-1.19 (m, 3H), 1.28-1.39 (m, 2H), 1.57-
1.62 (m, 1H), 1.66-1.71 (m, 2H), 1.79-1.86 (m, 2H), 1.89-1.93 (m, 2H), 2.65 (t, J = 8.00 Hz,
2H), 3.17-3.22 (m, 2H), 3.42-3.49 (m, 1H), 4.25 (t, J = 8.00 Hz, 1H), 4.37 (t, J = 8.00 Hz, 1H),
7.17-7.21 (m, 3H), 7.27-7.30 (m, 2H). 13C-NMR (CDCl3) δ 24.84, 25.50, 31.79, 33.15, 33.86,
40.01, 49.00, 125.95, 128.40, 128.46, 141.71, 157.70. HRMS Calcd for C16H24N2O m/z [M+H]
261.1968, found 261.1994.
6.1.1.12. 1-(Cyclohexylmethyl)-3-(3-phenylpropyl)urea (12); Prepared by general procedure.
Yield 49 %; White solid; mp 95-97 °C; Rf 0.35 (1 : 2 Ethylacetate : Hexane), IR (neat): 3322,
2940, 2866, 1667, 1623, 1568, 1511 cm-1; 1H-NMR (CDCl3) δ 0.85-0.94 (m, 2H), 1.12-1.27 (m,
3H), 1.37-1.43 (m, 1H), 1.65-1.72 (m, 5H), 1.81-1.88 (m, 2H), 2.67 (t, J = 8.00 Hz, 2H), 2.96 (t,
J = 6.00 Hz, 2H), 3.18-3.23 (m, 2H), 4.18-4.22 (m, 2H), 7.18-7.31 (m, 5H). 13C-NMR (CDCl3)
δ 25.89, 26.48, 30.89, 31.96, 33.28, 38.51, 40.15, 46.90, 126.07, 128.52, 128.58, 141.83, 158.77.
HRMS Calcd for C17H26N2O m/z [M+H] 275.2124, found 275.2153.
6.1.1.13. 1-(2-Cyclohexylethyl)-3-(3-phenylpropyl)urea (13); Prepared by general procedure.
Yield 98 %; Colorless oil; Rf 0.40 (1 : 2 Ethylacetate : Hexane), IR (neat): 3325, 2938, 2865,

ACCEPTED MANUSCRIPT
1662, 1618, 1572, 1510 cm-1; 1H-NMR (CDCl3) δ 0.86-0.94 (m, 2H), 1.12-1.29 (m, 4H), 1.34-
1.39 (m, 2H), 1.63-1.70 (m, 5H), 1.80-1.87 (m, 2H), 2.66 (t, J = 8.00 Hz, 2H), 3.10-3.15 (m,
2H), 3.18-3.23 (m, 2H), 4.18 (bs, 1H), 4.28 (bs, 1H), 7.17-7.31 (m, 5H). 13C-NMR (CDCl3) δ
26.13, 26.40, 31.78, 33.11, 33.15, 35.18, 37.57, 38.25, 40.04, 125.96, 128.40, 128.46, 141.68,
158.45. HRMS Calcd for C18H28N2O m/z [M+H] 289.2281, found 289.2304.
6.1.1.14. 1-(3-Phenylpropyl)-3-((tetrahydro-2H-pyran-4-yl)methyl)urea (14); Prepared by
general procedure. Yield 80 %; White solid; mp: 123-125 °C; Rf 0.35 (2 : 1 Ethylacetate :
Hexane), IR (neat): 3259, 1653, 1558, 1337, 1151, 1108, 1023 cm-1; 1H-NMR (CDCl3) δ 1.22-
1.33 (m, 2H), 1.58-1.61 (m, 2H), 1.68-1.72 (m, 1H), 1.80-1.87 (m, 2H), 2.66 (t, J = 7.60 Hz,
2H), 3.04 (t, J = 6.40 Hz, 2H), 3.17-3.22 (m, 2H), 3.32-3.39 (m, 2H), 3.94-3.98 (m, 2H), 4.32
(bs, 1H), 4.37 (bs, 1H), 7.17-7.21 (m, 3H), 7.27-7.30 (m, 2H). 13C-NMR (CDCl3) δ 30.57,
31.82, 33.16, 35.77, 40.06, 46.09, 67.59, 125.94, 128.30, 128.41, 141.49, 158.48. HRMS Calcd
for C16H24N2O2 m/z [M+H] 277.1917, found 277.1942.
