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Design and synthesis of sulfonamidophenylethylureas as novel cardiac myosin activator
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
DOI:
10.1016/j.ejmech.2017.10.077
Reference:
EJMECH 9869
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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Design and synthesis of sulfonamidophenylethylureas as novel cardiac myosin activator
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GRAPHICAL ABSTRACT
N,N-Dimethylsulfonamide group is important O N
O N H
N R1
Optimization
30.04 18.90
% increase
18.27 12.15
Fractional Ejection Cardiac myosin ATPase activation shortening Fraction % at 10 uM
1
EP AC C
2
R = H; R = H R1 = CH3; R2 = H R1 = H; R2 = CH3
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53.3 51.1
% increase
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Cardiac myosin Fractional Ejection ATPase activation shortening Fraction n=1 n=2
N R2
HBD is important Partial masking of urea HBD is crucial
Lead compound
% at 10 uM
O
SC
n N H
Flexible spacer is tolerated
O S
91.6 52.3 47.6
% increase
17.62 38.96 23.19
Ventricular cell contractility (%
% increase change at 5 µM)
11.55 24.19 15.47
47.9 + 3.2 45.5 + 2.4 63.5 + 2.2
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Design and synthesis of sulfonamidophenylethylureas as novel cardiac myosin activator Manoj Manickama, Hitesh B. Jalania, Thanigaimalai Pillaiyara, Pulla Reddy Boggua, Niti Sharmaa, Eeda Venkateswararaoa, You-Jung Leeb, Eun-Seok Jeonb, Min-Jeong Sona, Sun-Hee
a
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Wooa, Sang-Hun Junga*
College of Pharmacy and Institute of Drug Research and Development, Chungnam National
University, Daejeon 34134, Korea
Division of Cardiology, Samsung Medical Center, Samsung Biomedical Research Institute, School
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b
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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 N,N-dimethyl-4-(2-(3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide
91.6%,
FS
=
TE D
them
17.62%;
EF
=
11.55%),
phenylpropyl)ureido)ethyl)benzene sulfonamide
(13,
CMA=
N,N-dimethyl-4-(2-(1-methyl-3-(3-
(40, CMA = 52.3%, FS = 38.96%; EF =
EP
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
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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. Key words: Phenylpropylurea; Cardiac myosin activator; inotrope; Systolic heart failure
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*Corresponding author: [email protected]. Tel.; +82 42 821 5939; Fax +82 42 823 6566; Present address: College of Pharmacy and Institute of Drug Research and development,
AC C
EP
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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
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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
SC
aim at diverse targets including sodium retention, arterial and venous constriction, neuroendocrine activation increases heart rate, cardiac dyssynchrony and arrhythmias and often
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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
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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
EP
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].
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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
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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
SC
cAMP and calcium and decrease ejection time, OM increased myocardial contraction and stroke volume without increasing oxygen consumption, thereby increasing myocardial efficiency
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[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
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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
EP
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
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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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2. Chemistry
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Fig.1. Background and exploration of urea scaffold for novel cardiac myosin activator
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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
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corresponding urea derivatives 3-11, 14-21, 24-25. Urea 6,11 were prepared by the reaction of 4phenylbutylamine (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 H2 and Pd/C which was consequently dimethylated to 23 using methyliodide in presence of K2CO3 as base. In order to prepare the urea 26, 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
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yielded intermediate 52 which on reaction with 3-phenylpropylisocyanate (47) furnished the urea
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EP
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26.
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 CH3I, NaH, DMF, RT, 3 h, (d) H2/Pd/C, MeOH, RT, 10 h, (e) CH3I, K2CO3, acetone, reflux 2 h, (f) CH3COCl, TEA, CH2Cl2, 0 °C to RT, 3 h (g) (i) ClSO3H, CH2Cl2, 0 °C to RT, 3 h (ii) NH4OAc, THF, reflux, 5 h (h) 3N HCl, n-butanol, reflux, 8 h. (i) 47, acetonitrile, RT, 8 h. 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,
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34, the key intermediate 56 was reacted with appropriate amine (‘L’ group and ‘n’ are in Table 1) under triphosgene condition. The preparation of 56 was achieved by the transformation of 4fluorocinnamic acid (53) to its amide 54 by coupling with ammonium chloride. The amide 54
SC
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
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56 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 4methoxyphenylbutanoic acid 57a/4-chlorophenylbutanoic acid 57b were reacted with methyl
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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
EP
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. prepare
N,N-dimethylsulfonamide
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To
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.
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Reagents and conditions: (a) EDC.HCl, NH4Cl, TEA, CH2Cl2 , RT, 16 h, (b) (i) LiAlH4, THF, 70 °C 3 h (ii) Boc2O, 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) NaN3/H2O, RT, 1 h (iii) toluene, reflux, 3 h, (f) acetonitrile, RT, 8 h, (g) CH3COCl, TEA, CH2Cl2, 0°C to RT, 3 h
EP
(h) (i) ClSO3H, CH2Cl2, 0 °C to RT, 3 h (ii) dimethylamine.hydrochloride, NaHCO3, 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.
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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
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SC
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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 CH3I, NaH, DMF, RT, 3 h, (c)(i) CH3COCl, TEA, CH2Cl2, 0°C to RT, 3 h (ii) ClSO3H, CH2Cl2, 0 °C to RT, 3 h (iii) dimethylamine.hydrochloride, NaHCO3, THF, RT, 5 h (iv) 3N HCl, n-butanol, reflux, 8
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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
EP
methyl iodide in presence of NaH afforded 73. Deprotection of the acetyl group of 73 furnished 74. Finally, the amine 74 was reacted with 3-phenylpropylisocyanate 47 to give the urea 40. In
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order to prepare the urea 41, 3-phenylpropylamine 59 was converted to its N-methyl derivative 75 analogous to the preparation of 74. Further 75 was reacted with 65 to give the urea 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 K2CO3 to furnish 77a/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
AC C
EP
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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. Reagents and conditions: (a) CH3COCl, TEA, CH2Cl2, 0 °C to RT, 3 h (b) CH3I, 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 CH3I, NaH, DMF, RT, 3 h, (g) CH3I or CH3CH2I, NaH, DMF, RT 3-15 h (h) CH3CH2I, or (CH3)2CHI, K2CO3, DMF, RT, 12 h.
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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 %
SC
increase in myosin ATPase activity at 10 µM. The results are shown in Table 1 - 3. Compound
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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
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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
EP
echocardiography [35] in seven-week-old Sprague-Dawley male rats. The results are shown in
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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. 4. Results and Discussion
Based on our previous report [25], compound 1 (CMA at 10 µM = 53.3% FS = 30.04%; EF = 18.27%) 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
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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.
L
R
n
1
Phenyl
Phenyl
1
2
Phenyl
Phenyl
2
3
Phenyl
0
4
Phenyl
1
5
Phenyl
2
6
Phenyl
m
ATPase activity % at 10 µM
Fractional Shoretning % increase
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Comp. No
SC
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Table 1. Optimization of spacer and groups in the urea scaffold
Ejection Fraction % increase
CLogP
53.3
30.04
18.27
3.696
1
51.1
18.90
12.15
4.025
1
31.8
---
---
2.118
1
39.6
---
---
1.859
1
41.9
11.24
7.23
2.188
2
2
36.6
---
---
4.212
Phenyl
1
1
25.4
---
---
3.615
Phenyl
2
1
40.0
---
---
3.944
Phenyl
1
1
38.8
---
---
3.354
Phenyl
2
1
122.4
23.97
15.87
3.683
11
Phenyl
2
2
32.5
---
---
2.717
12
Phenyl
1
1
34.5
---
---
2.891
9 10
EP
8
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7
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1
Phenyl
2
1
91.6
17.62
11.55
3.220
14
Phenyl
2
1
95.1
20.47
13.58
3.944
15
Phenyl
2
1
35.0
---
---
3.944
16
Phenyl
2
1
31.0
---
---
4.738
17
Phenyl
2
1
37.3
---
---
5.451
18
Phenyl
2
1
73.8
19
Phenyl
2
1
33.3
---
---
5.452
20
Phenyl
2
1
40.6
---
---
3.768
21
Phenyl
2
1
56.2
---
---
3.768
22
Phenyl
2
1
55.2
6.37
4.16
2.798
23
Phenyl
2
1
45.3
---
---
4.190
24
Phenyl
2
1
38.6
---
---
4.168
Phenyl
2
1
65.4
12.52
8.76
4.168
Phenyl
2
1
29.4
---
---
1
1
9.5
---
---
3.839
1
1
30.5
---
---
3.615
29
1
1
20.8
---
---
2.891
30
2
1
19.1
---
---
2.331
27 28
Precipitated during formulation
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26
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25
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13
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4.534
2.432
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31
2
1
34.8
---
---
32
2
1
37.9
---
---
2.901
33
2
1
38.4
---
---
3.602
34
2
1
20.6
---
---
3.363
13.35
3.266
Omecamtiv mecarbil
58.0
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20.32
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---, activity not checked
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STD
2.107
In the first set of experiment (Table 1), compounds 3 (n = 0; m = 1, CMA = 31.8%), 4 (n = 1; m = 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
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(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
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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.
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4-Methoxy substitution on phenyl ring (L) as shown in compound 7 (n = 1; m = 1, CMA = 25.4%) 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 3,4dimethoxy substitution analogs as considering the superior activity of 10 (n = 2; m = 1, CMA = 122.4%) 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
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and phenyl ring (L) was further substantiated from the compounds 12 and 13 with N,Ndimethylsulfonamide substitution on phenyl ring (L). Compound 12 (n = 1; m = 1, CMA = 34.5%) showed weak activity, whereas compound 13 (n = 2; m = 1, CMA = 91.6%, FS =
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17.62%; EF = 11.55%) showed strong activity in vitro and in vivo. From these results, two
fixed as optimum chain length.
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carbon chain from the urea to the phenyl ring (L) and three carbon chain to phenyl ring (R) were
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
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= 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 =
EP
31.0%) 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
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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
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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)
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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-
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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
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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
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the phenyl ring (R) of the compound 5 were introduced as shown in compounds 30 (CMA = 19.1%), 31 (CMA = 34.8%) and 32 (CMA = 37.9%), respectively. All the compounds showed
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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 33 (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.
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Table 2. Ventricular cell contractility and Selectivity study in myosins ATPase % activity Cardiac myosin
Skeletal myosin
Smooth myosin
(at 10 µM)
(at 100 µM)
(at 100 µM)
1
25.3 + 2.2 at 5 µM
53.3
1.4
˗ 3.7
2
20.8 + 1.9 at 5 µM
51.1
˗ 2.1
˗ 4.7
10
22.2 + 3.1 at 5 µM
122.4
˗ 2.4
4.3
13
47.9 + 3.2 at 5 µM
91.6
14
19.7 + 3.2 at 5 µM
95.1
OM
33.8 + 4.1 at 1 µM
(˗)Blebbistatin
----
SC
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Ventricular cell contractility [rat] % change
˗ 3.8
˗ 1.9
3.5
5.3
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Compound No.
58.0
2.8
˗ 5.5
----
˗ 50.7
˗ 35.7
The potent compounds 10, 13 and 14 were analyzed for selectivity in cardiac myosin over
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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.
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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
AC C
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 activity %
13
91.6
17.62
35
27.6
36
40.7
37
Ejection Fraction % increase
---
3.072
---
3.321
3.072
SC
3.220
---
---
---
32.6
---
---
85.1
CLogP
11.55
36.9
TE D
38
39
---
M AN U
Structure of the Compound
RI PT
at 10 uM
Fractional Shoretning % increase
Comp. No
precipitated during formulation
3.321
3.620
38.96
24.19
2.956
47.6
23.19
15.47
2.956
69.0
0.46
0.27
3.502
43
39.5
---
---
3.485
44
30.3
---
---
4.560
AC C
41
EP
52.3
40
42
ACCEPTED MANUSCRIPT
38.1
---
---
3.794
46
40.6
---
---
4.340
58.0
20.32
STD
Omecamtiv mecarbil
---, activity not checked
RI PT
45
13.35
3.266
The SAR of compound 13 with N,N-dimethylsulfonamide group on phenyl ring of (L) was
SC
further explored by studying the importance of the flexible chain and hydrogen bonding donor
M AN U
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 =
TE D
36.9%), 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.
activity.
EP
The above results clearly indicate the importance of flexible carbon chains as spacers for the
To substantiate the importance of the HBD property of the urea function in highly active 13,
AC C
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 = 24.19%) 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
69.0%, 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.
RI PT
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 =
SC
30.3%) 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
ATPase in vitro and in vivo.
M AN U
either side of the urea NH with methyl group is much suitable for activating the cardiac myosin
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
TE D
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
EP
+ 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
AC C
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.
SC
RI PT
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)
M AN U
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
TE D
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
AC C
EP
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 = 23.97%; EF = 15.87%), 4-N,N-dimethyl sulfonamide moiety (13, CMA = 91.6%, FS = 17.62%;
RI PT
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).
SC
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
M AN U
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
TE D
(CMA = 47.6%, FS = 23.19%; EF = 15.47%; % change in ventricular cell contractility at 5 µM = 63.5 + 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,
EP
41 were highly selective for cardiac myosin over skeletal and smooth myosins and thus proving them these new urea derivatives is a novel scaffold for discovery of cardiac myosin activators for
AC C
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
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
RI PT
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-
SC
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
M AN U
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.
TE D
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
EP
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.
AC C
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,Ndiisopropylethylamine (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
RI PT
compounds.
6.1.1.1. 4-(3-(3-Phenylpropyl)ureido)benzenesulfonamide (3); Prepared by general procedure (i). Yield 85%; White solid; Rf 0.42 (Ethylacetate); mp 207-209 °C; IR (neat): 3354, 3321, 3032,
SC
2947, 2358, 2342, 1681cm-1; 1H-NMR (DMSO-d6) δ 1.71-1.78 (m, 2H), 2.61 (t, J = 7.80 Hz, 2H), 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,
M AN U
2H), 7.67 (d, J = 8.40 Hz, 2H), 8.81 (s, 1H). 13C-NMR (DMSO-d6) δ 31.32, 32.40, 38.66, 116.82, 125.79, 126.76, 128.31, 128.36, 136.04, 141.72, 143.76, 154.92; HRMS Calcd for C16H19N3O3S m/z [M+H] 334.1225, found 334.1248. HPLC purity 100% 6.1.1.2. 4-((3-(3-Phenylpropyl)ureido)methyl)benzenesulfonamide (4); Prepared by general
TE D
procedure (i). Yield 82%; White solid; Rf 0.35 (Ethylacetate); mp 209 °C; IR (neat): 3355, 2923, 2357, 2341, 1623 cm-1; 1H-NMR (DMSO-d6) δ 1.66-1.70 (m, 2H), 2.57 (t, J = 7.80 Hz, 2H), 3.02-3.06 (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),
EP
7.13 -7.30 (m, 7H), 7.40 (d, J = 8.40 Hz, 2H), 7.76 (d, J = 8.40 Hz, 2H); HRMS Calcd for C17H21N3O3S m/z [M+H] 348.1382, found 349.1405.
AC C
6.1.1.3. 4-(2-(3-(3-Phenylpropyl)ureido)ethyl)benzenesulfonamide (5); Prepared by general procedure (i). Yield 84%; White solid; Rf 0.37 (Ethylacetate); mp 146-148 °C; IR (neat): 3327, 2923, 2855, 2358, 2341, 1697 cm-1; 1H-NMR (DMSO-d6) δ 1.62-1.69 (m, 2H), 2.56 (t, J = 7.20 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), 5.90-5.92 (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 = 8.40 Hz, 2H); 13C-NMR (DMSO-d6) δ 31.74, 32.44, 35.83, 38.79, 40.49, 125.72, 128.32,
ACCEPTED MANUSCRIPT
129.16, 141.91, 142.04, 144.15, 158.07; HRMS Calcd for C18H23N3O3S m/z [M+H] 362.1538, found 362.1567. HPLC purity 100% 6.1.1.4. 4-(2-(3-(4-Phenylbutyl)ureido)ethyl)benzenesulfonamide (6); Prepared by general
RI PT
procedure (ii). Yield 68%; White solid; Rf 0.38 (Ethylacetate); mp: 134-137 °C; IR (neat): 3254, 2946, 1698, 1557, 1164, 829, 667 cm-1; 1H-NMR (DMSO-d6) δ 1.31-1.38 (m, 2H), 1.49-1.56 (m, 2H), 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
SC
(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, 2H), 7.36-7.38 (d, J = 8.40 Hz, 2H), 7.72-7.74 (d, J = 8.40 Hz, 2H);
13
C-NMR (DMSO-d6) δ
M AN U
28.88, 30.16, 35.33, 36.38, 39.45, 41.04, 126.09, 126.13, 128.69, 128.74, 129.57, 142.41, 142.69, 144.54, 158.42; HRMS Calcd for C19H25N3O3S m/z [M+H] 376.1695, found 376.1723. HPLC purity 99.25%
6.1.1.5. 1-(4-Methoxybenzyl)-3-(3-phenylpropyl)urea (7); Prepared by general procedure (i).
TE D
Yield 88%; White solid; Rf 0.42 (2 : 1 Ethylacetate : Hexane); mp 114-116 °C; IR (neat): 3327, 2923, 2357, 2341, 1618 cm-1; 1H-NMR (DMSO-d6) δ 1.63-1.71 (m, 2H), 2.56 (t, J = 7.80 Hz, 2H), 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,
EP
1H), 6.87 (d, J = 7.40 Hz, 2H), 7.16-7.20 (m, 5H), 7.26-7.30 (m, 2H); HRMS Calcd for C18H22N2O2 m/z [M+H] 299.1760, found 299.1784.
AC C
6.1.1.6. 1-(4-Methoxyphenethyl)-3-(3-phenylpropyl)urea (8); Prepared by general procedure (i). Yield 86%; White solid; Rf 0.44 (2 : 1 Ethylacetate : Hexane); mp: 93-96 °C; IR (neat): 3329, 3030, 2932, 1615, 1560, 1511, 1243, 1040, 748, 696 cm-1; 1H-NMR (CDCl3) δ 1.77-1.81 (m, 2H), 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), 3.78 (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), 7.157.20 (m, 3H), 7.27-7.30 (m, 2H). 13C-NMR (CDCl3) δ 31.72, 33.06, 35.35, 39.94, 41.74, 55.16,
ACCEPTED MANUSCRIPT
113.92, 125.94, 128.37, 128.45, 129.76, 131.13, 141.60, 158.16, 158.25; HRMS Calcd for C19H24N2O2 m/z [M+H] 313.1916, found 313.1942. HPLC purity 98.46% 6.1.1.7. 1-(3,4-Dimethoxybenzyl)-3-(3-phenylpropyl)urea (9); Prepared by general procedure (i).
