Design and synthesis of sulfonamidophenylethylureas as novel cardiac myosin activator

Article abstract of DOI:10.1016/j.ejmech.2017.10.077

To optimize the lead urea scaffold 1 and 2 as selective cardiac myosin ATPase activator, a series of urea derivatives have been synthesized to explore its structure activity relationship. Among them N,N-dimethyl-4-(2-(3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (13, CMA = 91.6%, FS = 17.62%; EF = 11.55%), N,N-dimethyl-4-(2-(1-methyl-3-(3-phenylpropyl)ureido)ethyl)benzene sulfonamide (40, CMA = 52.3%, FS = 38.96%; EF = 24.19%) and N,N-dimethyl-4-(2-(3-methyl-3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (41, CMA = 47.6%, FS = 23.19%; EF = 15.47%) proved to be efficient to activate the cardiac myosin in vitro and in vivo. Further the % change in ventricular cell contractility at 5 μM of 13 (47.9 ± 3.2), 40 (45.5 ± 2.4) and 41 (63.5 ± 2.2) showed positive inotropic effect in isolated rat ventricular myocytes. The potent compounds 13, 40, 41 were highly selective for cardiac myosin over skeletal and smooth muscle myosin, thus proving them these new urea derivatives is a novel scaffold for discovery of cardiac myosin activators for the treatment of systolic heart failure.

Full text of DOI:10.1016/j.ejmech.2017.10.077

Accepted Manuscript
Design and synthesis of sulfonamidophenylethylureas as novel cardiac myosin
activator
Manoj Manickam, Hitesh B. Jalani, Thanigaimalai Pillaiyar, Pulla Reddy Boggu, Niti
Sharma, Eeda Venkateswararao, You-Jung Lee, Eun-Seok Jeon, Min-Jeong Son,
Sun-Hee Woo, Sang-Hun Jung
PII:
S0223-5234(17)30879-6
10.1016/j.ejmech.2017.10.077
EJMECH 9869
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 August 2017
Revised Date: 18 October 2017
Accepted Date: 30 October 2017
Please cite this article as: M. Manickam, H.B. Jalani, T. Pillaiyar, P.R. Boggu, N. Sharma, E.
Venkateswararao, Y.-J. Lee, E.-S. Jeon, M.-J. Son, S.-H. Woo, S.-H. Jung, Design and synthesis
of sulfonamidophenylethylureas as novel cardiac myosin activator, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.10.077.
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ACCEPTED MANUSCRIPT
Design and synthesis of sulfonamidophenylethylureas as novel cardiac myosin activator
GRAPHICAL ABSTRACT
N,N-Dimethylsulfonamide
group is important
Flexible spacer is tolerated
O
O
S
O
N
O
n
N
N
N
R1
N
R2
H
H
Optimization
HBD is important
Partial masking of urea HBD is crucial
Lead compound
Fractional Ejection Ventricular cell
shortening Fraction contractility (%
Cardiac myosin Fractional Ejection
ATPase activation shortening Fraction
Cardiac myosin
ATPase activation
% at 10 uM
increase % increase change at 5 µM)
%
at 10 uM
% increase % increase
%
1
2
R = H; R = H
47.9 + 3.2
45.5 + 2.4
63.5 + 2.2
n = 1
n = 2
53.3
51.1
30.04
18.90
18.27
12.15
91.6
52.3
47.6
17.62
38.96
23.19
11.55
24.19
15.47
1
2
R = CH ; R = H
3
1
2
R = H; R = CH
3

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Design and synthesis of sulfonamidophenylethylureas as novel cardiac myosin activator
a
a
a
a
Manoj Manickam , Hitesh B. Jalani , Thanigaimalai Pillaiyar , Pulla Reddy Boggu , Niti
Sharma , Eeda Venkateswararao , You-Jung Lee , Eun-Seok Jeonb, Min-Jeong Son , Sun-Hee
a
a
b
a
a
a
Woo , Sang-Hun Jung *
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
To optimize the lead urea scaffold 1 and 2 as selective cardiac myosin ATPase activator, a series
of urea derivatives have been synthesized to explore its structure activity relationship. Among
them N,N-dimethyl-4-(2-(3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (13, CMA=
9
1.6%,
phenylpropyl)ureido)ethyl)benzene sulfonamide (40, CMA = 52.3%, FS = 38.96%; EF =
4.19%) and N,N-dimethyl-4-(2-(3-methyl-3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide
41, CMA = 47.6%, FS = 23.19%; EF = 15.47%) proved to be efficient to activate the cardiac
myosin in vitro and in vivo. Further the % change in ventricular cell contractility at 5 µM of 13
47.9 + 3.2), 40 (45.5 + 2.4) and 41 (63.5 + 2.2) showed positive inotropic effect in isolated rat
FS
=
17.62%;
EF
=
11.55%),
N,N-dimethyl-4-(2-(1-methyl-3-(3-
2
(
(
ventricular myocytes. The potent compounds 13, 40, 41 were highly selective for cardiac myosin
over skeletal and smooth muscle myosin, thus proving them these new urea derivatives is a novel
scaffold for discovery of cardiac myosin activators for the treatment of systolic heart failure.
Key words: Phenylpropylurea; 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

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1
.
Introduction
Heart failure (HF) is a major global public health concern with an estimated prevalence of 37.7
million individuals worldwide [1-3] and leads to substantial numbers of hospitalizations and
health care costs [4,5]. Heart failure is more common in older ages [6,7], and therefore, heart
failure prevalence will continue to increase with aging of the world population.
Treatment of heart failure still remains unsatisfactory for many patients. Available treatments
aim at diverse targets including sodium retention, arterial and venous constriction,
neuroendocrine activation increases heart rate, cardiac dyssynchrony and arrhythmias and often
fail to control symptoms or restore quality of life [8]. Moreover, mortality remains high in this
population. At least half the patients with heart failure have a low ejection fraction (40 % or less)
[9], thus leading to systolic heart failure [10]. In systolic heart failure, the reasons for reduced
myocardial contractility are complex and include the loss of cardiac myocytes [11], changes in
the extracellular matrix [12], reduced availability of high energy substrates such as ATP and
creatinine phosphate [13], impaired calcium recycling [14], and myofilament abnormalities [15].
In the treatment of heart failure due to reduced left ventricular systolic function, inotropic agents
have been used to improve myocardial contractility. Inotropic agents increase the velocity and
force of contraction but do not increase, in fact usually shorten, the duration of systole [16].
Cardiac myosin activators are a new mechanistic class designed specifically to increase
myocardial contractility. In contrast to existing inotropic drugs, they increase the duration of
systole (systolic ejection time) without changing the rate of left ventricular pressure
development, thereby increasing stroke volume and cardiac output [17,18].
Within the myofilament, cardiac myosin is central to myocardial contractility. During
myocardial contraction, myosin forms cross-bridges with actin. Initially myosin is weakly bound