6.1.1.15. 1-(3-Phenylpropyl)-3-(thiophen-2-ylmethyl)urea (15); Prepared by general procedure.
Yield 82 %; White solid; mp 89-91 °C; Rf 0.46 (2 : 1 Ethylacetate : Hexane),IR (neat): 3318,
2929, 1633, 1614, 1560, 1454 cm-1; 1H-NMR (CDCl3) δ 1.75-1.83 (m, 2H), 2.61 (t, J = 8.00 Hz,
2H), 3.15-3.20 (m, 2H), 4.49 (d, J = 8.00 Hz, 2H), 4.60-4.63 (m, 1H), 4.89-4.91 (m, 1H), 6.91-
6.93 (m, 2H), 7.13-7.19 (m, 4H), 7.24-7.28 (m, 2H). 13C-NMR (CDCl3) δ 31.70, 33.03, 39.27,
39.96, 124.82, 125.21, 125.93, 126.81, 128.40, 128.44, 141.63, 142.74, 158.15. HRMS Calcd for
C15H18N2OS m/z [M+H] 275.1219, found 275.1243.
6.1.1.16. 1-(Furan-2-ylmethyl)-3-(3-phenylpropyl)urea (16); Prepared by general procedure.
Yield 82 %; White solid; mp 70-72°C; Rf 0.42 (2 : 1 Ethylacetate : Hexane), IR (neat): 3319,
1626, 1561, 1496, 1457, 1332, 1183, 1153 cm-1; 1H-NMR (CDCl3) δ 1.78-1.85 (m, 2H), 2.64 (t,

ACCEPTED MANUSCRIPT
J = 7.60 Hz, 2H), 3.17-3.22 (m, 2H), 4.34 (d, J = 6.00 Hz, 2H), 4.49 (bs, 1H), 4.69 (bs, 1H),
6.19-6.20 (m, 1H), 6.30-6.31 (m, 1H), 7.15-7.20 (m, 3H), 7.25-7.29 (m, 2H), 7.32-7.33 (m, 1H);
13C-NMR (CDCl3) δ 31.70, 33.02, 37.32, 39.92, 106.78, 110.37, 125.92, 128.37, 128.43,
141.62, 141.93, 152.63, 158.25. HRMS Calcd for C15H18N2O2 m/z [M+H] 259.1447, found
259.1472.
6.1.1.17. 1-((1H-Indol-5-yl)methyl)-3-(3-phenylpropyl)urea (17); Prepared by general procedure.
Yield 86 %; White solid; mp 128-130 °C; Rf 0.44 (2 : 1 Ethylacetate : Hexane), IR (neat): 3341,
3280, 2982, 1651, 1560, 1492, 1215, 1120 cm-1; 1H-NMR (CDCl3) δ 1.63-1.71 (m, 2H), 2.56 (t,
J = 7.80 Hz, 2H), 3.00-3.05 (m, 2H), 4.25 (d, J = 6.00 Hz, 2H), 5.93 (bs, 1H), 6.18 (bs, 1H),
6.36-6.37 (m, 1H), 6.99 (dd, J = 8.40 Hz, J = 2.00 Hz, 1H), 7.15-7.33 (m, 7H), 7.40 (s, 1H),
11.01 (bs, 1H). 13C-NMR (CDCl3) δ 31.82, 32.45, 38.87, 43.52, 100.84, 111.15, 118.47, 120.96,
125.49, 125.71, 127.63, 128.31, 131.08, 131.08, 135.02, 141.93, 158.18. HRMS Calcd for
C19H21N3O m/z [M+H] 308.1764, found 308.1796.
6.1.1.18. 1-(3-Phenylpropyl)-3-(2-(pyridine-2-yl)ethyl)urea (18); Prepared by general procedure.