RI PT
Yield 82%; Pale yellow solid; Rf 0.40 (2 : 1 Ethylacetate : Hexane); mp: 110-112 °C; IR (neat): 3340, 1621, 1568, 1515, 1337, 1262, 1233, 1141, 1026 cm-1; 1H-NMR (CDCl3) δ 1.77-1.84 (m, 2H), 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
SC
(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 (CDCl3) δ 31.77, 33.06, 40.01, 44.31, 55.80, 55.87, 110.75, 111.11, 119.55, 125.89, 128.28,
M AN U
128.37, 131.77, 141.49, 148.23, 149.06, 158.35; HRMS Calcd for C19H24N2O3 m/z [M+H] 329.1865, found 329.1891. HPLC purity 100% 6.1.1.8.
1-(3,4-Dimethoxyphenethyl)-3-(3-phenylpropyl)urea
(10);
Prepared
by
general
procedure (i). Yield 90%; White solid; Rf 0.42 (2 : 1 Ethylacetate : Hexane); mp 102-104 °C; IR
TE D
(neat): 3338, 2973, 2860, 1667, 1618, 1572, 1508 cm-1; 1H-NMR (CDCl3) δ 1.76-1.83 (m, 2H), 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).
EP
13C-NMR (CDCl3) δ 31.71, 33.07, 35.87, 40.01, 41.67, 55.77, 55.83, 111.29, 111.96, 120.68, 125.97, 128.36, 128.45, 131.73, 141.58, 147.62, 149.01, 158.24; HRMS Calcd for C20H26N2O3
AC C
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; Rf 0.45 (2 : 1 Ethylacetate : Hexane); mp: 109-112 °C; IR (neat): 3244, 2944, 1683, 1558, 1540, 1508, 1235, 1030, 802, 701, 668 cm-1; 1H-NMR (DMSO-d6) δ 1.46-1.53 (m, 2H), 1.59-1.65 (m, 2H), 2.62 (t, J = 7.80 Hz, 2H), 2.75 (t, J = 7.80 Hz, 2H), 3.113.16 (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
(m, 2H), 6.79 (d, J = 8.00 Hz, 1H), 7.15-7.20 (m, 3H), 7.25-7.29 (m, 2H); 13C-NMR (CDCl3) δ 28.54, 29.69, 35.41, 35.86, 40.24, 41.65, 55.72, 55.80, 111.18, 111.85, 120.65, 125.82, 128.35, 128.40, 131.70, 142.18, 147.55, 148.94, 158.20; HRMS Calcd for C21H28N2O3 m/z [M+H]
RI PT
357.2178, found 357.2200.
6.1.1.10. 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
SC
at ambient temperature for 45 minutes. Then the reaction mixture was cooled and CH3I (2.20 mmol) was added The reaction mixture was further stirred at ambient temperature for 3 h. After
M AN U
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
TE D
cm-1; 1H-NMR (CDCl3) δ 1.78-1.86 (m, 2H), 2.62-2.68 (m, 2H), 2.65 (s, 6H), 3.19-3.24 (m, 2H), 4.40 (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), 7.35 (d, J = 8.20 Hz, 2H), 7.59 (d, J = 8.20 Hz, 2H). 13C-NMR (CDCl3) δ 31.78, 33.01, 37.80,
EP
39.91, 43.33, 125.96, 127.57, 127.80, 128.38, 128.45, 133.52, 141.59, 145.55, 158.43; HRMS Calcd for C19H25N3O3S m/z [M+H] 376.1695, found 376.1717. HPLC purity 99.61%
AC C
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; Rf 0.38 (Ethylacetate); mp 98-100 °C IR (neat): 3335, 3015, 2983, 1652, 1620, 1549, 1488 cm-1; 1H-NMR (CDCl3) 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 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 (CDCl3) δ 31.74, 33.07, 36.27, 37.84, 39.92, 41.04, 125.99, 127.96,
ACCEPTED MANUSCRIPT
128.39, 128.47, 129.58, 133.34, 141.61, 145.08, 158.16; HRMS Calcd for C20H27N3O3S 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
RI PT
(i). Yield 90%; Yellow powder; Rf 0.44 (2 : 1 Ethylacetate : Hexane); mp 91-93 °C; IR (neat): 3341, 2977, 2856, 1665, 1621, 1562, 1504 cm-1; 1H-NMR (CDCl3) δ 1.78-1.85 (m, 2H), 2.64 (t, 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),
SC
4.37 (bs, 1H), 4.44 (bs, 1H), 6.85-6.91 (m, 2H), 7.11-7.30 (m, 7H). 13C-NMR (CDCl3) δ 30.94, 31.71, 33.08, 40.03, 40.63, 55.25, 110.39, 120.70, 125.95, 127.45, 127.84, 128.40, 128.45,
313.1945. HPLC purity 99.21%
M AN U
130.67, 141.64, 157.54, 158.35; HRMS Calcd for C19H24N2O2 m/z [M+H] 313.1916, found
6.1.1.13. 1-(3-Methoxyphenethyl)-3-(3-phenylpropyl)urea (15); Prepared by general procedure (i). Yield 83%; White solid; Rf 0.44 (2 : 1 Ethylacetate : Hexane); mp: 66-69 °C; IR (neat): 3318,
TE D
3005, 2938, 1614, 1570, 1541, 1457, 1258, 1169, 1043, 789, 695 cm-1; 1H-NMR (DMSO-d6) δ 1.59-1.69 (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, 2H), 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),
EP
7.14-7.22 (m, 4H), 7.25-7.30 (m, 2H); 13C-NMR (DMSO-d6) δ 32.33, 32.98, 36.68, 39.30, 41.26, 55.31, 111.91, 114.74, 121.36, 126.13, 128.72, 129.73, 141.84, 142.31, 158.46, 159.74; HRMS
AC C
Calcd for C19H24N2O2 m/z [M+H] 313.1916, found 313.1940. 6.1.1.14. 1-(4-Chlorophenethyl)-3-(3-phenylpropyl)urea (16); Prepared by general procedure (i). Yield 74%; White solid; Rf 0.35 (1 : 1 Ethylacetate : Hexane); mp 120-122 °C; IR (neat): 3335, 2948, 2883, 1672, 1618, 1555, 1510 cm-1; 1H-NMR (CDCl3) δ 1.77-1.85 (m, 2H), 2.64 (t, J = 8.00 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), 7.11-7.30 (m, 9H). 13C-NMR (CDCl3) δ 31.68, 33.07, 35.69, 39.99, 41.37, 126.01, 128.37,
ACCEPTED MANUSCRIPT
128.48, 128.68, 130.19, 132.20, 137.71, 141.54, 158.17; HRMS Calcd for C18H21ClN2O m/z [M+H] 317.1421, found 317.1441. 6.1.1.15. 1-(2,4-Dichlorophenethyl)-3-(3-phenylpropyl)urea (17); Prepared by general procedure
RI PT
(i). Yield 80%; Yellow powder; Rf 0.38 (1 : 1 Ethylacetate : Hexane); mp: 102-104 °C; IR (neat): 3310, 2950, 2801, 1662, 1615, 1570, 1501 cm-1; 1H-NMR (CDCl3) δ 1.77-1.84 (m, 2H), 2.64 (t, J = 7.80 Hz, 2H), 2.90 (t, J = 7.20 Hz, 2H), 3.14-3.19 (m, 2H), 3.36-3.41 (m, 2H), 4.32-4.38 (m, 13
C-NMR (CDCl3) δ 31.68, 33.10,
SC
2H), 7.13-7.21 (m, 5H), 7.26-7.30 (m, 2H), 7.37 (s, 1H);
33.60, 39.89, 40.10, 126.04, 127.25, 128.40, 128.51, 129.37, 131.92, 132.96, 134.79, 135.49,
M AN U
141.54, 158.01; HRMS Calcd for C18H20Cl2N2O m/z [M+H] 351.1031, found 351.1049. 6.1.1.16. 1-(4-Methylphenethyl)-3-(3-phenylpropyl)urea (18); Prepared by general procedure (i). Yield 85%; White solid; Rf 0.36 (1 : 1 Ethylacetate : Hexane); mp 101-103 °C; IR (neat): 3319, 2937, 2861, 1633, 1615, 1569, 1494 cm-1; 1H-NMR (CDCl3) δ 1.75-1.82 (m, 2H), 2.31 (s, 3H),
TE D
2.63 (t, J = 7.20 Hz, 2H), 2.75 (t, J = 7.80 Hz, 2H), 3.12-3.17 (m, 2H), 3.36-3.40 (m, 2H), 4.274.28 (m, 2H), 7.06-7.20 (m, 7H), 7.26-7.29 (m, 2H). 13C-NMR (CDCl3) δ 20.90, 31.70, 33.10, 35.83, 40.03, 41.66, 125.96, 128.39, 128.46, 128.73, 129.30, 135.97, 136.07, 141.63, 158.22;
EP
HRMS Calcd for C19H24N2O m/z [M+H] 297.1967, found 297.1995. HPLC purity 99.08% 6.1.1.17. 1-(4-Isopropylphenethyl)-3-(3-phenylpropyl)urea (19); Prepared by general procedure
AC C
(i). Yield 82%; White solid; Rf 0.40 (1 : 1 Ethylacetate : Hexane); mp: 89-91 °C; IR (neat): 3338, 2973, 2860, 1667, 1618, 1572, 1508 cm-1; 1H-NMR (CDCl3) δ 1.24 (d, J = 7.20 Hz, 6H), 1.761.84 (m, 2H), 2.64 (t, J = 7.60 Hz, 2H), 2.76 (t, J = 7.80 Hz, 2H), 2.85-2.91 (m, 1H), 3.14-3.19 (m, 2H), 3.37-3.42 (m, 2H), 4.18-4.24 (m, 2H), 7.10-7.12 (m, 2H), 7.16-7.20 (m, 5H), 7.26-7.30 (m, 2H);
13
C-NMR (CDCl3) δ 23.94, 31.70, 33.13, 33.64, 35.82, 40.10, 41.64, 125.99, 126.70,
ACCEPTED MANUSCRIPT
128.40, 128.49, 128.79, 136.43, 141.63, 147.09, 158.08; HRMS Calcd for C21H28N2O m/z [M+H] 325.2280, found 325.2299. 6.1.1.18. 4-(2-(3-(3-Phenylpropyl)ureido)ethyl)benzoic acid
(20); Prepared by general
3580, 3115, 2940, 1670, 1620, 1524, 1463, 1270 cm-1;
1
RI PT
procedure (i). Yield 75%; Yellow powder; Rf 0.25 (Ethylacetate); mp: 142-145 °C; IR (neat): H-NMR (DMSO-d6) δ 1.61-1.66 (m,
2H), 2.54 (m, 2H), 2.74-2.78 (m, 2H), 2.91-3.01 (m, 2H), 3.22-3.29 (m, 2H), 5.78-5.82 (m, 1H),
SC
5.90-5.94 (m, 1H), 7.16-7.21 (m, 3H), 7.26-7.34 (m, 4H), 7.86-7.89 (m, 2H) 12.80 (bs, 1H); 13CNMR (DMSO-d6) δ 31.74, 32.43, 36.07, 38.78, 40.43, 125.71, 128.31, 128.75, 128.92, 129.39,
M AN U
141.92, 145.25, 158.07, 167.39; HRMS Calcd for C19H22N2O3 m/z [M+H] 327.1709, found 327.1737.
6.1.1.19. 1-(4-Nitrophenethyl)-3-(3-phenylpropyl)urea (21); Prepared by general procedure (i). Yield 84%; White solid; Rf 0.45 (2 : 1 Ethylacetate : Hexane); mp 101-103 °C; IR (neat): 3298,
TE D
3003, 2986, 1662, 1652, 1616, 1533, 1497 cm-1; 1H-NMR (CDCl3) δ 1.77-1.85 (m, 2H), 2.64 (t, J = 7.80 Hz, 2H), 2.91 (t, J = 8.00 Hz, 2H), 3.14-3.19 (m, 2H), 3.43-3.48 (m, 2H), 4.07 (bs, 2H), 7.17-7.36 (m, 7H), 8.12-8.15 (m, 2H); 13C-NMR (CDCl3) δ 31.70, 33.06, 36.39, 39.97, 40.98,
EP
123.71, 126.04, 128.35, 128.50, 129.75, 141.49, 146.62, 147.45, 158.10; HRMS Calcd for C18H21N3O3 m/z [M+H] 328.1661, found 328.1679. HPLC purity 100%
AC C
6.1.1.20. 1-(4-Aminophenethyl)-3-(3-phenylpropyl)urea (22); To a solution of compound 21 (0.65 mmol) in methanol, Pd/C (10%, 190 mg) was added under N2 atmosphere. The reaction mixture was hydrogenated using parr shaker for 10 h. After the formation of the reaction, the reaction mixture was filtered through celite and concentrated under vacuum. The product was purified by column chromatography. Yield 86%; Yellow powder; Rf 0.25 (2 : 1 Ethylacetate : Hexane); mp 93-95 °C; IR (neat): 3405, 3325, 2940, 2858, 1669, 1620, 1567 cm-1; 1H-NMR
ACCEPTED MANUSCRIPT
(CDCl3) δ 1.75-1.83 (m, 2H), 2.63 (t, J = 7.80 Hz, 2H), 2.68 (t, J = 8.00 Hz, 2H), 3.13-3.18 (m, 2H), 3.32-3.37 (m, 2H), 3.58 (bs, 2H), 4.17-4.19 (m, 2H), 6.63 (d, J = 8.00 Hz, 2H), 6.97 (d, J = 8.00 Hz, 2H), 7.16-7.30 (m, 5H); 13C-NMR (CDCl3) δ 31.70, 33.08, 35.33, 40.00, 41.80,
RI PT
115.36, 125.94, 128.39, 128.45, 128.99, 129.65, 141.68, 144.84, 158.29; HRMS Calcd for C18H23N3O m/z [M+H] 298.1919, found 298.1942. HPLC purity 99.84% 6.1.1.21. 1-(4-(Dimethylamino)phenethyl)-3-(3-phenylpropyl)urea
(23); Compound 22 (0.5
SC
mmol) was dissolved in acetone and K2CO3 (1.2 mmol) was added followed by the addition of CH3I (1.2 mmol). The reaction mixture was refluxed for 2 h. After the completion of the
M AN U
reaction, the reaction mixture was concentrated, water was added and extracted using ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The obtained crude residue was purified by column chromatography. Yield 72%; White solid; Rf 0.42 (2 : 1 Ethylacetate : Hexane); mp 90-92 °C; IR (neat): 3315, 2975, 2886, 1650,
TE D
1624, 1614, 1540, 1496 cm-1; 1H-NMR (CDCl3) δ 1.77-1.80 (m, 2H), 2.62 (t, J = 8.00 Hz, 2H), 2.69 (t, J = 8.00 Hz, 2H), 2.91 (s, 6H), 3.13-3.16 (m, 2H), 3.33-3.36 (m, 2H), 4.21-4.25 (m, 2H), 6.69 (d, J = 8.00 Hz, 2H), 7.06 (d, J = 8.00 Hz, 2H), 7.16-7.27 (m, 5H). 13C-NMR (CDCl3) δ
EP
32.02, 33.43, 35.48, 40.36, 41.03, 42.21, 113.34, 126.26, 128.72, 128.77, 129.79, 129.91, 141.98, 149.75, 158.57; HRMS Calcd for C20H27N3O m/z [M+H] 326.2232, found 326.2255.
AC C
6.1.1.22. 1-(4-Fluorophenethyl)-3-(3-phenylpropyl)urea (24); Prepared by general procedure (i). Yield 82%; White solid; Rf 0.32 (1 : 1 Ethylacetate : Hexane); mp: 83-86 °C; IR (neat): 3352, 3307, 3023, 2969, 1739, 1618, 1560, 1365, 1217, 696 cm-1; 1H-NMR (CDCl3) δ 1.76-1.83 (m, 2H), 2.63 (t, J = 7.80 Hz, 2H), 2.76 (t, J = 8.00 Hz, 2H), 3.13-3.18 (m, 2H), 3.35-3.40 (m, 2H), 4.30-4.31 (m, 2H), 6.95-7.00 (m, 2H), 7.11-7.20 (m, 5H), 7.25-7.30 (m, 2H). 13C-NMR (CDCl3)
ACCEPTED MANUSCRIPT
δ 31.70, 33.05, 35.50, 35.91, 41.55, 115.21, 115.42, 125.98, 128.36, 128.46, 130.24, 134.82, 141.54, 158.24, 162.80; HRMS Calcd for C18H21FN2O m/z [M+H] 301.1716, found 301.1739. 6.1.1.23. 1-(2-Fluorophenethyl)-3-(3-phenylpropyl)urea (25); Prepared by general procedure (i).
RI PT
Yield 85%; White solid; Rf 0.32 (1 : 1 Ethylacetate : Hexane); mp 82-83 °C; IR (neat): 3328, 3011, 2991, 1648, 1618, 1543, 1498 cm-1; 1H-NMR (CDCl3) δ 1.77-1.84 (m, 2H), 2.64 (t, J = 8.00 Hz, 2H), 2.84 (t, J = 8.00 Hz, 2H), 3.14-3.19 (m, 2H), 3.38-3.43 (m, 2H), 4.29-4.34 (m,
SC
2H), 7.00-7.29 (m, 9H); 13C-NMR (CDCl3) δ 29.81, 31.69, 33.08, 40.00, 40.45, 115.20, 115.41, 124.18, 125.96, 128.40, 128.46, 131.23, 141.63, 158.25, 160.13, 162.57; HRMS Calcd for
M AN U
C18H21FN2O m/z [M+H] 301.1716, found 301.1746. HPLC purity 100%
6.1.1.24. 4-Methoxy-2-(2-(3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (26): 6.1.1.24.1. Preparation of N-(3-methoxyphenethyl)acetamide (50): To a cooled solution of 2-(3 methoxyphenyl)ethanamine (49, 6.20 mmol) in methylene chloride at 0 °C triethylamine (9.30
TE D
mmol) was added. After 5 minutes, acetylchloride (9.30 mmol) was added at the same temperature and allowed to stir for 3 h at ambient temperature. The reaction was monitored by TLC. After the completion of the reaction, the reaction mixture was quenched with water and
EP
extracted using methylene chloride. The organic layer was washed with water twice, dried over anhydrous sodium sulfate and concentrated under vacuum to get the crude product 50. Without
AC C
purification the product was taken for next step. Yield 80%; 1H-NMR (CDCl3) δ 1.94 (s, 3H), 2.79 (t, J = 7.40 Hz, 2H), 3.49-3.54 (m, 2H), 3.80 (s, 3H), 6.73-6.84 (m, 3H), 7.24 (t, J = 7.60 Hz, 1H).