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to the actin filament and undergoes a transition to a strongly bound cross-bridge state in order to
produce a force-generating power stroke. Cardiac myosin activators increase the transition rate
from the weakly bound to the strongly bound force-generating state, increasing myocardial
contraction [19-21].
The selective cardiac myosin activator, omecamtiv mecarbil (OM), is currently completing late
phase II trials on the verge of phase III trials [22]. Unlike prior agents that increased intracellular
cAMP and calcium and decrease ejection time, OM increased myocardial contraction and stroke
volume without increasing oxygen consumption, thereby increasing myocardial efficiency
[
23,24]. Patients with ischemic cardiomyopathy and angina showed no adverse effects of OM
13]. In healthy volunteers OM produced dose-dependent and concentration-dependent increase
[
in systolic ejection time, fractional shortening, and ejection fraction [25,26].
Although OM is currently undergoing clinical trials, a broader range of drugs that can improve
cardiac function is urgently required. In our earlier report [27], a series of flexible
phenylpropylurea scaffold was discovered as novel cardiac myosin activators. Particularly,
compounds 1 and 2 (Figure 1) showed excellent cardiac myosin ATPase activation (CMA) in
vitro and potent fractional shortening (FS) and ejection fraction (EF) in vivo compared to OM.
To further explore the structure activity relationship of the lead molecules 1 and 2, the SAR
study focus to the optimization of the substituents on both the phenyl rings, importance of
hydrogen bonding donor characteristics of the urea NH, importance and optimization of the
flexible chain.

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Fig.1. Background and exploration of urea scaffold for novel cardiac myosin activator
2
. Chemistry
Scheme 1 represents the synthesis of compounds 3-26 denoted in Table 1. The reaction of
various amine (‘L’ group and ‘n’ are in Table 1) with 3-phenylpropylisocyanate (47) afforded the
corresponding urea derivatives 3-11, 14-21, 24-25. Urea 6,11 were prepared by the reaction of 4-
phenylbutylamine (48) with triphosgene in the presence of N,N-diisopropylethylamine for 0.5 h
stirring at 0 °C followed by the addition of amines (‘L’ group and ‘n’ are in Table 1). The
sulfonamide group of the ureas 4, 5 was dimethylated using 2 equivalents of methyliodide in
presence of NaH to afford the ureas 12, 13 respectively. The nitro group of the urea 21 was
catalytically reduced to amine 22 using H
2
and Pd/C which was consequently dimethylated to 23
as base. In order to prepare the urea 26, 3-
using methyliodide in presence of K CO
2
3

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methoxyphenethyl amine (49) was protected with acetyl group to get 50 which on
chlorosulfonation at para to methoxy group followed by the reaction with ammonium acetate in
THF under reflux afforded 51. Deprotection of the acetyl group of 51 using 3N HCl at reflux
yielded intermediate 52 which on reaction with 3-phenylpropylisocyanate (47) furnished the urea
2
6.
Scheme 1. Synthesis of compounds 3-26 denoted in Table 1.
Reagents and conditions: (a) Acetonitrile, RT, 8 h, (b) triphosgene, DIPEA, THF, 0 °C to RT, 8
h, (c) 2 eq CH I, NaH, DMF, RT, 3 h, (d) H /Pd/C, MeOH, RT, 10 h, (e) CH I, K CO , acetone,
3
2
3
2
3
reflux 2 h, (f) CH
3
COCl, TEA, CH
2
Cl
2
, 0 °C to RT, 3 h (g) (i) ClSO
3
H, CH
2
2
Cl , 0 °C to RT, 3 h
(
ii) NH OAc, THF, reflux, 5 h (h) 3N HCl, n-butanol, reflux, 8 h. (i) 47, acetonitrile, RT, 8 h.
4
The ‘L’ group of the amine and ‘n’ are denoted in Table 1.

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Scheme 2 denotes the preparation of urea derivatives 27-34 with substitution on the phenyl ring
(R) as mentioned in Table 1. In order to prepare the 4-fluoro substituted urea derivatives 27, 30,
3
4, the key intermediate 56 was reacted with appropriate amine (‘L’ group and ‘n’ are in Table
) under triphosgene condition. The preparation of 56 was achieved by the transformation of 4-
1
fluorocinnamic acid (53) to its amide 54 by coupling with ammonium chloride. The amide 54
was then reduced with lithium aluminum hydride to amine and subsequently reacted with Boc
anhydride to yield Boc-protected amine 55 for the purpose of purification. The key intermediate
5
6 was prepared by deprotection of Boc group of 55 with HCl in dioxane solution. To prepare
the 4-methoxy substituted ureas 28, 31, 33 and chloro substituted urea 32, the methoxy and
chloro substituted isocyanates 58a and 58b were prepared as follows; First the 4-
methoxyphenylbutanoic acid 57a/4-chlorophenylbutanoic acid 57b were reacted with methyl
chloroformate in presence of triethylamine as base to form mixed anhydride which on reaction
with sodium azide at ambient temperature afforded the respective acyl azide. The acyl azide,
without isolation was heated in refluxing toluene to give the 4-methoxy/4-chloro substituted
isocyanates 58a/58b. The reaction of the obtained isocyanates 58a/58b with appropriate amine
(‘L’ group and ‘n’ are in Table 1) afforded the methoxy and chloro substituted ureas 28,31,32,33.
To prepare N,N-dimethylsulfonamide substituted urea derivative 29, the N,N-
dimethylsulfonamide substituted amine 62 was reacted with benzyl amine 63 under triphosgene
condition. The amine 62 was in turn prepared from 3-phenylpropylamine 59 by protecting it
with acetyl group to give 60 followed by chlorosulfonation and subsequent reaction with
dimethylamine afforded intermediate 61. Deprotection of the acetyl group of 61 yielded the
amine 62.