Yield 82 %; Pale yellow powder; mp 58-60 °C; Rf 0.35 (1 : 9 MeOH : CH2Cl2), IR (neat): 3305,
3010, 2939, 1647, 1616, 1539, 1497 cm-1; 1H-NMR (CDCl3) δ 1.77-1.84 (m, 2H), 2.64 (t, J =
8.00 Hz, 2H), 2.98 (t, J = 8.00 Hz, 2H), 3.15-3.20 (m, 2H), 3.56-3.61 (m, 2H), 4.47 (bs, 1H),
5.17 (bs, 1H), 7.12-7.30 (m, 7H), 7.59-7.63 (m, 1H), 8.48-8.50 (m, 1H). 13C-NMR (CDCl3) δ
31.73, 33.08, 37.80, 39.68, 40.00, 121.51, 123.63, 125.89, 128.38, 128.41, 136.66, 141.68,
149.08, 158.46, 159.91. HRMS Calcd for C17H21N3O m/z [M+H] 284.1764, found 284.1792.
6.1.1.19. 1-(3-Phenylpropyl)-3-(2-(thiophen-2-yl)ethyl)urea (19); Prepared by general procedure.
Yield 80 %; White solid; mp 68-70 °C; Rf 0.55 (2 : 1 Ethylacetate : Hexane), IR (neat): 3328,
2935, 2856, 1650, 1624, 1560, 1496 cm-1; 1H-NMR (CDCl3) δ 1.77-1.84 (m, 2H), 2.64 (t, J =

ACCEPTED MANUSCRIPT
8.00 Hz, 2H), 3.01 (t, J = 6.00 Hz, 2H), 3.14-3.19 (m, 2H), 3.40-3.45 (m, 2H), 4.33-4.35 (m,
1H), 4.43-4.46 (m, 1H), 6.82-6.83 (m, 1H), 6.94 (dd, J = 4.00 Hz, 1H), 7.14-7.20 (m, 4H), 7.26-
7.29 (m, 2H). 13C-NMR (CDCl3) δ 30.57, 31.69, 33.09, 40.04, 41.71, 123.86, 125.35, 125.98,
127.05, 128.40, 128.48, 141.60, 141.74, 158.14. HRMS Calcd for C16H20N2OS m/z [M+H]
289.1375, found 289.1401.
6.1.1.20. 1-(2-(Furan-2-yl)ethyl)-3-(3-phenylpropyl)urea (20); Prepared by general procedure.
Yield 65 %; White solid; mp 81-83 °C; Rf 0.48 (2 : 1 Ethylacetate : Hexane), IR (neat): 3325,
1630, 1561, 1535, 1280, 1145, 1136, 995 cm-1; 1H-NMR (CDCl3) δ 1.78-1.85 (m, 2H), 2.63 (t, J
= 7.80 Hz, 2H), 2.92 (t, J = 7.80 Hz, 2H), 3.20-3.25 (m, 2H), 3.76 (t, J = 7.20 Hz, 2H), 5.90 (bs,
1H), 6.06-6.07 (m, 1H), 6.26-6.27 (m, 1H), 6.47 (s, 1H), 7.17-7.21 (m, 3H), 7.27-7.32 (m, 3H).
13C-NMR (CDCl3) δ 25.50, 31.46, 33.10, 39.65, 49.54, 106.09, 110.33, 125.95, 128.40, 128.48,
141.37, 141.59, 153.25, 160.99. HRMS Calcd for C16H20N2O2 m/z [M+H] 273.1604, found
273.1636.
6.1.1.21. 1-(2-Morpholinoethyl)-3-(3-phenylpropyl)urea (21); Prepared by general procedure.