6.1.1.24.2. Preparation of N-(5-methoxy-2-sulfamoylphenethyl)acetamide (51): To a cooled solution of chlorosulfuric acid at 0 °C, 50 (3.33 mmol) dissolved in methylene chloride was added slowly and allowed to stir for 3 h to attain ambient temperature. The reaction was
ACCEPTED MANUSCRIPT
monitored by TLC. After the completion of the reaction, the reaction mixture was slowly added to crushed ice with vigorous stirring. After all ice had melt down, the reaction mass was extracted using methylene chloride and concentrated to obtain the sulfonyl chloride. The crude
RI PT
sulfonyl chloride was dissolved in THF and NH4OAC (10.0 mmol) was added and refluxed for 5 h. After the completion of the reaction, water was added and the reaction mixture was extracted using ethyl acetate. The crude product 51 was taken for the next step. Yield 65%; 1H-NMR
SC
(DMSO-d6) δ 1.82 (s, 3H), 3.07-3.10 (m, 2H), 3.27-3.32 (m, 2H), 3.81 (s, 3H), 6.91-6.93 (m, 2H), 7.42 (s, 2H), 7.80 (d, J = 8.20 Hz, 1H), 8.13-8.15 (m, 1H).
M AN U
6.1.1.24.3. Preparation of 2-(2-aminoethyl)-4-methoxybenzenesulfonamide (52): To a solution of 51 (0.10 mmol) dissolved in n-butanol (2 mL), 3N HCl (100 mL) was added and stirred at reflux for 8 h. After the completion of the reaction, the reaction mixture was extracted using diethyl ether. The aqueous layer was neutralized using solid potassium carbonate and extracted using
TE D
ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated. The amine 52 was obtained as white solid. Yield 72%; 1H-NMR (DMSO-d6) δ 2.80-2.83 (m, 2H), 2.99-3.03 (m, 2H), 3.80 (s, 3H), 6.87-6.93 (m, 2H), 7.78 (d, J = 8.20 Hz, 1H).
EP
6.1.1.24.4. 4-Methoxy-2-(2-(3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (26): Prepared by the reaction of amine 52 and 3-phenylpropylisocyanate 47 according to the general
AC C
procedure (i). Yield 52%; White solid; Rf 0.35 (Ethylacetate); mp 182-184 °C; IR (neat): 3341, 1635, 1540, 1366, 1320, 1124, 1089, 940 cm-1; 1H-NMR (DMSO-d6) δ 1.63-1.70 (m, 2H), 2.552.59 (m, 2H), 2.99-3.07 (m, 4H), 3.18-3.22 (m, 2H), 3.80 (s, 3H), 6.09-6.12 (m, 2H), 6.89-6.91 (m, 2H), 7.17-7.21 (m, 3H), 7.26-7.30 (m, 2H), 7.50 (s, 2H, NH2-D2O exchangeable), 7.80 (d, J = 8.40 Hz, 1H). 13C-NMR (DMSO-d6) δ 31.20, 31.74, 32.47, 33.25, 40.78, 55.43, 111.13,
ACCEPTED MANUSCRIPT
117.11, 125.78, 125.79, 128.36, 129.66, 134.85, 139.47, 141.91, 158.70, 161.54; HRMS Calcd for C19H25N3O4S m/z [M+H] 392.1644, found 392.1669. 6.1.1.25. Preparation of 1-benzyl-3-(3-(4-fluorophenyl)propyl)urea (27)
RI PT
6.1.1.25.1. Preparation of (E)-3-(4-fluorophenyl)acrylamide (54): To a stirred solution of 4fluorocinnamic acid 53 (18.05 mmol) in methylene chloride at 0 °C, triethylamine (54.16 mmol), EDC.HCl (27.05 mmol) and NH4Cl (36.11 mmol) were added. The resulting reaction mixture
SC
was stirred at room temperature for 16 h. Completion of the reaction was confirmed by TLC. The reaction was diluted with methylene chloride and washed with 1N HCl. The organic layer was
M AN U
dried over anhydrous sodium sulfate and then concentrated under reduced pressure to afford 54 as brown sticky oil. The crude product was used for the next step without further purification. Yield 83%.
6.1.1.25.2. Preparation of tert-butyl-3-(4-fluorophenyl)propylcarbamate (55): To a stirred
TE D
solution of 54 (10.89 mmol) in THF (30 mL) at 0 °C, LiAlH4 (2.0 M solution in THF, 32.69 mmol) was added slowly. The resulting pale yellow suspension was heated at 70 oC for a further 3 h. After cooling to 0 °C, the reaction mixture was quenched with 1N NaOH solution followed
EP
by ice cold water (2 mL). To this reaction mixture, a solution of Boc2O (16.34 mmol) in THF was added and the resulting reaction mixture was stirred at ambient temperature for 16 h. The
AC C
reaction mixture was filtered through celite bed, washed with ethyl acetate (50 mL) and the filtrate was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The crude residue was purified by column chromatography to afford 55 as a light brown solid. Yield 40 %, 1H NMR (CDCl3) δ 1.45 (s, 9H), 1.79 (m, 2H), 2.62 (t, J = 7.68 Hz, 2H), 3.08-3.21 (m, 2H), 6.97 (d, J = 8.29 Hz, 2H), 7.13 (d, J = 7.93 Hz, 2H).
ACCEPTED MANUSCRIPT
6.1.1.25.3. Preparation of 3-(4-fluorophenyl)propan-1-amine hydrochloride (56): To a stirred solution of 55 (3.95 mmol) in methylene chloride HCl (4.0 M solution of in 1, 4-dioxane, 7.91 mmol) was slowly added. The resulting reaction mixture was stirred at ambient temperature for 2
RI PT
h. After the completion of the reaction, the solvent was evaporated from the reaction mixture to afford 56 as a light brown solid. Yield 80%. 1H NMR (DMSO-d6) δ 1.84 (m, 2H), 2.64 (t, J = 7.68 Hz, 2H), 2.71-2.80 (m, 2H), 7.10-7.16 (m, 2H), 7.24-7.29 (m, 2H), 8.00 (bs, 2H).
SC
6.1.1.25.4. Preparation of 1-benzyl-3-(3-(4-fluorophenyl)propyl)urea (27); Prepared from the reaction of 56 with amine (‘L’ and ‘n’ are mentioned in Table 1) according to the general
M AN U
procedure (ii). Yield 75%; White solid; Rf 0.30 (1 : 1 Ethylacetate : Hexane); mp: 100-103 °C; IR (neat): 3324, 3035, 1615, 1509, 1454, 1260, 1223, 1103, 1028, 816, 732 cm-1;
1
H NMR
(DMSO-d6) δ 1.60-1.71 (m, 2H), 2.52-2.59 (m, 2H), 3.01 (m, 2H), 4.20 (d, J = 5.85 Hz, 2H), 5.98 (bs, 1H), 6.28 (bs, 1H), 7.05-7.12 (m, 2H), 7.18-7.26 (m, 5H), 7.28-7.34 (m, 2H); 13C NMR
TE D
(DMSO-d6) δ 32.17, 32.49, 39.42, 43.51, 115.50, 115.70, 127.22, 127.70, 128.90, 130.61, 130.72, 138.63, 141.75, 158.82; HRMS Calcd for C17H19FN2O m/z [M+H] 287.1560, found 287.1588.
EP
6.1.1.26. Preparation of 1-Benzyl-3-(3-(4-methoxyphenyl)propyl)urea (28): 6.1.1.26.1. Preparation of 1-(3-isocyanatopropyl)-4-substituted benzene (58): To a solution of 4-
AC C
(4-substituted phenyl)butanoic acid 57 (15.5 mmol) in acetone (60 ml) at ambient temperature, triethylamine (18.6 mmol) was added slowly. Then the reaction mixture was cooled to -10 °C, and methyl chloroformate (18.6 mmol) in acetone (10 mL) was added within 20-30 minutes and the reaction was stirred for another 30 minutes. Then a solution of sodium azide (31.1 mmol) in water (6 mL) was added slowly and stirred for another 1 h. The reaction mixture was quenched with ice water (300 mL) and stirred for 5-10 minutes. To this aqueous solution toluene (250 mL)
ACCEPTED MANUSCRIPT
was added and stirred for another 20-30 minutes. The toluene layer was collected and aqueous layer was once again extracted with toluene (100 mL). The combined toluene layer was dried over anhydrous Na2SO4 and concentrated to about 100 mL and then refluxed for 3 h. After the
RI PT
formation of the product, the solvent was evaporated and the crude product 58a was used as such for the next step. Yield 65%.
6.1.1.26.2. Preparation of 1-Benzyl-3-(3-(4-methoxyphenyl)propyl)urea (28), Prepared by the
SC
reaction of 1-(3-isocyanatopropyl)-4-methoxybenzene (58a) and amine (‘L’ and ‘n’ are mentioned in Table 1) according to the general procedure (i). Yield 84%; White solid; Rf 0.40 (2
M AN U
: 1 Ethylacetate : Hexane); mp: 100-103 °C; IR (neat): 3274, 2941, 1698, 1557, 1538, 1253, 1033, 801, 696, 668 cm-1; 1H-NMR (DMSO-d6) δ 1.60-1.67 (m, 2H), 2.47 (t, J = 7.80 Hz 2H), 2.98-3.03 (m, 2H), 3.72 (s, 3H), 4.20 (d, J = 6.00 Hz 2H), 5.97-5.99 (m, 1H), 6.28-6.31 (m, 1H), 6.83-6.85 (d, J = 8.00 Hz 2H), 7.09-7.11 (d, J = 7.40 Hz 2H), 7.20-7.25 (m, 3H), 7.31-7.33 (m,
TE D
2H); 13C-NMR (DMSO-d6) δ 31.57, 32.07, 42.91, 54.96, 55.02, 113.81, 126.66, 127.12, 128.34, 129.32, 133.78, 141.18, 157.52, 158.25; HRMS Calcd for C18H22N2O2 m/z [M+H] 299.1760, found 299.1779.
EP
6.1.1.27. 4-(3-(3-Benzylureido)propyl)-N,N-dimethylbenzenesulfonamide (29): 6.1.1.27.1. Preparation of N-(3-phenylpropyl)acetamide (60): To a cooled solution of 3-
AC C
phenylpropylamine 59 (6.20 mmol) in methylene chloride at 0 °C, triethylamine (9.30 mmol) was added. After 5 minutes, acetylchloride (9.30 mmol) was added at the same temperature and allowed to stir for 3 h at ambient temperature. The reaction was monitored by TLC. After the completion of the reaction, the reaction mixture was quenched with water and extracted using methylene chloride. The organic layer was washed with water twice, dried over anhydrous sodium sulfate and concentrated to get the crude product. Without purification the product 60
ACCEPTED MANUSCRIPT
was taken for next step. Yield 80%; 1H-NMR (CDCl3) δ 1.81-1.87 (m, 2H), 1.95 (s, 3H), 2.66 (t, J = 7.20 Hz, 2H), 3.27-3.32 (m, 2H), 5.51 (bs, 1H), 7.18-7.21 (m, 3H), 7.28-7.31 (m, 2H). 6.1.1.27.2. Preparation of N-(3-(4-(N,N-dimethylsulfamoyl)phenyl)propyl)acetamide (61): To a
RI PT
cooled solution of chlorosulfuric acid (2 mL) at 0 °C, 60 (3.33 mmol) dissolved in methylene chloride (2 mL) was added slowly and allowed to stir for 3 h to attain ambient temperature. The reaction was monitored by TLC. After the completion of the reaction, the reaction mixture was
SC
slowly added to crushed ice with vigorous stirring. After all ice has melt down, the reaction mass was extracted using methylene chloride and concentrated to get sulfonyl chloride. In the next
M AN U
step, NaHCO3 (1.99 mmol) was added to a solution of N,N-dimethylamine hydrochloride (2.66 mmol) in THF (10 mL) at 0 °C to generate free amine. To this reaction mixture, the obtained sulfonyl chloride (1.33 mmol) dissolved in THF was added and stirred for 5 h at ambient temperature. After the completion of the reaction, water was added and the reaction mixture was
TE D
extracted using ethyl acetate. The product was purified by column chromatography. Yield 65%; 1H-NMR (CDCl3) δ 1.83-1.89 (m, 2H), 1.98 (s, 3H), 2.70 (s, 6H), 2.73-2.81 (m, 2H), 3.28-3.33 (m, 2H), 5.64 (bs, 1H), 7.35 (d, J = 8.20 Hz, 2H), 7.69 (d, J = 8.20 Hz, 2H).
EP
6.1.1.27.3. Preparation 4-(3-aminopropyl)-N,N-dimethylbenzenesulfonamide (62): To a solution of 61 (0.10 mmol) dissolved in n-butanol (2 mL), 3N HCl (100 mL) was added and stirred at
AC C
reflux for 8 h. After the completion of the reaction, the reaction mixture was extracted using diethyl ether. The aqueous layer was neutralized using solid potassium carbonate and extracted using ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The amine 62 was obtained as white solid which was used for the next step. Yield 75%; 1H-NMR (CDCl3) δ 1.80-1.84 (m, 2H), 2.70 (s, 6H), 2.74-2.81 (m, 4H), 5.64 (bs, 1H), 7.36 (d, J = 8.20 Hz, 2H), 7.69 (d, J = 8.20 Hz, 2H).
ACCEPTED MANUSCRIPT
6.1.1.27.4. 4-(3-(3-Benzylureido)propyl)-N,N-dimethylbenzenesulfonamide (29): Prepared by the reaction of 62 and benzylamine 63 according to the general procedure (ii). Yield 65%; White solid; Rf 0.36 (Ethylacetate); m.p; 105-107 °C; IR (neat): 3332, 1625, 1538, 1334, 1319, 1160,
RI PT
1088, 951 cm-1; 1H-NMR (CDCl3) δ 1.81-1.88 (m, 2H), 2.64-2.67 (m, 2H), 2.67 (s, 6H), 3.223.25 (m, 2H), 4.43 (s, 2H), 4.95-5.13 (bs, 2H), 7.17-7.21 (m, 3H), 7.27-7.30 (m, 2H), 7.39 (d, J = 8.20 Hz, 2H), 7.63 (d, J = 8.20 Hz, 2H);13C-NMR (CDCl3) δ 31.38, 32.82, 37.88, 39.84, 44.48,
SC
127.44, 127.95, 128.72, 129.02, 132.91, 139.08, 147.18, 158.31; HRMS Calcd for C19H25N3O3S m/z [M+H] 376.1695, found 376.1714.
M AN U
6.1.1.28. 4-(2-(3-(3-(4-Fluorophenyl)propyl)ureido)ethyl)benzenesulfonamide (30); Prepared from the reaction of 56 with amine (‘L’ and ‘n’ are mentioned in Table 1) according to the general procedure (ii) Yield 50%; White solid; Rf 0.38 (Ethylacetate); mp: 123-126 °C; IR (neat): 3307, 3182, 3071, 2922, 2869, 1623, 1560, 1507, 1326, 1257, 1208, 1152, 1098, 912, 819
TE D
cm-1; 1H NMR (DMSO-d6) δ 1.58-1.69 (m, 2H), 2.52-2.57 (m, 2H), 2.75 (t, J = 7.40 Hz, 2H), 2.97 (m, 2H), 3.25 (m, 2H), 5.82 (bs, 1H), 5.92 (bs, 1H), 7.05-7.13 (m, 2H), 7.19-7.26 (m, 2H), 7.28 (s, 2H), 7.38 (d, J = 8.40 Hz, 2H), 7.74 (d, J = 8.40 Hz, 2H); 13C NMR (DMSO-d6) δ 31.33,
EP
31.63, 35.64, 38.46, 40.30, 114.66, 114.86, 125.53, 128.98, 129.87, 137.79, 137.82, 141.84, 143.96, 157.87, 161.67; HRMS Calcd for C18H22FN3O3S m/z [M+H] 380.1445, found 380.1466.
AC C
6.1.1.29. 4-(2-(3-(3-(4-Methoxyphenyl)propyl)ureido)ethyl)benzenesulfonamide (31); Prepared from the reaction of 58a with amine (‘L’ and ‘n’ are mentioned in Table 1) according to the general procedure (i). Yield 81.7%; White powder; Rf 0.40 (Ethylacetate); mp: 128-131 °C; IR (neat): 3334, 3312, 3045, 2938, 2879, 2835, 1633, 1573, 1514, 1335, 1265,1157, 1030, 898, 809, 731, 690 cm-1; 1H-NMR (DMSO-d6) δ 1.59-1.62 (m, 2H), 2.53-2.58 (m, 2H), 2.74 (t, J = 7.80 Hz 2H), 2.94-2.99 (m, 2H), 3.22-3.27 (m, 2H), 3.71 (s, 3H), 5.80-5.83 (m, 1H), 5.89-5.92 (m, 1H),
ACCEPTED MANUSCRIPT
6.84 (d, J = 6.80 Hz 2H), 7.10 (d, J = 7.00 Hz 2H), 7.28 (s, 2H), 7.38 (d, J = 8.20 Hz 2H), 7.74 (d, J = 8.20 Hz 2H);
13
C-NMR (DMSO-d6) δ 31.56, 32.02, 35.89, 38.77, 40.54, 54.95, 113.69,
125.61, 125.70, 129.17, 133.63, 141.93, 144.08, 157.35, 157.97; HRMS Calcd for C19H25N3O4S
RI PT
m/z [M+H] 392.1644, found 392.1665.