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Scheme 2. Synthesis of compounds 27-34 denoted in Table 1.
Reagents and conditions: (a) EDC.HCl, NH Cl, TEA, CH Cl , RT, 16 h, (b) (i) LiAlH , THF,
4
2
2
4
7
0 °C 3 h (ii) Boc
2
O, RT, 16 h, (c) 4M HCl in 1,4-dioxane, RT, 2 h (d) triphosgene, DIPEA,
THF, 0 °C to RT, 8 h, (e) (i) methyl chloroformate, acetone, TEA, RT, 0.5 h (ii) NaN /H O, RT,
3
2
1
h (iii) toluene, reflux, 3 h, (f) acetonitrile, RT, 8 h, (g) CH COCl, TEA, CH Cl , 0°C to RT, 3 h
3 2 2
3 2 2 3
(h) (i) ClSO H, CH Cl , 0 °C to RT, 3 h (ii) dimethylamine.hydrochloride, NaHCO , THF, RT, 5
h (i) 3N HCl, n-butanol, reflux, 8 h. The ‘L’ group of the amine and ‘n’ are denoted in Table 1.
To study the effect of rigidification of the flexible carbon chain, compounds 35-39 (Table 3)
were prepared as depicted in scheme 3. The reaction of sulfonamide substituted amine 64, 65
with 4-phenylpiperidine 66 afforded the intermediate 67, 68 respectively, which further on
methylation of the sulfonamide group with 2 equivalents of methyliodide in presence of NaH as
base furnished the respective N,N-dimethylsulfonamide substituted rigid ureas 35 and 36. In
order to prepare the rigid ureas 37,38 and 39, the respective isocyanates 70,71,47 were reacted

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with 4-N,N-dimethylsulfonamide substituted 4-phenylpiperidine 69 which in turn prepared from
4
-phenylpiperidine 66 similar to the procedure mentioned in scheme 2.
Scheme 3. Synthesis of compounds 35-39 denoted in Table 3.
Reagents and conditions: (a) triphosgene, DIPEA, THF, 0 °C to RT, 8 h, (b) 2 eq CH
3
I, NaH,
DMF, RT, 3 h, (c)(i) CH COCl, TEA, CH Cl , 0°C to RT, 3 h (ii) ClSO H, CH Cl , 0 °C to RT,
3
2
2
3
2
2
3
h (iii) dimethylamine.hydrochloride, NaHCO , THF, RT, 5 h (iv) 3N HCl, n-butanol, reflux, 8
3
h, (d) acetonitrile, RT, 8 h.
Scheme 4 represents the synthesis of compounds 40-46 denoted in Table 3. 65 was protected
with acetyl group using acetyl chloride to give 72. Methylation of 72 with 3 equivalents of
methyl iodide in presence of NaH afforded 73. Deprotection of the acetyl group of 73 furnished
7
4. Finally, the amine 74 was reacted with 3-phenylpropylisocyanate 47 to give the urea 40. In
order to prepare the urea 41, 3-phenylpropylamine 59 was converted to its N-methyl derivative
5 analogous to the preparation of 74. Further 75 was reacted with 65 to give the urea
7
intermediate 76 which was methylated with 2 equivalents of methyl iodide to 41. Reaction of 13
with methyl iodide and ethyl iodide afforded 42 and 44, respectively. In another set of
experiment 65 was reacted with ethyl iodide/isopropyl iodide in presence of K
2 3
CO to furnish
7
7a/77b along with their dialkylated derivatives. Further 77a/77b were reacted with 47 to give

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the urea intermediate 78a/78b which were reacted with 2 equivalents of methyl iodide to afford
the urea 43, 45. Compound 46 was prepared by the methylation of the urea NH of 45.
Scheme 4. Synthesis of compounds 40-46 denoted in Table 3.
3 2 2 3
Reagents and conditions: (a) CH COCl, TEA, CH Cl , 0 °C to RT, 3 h (b) CH I, NaH, DMF, 3 h
(c) 3N HCl, n-butanol, reflux, 8 h (d) acetonitrile, RT, 8 h (e) triphosgene, THF, 0 °C to RT, 8 h

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(f) 2 eq CH
3 3 3 2 3 2
I, NaH, DMF, RT, 3 h, (g) CH I or CH CH I, NaH, DMF, RT 3-15 h (h) CH CH I,
or (CH ) CHI, K CO , DMF, RT, 12 h.
3
2
2
3
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
[28] with modifications. Compounds that activate the sarcomere were identified by measuring %
increase in myosin ATPase activity at 10 µM. The results are shown in Table 1 - 3. 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)) [28], skeletal (rabbit (100 µM)) [29-31] and smooth muscle (chicken gizzard (100 µM))
[29,32,33] 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 [34]. The results are shown
in Table 2. Positive inotropic effect was tested by measuring % increase of left ventricle
fractional shortening (FS) and ejection fraction (EF) with/without samples using
echocardiography [35] in seven-week-old Sprague-Dawley male rats. The results are shown in
Table 1 and Table 3. In addition to this, positive inotropic effect was measured by the change in
ventricular cell contractility in rat ventricular myocytes isolated from male Sprague-Dawley rats
[
36]. The results are shown in Table 2 and Fig.2.
. Results and Discussion
Based on our previous report [25], compound 1 (CMA at 10 µM = 53.3% FS = 30.04%; EF =
8.27%) and compound 2 (CMA at 10 µM = 51.1% FS = 18.90%; EF = 12.15%) showed
excellent in vitro and in vivo activity and were taken as lead compounds (Table 1). Therefore to
4
1

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optimize the lead, the structure activity relationship was established on the compounds 1 and 2.
In this respect, the substituents on both the phenyl rings, variation of the chain length, hydrogen
bonding property of the urea moiety, importance of the flexible chain were investigated.
Table 1. Optimization of spacer and groups in the urea scaffold
ATPase
activity
Fractional
Shoretning
Ejection
Fraction
Comp.
No
L
R
n
m
CLogP
%
at 10 µM % increase % increase
1
2
Phenyl
Phenyl
Phenyl
Phenyl
1
2
1
1
53.3
51.1
30.04
18.90
18.27
12.15
3.696
4.025
3
4
5
Phenyl
Phenyl
Phenyl
0
1
2
1
1
1
31.8
39.6
41.9
---
---
---
---
2.118
1.859
2.188
11.24
7.23
6
Phenyl
2
2
36.6
---
---
4.212
7
8
9
Phenyl
Phenyl
Phenyl
1
2
1
1
1
1
25.4
40.0
38.8
---
---
---
---
---
---
3.615
3.944
3.354
1
0
Phenyl
2
1
122.4
23.97
15.87
3.683
1
1
1
2
Phenyl
Phenyl
2
1
2
1
32.5
34.5
---
---
---
---
2.717
2.891

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1
1
1
3
4
5
Phenyl
Phenyl
Phenyl
2
2
2
1
1
1
91.6
95.1
35.0
17.62
20.47
---
11.55
13.58
---
3.220
3.944
3.944
1
6
Phenyl
2
1
31.0
---
---
---
---
4.738
1
1
7
8
Phenyl
Phenyl
2
2
1
1
37.3
73.8
5.451
4.534
Precipitated during
formulation
1
2
9
0
Phenyl
Phenyl
2
2
1
1
33.3
40.6
---
---
---
---
5.452
3.768
2
2
1
2
Phenyl
Phenyl
2
2
1
1
56.2
55.2
---
---
3.768
2.798
6.37
4.16
2
3
Phenyl
2
1
45.3
---
---
4.190
2
2
4
5
Phenyl
Phenyl
2
2
1
1
38.6
65.4
---
---
4.168
4.168
12.52
8.76
2
6
Phenyl
2
1
29.4
---
---
2
.432
2
2
2
3
7
8
9
0
1
1
1
2
1
1
1
1
9.5
---
---
---
---
---
---
---
---
3.839
3.615
2.891
2.331
30.5
20.8
19.1