Yield 83 %; pale yellow powder; mp: 92-94 °C; Rf 0.25 (9 : 1 Ethylacetate : Hexane), IR (neat):
1
3230, 2912, 2790, 1660, 1611, 1575, 1508 cm-1; H-NMR (CDCl3) δ 1.81-1.88 (m, 2H), 2.42-
2.48 (m, 6H), 2.66 (t, J = 7.80 Hz, 2H), 3.19-3.26 (m, 4H), 3.68-3.70 (m, 4H), 4.82-4.88 (m, 2H),
13
7.17-7.21 (m, 3H), 7.27-7.31 (m, 2H); C-NMR (CDCl3) δ 31.94, 33.29, 36.97, 40.23, 53.47,
58.10, 67.04, 126.11, 128.51, 128.60, 141.78, 158.63. HRMS Calcd for C16H25N3O2 m/z [M+H]
292.2026, found 292.2055.
6.1.1.22. 1-(2-Morpholinoethyl)-3-(4-phenylbutyl)urea (22); Prepared by general procedure (i).
o
Yield 36 %; Cream solid; mp: 73-76 C; Rf 0.28 (9 : 1 Ethylacetate : Hexane), IR (neat): 3309,
1
2940, 1624, 1576, 1115, 867, 745, 697 cm-1; H-NMR (CDCl3) δ 1.50-1.57 (m, 2H), 1.62-1.70

ACCEPTED MANUSCRIPT
(m, 2H), 2.45-2.49 (m, 6H), 2.61-2.65 (t, 2H), 3.16-3.21 (m, 2H), 3.23-3.27 (m, 2H), 3.67-3.73
13
(m, 4H), 4.82-4.87 (bs, 2H), 7.16-7.20 (m, 3H), 7.28-7.30 (d, 2H); C-NMR (CDCl3) δ 28.90,
30.20, 35.35, 36.75, 39.45, 53.76, 58.77, 66.64, 126.09, 128.68, 128.74, 142.69, 158.48. HRMS
Calcd for C17H27N3O2 m/z [M+H] 306.2182, found 306.2207.
6.1.1.23. 1-(3-Cyclohexylpropyl)-3-phenethylurea (23);
6.1.1.23.1. Preparation of 3-cyclohexylpropan-1-amine (47); 3-Cyclohexylpropan-1-ol (10.0
mmol) was refluxed with hydrobromic acid (10.0 mmol) for 5 h. After the addition of ice water,
the reaction mixture was extracted with ethyl acetate to get 3-(bromopropyl)cyclohexane. The
crude mixture was reacted with phthalimide (15.0 mmol) in THF (20 mL) under reflux for 2 h to
get a the phthalimide intermediate as white powder. It was filtered and further reacted with
hydrazine (98 %, 63 mmol) in absolute ethanol (15 mL) at 60 °C for 30 minutes. The reaction
mixture was diluted with water and extracted using ethyl acetate to get the 3-cyclohexylpropan-
1-amine 47. The amine 47 was reacted with phenethylisocyanate according to the general
procedure to get 23. Yield 87 %; White solid; mp 84-86 °C; Rf 0.40 (1 : 2 Ethylacetate :
Hexane), IR (neat): 3359, 3307, 2911, 2849, 1625, 1560, 1496 cm-1; 1H-NMR (CDCl3) δ 0.81-
0.89 (m, 2H), 1.11-1.26 (m, 6H), 1.41-1.49 (m, 2H), 1.62-1.69 (m, 5H), 2.81 (t, J = 8.00 Hz,
2H), 3.05-3.10 (m, 2H), 3.41-3.46 (m, 2H), 4.26-4.33 (m, 2H), 7.19-7.24 (m, 3H), 7.28-7.33 (m,
2H). 13C-NMR (CDCl3) δ 26.24, 26.53, 27.45, 33.24, 34.51, 36.37, 37.33, 40.82, 41.57, 126.40,
128.59, 128.86, 139.30, 158.31. HRMS Calcd for C18H28N2O2 m/z [M+H] 289.2281, found
289.2308.
6.1.1.24. 1-(3-1H-Imidazol-1-yl)propyl)-3-phenethylurea (24); Prepared by general procedure.
Yield 80 %; White solid; mp 72-74 °C; Rf 0.22 (9 : 1 Ethylacetate : Hexane), IR (neat): 3301,
2938, 1637, 1558, 1508, 1475, 1230, 1108 cm-1; 1H-NMR (CDCl3) δ 1.90-1.97 (m, 2H), 2.80 (t,