6.1.1.30. 4-(2-(3-(3-(4-Chlorophenyl)propyl)ureido)ethyl)benzenesulfonamide (32); Prepared from the reaction of 1-(3-isocyanatopropyl)-4-chlorobenzene (58b) with amine (‘L’ and ‘n’ are
SC
mentioned in Table 1) according to the general procedure (i). Yield 52%; White solid; Rf 0.42 (Ethylacetate); mp: 153-156 °C; IR (neat): 3273, 2948, 1698, 1557, 1539, 1154, 894, 744, 668
M AN U
cm-1; 1H-NMR (DMSO-d6) δ 1.59-1.66 (m, 2H), 2.54 (t, J = 8.00 Hz, 2H), 2.75 (t, J = 8.00 Hz 2H), 2.94-2.99 (m, 2H), 3.22-3.27 (m, 2H), 5.82-5.85 (m, 1H), 5.92-5.95 (m, 1H), 7.21-7.23 (m, 2H), 7.30 (s, 2H), 7.32-7.34 (m, 2H), 7.37-3.39 (m, 2H), 7.74 (d, J = 8.20 Hz 2H);
13
C-NMR
(DMSO-d6) δ 32.10, 32.20, 36.36, 39.15, 41.01, 126.13, 128.65, 129.58, 130.63, 130.75, 141.34,
TE D
142.42, 144.53, 158.43; HRMS Calcd for C18H22ClN3O3S m/z [M+H] 396.1149, found 396.1174. HPLC purity 99.39%
6.1.1.31. 1-(3,4-Dimethoxyphenethyl)-3-(3-(4-methoxyphenyl)propyl)urea (33); Prepared from
EP
the reaction of 58a with amine (‘L’ and ‘n’ are mentioned in Table 1) according to the general procedure (i). Yield 33%; White solid; Rf 0.45 (2 : 1 Ethylacetate : Hexane); mp: 103-106 °C; IR
AC C
(neat): 3308, 2932, 1616, 1558, 1513, 1227, 1139, 1026, 807, 757 cm-1; 1H-NMR (DMSO-d6) δ 1.58-1.62 (m, 2H), 2.47 (t, J = 8.00 Hz 2H), 2.60 (t, J = 7.80 Hz 2H), 2.94-2.99 (m, 2H), 3.173.22 (m, 2H), 3.70 (s, 3H), 3.71 (s, 3H), 3.73 (s, 3H), 5.72-5,75 (m, 1H), 5.90-5,93 (m, 1H), 6.68-6.71 (m, 1H), 6.78 (s, 1H), 6.83-6.86 (m, 3H), 7.09-7.11 (m, 2H); 13C-NMR (DMSO-d6) δ 31.59, 32.11, 35.75, 38.78, 41.07, 54.98, 55.39, 55.53, 111.96, 112.64, 113.83, 120.57, 129.36,
ACCEPTED MANUSCRIPT
132.35, 133.81, 147.28, 148.74, 157.54, 158.20; HRMS Calcd for C21H28N2O4 m/z [M+H] 373.2127, found 373.2140. 6.1.1.32.
4-(2-(3-(3-(4-Fluorophenyl)propyl)ureido)ethyl)-N,N-dimethylbenzenesulfonamide
RI PT
(34); Prepared by methylation of compound 30. The methylation procedure was analogous to the preparation of compound 12. Yield 75%; White solid; Rf 0.40 (Ethylacetate); mp: 64-68 °C; IR (neat): 3315, 2934, 2859, 1624, 1560, 1508, 1409, 1334, 1219, 1156, 1089, 952, 822, 704 cm-1;
SC
1H NMR (CDCl3) δ 1.72-1.82 (m, 2H), 2.57-2.66 (m, 2H), 2.69 (s, 6H), 2.88 (t, J = 7.83 Hz, 2H), 3.15 (m, 2H), 3.43 (m, 2H), 4.34-4.48 (m, 2H), 6.96 (m, 2H), 7.12 (m, 2H), 7.35 (d, J =
M AN U
8.40 Hz, 2H), 7.68 (d, J = 8.40 Hz, 2H); 13C NMR (CDCl3) δ 32.12, 32.50, 36.51, 38.17, 40.19, 41.44, 115.40, 115.61, 128.31, 129.91, 130.00, 130.08, 133.78, 137.42, 145.25, 158.55, 162.90; HRMS Calcd for C20H26FN3O3S m/z [M+H] 408.1757, found 408.1785. 6.1.1.33.
N-(4-(N,N-Dimethylsulfamoyl)benzyl)-4-phenylpiperidine-1-carboxamide
(35);
TE D
6.1.1.33.1. Preparation of 4-Phenyl-N-(4-sulfamoylbenzyl)piperidine-1-carboxamide (67); The reaction of 4-(aminomethyl)benzenesulfonamide (64) and 4-phenylpiperidine (66) according to the general procedure (ii) afforded 4-phenyl-N-(4-sulfamoylbenzyl)piperidine-1-carboxamide
EP
(67). 1H-NMR (DMSO-d6) δ 1.47-1.55 (m, 2H), 1.73-1.77 (m, 2H), 2.68-2.70 (m, 1H), 2.762.82 (m, 2H), 4.11-4.14 (m, 2H), 4.31 (d, J = 6.00 Hz, 2H), 7.17-7.20 (m, 4H), 7.23-7.29 (m,
AC C
4H), 7.44 (d, J = 8.40 Hz, 2H), 7.76 (d, J = 8.40 Hz, 2H). 6.1.1.33.2.
N-(4-(N,N-Dimethylsulfamoyl)benzyl)-4-phenylpiperidine-1-carboxamide
(35);
Prepared by the methylation of 67. Methylation procedure is analogous to the preparation of compound 12. Yield 82%; Colourless viscous oil; Rf 0.42 (Ethylacetate); IR (neat): 3325, 1647, 1535, 1321, 1301, 1303, 1152, 1078, 944 cm-1; 1H-NMR (CDCl3) δ 1.65-1.76 (m, 2H), 1.891.92 (m, 2H), 2.67-2.74 (m, 1H), 2.70 (s, 6H), 2.92-2.99 (m, 2H), 4.13-4.16 (m, 2H), 4.56 (d, J =
ACCEPTED MANUSCRIPT
6.00 Hz, 2H), 5.05-5.07 (bs, 1H), 7.21-7.25 (m, 3H), 7.31-7.35 (m, 2H), 7.48 (d, J = 8.40 Hz, 2H), 7.72 (d, J = 8.40 Hz, 2H); 13C-NMR (CDCl3) δ 32.85, 36.33, 37.85, 42.44, 44.61, 126.49, 126.73, 128.01, 128.59, 129.53, 133.38, 145.07, 145.38, 157.40; HRMS Calcd for C21H27N3O3S
6.1.1.34.
RI PT
m/z [M+H] 402.1851, found 402.1877.
N-(4-(N,N-Dimethylsulfamoyl)phenethyl)-4-phenylpiperidine-1-carboxamide
(36);
6.1.1.33.1. Preparation of 4-phenyl-N-(4-sulfamoylphenethyl)piperidine-1-carboxamide (68);
SC
The reaction of 4-(2-aminoethyl)benzenesulfonamide (65) and 4-phenylpiperidine (66) according to the general procedure (ii) afforded 4-phenyl-N-(4-sulfamoylphenethyl)piperidine-1-
M AN U
carboxamide 68. 1H-NMR (DMSO-d6) δ 1.43-1.47 (m, 2H), 1.70-1.73 (m, 2H), 2.65-2.75 (m, 3H), 2.81 (t, J = 7.20 Hz, 2H), 3.25-3.29 (m, 2H), 4.05-4.08 (m, 2H), 6.60-6.63 (m, 1H), 7.197.30 (m, 7H), 7.39 (d, J = 8.40 Hz, 2H), 7.75 (d, J = 8.40 Hz, 2H). 6.1.1.34.
N-(4-(N,N-Dimethylsulfamoyl)phenethyl)-4-phenylpiperidine-1-carboxamide
(36);
TE D
Prepared by the methylation of 68. Methylation procedure is analogous to the preparation of compound 12. Yield 80%; Colorless viscous oil; Rf 0.44 (Ethylacetate); IR (neat): 3332, 1625, 1538, 1334, 1319, 1160, 1088, 951 cm-1; 1H-NMR (CDCl3) δ 1.62-1.69 (m, 2H), 1.85-1.88 (m,
EP
2H), 2.64-2.68 (m, 1H), 2.70 (s, 6H), 2.84-2.91 (m, 2H), 2.95 (t, J = 7.20 Hz, 2H), 3.51-3.58 (m, 2H), 4.00-4.04 (m, 2H), 4.56 (bs, 1H), 7.19-7.24 (m, 3H), 7.30-7.34 (m, 2H), 7.39 (d, J = 8.40
AC C
Hz, 2H), 7.72 (d, J = 8.40 Hz, 2H). 13C-NMR (CDCl3) δ 32.92, 36.39, 37.86, 41.81, 42.52, 44.83, 128.53, 126.76, 128.07, 128.63, 129.54, 133.64, 145.06, 145.42, 157.48; HRMS Calcd for C22H29N3O3S m/z [M+H] 416.2008, found 416.2029. 6.1.1.35.
N-Benzyl-4-(4-(N,N-dimethylsulfamoyl)phenylpiperidine-1-carboxamide
(37);
6.1.1.35.1. Preparation of N,N-dimethyl-4-(piperidin-4-yl)benzenesulfonamide (69); Prepared by the procedure analogous to the preparation of intermediate 62. 1H-NMR (CDCl3) δ 1.67-1.71 (m,
ACCEPTED MANUSCRIPT
2H), 1.87-1.88 (m, 2H), 2.69-2.71 (m, 1H), 2.71 (s, 6H), 2.78-2.81 (m, 2H), 3.22-3.26 (m, 2H), 7.39 (d, J = 8.40 Hz, 2H), 7.72 (d, J = 8.40 Hz, 2H). 6.1.1.35.2.
N-Benzyl-4-(4-(N,N-dimethylsulfamoyl)phenylpiperidine-1-carboxamide
(37);
RI PT
Prepared from the reaction of 69 and (isocyanatomethyl)benzene (70) according to the general procedure (i). Yield 88%; White solid; Rf 0.40 (Ethylacetate); mp: 158-160 °C; IR (neat): 3330, 1625, 1538, 1379, 1319, 1160, 1088, 951 cm-1; 1H-NMR (CDCl3) δ 1.64-1.75 (m, 2H), 1.87-
SC
1.90 (m, 2H), 2.72 (s, 6H), 2.74-2.81 (m, 1H), 2.89-2.96 (m, 2H), 4.12-4.15 (m, 2H), 4.47 (d, J = 4.80 Hz, 2H), 4.80 (bs, 1H), 7.22-7.26 (m, 3H), 7.31-7.32 (m, 2H), 7.35 (d, J = 8.40 Hz, 2H),
M AN U
7.72 (d, J = 8.40 Hz, 2H); 13C-NMR (CDCl3) δ 32.59, 37.86, 42.53, 44.55, 45.07, 127.41, 127.47, 127.89, 128.17, 128.69, 133.71, 139.47, 150.61, 157.46; HRMS Calcd for C21H27N3O3S m/z [M+H] 402.1851, found 402.1871. 6.1.1.36.
4-(4-(N,N-Dimethylsulfamoyl)phenyl)-N-phenethylpiperidine-1-carboxamide
(38);
TE D
Prepared from the reaction of 69 and (2-isocyanatoethyl)benzene (71) according to the general procedure (i). Yield 86%; Pale yellow oil; Rf 0.42 (Ethylacetate); IR (neat): 3349, 1624, 1538, 1361, 1322, 1235, 1157, 1089, 950 cm-1; 1H-NMR (CDCl3) δ 1.58-1.69 (m, 2H), 1.83-1.87 (m,
EP
2H), 2.73-2.78 (m, 1H), 2.72 (s, 6H), 2.83-2.90 (m, 4H), 3.51-3.56 (m, 2H), 4.02-4.05 (m, 2H), 4.51 (bs, 1H), 7.22-7.26 (m, 3H), 7.31-7.32 (m, 2H), 7.35 (d, J = 8.40 Hz, 2H), 7.72 (d, J = 8.40
AC C
Hz, 2H); 13C-NMR (CDCl3) δ 32.63, 36.33, 37.92, 42.07, 42.62, 44.46, 126.39, 127.40, 128.11, 128.59, 128.84, 133.67, 139.37, 150.56, 157.41; HRMS Calcd for C22H29N3O3S m/z [M+H] 416.2008, found 416.2032. 6.1.1.37.
4-(4-(N,N-Dimethylsulfamoyl)phenyl)-N-(3-phenylpropyl)piperidine-1-carboxamide
(39); Prepared from the reaction of 69 and (3-isocyanatopropyl)benzene (47) according to the general procedure (i). Yield 86%; Pale white solid; Rf 0.44 (Ethylacetate); mp: 125-127 °C; IR
ACCEPTED MANUSCRIPT
(neat): 3358, 1608, 1538, 1339, 1324, 1160, 1089, 980 cm-1; 1H-NMR (CDCl3) δ 1.60-1.68 (m, 2H), 1.82-1.93 (m, 4H), 2.68-2.74 (m, 3H), 2.71 (s, 6H), 2.79-2.85 (m, 2H), 3.30-3.35 (m, 2H), 3.95-3.99 (m, 2H), 4.40 (bs, 1H), 7.19-7.22 (m, 3H), 7.27-7.31 (m, 2H), 7.35 (d, J = 8.40 Hz,
RI PT
2H), 7.72 (d, J = 8.40 Hz, 2H); 13C-NMR (CDCl3) δ 31.64, 32.64, 33.61, 37.90, 40.84, 42.60, 44.36, 125.90, 127.39, 128.07, 128.38, 128.45, 133.60, 141.84, 150.62, 157.47; HRMS Calcd for C23H31N3O3S m/z [M+H] 430.2164, found 430.2183. HPLC purity 100%
SC
6.1.1.38. Preparation of N,N-Dimethyl-4-(2-(1-methyl-3-(3-phenylpropyl)ureido)ethyl)benzene sulfonamide (40);
M AN U
6.1.1.38.1. Preparation of N-(4-sulfamoylphenethyl)acetamide (72): To a cooled solution of 65 (10 mmol) in methylene chloride at 0 °C, triethylamine (15 mmol) was added and stirred for 5 minutes. Then, acetyl chloride (15 mmol) was added at 0 °C and the whole reaction mixture was slowly allowed to attain ambient temperature and further stirred for 3 h. After the completion of
TE D
the reaction, water was added and the reaction mixture was extracted using methylene chloride. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. Without further purification, the crude product 72 was taken for the next step. Yield 85%
EP
6.1.1.38.2. Preparation of N-(4-(N,N-dimethylsulfamoyl)phenethyl)-N-methylacetamide (73): To a suspension of NaH (16.20 mmol) in DMF at 0 °C, 72 (5.40 mmol) was added and stirred for 45
AC C
minutes at ambient temperature. The reaction mixture was cooled and methyl iodide (16.20 mmol) was added and further stirred at ambient temperature for 3 h. After the completion of the reaction, 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 product 73 was purified by column chromatography. Yield 87%
ACCEPTED MANUSCRIPT
6.1.1.38.3. Preparation of N,N-dimethyl-4-(2-(methylamino)ethyl)benzenesulfonamide (74): The above obtained 73 (3.50 mmol) was dissolved in n-butanol (3 mL) and 3N HCl (100 mL) was added. The reaction mixture was refluxed for 8 h. After the completion of the reaction, the
RI PT
solution was extracted using diethyl ether and the aqueous layer was neutralized with solid K2CO3. Then it was extracted using ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum to get the amine 74. Yield 75%; 1H-NMR
SC
(CDCl3) δ 2.28 (s, 3H), 2.59 (s, 6H), 2.71-2.74 (m, 2H), 2.76-2.81 (m, 2H), 3.32 (bs, 1H), 7.49 (d, J = 8.20 Hz, 2H), 7.65 (d, J = 8.20 Hz, 2H).
M AN U
6.1.1.38.4. N,N-Dimethyl-4-(2-(1-methyl-3-(3-phenylpropyl)ureido)ethyl)benzene sulfonamide (40); Prepared from the reaction of the above obtained amine 74 and 47 according to the general procedure (i). Yield 78%; White solid; Rf 0.40 (Ethylacetate); mp 72-74 °C; IR (neat): 3442, 2916, 1647, 1538, 1490, 1363, 1158, 1088 cm-1; 1H-NMR (CDCl3) δ 1.81-1.89 (m, 2H), 2.65-
TE D
2.71 (m, 2H), 2.67 (s, 3H), 2.68 (s, 6H), 2.90 (t, J = 7.20 Hz, 2H), 3.25-3.30 (m, 2H), 3.51 (t, J = 7.80 Hz, 2H), 4.22 (bs, 1H), 7.19-7.31 (m, 5H), 7.38 (d, J = 8.20 Hz, 2H), 7.70 (d, J = 8.20 Hz, 2H). 13C-NMR (CDCl3) δ 31.74, 33.44, 34.51, 34.67, 37.86, 40.62, 50.39, 125.96, 128.01,
EP
128.40, 128.50, 129.51, 133.26, 141.82, 144.92, 157.78; HRMS Calcd for C21H29N3O3S m/z [M+H] 404.2008, found 404.2027. HPLC purity 99.01% Preparation
AC C
6.1.1.39.
of
N,N-Dimethyl-4-(2-(3-methyl-3-(3-
phenylpropyl)ureido)ethyl)benzenesulfonamide (41); 6.1.1.39.1. Preparation of N-methyl-3-phenylpropan-1-amine (75); The intermediate 75 was prepared from 59 analogous to the preparation of 74. 1H-NMR (CDCl3) δ 1.80-1.85 (m, 2H), 2.41 (s, 3H), 2.58-2.68 (m, 4H), 7.17-7.21 (m, 3H), 7.27-7.31 (m, 2H).
ACCEPTED MANUSCRIPT
6.1.1.39.2. 4-(2-(3-Methyl-3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (76); Prepared by the reaction of 75 and 65 according to the general procedure (ii). Yield 75%; 1H-NMR (DMSO-d6) δ 1.67-1.75 (m, 2H), 2.50-2.54 (m, 2H), 2.75 (s, 3H), 2.79 (t, J = 7.20 Hz, 2H),
RI PT
3.18-3.27 (m, 4H), 6.33 (bs, 1H), 7.15-7.21 (m, 3H), 7.27-7.30 (m, 4H), 7.37 (d, J = 8.20 Hz, 2H), 7.74 (d, J = 8.20 Hz, 2H). 6.1.1.39.3.