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3
3
1
2
2
2
1
1
34.8
37.9
---
---
---
---
2
.107
2.901
3.602
3
3
3
4
2
2
1
1
38.4
20.6
58.0
---
---
---
---
3.363
3.266
STD
--, activity not checked
In the first set of experiment (Table 1), compounds 3 (n = 0; m = 1, CMA = 31.8%), 4 (n = 1; m
Omecamtiv mecarbil
20.32
13.35
-
=
=
1, CMA = 39.6%), 5 (n = 2; m = 1, CMA = 41.9% FS = 11.24%; EF = 7.23%) and 6 (n = 2; m
2, CMA = 36.6%) were synthesized with sulfonamide substitution on phenyl ring (L) and
varying the length of the carbon chain between urea functional group and both the phenyl rings
L and R). The results proved that none of the compounds with sulfonamide substitution at para
(
position of the phenyl ring (L) improved cardiac myosin ATPase activity compared to 1, 2 or
OM. Despite the compound 5 with two carbon spacer on one side (L) and three carbon spacer on
other side (R) showed moderate activity in vitro, the in vivo activity did not prove effectiveness.
4
2
-Methoxy substitution on phenyl ring (L) as shown in compound 7 (n = 1; m = 1, CMA =
5.4%) and 8 (n = 2; m = 1, CMA = 40.0%) did not improve the activity. However, two carbon
spacer between urea and phenyl ring (L) as shown in 8 improved the activity compared to one
carbon spacer. This effective spacing pattern of flexible chain was also recognized in 3,4-
dimethoxy substitution analogs as considering the superior activity of 10 (n = 2; m = 1, CMA =
1
22.4%) to those of 9 (n = 1; m = 1, CMA = 38.8%), and 11 (n = 2; m = 2, CMA = 32.5%) and
thus the suitable spacers between urea and phenyl ring (L) and between urea and phenyl ring (R)

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should be two and three, respectively. Further, compound 10 showed highly effective in vivo
activity (FS = 23.97%; EF = 15.87%) proving dimethoxy substitution is suitable for activating
cardiac myosin. The priority of two carbon spacer rather than one carbon spacer between urea
and phenyl ring (L) was further substantiated from the compounds 12 and 13 with N,N-
dimethylsulfonamide substitution on phenyl ring (L). Compound 12 (n = 1; m = 1, CMA =
3
1
4.5%) showed weak activity, whereas compound 13 (n = 2; m = 1, CMA = 91.6%, FS =
7.62%; EF = 11.55%) showed strong activity in vitro and in vivo. From these results, two
carbon chain from the urea to the phenyl ring (L) and three carbon chain to phenyl ring (R) were
fixed as optimum chain length.
In the next set of experiment (Table 1), the effect of substituents on the phenyl ring (L) were
further explored as shown in compounds 14-26 with the optimum chain length. Methoxy
substitution at ortho- position as represented in compound 14 (CMA = 95.1%, FS = 20.47%; EF
=
13.58%) showed potent activity both in vitro and in vivo whereas the meta- substitution as
demonstrated in compound 15 (CMA = 35.0%) did not improve the activity. The effect of chloro
substituent was explored in compounds 16 and 17. Compound 16 with 4-chloro moiety (CMA =
3
1.0%) and 17 with 2,4-dichloro moiety (CMA = 37.3%) showed poor activity. The methyl
substituent at para position as represented in compound 18 (CMA = 73.8%) showed excellent
activity. However, it had poor water solubility due to its hydrophobic nature (Clog P = 4.534)
and precipitated during in vivo formulation. Increase in bulkiness with 4-isopropyl group as
mentioned in compound 19 (CMA = 33.3%) unfortunately showed weak activity. Electron
withdrawing groups at para position such as carboxylic acid (20, CMA = 40.6%) or nitro group
(21, CMA = 56.2%) did not influence the activity. The electron donating groups at para position
of the phenyl ring (L) such as primary amino (22, CMA = 55.2%, FS = 6.37%; EF = 4.16%) or

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N,N-dimethyl amino (23, CMA = 45.3%) also failed to improve the activity in vitro and in vivo.
The introduction of small group such as fluorine at para position of the phenyl ring (L) as shown
in 24 (CMA = 38.6%) did not improve the activity. However, when switching the fluorine to
ortho position as represented in 25 (CMA = 65.4%, FS = 12.52%; EF = 8.76%) fetched valuable
activity in vitro. But the in vivo efficacy was poor compared to compounds 1, 2 or OM. Next,
introduction of two functional groups like methoxy and sulfonamido in the same phenyl ring (L)
as demonstrated in 26 (CMA = 29.4%) did not enhance the activity.
The above results denote that substituents like 4-N,N-dimethylsulfonamido, 2-methoxy and 3,4-
dimethoxy groups on the phenyl ring (L) are suitable for activating cardiac myosin in vitro as
well as in vivo.
The next set of series was focused to explore the substituents on the other phenyl ring (R). First,
the active compound 1 was substituted with various groups on the para position of the phenyl
ring (R). The introduction of fluoro, methoxy and N,N-dimethylsulfonamido groups as
represented in compounds 27 (CMA = 9.5%), 28 (CMA = 30.5%) and 29 (CMA = 20.8%),
respectively, decreased the activity. The fluoro, methoxy and chloro group at the para position of
the phenyl ring (R) of the compound 5 were introduced as shown in compounds 30 (CMA =
1
9.1%), 31 (CMA = 34.8%) and 32 (CMA = 37.9%), respectively. All the compounds showed
weak activity compared to compound 5, 1 or 2. In another attempt, the substitution of highly
active compound 10 with methoxy group at the para position of the phenyl ring (R) as shown in
3
3 (CMA = 38.4%) and 13 with simple fluoro group as shown in compound 34 (CMA = 20.6%)
led to the reduced activity. All the above results clearly attest that the substitution on the other
phenyl ring (R) is not tolerated.

ACCEPTED MANUSCRIPT
Table 2. Ventricular cell contractility and Selectivity study in myosins
ATPase % activity
Compound
Ventricular cell
contractility [rat] %
change
Cardiac myosin Skeletal myosin Smooth myosin
No.
(
at 10 µM)
(at 100 µM)
1.4
(at 100 µM)
˗ 3.7
1
2
25.3 + 2.2 at 5 µM
20.8 + 1.9 at 5 µM
22.2 + 3.1 at 5 µM
47.9 + 3.2 at 5 µM
19.7 + 3.2 at 5 µM
33.8 + 4.1 at 1 µM
----
53.3
51.1
˗ 2.1
˗ 4.7
1
1
1
0
3
4
122.4
91.6
˗ 2.4
4.3
˗ 3.8
˗ 1.9
95.1
3.5
5.3
OM
58.0
2.8
˗ 5.5
(
˗)Blebbistatin
----
˗ 50.7
˗ 35.7
The potent compounds 10, 13 and 14 were analyzed for selectivity in cardiac myosin over
skeletal and smooth myosins (Table 2). None of the compound showed significant activity for
myosin ATPase of skeletal or smooth myosin S1. Thus, from these results it is evident that these
urea derivatives are selective for cardiac myosin S1.
Further, along with these three compounds 10, 13 and 14, the lead compounds 1 and 2 were
subjected to ventricular cell contractility assay, in order to provide evidence for their positive
inotropic effect in the ventricle cells isolated from rat (Table 2). All the five compounds showed
positive inotropic effect by reversibly increasing ventricular cell shortening. Among them,
compound 13 showed potent activity in the assay (% change in ventricular cell contractility at 5
µM = 47.9 + 3.2).

ACCEPTED MANUSCRIPT
Table 3. SAR of compound 13
ATPase
Fractional
Shoretning
Ejection
Fraction %
increase
Comp.
No
Structure of the Compound
activity
%
CLogP
%
increase
at 10 uM
1
3
3
5
91.6
27.6
17.62
11.55
---
3.220
3.072
---
---
3
3
6
7
40.7
36.9
---
---
---
3.321
3.072
---
---
3
3
8
9
32.6
85.1
3.321
precipitated during
formulation
3.620
4
4
4
4
0
1
2
3
52.3
47.6
38.96
23.19
24.19
15.47
2.956
2.956
69.0
0.46
---
0.27
---
3.502
3.485
39.5
30.3
4
4
---
---
4.560

ACCEPTED MANUSCRIPT
4
4
5
6
38.1
---
---
3.794
40.6
58.0
---
---
4.340
3.266
STD
Omecamtiv mecarbil
20.32
13.35
---, activity not checked
The SAR of compound 13 with N,N-dimethylsulfonamide group on phenyl ring of (L) was
further explored by studying the importance of the flexible chain and hydrogen bonding donor
property of the urea NH (Table 3). Initially the flexible chain between the urea and phenyl ring
(R) was rigidified with piperidine moiety as represented in compound 35 (CMA = 27.6%) and 36
(CMA = 40.7%), respectively. The results clearly show that rigidification of the flexible chain
has adverse effect for cardiac myosin activation. To prove the same, the flexible chain between
urea and ring (L) were rigidified with piperidine moiety as shown in examples 37 (CMA =
3
6.9%), 38 (CMA = 32.6%) and 39 (CMA = 85.1%), respectively. Only 39 demonstrated potent
ATPase activation. However, it was precipitated during aqueous formulation for in vivo study.
The above results clearly indicate the importance of flexible carbon chains as spacers for the
activity.
To substantiate the importance of the HBD property of the urea function in highly active 13,
compounds 40-46 were prepared. Masking systematically the HBD of urea group of 13 on either
sides with methyl group as demonstrated in compound 40 (CMA = 52.3%, FS = 38.96%; EF =
2
4.19%) and 41 (CMA = 47.6%, FS = 23.19%; EF = 15.47%) showed promising results both in
vitro and in vivo (Table 3). Compound 40, besides retaining the ATPase activity, showed two
folds increase in the FS and EF in the animal model when compared to 13. Much curiosity to
completely masked both the HBD of the urea of 13 as represented in compound 42 (CMA =

ACCEPTED MANUSCRIPT
6
9.0%, FS = 0.46%; EF = 0.27%). Even though improved its in vitro ATPase activity, its activity
in the animal model was far deprived. This result emphasizes the importance of minimum HBD
site required for effective binding.
The above results brought few more ideas to partially mask the HBD with bulkier groups like
ethyl group 43 (CMA = 39.5%), and isopropyl group 45 (CMA = 38.1%), which proved to be
ineffective for activity. Complete masking the HBD as shown in 44 with ethyl group (CMA =
3
0.3%) and 46 with one isopropyl and one methyl groups (CMA = 40.6%) also exerted adverse
effect on activating cardiac myosin ATPase. All the above facts confirm partial masking on
either side of the urea NH with methyl group is much suitable for activating the cardiac myosin
ATPase in vitro and in vivo.
Compounds 40 (ATPase activation % at 100 µM: skeletal myosin = 3.5, smooth myosin = -2.2)
and 41 (ATPase activation % at 100 µM: skeletal myosin = 2.9, smooth myosin = 4.1) showed
selective activation for cardiac myosin S1 over skeletal or smooth myosin S1 ATPase.
Consequently compounds 40 and 41 were subjected to ventricle cell contractility assay.
Compound 40 showed similar activity (% change in ventricular cell contractility at 5 µM is 45.5
+
2.4) to compound 13 whereas compound 41 showed excellent positive inotropic effect by
reversibly increasing ventricular cell shortening (% change in ventricular cell contractility at 5
µM is 63.5 + 2.2) (Fig. 2). These results once again insist that partial masking of the HBD of
urea of 13 leads to excellent positive inotropic effect together with potent cardiac myosin
activation in vitro and in vivo.

ACCEPTED MANUSCRIPT
Figure 2. Effect of compound 41 on cell shortening in rat ventricular myocytes
(A) Representative contraction traces recorded immediately before the exposure to 41 (5 µM)
and at the time when a maximal increase in cell shortening by 41 was observed. (B)
Comparisons of mean changes in cell shortenings (% of control) between control and 41 (n = 3).
*
P < 0.05 vs. Control. Tyrode buffer solution is used as control.
5
. Conclusion
Structure activity relationship study of the lead phenylpropylurea scaffold 1 and 2 was well
explored. The SAR study (Figure 3) demonstrated that the optimum distance between the urea
functionality and both the phenyl rings should be two on one side (L) and three on other side (R).
Fig. 3. SAR of urea as a selective cardiac myosin activator