N,N-Dimethyl-4-(2-(3-methyl-3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide
SC
(41); Prepared by the methylation of 76 with 2 equivalents of methyl iodide. Methylation procedure is analogous to the preparation of compound 12. Yield 80%; White solid; Rf 0.40
M AN U
(Ethylacetate); mp 67-69 °C; IR (neat): 3430, 2928, 1632, 1527, 1386, 1185, 1157, 1090 cm-1; 1H-NMR (CDCl3) δ 1.81-1.88 (m, 2H), 2.61 (t, J = 7.60 Hz, 2H), 2.69 (s, 6H), 2.81 (s, 3H), 2.89 (t, J = 7.80 Hz, 2H), 3.26 (t, J = 7.80 Hz, 2H), 3.44-3.49 (m, 2H), 4.26 (bs, 1H), 7.16-7.22 (m, 3H), 7.27-7.31 (m, 2H), 7.36 (d, J = 8.40 Hz, 2H), 7.71 (d, J = 8.40 Hz, 2H). 13C-NMR (CDCl3)
TE D
δ 29.39, 32.82, 34.07, 36.37, 37.89, 41.75, 48.12, 126.03, 128.03, 128.31, 128.50, 129.53, 133.20, 141.47, 145.07, 157.77; HRMS Calcd for C21H29N3O3S m/z [M+H] 404.2008, found 404.2030. HPLC purity 100%
(42);
EP
6.1.1.40. 4-(2-(1,3-Dimethyl-3-(3-phenylpropyl)ureido)ethyl)N,N-dimethylbenzene sulfonamide
AC C
Prepared by the methylation of compound 13. Methylation procedure is analogous to the preparation of compound 12. Yield 59%; Pale yellow oil; Rf 0.42 (Ethylacetate); IR (neat): 3105, 3020, 2987, 1655, 1617, 1554, 1488 cm-1; 1H-NMR (CDCl3) δ 1.83-1.90 (m, 2H), 2.59 (t, J = 8.00 Hz, 2H), 2.69 (s, 6H), 2.72 (s, 3H), 2.77 (s, 3H), 2.92 (t, J = 8.00 Hz, 2H), 3.14 (t, J = 8.00 Hz, 2H), 3.39 (t, J = 8.00 Hz, 2H), 7.16-7.21 (m, 3H), 7.26-7.30 (m, 2H), 7.36 (d, J = 8.20 Hz, 2H), 7.69 (d, J = 8.20 Hz, 2H); 13C-NMR (CDCl3) δ 29.09, 33.08, 33.81, 36.43, 37.34, 37.86,
ACCEPTED MANUSCRIPT
49.90, 51.32, 125.97, 127.98, 128.35, 128.45, 129.39, 133.37, 141.64, 145.07, 165.08; HRMS Calcd for C22H31N3O3S m/z [M+H] 418.2164, found 418.2186. HPLC purity 99.21% 6.1.1.41. Preparation of 4-(2-(1-Ethyl-3-(3-phenylpropyl)ureido)ethyl)-N,N-dimethylbenzene
RI PT
sulfonamide (43):
6.1.1.41.1. Preparation of 4-(2-ethylamino)ethyl)benzenesulfonamide (77a): To a solution of 65 (0.5 mmol) in DMF, K2CO3 (0.5 mmol) was added and stirred for 15 minutes. After that
SC
ethyliodide (0.5 mmol) was added and allowed to stir for 12 h at ambient temperature. After the formation of the product by TLC, water was added to the reaction mixture and extracted using
M AN U
ethyl acetate. The intermediate 77a was separated by column chromatography. Yield 55%. (The other product was the dialkylated derivative).
6.1.1.41.2. Preparation of 4-(2-(1-ethyl-3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (78a); Prepared by the reaction of 77a and 47 according to the general procedure (i). Yield 40%;
TE D
1H-NMR (CDCl3) δ 1.07 (t, J = 7.20 Hz, 3H), 1.81-1.89 (m, 2H), 2.65-2.70 (m, 2H), 2.94 (t, J = 7.60 Hz, 2H), 3.24-3.35 (m, 4H), 3.40-3.45 (m, 2H), 4.30 (bs, 1H), 4.94 (s, 2H), 7.18-7.34 (m, 5H), 7.33 (d, J = 8.20 Hz, 2H), 7.79 (d, J = 8.20 Hz, 2H).
(43);
4-(2-(1-Ethyl-3-(3-phenylpropyl)ureido)ethyl)-N,N-dimethylbenzene
sulfonamide
EP
6.1.1.41.3.
Prepared by the methylation of 78a by 2 equivalents of methyl iodide. Methylation
AC C
procedure is analogous to the preparation of compound 12. Yield 30%; Colourless oil; Rf 0.42 (2 : 1 Ethylacetate : Hexane); IR (neat): 3334, 2923, 1642, 1540, 1495, 1339, 1213, 1189 cm-1; 1HNMR (CDCl3) δ 1.05 (t, J = 7.20 Hz, 3H), 1.83-1.90 (m, 2H), 2.65-2.72 (m, 2H), 2.68 (s, 6H), 2.92 (t, J = 7.60 Hz, 2H), 2.99-3.04 (m, 2H), 3.26-3.31 (m, 2H), 3.44 (t, J = 7.80 Hz, 2H), 4.23 (bs, 1H), 7.17-7.21 (m, 2H), 7.27-7.34 (m, 3H), 7.38 (d, J = 8.40 Hz, 2H), 7.70 (d, J = 8.40 Hz, 2H). 13C-NMR (CDCl3) δ 13.62, 31.98, 33.62, 35.19, 37.99, 40.70, 42.36, 48.64, 126.11,
ACCEPTED MANUSCRIPT
128.15, 128.54, 128.64, 129.64, 133.52, 141.95, 145.12, 157.43; HRMS Calcd for C23H33N3O3S m/z [M+H] 432.2321, found 432.2341. 6.1.1.42. 4-(2-(1-Ethyl-3-(3-phenylpropyl)ureido)ethyl)-N,N-dimethylbenzene sulfonamide (44);
RI PT
To a cooled suspension of NaH (3.08 mmol) in DMF, 13 (0.51 mmol) was added and stirred at ambient temperature for 45 minutes. Then the reaction mixture was cooled and ethyliodide (2.06 mmol) was added. The reaction mixture was further stirred at ambient temperature for 15 h.
SC
After the formation of product, saturated NH4Cl solution was added to neutralize excess NaH and the reaction mixture was extracted with ethyl acetate. The organic layer was dried over
M AN U
anhydrous sodium sulfate and concentrated under vacuum to get 44. The product was purified by column chromatography. Yield 30%; Colourless oil; Rf 0.50 (2 : 1 Ethylacetate : Hexane); IR (neat): 3340, 2923, 1642, 1555, 1488, 1341, 1211, 1187 cm-1; 1H-NMR (CDCl3) δ 1.02-1.08 (m, 6H), 1.82-1.89 (m, 2H), 2.59 (t, J = 7.80 Hz, 2H), 2.69 (s, 6H), 2.88 (t, J = 7.20 Hz, 2H), 3.08-
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3.16 (m, 6H), 3.34 (t, J = 7.20 Hz, 2H), 7.16-7.20 (m, 3H), 7.26-7.30 (m, 2H), 7.35 (d, J = 8.40 Hz, 2H), 7.69 (d, J = 8.40 Hz, 2H); 13C-NMR (CDCl3) δ 13.14, 13.25, 29.68, 33.40, 34.42, 37.99, 43.11, 44.40, 47.12, 48.59, 126.09, 128.05, 128.47, 128.57, 129.58, 133.37, 141.80,
purity 99.64%
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145.58, 164.98; HRMS Calcd for C24H35N3O3S m/z [M+H] 446.2477, found 446.2499. HPLC
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6.1.1.43. Preparation of 4-(2(1-isopropyl-3-(3-phenylpropyl)ureido)ethyl)-N,N-dimethylbenzene sulfonamide (45);
6.1.1.43.1. Preparation of 4-(2-(isopropylamino)ethyl)benzenesulfonamide (77b): To a solution of 65 (0.5 mmol) in DMF, K2CO3 (0.5 mmol) was added and stirred for 15 minutes. After that isopropyl iodide (0.5 mmol) was added and allowed to stir for 12 h at ambient temperature. After the formation of the product by TLC, water was added to the reaction mixture and extracted
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using ethyl acetate. The intermediate 77b was separated by column chromatography. Yield 50%. (The other product was the dialkylated derivative). 6.1.1.43.2. Preparation of 4-(2-(1-isopropyl-3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide
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(78b): Prepared by the reaction of 77b and 47 according to the general procedure (i). Yield 40%; 1H-NMR (CDCl3) δ 1.12 (d, J = 6.80 Hz, 6H), 1.80-1.86 (m, 2H), 2.66 (t, J = 7.60 Hz, 2H), 2.90 (t, J = 7.60 Hz, 2H), 3.24-3.31 (m, 4H), 3.84-3.90 (m, 1H), 4.24 (bs, 1H), 4.91 (s, 2H), 7.19-7.31
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(m, 5H), 7.35 (d, J = 8.20 Hz, 2H), 7.85 (d, J = 8.20 Hz, 2H).
6.1.1.43.3. 4-(2(1-Isopropyl-3-(3-phenylpropyl)ureido)ethyl)-N,N-dimethylbenzene sulfonamide
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(45); Prepared by the methylation of 78b by 2 equivalents of methyl iodide. Methylation procedure is analogous to the preparation of compound 12. Yield 70%; Colourless oil; Rf 0.32 (2 : 1 Ethylacetate : Hexane); IR (neat): 3330, 2984, 1638, 1540, 1490, 1337, 1210, 1188 cm-1; 1HNMR (CDCl3) δ 1.10 (d, J = 6.80 Hz, 6H), 1.84-1.91 (m, 2H), 2.64-2.72 (m, 2H), 2.69 (s, 6H),
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2.90-2.96 (m, 2H), 3.26-3.32 (m, 4H), 3.87-3.90 (m, 1H), 4.28 (bs, 1H), 7.17-7.21 (m, 3H), 7.277.31 (m, 2H), 7.38 (d, J = 8.40 Hz, 2H), 7.70 (d, J = 8.40 Hz, 2H); 13C-NMR (CDCl3) δ 20.91, 31.80, 33.57, 36.72, 37.87, 40.71, 43.21, 46.85, 126.01, 128.09, 128.44, 128.54, 129.48, 133.57,
6.1.1.44.
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141.87, 145.11, 157.61; HRMS Calcd for C23H33N3O3S m/z [M+H] 432.2321, found 432.2340. 4-(2-(1-Isopropyl-3-methyl-3-(3-phenylpropyl)ureido)ethyl)-N,N-dimethylbenzene
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sulfonamide (46); Prepared by the methylation of compound 45 with 1 equivalent of methyliodide and NaH. Methylation procedure is analogous to the preparation of compound 12. Yield 60%; White solid; Rf 0.35 (2 : 1 Ethylacetate : Hexane); mp 57-59 °C; IR (neat): 3010, 2989, 1641, 1550, 1496, 1335, 1211, 1180 cm-1; 1H-NMR (CDCl3) δ 0.95 (d, J = 6.80 Hz, 6H), 1.87-1.90 (m, 2H), 2.60 (t, J = 7.60 Hz, 2H), 2.68 (s, 6H), 2.76 (s, 3H), 2.82 (t, J = 7.20 Hz, 2H), 3.19-3.24 (m, 4H), 3.57-3.64 (m, 1H), 7.17-7.21 (m, 3H), 7.27-7.31 (m, 2H), 7.33 (d, J = 8.20
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Hz, 2H), 7.68 (d, J = 8.20 Hz, 2H); 13C-NMR (CDCl3) δ 19.98, 29.23, 33.16, 35.29, 36.36, 37.89, 42.44, 49.71, 50.60, 126.02, 127.77, 128.37, 128.50, 129.65, 133.04, 141.67, 146.13, 164.98; HRMS Calcd for C24H35N3O3S m/z [M+H] 446.2477, found 446.2498.
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6.2. Pharmacology
6.2.1. Sarcomere assay procedure for the measurement of myosin ATPase activity:
In the sarcomere, force generation is directly coupled to ATP hydrolysis. Compounds that
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activate the sarcomere were identified by measuring the increase in myosin ATPase activity in a sarcomere assay at 10 µM.
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Actin stimulated ATPase activity was assayed spectrophotometrically as reported previously [28] with modifications. The standard reaction mixture contained 20 mM Tris HCl (pH 7.5), 15 mM KCl, 6 mM MgCl2, 1 mM ATP, S1 myosin (CS-MYS03) and actin thin filament complex (CSTFC01) with pCa = 6.5 [29]. The reaction was stopped by addition of Cytophos reagent
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(Cytoskeleton BK054 kit) after 10 min incubation at room temperature, samples were analyzed for inorganic phosphate liberated by taking the absorbance at 650 nm on TECAN Infinite. Omecamtiv was used as a positive control for the selectivity assay. Blank had buffer, ATP and
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Cytophos mixture while DMSO was used as a negative control. % Activity = (Mean A - B)-(NC -B)/ (NC-B) x100
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Where, NC=Negative control; B=Blank, A=Absorbance 6.2.2. Selectivity Studies
Compound specificity with respect to muscle type were evaluated by comparing the effect of the compound on actin stimulated ATPase activity of a panel of myosin isoforms including cardiac (bovine), skeletal (rabbit) and smooth muscle (chicken gizzard) at a single higher dose (100 uM ) of the compound.
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6.2.2.1. For Skeletal muscle: Actin stimulated ATPase activity was assayed spectrophotometrically as reported previously [2831] with modifications. The standard reaction mixture contained 20 mM Tris HCl (pH 7.5), 15
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mM KCl, 6 mM MgCl2, 1 mM ATP, S1 myosin (CS-MYS04) and actin thin filament complex (CS-TFC01) with pCa = 6.5. The reaction was stopped by addition of Cytophos reagent (Cytoskeleton BK054 kit) After 10 min incubation at room temperature, samples were analyzed
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for inorganic phosphate liberated by taking absorbance at 650 nm on TECAN Infinite.
6.2.2.2. For Smooth muscle:
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Blebbistatin was used as a negative control for selectivity assay [34].
Actin stimulated ATPase activity was assayed spectrophotometrically as reported previously [28,29,32,33] with modifications. The standard reaction mixture contained 20 mM Tris HCl (pH 7.5), 15 mM KCl, 6 mM MgCl2, 1 mM ATP, S1 myosin (CS-MYS05), actin (AD 99) and
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Tropomyosin (T2400) with pCa = 6.5. The reaction was stopped by addition of Cytophos reagent (Cytoskeleton BK054 kit) After 10 min incubation at room temperature, samples were analyzed for inorganic phosphate liberated by taking absorbance at 650 nm on TECAN Infinite.
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Blebbistatin was used as a negative control for selectivity assay [34]. 6.2.3. Animal study (in vivo)
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6.2.3.1. The formulation of sample
The stock solution was prepared by dissolving 8 mg of respective compound in 2 mL of DMSO (i.e. 4 mg/mL or 4000 µg/mL of DMSO). It was diluted 100 times with saline solution to give 40 µg/mL final solution (% of DMSO is 1 %). The maximum % of DMSO in the final solution was limited to 10 %. The concentration of the final solution (unknown concentration) was measured by HPLC along with three standards (known concentration).
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6.2.3.2. Measurement of fractional shortening (FS) and ejection fraction (EF) by echocardiography 6.2.3.2.1. Animals
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Seven-week-old Sprague-Dawley male Rats were purchased from the Orient Bio (South Korea). The protocols used in this study [35] conformed to the Guide for the Care and use of Laboratory Animals published by National Institutes of Health (NIH Publication 85-23, revised 1996). All
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animal experiments were approved by the Institutional Animal Care and Use Committee of Samsung Biomedical Research Institute (SBRI). SBRI is accredited by the Association for
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Assessment and Accreditation of Laboratory Animal Care International and abided by the Institute of Laboratory Animal Resources guide. 6.2.3.2.2. Echocardiography
The rats were anesthetized by 1.5 % isoflurane inhalation method with nosecone. The right
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internal jugular vein was used as a central venous line for drug administration. Rats were infused with samples at 0, 2, 4, 8, and 16 µg/kg/min for 3 minutes using 40 µg/mL final solution. Mmode echocardiograms were performed at baseline and 16 µg/kg/min. Images were acquired
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with a MS250 transducer operated at 25 MHz connected to a VisualSonics Vevo2100 (VisualSonics Inc., Toronto, Ontario, Canada). LV end-diastolic interventricular septum,
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posterior wall thickness, LV dimension, LV fractional shortening (FS) and ejection fraction (EF) were measured. % increase value was calculated according to following formula: % Increase FS(EF) = (FS(EF) data of 16 µg/kg/min - FS(EF) data of 0 µg/kg/min) / baseline data of FS(EF) x 100 %. Three rats per drug were used. A single sonographer who was blinded to treatment information performed all echacardiograms for data acquisition.
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6.2.4. Ventricular cell contractility 6.2.4.1. Single-cell Isolation Rat ventricular myocytes were enzymatically isolated from male Sprague-Dawley rats (200–300
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g) as described previously [36]. Briefly, rats were deeply anesthetized with sodium pentobarbital (150 mg/kg, intraperitoneally), chest cavities were opened, and hearts were excised. This surgical procedure was carried out in accordance with university ethical guidelines. The excised hearts
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were retrogradely perfused at 7 mL/min through the aorta (at 36.5°C), first for 3 minutes with Ca2+-free Tyrode solution composed of (in millimolar) 137 NaCl, 5.4 KCl, 10 HEPES, 1 MgCl2,
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and 10 glucose (pH 7.3), and then with Ca2+ free Tyrode solution containing collagenase (1.4 mg/mL, Type I; Roche, Indianapolis, IN) and protease (0.14 mg/mL, Type XIV; Sigma-Aldrich, St. Louis, MO) for 12 minutes, and finally with Tyrode solution containing 0.2 mM CaCl2 for 8 minutes. The ventricles of the digested heart were then cut into several sections and subjected to
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gentle agitation to dissociate the cells.
6.2.4.2. Measurement of Cell Shortenings
Isolated myocytes were continuously superfused with normal Tyrode solution (see above)
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containing 2 mM Ca2+. Cells were field stimulated with 2 paralleled platinum wires connected with an electrical stimulator (Stimulator I; Hugo Sach Elektronik, March-Hugstetten, Germany)
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at 1 Hz. Single-cell shortenings were detected with a video edge detector (Model VED-105; Crescent Electronics, Sandy, UT) connected with a CCD camera (LCL902C; Till Photonics, Graefelting, Germany) and video monitor (ViewFinder III, Polychrome V system; Till Photonics). Analog signals from the edge detector were converted into digital signals by an A/D converter (Digidata 1322A; Molecular Devices). The digitized cell shortening signals were recorded with a PC program, pClamp 9 (Molecular Devices, Sunnyvale, CA).
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A Stock solution of the compounds were made in dimethyl sulfoxide (DMSO), which was diluted in the external normal Tyrode solution to make the final testing solutions. The drug solutions were applied to the cells by super fusion using custom-made solution switching
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apparatus. The experiments were all performed at room temperature (22-25 oC). ACKNOWLEDGMENT
This work was supported by Priority Research Centers Program through the National Research
(2009-0093815) and KDDF (201202-09).
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REFERENCES
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Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology
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Highlights SAR study of sulfonamidophenylethylureas discovered highly potent inotrope.
•
These urea analogs are selective cardiac myosin ATPase activators.
•
Compound 13, 40 and 41 shows 17.6, 38.9 and 23.2% fractional shortening in the echocardiographic study with rat, respectively.
The cell contractility of 13, 40, and 41 shows 47.9, 45.5 and 63.5% at 5 µM in rat
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ventricle cells, respectively.