ACCEPTED MANUSCRIPT
Substitution on phenyl ring (R) is not favorable for cardiac myosin activation. On the other hand,
substitution on the phenyl ring (L) with 3,4-dimethoxy moiety (10, CMA = 122.4%, FS =
2
3.97%; EF = 15.87%), 4-N,N-dimethyl sulfonamide moiety (13, CMA = 91.6%, FS = 17.62%;
EF = 11.55%) and 2-methoxy moiety (14, CMA = 95.1%, FS = 20.47%; EF = 13.58%) are
suitable. Among them compound 13 showed potent positive inotropic effect in ventricular cell
contractility experiment (% change in ventricular cell contractility at 5 µM = 47.9 + 3.2).
Further, the SAR of compound 13 was explored. 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 both the NH of the urea is not
favorable for cardiac myosin ATPase activity, whereas partial masking of the urea NH on either
side with simple methyl group as demonstrated in compound 40 (CMA = 52.3%, FS = 38.96%;
EF = 24.19%; % change in ventricular cell contractility at 5 µM = 45.5 + 2.4) and compound 41
(
CMA = 47.6%, FS = 23.19%; EF = 15.47%; % change in ventricular cell contractility at 5 µM =
3.5 + 2.2) showed excellent activity in all the three experiments. In particular, compound 41
showed the best activity among all the compounds synthesized. The potent compounds 13, 40,
1 were highly selective for cardiac myosin over skeletal and smooth myosins and thus proving
6
4
them these new urea derivatives is a novel scaffold for discovery of cardiac myosin activators for
the treatment of systolic heart failure.
6
6
. Experimental section
.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

ACCEPTED MANUSCRIPT
the standard procedures [37] 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. HPLC analysis for purity was performed
using a LC-10AD series system (Shimadzu, Kyoto, Japan) and LC-20AD series system
(Shimadzu, Kyoto, Japan) with SPD-10A UV/Vis detector. ACE-C18 column (250 × 4.6mm)
was used as the stationary phase. HPLC conditions include a flow rate of 1.0 mL/min using
water and methanol as solvents and a detection wavelength of 254 nm.
6
.1.1. General synthetic procedure for the preparation of Urea derivatives:
(i) Preparation of urea from the reaction of amines and isocyanates: To the corresponding amine
(1.10 mmol) in acetonitrile, 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.
(ii) Preparation of urea from the reaction of two amines: To a cooled solution of triphosgene
(1.41 mmol) in THF at 0 °C, a mixture of primary amine (3.52 mmol) and N,N-
diisopropylethylamine (DIPEA, 7.04 mmol) was added. The reaction mixture was stirred at the
same temperature for 30 minutes and then the other amine (3.52 mmol) was added. The reaction
mixture was slowly allowed to attain ambient temperature and further stirred for 8 h. After the

ACCEPTED MANUSCRIPT
completion of the reaction, water was added to the reaction mixture and extracted using 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. 4-(3-(3-Phenylpropyl)ureido)benzenesulfonamide (3); Prepared by general procedure (i).
Yield 85%; White solid; R 0.42 (Ethylacetate); mp 207-209 °C; IR (neat): 3354, 3321, 3032,
f
-
1
2
2
2
1
947, 2358, 2342, 1681cm ; 1H-NMR (DMSO-d
H), 3.09-3.14 (m, 2H), 6.33-6.36 (m, 1H), 7.12 (s, 2H), 7.16-7.31 (m, 5H), 7.54 (d, J = 8.40 Hz,
H), 7.67 (d, J = 8.40 Hz, 2H), 8.81 (s, 1H). 13C-NMR (DMSO-d ) δ 31.32, 32.40, 38.66,
16.82, 125.79, 126.76, 128.31, 128.36, 136.04, 141.72, 143.76, 154.92; HRMS Calcd for
6
) δ 1.71-1.78 (m, 2H), 2.61 (t, J = 7.80 Hz,
6
C H N O S m/z [M+H] 334.1225, found 334.1248. HPLC purity 100%
16
19
3
3
6
.1.1.2. 4-((3-(3-Phenylpropyl)ureido)methyl)benzenesulfonamide (4); Prepared by general
procedure (i). Yield 82%; White solid; R 0.35 (Ethylacetate); mp 209 °C; IR (neat): 3355, 2923,
f
-
1
2
3
7
357, 2341, 1623 cm ; 1H-NMR (DMSO-d ) δ 1.66-1.70 (m, 2H), 2.57 (t, J = 7.80 Hz, 2H),
6
.02-3.06 (m, 2H), 4.27 (d, J = 4.00 Hz, 2H), 6.43 (t, J = 4.00 Hz, 1H), 6.08 (t, J = 4.00 Hz, 1H),
.13 -7.30 (m, 7H), 7.40 (d, J = 8.40 Hz, 2H), 7.76 (d, J = 8.40 Hz, 2H); HRMS Calcd for
C H N O S m/z [M+H] 348.1382, found 349.1405.
17
21
3
3
6
.1.1.3. 4-(2-(3-(3-Phenylpropyl)ureido)ethyl)benzenesulfonamide (5); Prepared by general
procedure (i). Yield 84%; White solid; R 0.37 (Ethylacetate); mp 146-148 °C; IR (neat): 3327,
f
-1
2
923, 2855, 2358, 2341, 1697 cm ; 1H-NMR (DMSO-d ) δ 1.62-1.69 (m, 2H), 2.56 (t, J = 7.20
6
Hz, 2H), 2.76 (t, J = 7.20 Hz, 2H), 2.96-3.01 (m, 2H), 3.23-3.28 (m, 2H), 5.79-5.82 (m, 1H),
5
8
.90-5.92 (m, 1H), 7.15-7.20 (m, 3H), 7.26-7.30 (m, 4H), 7.38 (d, J = 8.40 Hz, 2H), 7.74 (d, J =
.40 Hz, 2H); 13C-NMR (DMSO-d ) δ 31.74, 32.44, 35.83, 38.79, 40.49, 125.72, 128.32,
6

ACCEPTED MANUSCRIPT
1
29.16, 141.91, 142.04, 144.15, 158.07; HRMS Calcd for C18
found 362.1567. HPLC purity 100%
.1.1.4. 4-(2-(3-(4-Phenylbutyl)ureido)ethyl)benzenesulfonamide (6); Prepared by general
procedure (ii). Yield 68%; White solid; R 0.38 (Ethylacetate); mp: 134-137 °C; IR (neat): 3254,
23 3 3
H N O S m/z [M+H] 362.1538,
6
f
-
1 1
2
2
946, 1698, 1557, 1164, 829, 667 cm ; H-NMR (DMSO-d ) δ 1.31-1.38 (m, 2H), 1.49-1.56 (m,
6
H), 2.54-2.58 (t, J = 7.20 Hz, 2H), 2.71-2.75 (t, J = 7.80 Hz, 2H), 2.96-3.01 (m, 2H), 3.20-3.25
(m, 2H), 5.77-5.80 (m, 1H), 5.85-5.88 (m, 1H), 7.14-7.19 (m, 3H), 7.25-7.29 (t, 2H), 7.30 (s,
1
3
2
6
H), 7.36-7.38 (d, J = 8.40 Hz, 2H), 7.72-7.74 (d, J = 8.40 Hz, 2H); C-NMR (DMSO-d ) δ
2
8.88, 30.16, 35.33, 36.38, 39.45, 41.04, 126.09, 126.13, 128.69, 128.74, 129.57, 142.41, 142.69,
1
44.54, 158.42; HRMS Calcd for C H N O S m/z [M+H] 376.1695, found 376.1723. HPLC
19
25
3
3
purity 99.25%
6
.1.1.5. 1-(4-Methoxybenzyl)-3-(3-phenylpropyl)urea (7); Prepared by general procedure (i).
Yield 88%; White solid; R 0.42 (2 : 1 Ethylacetate : Hexane); mp 114-116 °C; IR (neat): 3327,
f
-
1
2
2
1
923, 2357, 2341, 1618 cm ; 1H-NMR (DMSO-d ) δ 1.63-1.71 (m, 2H), 2.56 (t, J = 7.80 Hz,
6
H), 2.99-3.04 (m, 2H), 3.72 (s, 3H), 4.13 (d, J = 6.00 Hz, 2H), 5.91-5.94 (m, 1H), 6.16-6.19 (m,
H), 6.87 (d, J = 7.40 Hz, 2H), 7.16-7.20 (m, 5H), 7.26-7.30 (m, 2H); HRMS Calcd for
C H N O m/z [M+H] 299.1760, found 299.1784.
18
22
2
2
6
.1.1.6. 1-(4-Methoxyphenethyl)-3-(3-phenylpropyl)urea (8); Prepared by general procedure (i).
Yield 86%; White solid; R 0.44 (2 : 1 Ethylacetate : Hexane); mp: 93-96 °C; IR (neat): 3329,
f
-
1
1
3
2
3
7
030, 2932, 1615, 1560, 1511, 1243, 1040, 748, 696 cm ; H-NMR (CDCl ) δ 1.77-1.81 (m,
3
H), 2.63 (t, J = 7.80 Hz 2H), 2.73 (t, J = 7.80 Hz, 2H), 3.13-3.18 (m, 2H), 3.34-3.39 (m, 2H),
.78 (s, 3H), 4.27-4.30 (m, 2H), 6.84 (d, J = 7.40 Hz, 2H), 7.09-7.11 (d, J = 7.80 Hz, 2H), 7.15-
1
3
3
.20 (m, 3H), 7.27-7.30 (m, 2H). C-NMR (CDCl ) δ 31.72, 33.06, 35.35, 39.94, 41.74, 55.16,

ACCEPTED MANUSCRIPT
1
13.92, 125.94, 128.37, 128.45, 129.76, 131.13, 141.60, 158.16, 158.25; HRMS Calcd for
C H N O m/z [M+H] 313.1916, found 313.1942. HPLC purity 98.46%
19
24
2
2
6
.1.1.7. 1-(3,4-Dimethoxybenzyl)-3-(3-phenylpropyl)urea (9); Prepared by general procedure (i).
Yield 82%; Pale yellow solid; R 0.40 (2 : 1 Ethylacetate : Hexane); mp: 110-112 °C; IR (neat):
f
-
1
3
2
340, 1621, 1568, 1515, 1337, 1262, 1233, 1141, 1026 cm ; 1H-NMR (CDCl ) δ 1.77-1.84 (m,
3
H), 2.62 (t, J = 7.60 Hz, 2H), 3.17-3.22 (m, 2H), 3.85 (s, 6H), 4.26 (d, J = 6.00 Hz, 2H), 4.39
(
bs, 1H), 4.62 (bs, 1H), 6.78-6.83 (m, 3H), 7.13-7.19 (m, 3H), 7.24-7.28 (m, 2H); 13C-NMR
CDCl ) δ 31.77, 33.06, 40.01, 44.31, 55.80, 55.87, 110.75, 111.11, 119.55, 125.89, 128.28,
28.37, 131.77, 141.49, 148.23, 149.06, 158.35; HRMS Calcd for C19 m/z [M+H]
29.1865, found 329.1891. HPLC purity 100%
.1.1.8. 1-(3,4-Dimethoxyphenethyl)-3-(3-phenylpropyl)urea (10); Prepared by general
(
3
1
3
6
24 2 3
H N O
procedure (i). Yield 90%; White solid; R 0.42 (2 : 1 Ethylacetate : Hexane); mp 102-104 °C; IR
f
-1
(
neat): 3338, 2973, 2860, 1667, 1618, 1572, 1508 cm ; 1H-NMR (CDCl ) δ 1.76-1.83 (m, 2H),
3
2
.63 (t, J = 7.80 Hz, 2H), 2.74 (t, J = 7.80 Hz, 2H), 3.13-3.18 (m, 2H), 3.37-3.42 (m, 2H), 3.85
(s, 6H), 4.27-4.30 (m, 2H), 6.71-6.73 (m, 2H), 6.79 (d, J = 8.00 Hz, 1H), 7.11-7.30 (m, 5H).
1
3C-NMR (CDCl
25.97, 128.36, 128.45, 131.73, 141.58, 147.62, 149.01, 158.24; HRMS Calcd for C H N O
3
3
) δ 31.71, 33.07, 35.87, 40.01, 41.67, 55.77, 55.83, 111.29, 111.96, 120.68,
1
20
26
2
m/z [M+H] 343.2022, found 343.2038. HPLC purity 98.58%
6
.1.1.9. 1-(3,4-Dimethoxyphenethyl)-3-(4-phenylbutyl)urea (11); Prepared by general procedure
(
ii). Yield 63%; White solid; R 0.45 (2 : 1 Ethylacetate : Hexane); mp: 109-112 °C; IR (neat):
f
-
1 1
3
1
3
244, 2944, 1683, 1558, 1540, 1508, 1235, 1030, 802, 701, 668 cm ; H-NMR (DMSO-d ) δ
6
.46-1.53 (m, 2H), 1.59-1.65 (m, 2H), 2.62 (t, J = 7.80 Hz, 2H), 2.75 (t, J = 7.80 Hz, 2H), 3.11-
.16 (m, 2H), 3.38-3.43 (m, 2H), 3.85 (s, 6H), 4.22-4.25 (m, 1H), 4.27-4.30 (m, 1H), 6.71-6.73