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•
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S0223-5234(17)30879-6
DOI:
10.1016/j.ejmech.2017.10.077
Reference:
EJMECH 9869
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. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Design and synthesis of sulfonamidophenylethylureas as novel cardiac myosin activator
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GRAPHICAL ABSTRACT
N,N-Dimethylsulfonamide group is important O N
O N H
N R1
Optimization
30.04 18.90
% increase
18.27 12.15
Fractional Ejection Cardiac myosin ATPase activation shortening Fraction % at 10 uM
1
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2
R = H; R = H R1 = CH3; R2 = H R1 = H; R2 = CH3
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53.3 51.1
% increase
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Cardiac myosin Fractional Ejection ATPase activation shortening Fraction n=1 n=2
N R2
HBD is important Partial masking of urea HBD is crucial
Lead compound
% at 10 uM
O
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n N H
Flexible spacer is tolerated
O S
91.6 52.3 47.6
% increase
17.62 38.96 23.19
Ventricular cell contractility (%
% increase change at 5 µM)
11.55 24.19 15.47
47.9 + 3.2 45.5 + 2.4 63.5 + 2.2
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Design and synthesis of sulfonamidophenylethylureas as novel cardiac myosin activator Manoj Manickama, Hitesh B. Jalania, Thanigaimalai Pillaiyara, Pulla Reddy Boggua, Niti Sharmaa, Eeda Venkateswararaoa, You-Jung Leeb, Eun-Seok Jeonb, Min-Jeong Sona, Sun-Hee
a
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Wooa, Sang-Hun Junga*
College of Pharmacy and Institute of Drug Research and Development, Chungnam National
University, Daejeon 34134, Korea
Division of Cardiology, Samsung Medical Center, Samsung Biomedical Research Institute, School
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b
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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 N,N-dimethyl-4-(2-(3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide
91.6%,
FS
=
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them
17.62%;
EF
=
11.55%),
phenylpropyl)ureido)ethyl)benzene sulfonamide
(13,
CMA=
N,N-dimethyl-4-(2-(1-methyl-3-(3-
(40, CMA = 52.3%, FS = 38.96%; EF =
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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
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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. Key words: Phenylpropylurea; Cardiac myosin activator; inotrope; Systolic heart failure
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*Corresponding author: [email protected]. Tel.; +82 42 821 5939; Fax +82 42 823 6566; Present address: College of Pharmacy and Institute of Drug Research and development,
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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
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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
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aim at diverse targets including sodium retention, arterial and venous constriction, neuroendocrine activation increases heart rate, cardiac dyssynchrony and arrhythmias and often
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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
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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
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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].
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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
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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
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cAMP and calcium and decrease ejection time, OM increased myocardial contraction and stroke volume without increasing oxygen consumption, thereby increasing myocardial efficiency
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[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
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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
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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
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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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2. Chemistry
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Fig.1. Background and exploration of urea scaffold for novel cardiac myosin activator
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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
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corresponding urea derivatives 3-11, 14-21, 24-25. Urea 6,11 were prepared by the reaction of 4phenylbutylamine (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 H2 and Pd/C which was consequently dimethylated to 23 using methyliodide in presence of K2CO3 as base. In order to prepare the urea 26, 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
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yielded intermediate 52 which on reaction with 3-phenylpropylisocyanate (47) furnished the urea
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26.
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 CH3I, NaH, DMF, RT, 3 h, (d) H2/Pd/C, MeOH, RT, 10 h, (e) CH3I, K2CO3, acetone, reflux 2 h, (f) CH3COCl, TEA, CH2Cl2, 0 °C to RT, 3 h (g) (i) ClSO3H, CH2Cl2, 0 °C to RT, 3 h (ii) NH4OAc, THF, reflux, 5 h (h) 3N HCl, n-butanol, reflux, 8 h. (i) 47, acetonitrile, RT, 8 h. 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,
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34, the key intermediate 56 was reacted with appropriate amine (‘L’ group and ‘n’ are in Table 1) under triphosgene condition. The preparation of 56 was achieved by the transformation of 4fluorocinnamic acid (53) to its amide 54 by coupling with ammonium chloride. The amide 54
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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
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56 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 4methoxyphenylbutanoic acid 57a/4-chlorophenylbutanoic acid 57b were reacted with methyl
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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
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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. prepare
N,N-dimethylsulfonamide
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To
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.
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Reagents and conditions: (a) EDC.HCl, NH4Cl, TEA, CH2Cl2 , RT, 16 h, (b) (i) LiAlH4, THF, 70 °C 3 h (ii) Boc2O, 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) NaN3/H2O, RT, 1 h (iii) toluene, reflux, 3 h, (f) acetonitrile, RT, 8 h, (g) CH3COCl, TEA, CH2Cl2, 0°C to RT, 3 h
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(h) (i) ClSO3H, CH2Cl2, 0 °C to RT, 3 h (ii) dimethylamine.hydrochloride, NaHCO3, 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.
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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
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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 CH3I, NaH, DMF, RT, 3 h, (c)(i) CH3COCl, TEA, CH2Cl2, 0°C to RT, 3 h (ii) ClSO3H, CH2Cl2, 0 °C to RT, 3 h (iii) dimethylamine.hydrochloride, NaHCO3, THF, RT, 5 h (iv) 3N HCl, n-butanol, reflux, 8
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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
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methyl iodide in presence of NaH afforded 73. Deprotection of the acetyl group of 73 furnished 74. Finally, the amine 74 was reacted with 3-phenylpropylisocyanate 47 to give the urea 40. In
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order to prepare the urea 41, 3-phenylpropylamine 59 was converted to its N-methyl derivative 75 analogous to the preparation of 74. Further 75 was reacted with 65 to give the urea 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 K2CO3 to furnish 77a/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
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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. Reagents and conditions: (a) CH3COCl, TEA, CH2Cl2, 0 °C to RT, 3 h (b) CH3I, 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 CH3I, NaH, DMF, RT, 3 h, (g) CH3I or CH3CH2I, NaH, DMF, RT 3-15 h (h) CH3CH2I, or (CH3)2CHI, K2CO3, DMF, RT, 12 h.
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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 %
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increase in myosin ATPase activity at 10 µM. The results are shown in Table 1 - 3. Compound
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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
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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
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echocardiography [35] in seven-week-old Sprague-Dawley male rats. The results are shown in
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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. 4. Results and Discussion
Based on our previous report [25], compound 1 (CMA at 10 µM = 53.3% FS = 30.04%; EF = 18.27%) 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
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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.
L
R
n
1
Phenyl
Phenyl
1
2
Phenyl
Phenyl
2
3
Phenyl
0
4
Phenyl
1
5
Phenyl
2
6
Phenyl
m
ATPase activity % at 10 µM
Fractional Shoretning % increase
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Comp. No
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Table 1. Optimization of spacer and groups in the urea scaffold
Ejection Fraction % increase
CLogP
53.3
30.04
18.27
3.696
1
51.1
18.90
12.15
4.025
1
31.8
---
---
2.118
1
39.6
---
---
1.859
1
41.9
11.24
7.23
2.188
2
2
36.6
---
---
4.212
Phenyl
1
1
25.4
---
---
3.615
Phenyl
2
1
40.0
---
---
3.944
Phenyl
1
1
38.8
---
---
3.354
Phenyl
2
1
122.4
23.97
15.87
3.683
11
Phenyl
2
2
32.5
---
---
2.717
12
Phenyl
1
1
34.5
---
---
2.891
9 10
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8
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7
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1
Phenyl
2
1
91.6
17.62
11.55
3.220
14
Phenyl
2
1
95.1
20.47
13.58
3.944
15
Phenyl
2
1
35.0
---
---
3.944
16
Phenyl
2
1
31.0
---
---
4.738
17
Phenyl
2
1
37.3
---
---
5.451
18
Phenyl
2
1
73.8
19
Phenyl
2
1
33.3
---
---
5.452
20
Phenyl
2
1
40.6
---
---
3.768
21
Phenyl
2
1
56.2
---
---
3.768
22
Phenyl
2
1
55.2
6.37
4.16
2.798
23
Phenyl
2
1
45.3
---
---
4.190
24
Phenyl
2
1
38.6
---
---
4.168
Phenyl
2
1
65.4
12.52
8.76
4.168
Phenyl
2
1
29.4
---
---
1
1
9.5
---
---
3.839
1
1
30.5
---
---
3.615
29
1
1
20.8
---
---
2.891
30
2
1
19.1
---
---
2.331
27 28
Precipitated during formulation
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26
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25
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13
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4.534
2.432
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31
2
1
34.8
---
---
32
2
1
37.9
---
---
2.901
33
2
1
38.4
---
---
3.602
34
2
1
20.6
---
---
3.363
13.35
3.266
Omecamtiv mecarbil
58.0
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20.32
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---, activity not checked
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STD
2.107
In the first set of experiment (Table 1), compounds 3 (n = 0; m = 1, CMA = 31.8%), 4 (n = 1; m = 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
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(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
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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.
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4-Methoxy substitution on phenyl ring (L) as shown in compound 7 (n = 1; m = 1, CMA = 25.4%) 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 3,4dimethoxy substitution analogs as considering the superior activity of 10 (n = 2; m = 1, CMA = 122.4%) 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
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and phenyl ring (L) was further substantiated from the compounds 12 and 13 with N,Ndimethylsulfonamide substitution on phenyl ring (L). Compound 12 (n = 1; m = 1, CMA = 34.5%) showed weak activity, whereas compound 13 (n = 2; m = 1, CMA = 91.6%, FS =
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17.62%; EF = 11.55%) showed strong activity in vitro and in vivo. From these results, two
fixed as optimum chain length.
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carbon chain from the urea to the phenyl ring (L) and three carbon chain to phenyl ring (R) were
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
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= 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 =
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31.0%) 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
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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
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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)
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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-
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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
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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
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the phenyl ring (R) of the compound 5 were introduced as shown in compounds 30 (CMA = 19.1%), 31 (CMA = 34.8%) and 32 (CMA = 37.9%), respectively. All the compounds showed
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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 33 (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.
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Table 2. Ventricular cell contractility and Selectivity study in myosins ATPase % activity Cardiac myosin
Skeletal myosin
Smooth myosin
(at 10 µM)
(at 100 µM)
(at 100 µM)
1
25.3 + 2.2 at 5 µM
53.3
1.4
˗ 3.7
2
20.8 + 1.9 at 5 µM
51.1
˗ 2.1
˗ 4.7
10
22.2 + 3.1 at 5 µM
122.4
˗ 2.4
4.3
13
47.9 + 3.2 at 5 µM
91.6
14
19.7 + 3.2 at 5 µM
95.1
OM
33.8 + 4.1 at 1 µM
(˗)Blebbistatin
----
SC
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Ventricular cell contractility [rat] % change
˗ 3.8
˗ 1.9
3.5
5.3
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Compound No.
58.0
2.8
˗ 5.5
----
˗ 50.7
˗ 35.7
The potent compounds 10, 13 and 14 were analyzed for selectivity in cardiac myosin over
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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.
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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
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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).
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Table 3. SAR of compound 13 ATPase activity %
13
91.6
17.62
35
27.6
36
40.7
37
Ejection Fraction % increase
---
3.072
---
3.321
3.072
SC
3.220
---
---
---
32.6
---
---
85.1
CLogP
11.55
36.9
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38
39
---
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Structure of the Compound
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at 10 uM
Fractional Shoretning % increase
Comp. No
precipitated during formulation
3.321
3.620
38.96
24.19
2.956
47.6
23.19
15.47
2.956
69.0
0.46
0.27
3.502
43
39.5
---
---
3.485
44
30.3
---
---
4.560
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41
EP
52.3
40
42
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38.1
---
---
3.794
46
40.6
---
---
4.340
58.0
20.32
STD
Omecamtiv mecarbil
---, activity not checked
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45
13.35
3.266
The SAR of compound 13 with N,N-dimethylsulfonamide group on phenyl ring of (L) was
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further explored by studying the importance of the flexible chain and hydrogen bonding donor
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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 =
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36.9%), 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.
activity.
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The above results clearly indicate the importance of flexible carbon chains as spacers for the
To substantiate the importance of the HBD property of the urea function in highly active 13,
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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 = 24.19%) 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 =
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69.0%, 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.
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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 =
SC
30.3%) 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
ATPase in vitro and in vivo.
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either side of the urea NH with methyl group is much suitable for activating the cardiac myosin
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
TE D
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
EP
+ 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
AC C
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.
SC
RI PT
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)
M AN U
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
TE D
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
AC C
EP
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 = 23.97%; EF = 15.87%), 4-N,N-dimethyl sulfonamide moiety (13, CMA = 91.6%, FS = 17.62%;
RI PT
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).
SC
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
M AN U
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
TE D
(CMA = 47.6%, FS = 23.19%; EF = 15.47%; % change in ventricular cell contractility at 5 µM = 63.5 + 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,
EP
41 were highly selective for cardiac myosin over skeletal and smooth myosins and thus proving them these new urea derivatives is a novel scaffold for discovery of cardiac myosin activators for
AC C
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
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
RI PT
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-
SC
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
M AN U
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.
TE D
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
EP
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.
AC C
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,Ndiisopropylethylamine (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
RI PT
compounds.
6.1.1.1. 4-(3-(3-Phenylpropyl)ureido)benzenesulfonamide (3); Prepared by general procedure (i). Yield 85%; White solid; Rf 0.42 (Ethylacetate); mp 207-209 °C; IR (neat): 3354, 3321, 3032,
SC
2947, 2358, 2342, 1681cm-1; 1H-NMR (DMSO-d6) δ 1.71-1.78 (m, 2H), 2.61 (t, J = 7.80 Hz, 2H), 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,
M AN U
2H), 7.67 (d, J = 8.40 Hz, 2H), 8.81 (s, 1H). 13C-NMR (DMSO-d6) δ 31.32, 32.40, 38.66, 116.82, 125.79, 126.76, 128.31, 128.36, 136.04, 141.72, 143.76, 154.92; HRMS Calcd for C16H19N3O3S m/z [M+H] 334.1225, found 334.1248. HPLC purity 100% 6.1.1.2. 4-((3-(3-Phenylpropyl)ureido)methyl)benzenesulfonamide (4); Prepared by general
TE D
procedure (i). Yield 82%; White solid; Rf 0.35 (Ethylacetate); mp 209 °C; IR (neat): 3355, 2923, 2357, 2341, 1623 cm-1; 1H-NMR (DMSO-d6) δ 1.66-1.70 (m, 2H), 2.57 (t, J = 7.80 Hz, 2H), 3.02-3.06 (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),
EP
7.13 -7.30 (m, 7H), 7.40 (d, J = 8.40 Hz, 2H), 7.76 (d, J = 8.40 Hz, 2H); HRMS Calcd for C17H21N3O3S m/z [M+H] 348.1382, found 349.1405.
AC C
6.1.1.3. 4-(2-(3-(3-Phenylpropyl)ureido)ethyl)benzenesulfonamide (5); Prepared by general procedure (i). Yield 84%; White solid; Rf 0.37 (Ethylacetate); mp 146-148 °C; IR (neat): 3327, 2923, 2855, 2358, 2341, 1697 cm-1; 1H-NMR (DMSO-d6) δ 1.62-1.69 (m, 2H), 2.56 (t, J = 7.20 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), 5.90-5.92 (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 = 8.40 Hz, 2H); 13C-NMR (DMSO-d6) δ 31.74, 32.44, 35.83, 38.79, 40.49, 125.72, 128.32,
ACCEPTED MANUSCRIPT
129.16, 141.91, 142.04, 144.15, 158.07; HRMS Calcd for C18H23N3O3S m/z [M+H] 362.1538, found 362.1567. HPLC purity 100% 6.1.1.4. 4-(2-(3-(4-Phenylbutyl)ureido)ethyl)benzenesulfonamide (6); Prepared by general
RI PT
procedure (ii). Yield 68%; White solid; Rf 0.38 (Ethylacetate); mp: 134-137 °C; IR (neat): 3254, 2946, 1698, 1557, 1164, 829, 667 cm-1; 1H-NMR (DMSO-d6) δ 1.31-1.38 (m, 2H), 1.49-1.56 (m, 2H), 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
SC
(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, 2H), 7.36-7.38 (d, J = 8.40 Hz, 2H), 7.72-7.74 (d, J = 8.40 Hz, 2H);
13
C-NMR (DMSO-d6) δ
M AN U
28.88, 30.16, 35.33, 36.38, 39.45, 41.04, 126.09, 126.13, 128.69, 128.74, 129.57, 142.41, 142.69, 144.54, 158.42; HRMS Calcd for C19H25N3O3S m/z [M+H] 376.1695, found 376.1723. HPLC purity 99.25%
6.1.1.5. 1-(4-Methoxybenzyl)-3-(3-phenylpropyl)urea (7); Prepared by general procedure (i).
TE D
Yield 88%; White solid; Rf 0.42 (2 : 1 Ethylacetate : Hexane); mp 114-116 °C; IR (neat): 3327, 2923, 2357, 2341, 1618 cm-1; 1H-NMR (DMSO-d6) δ 1.63-1.71 (m, 2H), 2.56 (t, J = 7.80 Hz, 2H), 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,
EP
1H), 6.87 (d, J = 7.40 Hz, 2H), 7.16-7.20 (m, 5H), 7.26-7.30 (m, 2H); HRMS Calcd for C18H22N2O2 m/z [M+H] 299.1760, found 299.1784.
AC C
6.1.1.6. 1-(4-Methoxyphenethyl)-3-(3-phenylpropyl)urea (8); Prepared by general procedure (i). Yield 86%; White solid; Rf 0.44 (2 : 1 Ethylacetate : Hexane); mp: 93-96 °C; IR (neat): 3329, 3030, 2932, 1615, 1560, 1511, 1243, 1040, 748, 696 cm-1; 1H-NMR (CDCl3) δ 1.77-1.81 (m, 2H), 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), 3.78 (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), 7.157.20 (m, 3H), 7.27-7.30 (m, 2H). 13C-NMR (CDCl3) δ 31.72, 33.06, 35.35, 39.94, 41.74, 55.16,
ACCEPTED MANUSCRIPT
113.92, 125.94, 128.37, 128.45, 129.76, 131.13, 141.60, 158.16, 158.25; HRMS Calcd for C19H24N2O2 m/z [M+H] 313.1916, found 313.1942. HPLC purity 98.46% 6.1.1.7. 1-(3,4-Dimethoxybenzyl)-3-(3-phenylpropyl)urea (9); Prepared by general procedure (i).
RI PT
Yield 82%; Pale yellow solid; Rf 0.40 (2 : 1 Ethylacetate : Hexane); mp: 110-112 °C; IR (neat): 3340, 1621, 1568, 1515, 1337, 1262, 1233, 1141, 1026 cm-1; 1H-NMR (CDCl3) δ 1.77-1.84 (m, 2H), 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
SC
(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 (CDCl3) δ 31.77, 33.06, 40.01, 44.31, 55.80, 55.87, 110.75, 111.11, 119.55, 125.89, 128.28,
M AN U
128.37, 131.77, 141.49, 148.23, 149.06, 158.35; HRMS Calcd for C19H24N2O3 m/z [M+H] 329.1865, found 329.1891. HPLC purity 100% 6.1.1.8.
1-(3,4-Dimethoxyphenethyl)-3-(3-phenylpropyl)urea
(10);
Prepared
by
general
procedure (i). Yield 90%; White solid; Rf 0.42 (2 : 1 Ethylacetate : Hexane); mp 102-104 °C; IR
TE D
(neat): 3338, 2973, 2860, 1667, 1618, 1572, 1508 cm-1; 1H-NMR (CDCl3) δ 1.76-1.83 (m, 2H), 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).
EP
13C-NMR (CDCl3) δ 31.71, 33.07, 35.87, 40.01, 41.67, 55.77, 55.83, 111.29, 111.96, 120.68, 125.97, 128.36, 128.45, 131.73, 141.58, 147.62, 149.01, 158.24; HRMS Calcd for C20H26N2O3
AC C
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; Rf 0.45 (2 : 1 Ethylacetate : Hexane); mp: 109-112 °C; IR (neat): 3244, 2944, 1683, 1558, 1540, 1508, 1235, 1030, 802, 701, 668 cm-1; 1H-NMR (DMSO-d6) δ 1.46-1.53 (m, 2H), 1.59-1.65 (m, 2H), 2.62 (t, J = 7.80 Hz, 2H), 2.75 (t, J = 7.80 Hz, 2H), 3.113.16 (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
(m, 2H), 6.79 (d, J = 8.00 Hz, 1H), 7.15-7.20 (m, 3H), 7.25-7.29 (m, 2H); 13C-NMR (CDCl3) δ 28.54, 29.69, 35.41, 35.86, 40.24, 41.65, 55.72, 55.80, 111.18, 111.85, 120.65, 125.82, 128.35, 128.40, 131.70, 142.18, 147.55, 148.94, 158.20; HRMS Calcd for C21H28N2O3 m/z [M+H]
RI PT
357.2178, found 357.2200.
6.1.1.10. 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
SC
at ambient temperature for 45 minutes. Then the reaction mixture was cooled and CH3I (2.20 mmol) was added The reaction mixture was further stirred at ambient temperature for 3 h. After
M AN U
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
TE D
cm-1; 1H-NMR (CDCl3) δ 1.78-1.86 (m, 2H), 2.62-2.68 (m, 2H), 2.65 (s, 6H), 3.19-3.24 (m, 2H), 4.40 (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), 7.35 (d, J = 8.20 Hz, 2H), 7.59 (d, J = 8.20 Hz, 2H). 13C-NMR (CDCl3) δ 31.78, 33.01, 37.80,
EP
39.91, 43.33, 125.96, 127.57, 127.80, 128.38, 128.45, 133.52, 141.59, 145.55, 158.43; HRMS Calcd for C19H25N3O3S m/z [M+H] 376.1695, found 376.1717. HPLC purity 99.61%
AC C
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; Rf 0.38 (Ethylacetate); mp 98-100 °C IR (neat): 3335, 3015, 2983, 1652, 1620, 1549, 1488 cm-1; 1H-NMR (CDCl3) 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 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 (CDCl3) δ 31.74, 33.07, 36.27, 37.84, 39.92, 41.04, 125.99, 127.96,
ACCEPTED MANUSCRIPT
128.39, 128.47, 129.58, 133.34, 141.61, 145.08, 158.16; HRMS Calcd for C20H27N3O3S 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
RI PT
(i). Yield 90%; Yellow powder; Rf 0.44 (2 : 1 Ethylacetate : Hexane); mp 91-93 °C; IR (neat): 3341, 2977, 2856, 1665, 1621, 1562, 1504 cm-1; 1H-NMR (CDCl3) δ 1.78-1.85 (m, 2H), 2.64 (t, 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),
SC
4.37 (bs, 1H), 4.44 (bs, 1H), 6.85-6.91 (m, 2H), 7.11-7.30 (m, 7H). 13C-NMR (CDCl3) δ 30.94, 31.71, 33.08, 40.03, 40.63, 55.25, 110.39, 120.70, 125.95, 127.45, 127.84, 128.40, 128.45,
313.1945. HPLC purity 99.21%
M AN U
130.67, 141.64, 157.54, 158.35; HRMS Calcd for C19H24N2O2 m/z [M+H] 313.1916, found
6.1.1.13. 1-(3-Methoxyphenethyl)-3-(3-phenylpropyl)urea (15); Prepared by general procedure (i). Yield 83%; White solid; Rf 0.44 (2 : 1 Ethylacetate : Hexane); mp: 66-69 °C; IR (neat): 3318,
TE D
3005, 2938, 1614, 1570, 1541, 1457, 1258, 1169, 1043, 789, 695 cm-1; 1H-NMR (DMSO-d6) δ 1.59-1.69 (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, 2H), 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),
EP
7.14-7.22 (m, 4H), 7.25-7.30 (m, 2H); 13C-NMR (DMSO-d6) δ 32.33, 32.98, 36.68, 39.30, 41.26, 55.31, 111.91, 114.74, 121.36, 126.13, 128.72, 129.73, 141.84, 142.31, 158.46, 159.74; HRMS
AC C
Calcd for C19H24N2O2 m/z [M+H] 313.1916, found 313.1940. 6.1.1.14. 1-(4-Chlorophenethyl)-3-(3-phenylpropyl)urea (16); Prepared by general procedure (i). Yield 74%; White solid; Rf 0.35 (1 : 1 Ethylacetate : Hexane); mp 120-122 °C; IR (neat): 3335, 2948, 2883, 1672, 1618, 1555, 1510 cm-1; 1H-NMR (CDCl3) δ 1.77-1.85 (m, 2H), 2.64 (t, J = 8.00 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), 7.11-7.30 (m, 9H). 13C-NMR (CDCl3) δ 31.68, 33.07, 35.69, 39.99, 41.37, 126.01, 128.37,
ACCEPTED MANUSCRIPT
128.48, 128.68, 130.19, 132.20, 137.71, 141.54, 158.17; HRMS Calcd for C18H21ClN2O m/z [M+H] 317.1421, found 317.1441. 6.1.1.15. 1-(2,4-Dichlorophenethyl)-3-(3-phenylpropyl)urea (17); Prepared by general procedure
RI PT
(i). Yield 80%; Yellow powder; Rf 0.38 (1 : 1 Ethylacetate : Hexane); mp: 102-104 °C; IR (neat): 3310, 2950, 2801, 1662, 1615, 1570, 1501 cm-1; 1H-NMR (CDCl3) δ 1.77-1.84 (m, 2H), 2.64 (t, J = 7.80 Hz, 2H), 2.90 (t, J = 7.20 Hz, 2H), 3.14-3.19 (m, 2H), 3.36-3.41 (m, 2H), 4.32-4.38 (m, 13
C-NMR (CDCl3) δ 31.68, 33.10,
SC
2H), 7.13-7.21 (m, 5H), 7.26-7.30 (m, 2H), 7.37 (s, 1H);
33.60, 39.89, 40.10, 126.04, 127.25, 128.40, 128.51, 129.37, 131.92, 132.96, 134.79, 135.49,
M AN U
141.54, 158.01; HRMS Calcd for C18H20Cl2N2O m/z [M+H] 351.1031, found 351.1049. 6.1.1.16. 1-(4-Methylphenethyl)-3-(3-phenylpropyl)urea (18); Prepared by general procedure (i). Yield 85%; White solid; Rf 0.36 (1 : 1 Ethylacetate : Hexane); mp 101-103 °C; IR (neat): 3319, 2937, 2861, 1633, 1615, 1569, 1494 cm-1; 1H-NMR (CDCl3) δ 1.75-1.82 (m, 2H), 2.31 (s, 3H),
TE D
2.63 (t, J = 7.20 Hz, 2H), 2.75 (t, J = 7.80 Hz, 2H), 3.12-3.17 (m, 2H), 3.36-3.40 (m, 2H), 4.274.28 (m, 2H), 7.06-7.20 (m, 7H), 7.26-7.29 (m, 2H). 13C-NMR (CDCl3) δ 20.90, 31.70, 33.10, 35.83, 40.03, 41.66, 125.96, 128.39, 128.46, 128.73, 129.30, 135.97, 136.07, 141.63, 158.22;
EP
HRMS Calcd for C19H24N2O m/z [M+H] 297.1967, found 297.1995. HPLC purity 99.08% 6.1.1.17. 1-(4-Isopropylphenethyl)-3-(3-phenylpropyl)urea (19); Prepared by general procedure
AC C
(i). Yield 82%; White solid; Rf 0.40 (1 : 1 Ethylacetate : Hexane); mp: 89-91 °C; IR (neat): 3338, 2973, 2860, 1667, 1618, 1572, 1508 cm-1; 1H-NMR (CDCl3) δ 1.24 (d, J = 7.20 Hz, 6H), 1.761.84 (m, 2H), 2.64 (t, J = 7.60 Hz, 2H), 2.76 (t, J = 7.80 Hz, 2H), 2.85-2.91 (m, 1H), 3.14-3.19 (m, 2H), 3.37-3.42 (m, 2H), 4.18-4.24 (m, 2H), 7.10-7.12 (m, 2H), 7.16-7.20 (m, 5H), 7.26-7.30 (m, 2H);
13
C-NMR (CDCl3) δ 23.94, 31.70, 33.13, 33.64, 35.82, 40.10, 41.64, 125.99, 126.70,
ACCEPTED MANUSCRIPT
128.40, 128.49, 128.79, 136.43, 141.63, 147.09, 158.08; HRMS Calcd for C21H28N2O m/z [M+H] 325.2280, found 325.2299. 6.1.1.18. 4-(2-(3-(3-Phenylpropyl)ureido)ethyl)benzoic acid
(20); Prepared by general
3580, 3115, 2940, 1670, 1620, 1524, 1463, 1270 cm-1;
1
RI PT
procedure (i). Yield 75%; Yellow powder; Rf 0.25 (Ethylacetate); mp: 142-145 °C; IR (neat): H-NMR (DMSO-d6) δ 1.61-1.66 (m,
2H), 2.54 (m, 2H), 2.74-2.78 (m, 2H), 2.91-3.01 (m, 2H), 3.22-3.29 (m, 2H), 5.78-5.82 (m, 1H),
SC
5.90-5.94 (m, 1H), 7.16-7.21 (m, 3H), 7.26-7.34 (m, 4H), 7.86-7.89 (m, 2H) 12.80 (bs, 1H); 13CNMR (DMSO-d6) δ 31.74, 32.43, 36.07, 38.78, 40.43, 125.71, 128.31, 128.75, 128.92, 129.39,
M AN U
141.92, 145.25, 158.07, 167.39; HRMS Calcd for C19H22N2O3 m/z [M+H] 327.1709, found 327.1737.
6.1.1.19. 1-(4-Nitrophenethyl)-3-(3-phenylpropyl)urea (21); Prepared by general procedure (i). Yield 84%; White solid; Rf 0.45 (2 : 1 Ethylacetate : Hexane); mp 101-103 °C; IR (neat): 3298,
TE D
3003, 2986, 1662, 1652, 1616, 1533, 1497 cm-1; 1H-NMR (CDCl3) δ 1.77-1.85 (m, 2H), 2.64 (t, J = 7.80 Hz, 2H), 2.91 (t, J = 8.00 Hz, 2H), 3.14-3.19 (m, 2H), 3.43-3.48 (m, 2H), 4.07 (bs, 2H), 7.17-7.36 (m, 7H), 8.12-8.15 (m, 2H); 13C-NMR (CDCl3) δ 31.70, 33.06, 36.39, 39.97, 40.98,
EP
123.71, 126.04, 128.35, 128.50, 129.75, 141.49, 146.62, 147.45, 158.10; HRMS Calcd for C18H21N3O3 m/z [M+H] 328.1661, found 328.1679. HPLC purity 100%
AC C
6.1.1.20. 1-(4-Aminophenethyl)-3-(3-phenylpropyl)urea (22); To a solution of compound 21 (0.65 mmol) in methanol, Pd/C (10%, 190 mg) was added under N2 atmosphere. The reaction mixture was hydrogenated using parr shaker for 10 h. After the formation of the reaction, the reaction mixture was filtered through celite and concentrated under vacuum. The product was purified by column chromatography. Yield 86%; Yellow powder; Rf 0.25 (2 : 1 Ethylacetate : Hexane); mp 93-95 °C; IR (neat): 3405, 3325, 2940, 2858, 1669, 1620, 1567 cm-1; 1H-NMR
ACCEPTED MANUSCRIPT
(CDCl3) δ 1.75-1.83 (m, 2H), 2.63 (t, J = 7.80 Hz, 2H), 2.68 (t, J = 8.00 Hz, 2H), 3.13-3.18 (m, 2H), 3.32-3.37 (m, 2H), 3.58 (bs, 2H), 4.17-4.19 (m, 2H), 6.63 (d, J = 8.00 Hz, 2H), 6.97 (d, J = 8.00 Hz, 2H), 7.16-7.30 (m, 5H); 13C-NMR (CDCl3) δ 31.70, 33.08, 35.33, 40.00, 41.80,
RI PT
115.36, 125.94, 128.39, 128.45, 128.99, 129.65, 141.68, 144.84, 158.29; HRMS Calcd for C18H23N3O m/z [M+H] 298.1919, found 298.1942. HPLC purity 99.84% 6.1.1.21. 1-(4-(Dimethylamino)phenethyl)-3-(3-phenylpropyl)urea
(23); Compound 22 (0.5
SC
mmol) was dissolved in acetone and K2CO3 (1.2 mmol) was added followed by the addition of CH3I (1.2 mmol). The reaction mixture was refluxed for 2 h. After the completion of the
M AN U
reaction, the reaction mixture was concentrated, water was added and extracted using ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The obtained crude residue was purified by column chromatography. Yield 72%; White solid; Rf 0.42 (2 : 1 Ethylacetate : Hexane); mp 90-92 °C; IR (neat): 3315, 2975, 2886, 1650,
TE D
1624, 1614, 1540, 1496 cm-1; 1H-NMR (CDCl3) δ 1.77-1.80 (m, 2H), 2.62 (t, J = 8.00 Hz, 2H), 2.69 (t, J = 8.00 Hz, 2H), 2.91 (s, 6H), 3.13-3.16 (m, 2H), 3.33-3.36 (m, 2H), 4.21-4.25 (m, 2H), 6.69 (d, J = 8.00 Hz, 2H), 7.06 (d, J = 8.00 Hz, 2H), 7.16-7.27 (m, 5H). 13C-NMR (CDCl3) δ
EP
32.02, 33.43, 35.48, 40.36, 41.03, 42.21, 113.34, 126.26, 128.72, 128.77, 129.79, 129.91, 141.98, 149.75, 158.57; HRMS Calcd for C20H27N3O m/z [M+H] 326.2232, found 326.2255.
AC C
6.1.1.22. 1-(4-Fluorophenethyl)-3-(3-phenylpropyl)urea (24); Prepared by general procedure (i). Yield 82%; White solid; Rf 0.32 (1 : 1 Ethylacetate : Hexane); mp: 83-86 °C; IR (neat): 3352, 3307, 3023, 2969, 1739, 1618, 1560, 1365, 1217, 696 cm-1; 1H-NMR (CDCl3) δ 1.76-1.83 (m, 2H), 2.63 (t, J = 7.80 Hz, 2H), 2.76 (t, J = 8.00 Hz, 2H), 3.13-3.18 (m, 2H), 3.35-3.40 (m, 2H), 4.30-4.31 (m, 2H), 6.95-7.00 (m, 2H), 7.11-7.20 (m, 5H), 7.25-7.30 (m, 2H). 13C-NMR (CDCl3)
ACCEPTED MANUSCRIPT
δ 31.70, 33.05, 35.50, 35.91, 41.55, 115.21, 115.42, 125.98, 128.36, 128.46, 130.24, 134.82, 141.54, 158.24, 162.80; HRMS Calcd for C18H21FN2O m/z [M+H] 301.1716, found 301.1739. 6.1.1.23. 1-(2-Fluorophenethyl)-3-(3-phenylpropyl)urea (25); Prepared by general procedure (i).
RI PT
Yield 85%; White solid; Rf 0.32 (1 : 1 Ethylacetate : Hexane); mp 82-83 °C; IR (neat): 3328, 3011, 2991, 1648, 1618, 1543, 1498 cm-1; 1H-NMR (CDCl3) δ 1.77-1.84 (m, 2H), 2.64 (t, J = 8.00 Hz, 2H), 2.84 (t, J = 8.00 Hz, 2H), 3.14-3.19 (m, 2H), 3.38-3.43 (m, 2H), 4.29-4.34 (m,
SC
2H), 7.00-7.29 (m, 9H); 13C-NMR (CDCl3) δ 29.81, 31.69, 33.08, 40.00, 40.45, 115.20, 115.41, 124.18, 125.96, 128.40, 128.46, 131.23, 141.63, 158.25, 160.13, 162.57; HRMS Calcd for
M AN U
C18H21FN2O m/z [M+H] 301.1716, found 301.1746. HPLC purity 100%
6.1.1.24. 4-Methoxy-2-(2-(3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (26): 6.1.1.24.1. Preparation of N-(3-methoxyphenethyl)acetamide (50): To a cooled solution of 2-(3 methoxyphenyl)ethanamine (49, 6.20 mmol) in methylene chloride at 0 °C triethylamine (9.30
TE D
mmol) was added. After 5 minutes, acetylchloride (9.30 mmol) was added at the same temperature and allowed to stir for 3 h at ambient temperature. The reaction was monitored by TLC. After the completion of the reaction, the reaction mixture was quenched with water and
EP
extracted using methylene chloride. The organic layer was washed with water twice, dried over anhydrous sodium sulfate and concentrated under vacuum to get the crude product 50. Without
AC C
purification the product was taken for next step. Yield 80%; 1H-NMR (CDCl3) δ 1.94 (s, 3H), 2.79 (t, J = 7.40 Hz, 2H), 3.49-3.54 (m, 2H), 3.80 (s, 3H), 6.73-6.84 (m, 3H), 7.24 (t, J = 7.60 Hz, 1H).
6.1.1.24.2. Preparation of N-(5-methoxy-2-sulfamoylphenethyl)acetamide (51): To a cooled solution of chlorosulfuric acid at 0 °C, 50 (3.33 mmol) dissolved in methylene chloride was added slowly and allowed to stir for 3 h to attain ambient temperature. The reaction was
ACCEPTED MANUSCRIPT
monitored by TLC. After the completion of the reaction, the reaction mixture was slowly added to crushed ice with vigorous stirring. After all ice had melt down, the reaction mass was extracted using methylene chloride and concentrated to obtain the sulfonyl chloride. The crude
RI PT
sulfonyl chloride was dissolved in THF and NH4OAC (10.0 mmol) was added and refluxed for 5 h. After the completion of the reaction, water was added and the reaction mixture was extracted using ethyl acetate. The crude product 51 was taken for the next step. Yield 65%; 1H-NMR
SC
(DMSO-d6) δ 1.82 (s, 3H), 3.07-3.10 (m, 2H), 3.27-3.32 (m, 2H), 3.81 (s, 3H), 6.91-6.93 (m, 2H), 7.42 (s, 2H), 7.80 (d, J = 8.20 Hz, 1H), 8.13-8.15 (m, 1H).