ACCEPTED MANUSCRIPT
1
3
(
m, 2H), 6.79 (d, J = 8.00 Hz, 1H), 7.15-7.20 (m, 3H), 7.25-7.29 (m, 2H); C-NMR (CDCl
8.54, 29.69, 35.41, 35.86, 40.24, 41.65, 55.72, 55.80, 111.18, 111.85, 120.65, 125.82, 128.35,
28.40, 131.70, 142.18, 147.55, 148.94, 158.20; HRMS Calcd for C H N O m/z [M+H]
3
) δ
2
1
3
6
2
1
28
2
3
57.2178, found 357.2200.
.1.1.10. N,N-Dimethyl-4-((3-(3-phenylpropyl)ureido)methyl)benzenesulfonamide (12); To a
cooled suspension of NaH (2.20 mmol) in DMF, compound 4 (1.10 mmol) was added and stirred
at ambient temperature for 45 minutes. Then the reaction mixture was cooled and CH I (2.20
3
mmol) was added The reaction mixture was further stirred at ambient temperature for 3 h. After
the completion of the reaction, water was added to the reaction mixture was extracted using ethyl
acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuum.
The crude product was purified by column chromatography to get 12. Yield 84%; White solid; Rf
0
.36 (Ethylacetate); mp 102-104 °C; IR (neat): 3330, 1626, 1560, 1535, 1332, 1149, 1090, 951
-1
cm ; 1H-NMR (CDCl ) δ 1.78-1.86 (m, 2H), 2.62-2.68 (m, 2H), 2.65 (s, 6H), 3.19-3.24 (m, 2H),
3
4
7
3
.40 (d, J = 6.00 Hz, 2H), 4.83 (bs, 1H), 5.23 (bs, 1H), 7.15-7.20 (m, 3H), 7.25-7.29 (m, 2H),
.35 (d, J = 8.20 Hz, 2H), 7.59 (d, J = 8.20 Hz, 2H). 13C-NMR (CDCl ) δ 31.78, 33.01, 37.80,
9.91, 43.33, 125.96, 127.57, 127.80, 128.38, 128.45, 133.52, 141.59, 145.55, 158.43; HRMS
3
Calcd for C H N O S m/z [M+H] 376.1695, found 376.1717. HPLC purity 99.61%
1
9
25
3
3
6
.1.1.11. N,N-Dimethyl-4-(2-(3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (13); Prepared
from compound 5 by the procedure analogous to the preparation of 12. Yield 78%; White solid;
-1
R 0.38 (Ethylacetate); mp 98-100 °C IR (neat): 3335, 3015, 2983, 1652, 1620, 1549, 1488 cm ;
f
1
H-NMR (CDCl ) δ 1.79-1.83 (m, 2H), 2.64 (t, J = 7.80 Hz, 2H), 2.69 (s, 6H), 2.87 (t, J = 7.80
3
Hz, 2H), 3.14-3.19 (m, 2H), 3.40-3.45 (m, 2H), 4.36-4.40 (m, 2H), 7.16-7.36 (m, 7H), 7.68 (d, J
8.20 Hz, 2H). 13C-NMR (CDCl ) δ 31.74, 33.07, 36.27, 37.84, 39.92, 41.04, 125.99, 127.96,
=
3

ACCEPTED MANUSCRIPT
1
27 3 3
28.39, 128.47, 129.58, 133.34, 141.61, 145.08, 158.16; HRMS Calcd for C20H N O S m/z
[M+H] 390.1851, found 390.1878. HPLC purity 99.53%
6
.1.1.12. 1-(2-Methoxyphenethyl)-3-(3-phenylpropyl)urea (14); Prepared by general procedure
(
i). Yield 90%; Yellow powder; R 0.44 (2 : 1 Ethylacetate : Hexane); mp 91-93 °C; IR (neat):
f
-1
3
341, 2977, 2856, 1665, 1621, 1562, 1504 cm ; 1H-NMR (CDCl ) δ 1.78-1.85 (m, 2H), 2.64 (t,
3
J = 7.80 Hz, 2H), 2.82 (t, J = 8.00 Hz, 2H), 3.15-3.20 (m, 2H), 3.32-3.37 (m, 2H), 3.81 (s, 3H),
4
3
1
3
6
.37 (bs, 1H), 4.44 (bs, 1H), 6.85-6.91 (m, 2H), 7.11-7.30 (m, 7H). 13C-NMR (CDCl
3
) δ 30.94,
1.71, 33.08, 40.03, 40.63, 55.25, 110.39, 120.70, 125.95, 127.45, 127.84, 128.40, 128.45,
30.67, 141.64, 157.54, 158.35; HRMS Calcd for C19
13.1945. HPLC purity 99.21%
24 2 2
H N O m/z [M+H] 313.1916, found
.1.1.13. 1-(3-Methoxyphenethyl)-3-(3-phenylpropyl)urea (15); Prepared by general procedure
i). Yield 83%; White solid; R 0.44 (2 : 1 Ethylacetate : Hexane); mp: 66-69 °C; IR (neat): 3318,
(
f
-
1 1
3
1
2
7
5
005, 2938, 1614, 1570, 1541, 1457, 1258, 1169, 1043, 789, 695 cm ; H-NMR (DMSO-d ) δ
6
.59-1.69 (m, 2H), 2.52-2.57 (t, J = 7.80 Hz, 2H), 2.62-2.67 (t, J = 7.80 Hz, 2H), 2.95-3.02 (m,
H), 3.19-3.25 (m, 2H), 3.72 (s, 3H), 5.74-5.78 (m, 1H), 5.91-5.94 (m, 1H), 6.75-6.78 (m, 3H),
1
3
.14-7.22 (m, 4H), 7.25-7.30 (m, 2H); C-NMR (DMSO-d
6
) δ 32.33, 32.98, 36.68, 39.30, 41.26,
5.31, 111.91, 114.74, 121.36, 126.13, 128.72, 129.73, 141.84, 142.31, 158.46, 159.74; HRMS
Calcd for C H N O m/z [M+H] 313.1916, found 313.1940.
1
9
24
2
2
6
.1.1.14. 1-(4-Chlorophenethyl)-3-(3-phenylpropyl)urea (16); Prepared by general procedure (i).
Yield 74%; White solid; R 0.35 (1 : 1 Ethylacetate : Hexane); mp 120-122 °C; IR (neat): 3335,
f
-
1
2
8
7
948, 2883, 1672, 1618, 1555, 1510 cm ; 1H-NMR (CDCl ) δ 1.77-1.85 (m, 2H), 2.64 (t, J =
3
.00 Hz, 2H), 2.77 (t, J = 8.00 Hz, 2H), 3.14-3.19 (m, 2H), 3.37-3.42 (m, 2H), 4.15 (bs, 2H),
3
.11-7.30 (m, 9H). 13C-NMR (CDCl ) δ 31.68, 33.07, 35.69, 39.99, 41.37, 126.01, 128.37,