M AN U
6.1.1.24.3. Preparation of 2-(2-aminoethyl)-4-methoxybenzenesulfonamide (52): To a solution of 51 (0.10 mmol) dissolved in n-butanol (2 mL), 3N HCl (100 mL) was added and stirred at reflux for 8 h. After the completion of the reaction, the reaction mixture was extracted using diethyl ether. The aqueous layer was neutralized using solid potassium carbonate and extracted using
TE D
ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated. The amine 52 was obtained as white solid. Yield 72%; 1H-NMR (DMSO-d6) δ 2.80-2.83 (m, 2H), 2.99-3.03 (m, 2H), 3.80 (s, 3H), 6.87-6.93 (m, 2H), 7.78 (d, J = 8.20 Hz, 1H).
EP
6.1.1.24.4. 4-Methoxy-2-(2-(3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (26): Prepared by the reaction of amine 52 and 3-phenylpropylisocyanate 47 according to the general
AC C
procedure (i). Yield 52%; White solid; Rf 0.35 (Ethylacetate); mp 182-184 °C; IR (neat): 3341, 1635, 1540, 1366, 1320, 1124, 1089, 940 cm-1; 1H-NMR (DMSO-d6) δ 1.63-1.70 (m, 2H), 2.552.59 (m, 2H), 2.99-3.07 (m, 4H), 3.18-3.22 (m, 2H), 3.80 (s, 3H), 6.09-6.12 (m, 2H), 6.89-6.91 (m, 2H), 7.17-7.21 (m, 3H), 7.26-7.30 (m, 2H), 7.50 (s, 2H, NH2-D2O exchangeable), 7.80 (d, J = 8.40 Hz, 1H). 13C-NMR (DMSO-d6) δ 31.20, 31.74, 32.47, 33.25, 40.78, 55.43, 111.13,
ACCEPTED MANUSCRIPT
117.11, 125.78, 125.79, 128.36, 129.66, 134.85, 139.47, 141.91, 158.70, 161.54; HRMS Calcd for C19H25N3O4S m/z [M+H] 392.1644, found 392.1669. 6.1.1.25. Preparation of 1-benzyl-3-(3-(4-fluorophenyl)propyl)urea (27)
RI PT
6.1.1.25.1. Preparation of (E)-3-(4-fluorophenyl)acrylamide (54): To a stirred solution of 4fluorocinnamic acid 53 (18.05 mmol) in methylene chloride at 0 °C, triethylamine (54.16 mmol), EDC.HCl (27.05 mmol) and NH4Cl (36.11 mmol) were added. The resulting reaction mixture
SC
was stirred at room temperature for 16 h. Completion of the reaction was confirmed by TLC. The reaction was diluted with methylene chloride and washed with 1N HCl. The organic layer was
M AN U
dried over anhydrous sodium sulfate and then concentrated under reduced pressure to afford 54 as brown sticky oil. The crude product was used for the next step without further purification. Yield 83%.
6.1.1.25.2. Preparation of tert-butyl-3-(4-fluorophenyl)propylcarbamate (55): To a stirred
TE D
solution of 54 (10.89 mmol) in THF (30 mL) at 0 °C, LiAlH4 (2.0 M solution in THF, 32.69 mmol) was added slowly. The resulting pale yellow suspension was heated at 70 oC for a further 3 h. After cooling to 0 °C, the reaction mixture was quenched with 1N NaOH solution followed
EP
by ice cold water (2 mL). To this reaction mixture, a solution of Boc2O (16.34 mmol) in THF was added and the resulting reaction mixture was stirred at ambient temperature for 16 h. The
AC C
reaction mixture was filtered through celite bed, washed with ethyl acetate (50 mL) and the filtrate was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The crude residue was purified by column chromatography to afford 55 as a light brown solid. Yield 40 %, 1H NMR (CDCl3) δ 1.45 (s, 9H), 1.79 (m, 2H), 2.62 (t, J = 7.68 Hz, 2H), 3.08-3.21 (m, 2H), 6.97 (d, J = 8.29 Hz, 2H), 7.13 (d, J = 7.93 Hz, 2H).
ACCEPTED MANUSCRIPT
6.1.1.25.3. Preparation of 3-(4-fluorophenyl)propan-1-amine hydrochloride (56): To a stirred solution of 55 (3.95 mmol) in methylene chloride HCl (4.0 M solution of in 1, 4-dioxane, 7.91 mmol) was slowly added. The resulting reaction mixture was stirred at ambient temperature for 2
RI PT
h. After the completion of the reaction, the solvent was evaporated from the reaction mixture to afford 56 as a light brown solid. Yield 80%. 1H NMR (DMSO-d6) δ 1.84 (m, 2H), 2.64 (t, J = 7.68 Hz, 2H), 2.71-2.80 (m, 2H), 7.10-7.16 (m, 2H), 7.24-7.29 (m, 2H), 8.00 (bs, 2H).
SC
6.1.1.25.4. Preparation of 1-benzyl-3-(3-(4-fluorophenyl)propyl)urea (27); Prepared from the reaction of 56 with amine (‘L’ and ‘n’ are mentioned in Table 1) according to the general
M AN U
procedure (ii). Yield 75%; White solid; Rf 0.30 (1 : 1 Ethylacetate : Hexane); mp: 100-103 °C; IR (neat): 3324, 3035, 1615, 1509, 1454, 1260, 1223, 1103, 1028, 816, 732 cm-1;
1
H NMR
(DMSO-d6) δ 1.60-1.71 (m, 2H), 2.52-2.59 (m, 2H), 3.01 (m, 2H), 4.20 (d, J = 5.85 Hz, 2H), 5.98 (bs, 1H), 6.28 (bs, 1H), 7.05-7.12 (m, 2H), 7.18-7.26 (m, 5H), 7.28-7.34 (m, 2H); 13C NMR
TE D
(DMSO-d6) δ 32.17, 32.49, 39.42, 43.51, 115.50, 115.70, 127.22, 127.70, 128.90, 130.61, 130.72, 138.63, 141.75, 158.82; HRMS Calcd for C17H19FN2O m/z [M+H] 287.1560, found 287.1588.
EP
6.1.1.26. Preparation of 1-Benzyl-3-(3-(4-methoxyphenyl)propyl)urea (28): 6.1.1.26.1. Preparation of 1-(3-isocyanatopropyl)-4-substituted benzene (58): To a solution of 4-
AC C
(4-substituted phenyl)butanoic acid 57 (15.5 mmol) in acetone (60 ml) at ambient temperature, triethylamine (18.6 mmol) was added slowly. Then the reaction mixture was cooled to -10 °C, and methyl chloroformate (18.6 mmol) in acetone (10 mL) was added within 20-30 minutes and the reaction was stirred for another 30 minutes. Then a solution of sodium azide (31.1 mmol) in water (6 mL) was added slowly and stirred for another 1 h. The reaction mixture was quenched with ice water (300 mL) and stirred for 5-10 minutes. To this aqueous solution toluene (250 mL)
ACCEPTED MANUSCRIPT
was added and stirred for another 20-30 minutes. The toluene layer was collected and aqueous layer was once again extracted with toluene (100 mL). The combined toluene layer was dried over anhydrous Na2SO4 and concentrated to about 100 mL and then refluxed for 3 h. After the
RI PT
formation of the product, the solvent was evaporated and the crude product 58a was used as such for the next step. Yield 65%.
6.1.1.26.2. Preparation of 1-Benzyl-3-(3-(4-methoxyphenyl)propyl)urea (28), Prepared by the
SC
reaction of 1-(3-isocyanatopropyl)-4-methoxybenzene (58a) and amine (‘L’ and ‘n’ are mentioned in Table 1) according to the general procedure (i). Yield 84%; White solid; Rf 0.40 (2
M AN U
: 1 Ethylacetate : Hexane); mp: 100-103 °C; IR (neat): 3274, 2941, 1698, 1557, 1538, 1253, 1033, 801, 696, 668 cm-1; 1H-NMR (DMSO-d6) δ 1.60-1.67 (m, 2H), 2.47 (t, J = 7.80 Hz 2H), 2.98-3.03 (m, 2H), 3.72 (s, 3H), 4.20 (d, J = 6.00 Hz 2H), 5.97-5.99 (m, 1H), 6.28-6.31 (m, 1H), 6.83-6.85 (d, J = 8.00 Hz 2H), 7.09-7.11 (d, J = 7.40 Hz 2H), 7.20-7.25 (m, 3H), 7.31-7.33 (m,
TE D
2H); 13C-NMR (DMSO-d6) δ 31.57, 32.07, 42.91, 54.96, 55.02, 113.81, 126.66, 127.12, 128.34, 129.32, 133.78, 141.18, 157.52, 158.25; HRMS Calcd for C18H22N2O2 m/z [M+H] 299.1760, found 299.1779.
EP
6.1.1.27. 4-(3-(3-Benzylureido)propyl)-N,N-dimethylbenzenesulfonamide (29): 6.1.1.27.1. Preparation of N-(3-phenylpropyl)acetamide (60): To a cooled solution of 3-
AC C
phenylpropylamine 59 (6.20 mmol) in methylene chloride at 0 °C, triethylamine (9.30 mmol) was added. After 5 minutes, acetylchloride (9.30 mmol) was added at the same temperature and allowed to stir for 3 h at ambient temperature. The reaction was monitored by TLC. After the completion of the reaction, the reaction mixture was quenched with water and extracted using methylene chloride. The organic layer was washed with water twice, dried over anhydrous sodium sulfate and concentrated to get the crude product. Without purification the product 60
ACCEPTED MANUSCRIPT
was taken for next step. Yield 80%; 1H-NMR (CDCl3) δ 1.81-1.87 (m, 2H), 1.95 (s, 3H), 2.66 (t, J = 7.20 Hz, 2H), 3.27-3.32 (m, 2H), 5.51 (bs, 1H), 7.18-7.21 (m, 3H), 7.28-7.31 (m, 2H). 6.1.1.27.2. Preparation of N-(3-(4-(N,N-dimethylsulfamoyl)phenyl)propyl)acetamide (61): To a
RI PT
cooled solution of chlorosulfuric acid (2 mL) at 0 °C, 60 (3.33 mmol) dissolved in methylene chloride (2 mL) was added slowly and allowed to stir for 3 h to attain ambient temperature. The reaction was monitored by TLC. After the completion of the reaction, the reaction mixture was
SC
slowly added to crushed ice with vigorous stirring. After all ice has melt down, the reaction mass was extracted using methylene chloride and concentrated to get sulfonyl chloride. In the next
M AN U
step, NaHCO3 (1.99 mmol) was added to a solution of N,N-dimethylamine hydrochloride (2.66 mmol) in THF (10 mL) at 0 °C to generate free amine. To this reaction mixture, the obtained sulfonyl chloride (1.33 mmol) dissolved in THF was added and stirred for 5 h at ambient temperature. After the completion of the reaction, water was added and the reaction mixture was
TE D
extracted using ethyl acetate. The product was purified by column chromatography. Yield 65%; 1H-NMR (CDCl3) δ 1.83-1.89 (m, 2H), 1.98 (s, 3H), 2.70 (s, 6H), 2.73-2.81 (m, 2H), 3.28-3.33 (m, 2H), 5.64 (bs, 1H), 7.35 (d, J = 8.20 Hz, 2H), 7.69 (d, J = 8.20 Hz, 2H).
EP
6.1.1.27.3. Preparation 4-(3-aminopropyl)-N,N-dimethylbenzenesulfonamide (62): To a solution of 61 (0.10 mmol) dissolved in n-butanol (2 mL), 3N HCl (100 mL) was added and stirred at
AC C
reflux for 8 h. After the completion of the reaction, the reaction mixture was extracted using diethyl ether. The aqueous layer was neutralized using solid potassium carbonate and extracted using ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The amine 62 was obtained as white solid which was used for the next step. Yield 75%; 1H-NMR (CDCl3) δ 1.80-1.84 (m, 2H), 2.70 (s, 6H), 2.74-2.81 (m, 4H), 5.64 (bs, 1H), 7.36 (d, J = 8.20 Hz, 2H), 7.69 (d, J = 8.20 Hz, 2H).
ACCEPTED MANUSCRIPT
6.1.1.27.4. 4-(3-(3-Benzylureido)propyl)-N,N-dimethylbenzenesulfonamide (29): Prepared by the reaction of 62 and benzylamine 63 according to the general procedure (ii). Yield 65%; White solid; Rf 0.36 (Ethylacetate); m.p; 105-107 °C; IR (neat): 3332, 1625, 1538, 1334, 1319, 1160,
RI PT
1088, 951 cm-1; 1H-NMR (CDCl3) δ 1.81-1.88 (m, 2H), 2.64-2.67 (m, 2H), 2.67 (s, 6H), 3.223.25 (m, 2H), 4.43 (s, 2H), 4.95-5.13 (bs, 2H), 7.17-7.21 (m, 3H), 7.27-7.30 (m, 2H), 7.39 (d, J = 8.20 Hz, 2H), 7.63 (d, J = 8.20 Hz, 2H);13C-NMR (CDCl3) δ 31.38, 32.82, 37.88, 39.84, 44.48,
SC
127.44, 127.95, 128.72, 129.02, 132.91, 139.08, 147.18, 158.31; HRMS Calcd for C19H25N3O3S m/z [M+H] 376.1695, found 376.1714.
M AN U
6.1.1.28. 4-(2-(3-(3-(4-Fluorophenyl)propyl)ureido)ethyl)benzenesulfonamide (30); Prepared from the reaction of 56 with amine (‘L’ and ‘n’ are mentioned in Table 1) according to the general procedure (ii) Yield 50%; White solid; Rf 0.38 (Ethylacetate); mp: 123-126 °C; IR (neat): 3307, 3182, 3071, 2922, 2869, 1623, 1560, 1507, 1326, 1257, 1208, 1152, 1098, 912, 819
TE D
cm-1; 1H NMR (DMSO-d6) δ 1.58-1.69 (m, 2H), 2.52-2.57 (m, 2H), 2.75 (t, J = 7.40 Hz, 2H), 2.97 (m, 2H), 3.25 (m, 2H), 5.82 (bs, 1H), 5.92 (bs, 1H), 7.05-7.13 (m, 2H), 7.19-7.26 (m, 2H), 7.28 (s, 2H), 7.38 (d, J = 8.40 Hz, 2H), 7.74 (d, J = 8.40 Hz, 2H); 13C NMR (DMSO-d6) δ 31.33,
EP
31.63, 35.64, 38.46, 40.30, 114.66, 114.86, 125.53, 128.98, 129.87, 137.79, 137.82, 141.84, 143.96, 157.87, 161.67; HRMS Calcd for C18H22FN3O3S m/z [M+H] 380.1445, found 380.1466.
AC C
6.1.1.29. 4-(2-(3-(3-(4-Methoxyphenyl)propyl)ureido)ethyl)benzenesulfonamide (31); Prepared from the reaction of 58a with amine (‘L’ and ‘n’ are mentioned in Table 1) according to the general procedure (i). Yield 81.7%; White powder; Rf 0.40 (Ethylacetate); mp: 128-131 °C; IR (neat): 3334, 3312, 3045, 2938, 2879, 2835, 1633, 1573, 1514, 1335, 1265,1157, 1030, 898, 809, 731, 690 cm-1; 1H-NMR (DMSO-d6) δ 1.59-1.62 (m, 2H), 2.53-2.58 (m, 2H), 2.74 (t, J = 7.80 Hz 2H), 2.94-2.99 (m, 2H), 3.22-3.27 (m, 2H), 3.71 (s, 3H), 5.80-5.83 (m, 1H), 5.89-5.92 (m, 1H),
ACCEPTED MANUSCRIPT
6.84 (d, J = 6.80 Hz 2H), 7.10 (d, J = 7.00 Hz 2H), 7.28 (s, 2H), 7.38 (d, J = 8.20 Hz 2H), 7.74 (d, J = 8.20 Hz 2H);
13
C-NMR (DMSO-d6) δ 31.56, 32.02, 35.89, 38.77, 40.54, 54.95, 113.69,
125.61, 125.70, 129.17, 133.63, 141.93, 144.08, 157.35, 157.97; HRMS Calcd for C19H25N3O4S
RI PT
m/z [M+H] 392.1644, found 392.1665.
6.1.1.30. 4-(2-(3-(3-(4-Chlorophenyl)propyl)ureido)ethyl)benzenesulfonamide (32); Prepared from the reaction of 1-(3-isocyanatopropyl)-4-chlorobenzene (58b) with amine (‘L’ and ‘n’ are
SC
mentioned in Table 1) according to the general procedure (i). Yield 52%; White solid; Rf 0.42 (Ethylacetate); mp: 153-156 °C; IR (neat): 3273, 2948, 1698, 1557, 1539, 1154, 894, 744, 668
M AN U
cm-1; 1H-NMR (DMSO-d6) δ 1.59-1.66 (m, 2H), 2.54 (t, J = 8.00 Hz, 2H), 2.75 (t, J = 8.00 Hz 2H), 2.94-2.99 (m, 2H), 3.22-3.27 (m, 2H), 5.82-5.85 (m, 1H), 5.92-5.95 (m, 1H), 7.21-7.23 (m, 2H), 7.30 (s, 2H), 7.32-7.34 (m, 2H), 7.37-3.39 (m, 2H), 7.74 (d, J = 8.20 Hz 2H);
13
C-NMR
(DMSO-d6) δ 32.10, 32.20, 36.36, 39.15, 41.01, 126.13, 128.65, 129.58, 130.63, 130.75, 141.34,
TE D
142.42, 144.53, 158.43; HRMS Calcd for C18H22ClN3O3S m/z [M+H] 396.1149, found 396.1174. HPLC purity 99.39%
6.1.1.31. 1-(3,4-Dimethoxyphenethyl)-3-(3-(4-methoxyphenyl)propyl)urea (33); Prepared from
EP
the reaction of 58a with amine (‘L’ and ‘n’ are mentioned in Table 1) according to the general procedure (i). Yield 33%; White solid; Rf 0.45 (2 : 1 Ethylacetate : Hexane); mp: 103-106 °C; IR
AC C
(neat): 3308, 2932, 1616, 1558, 1513, 1227, 1139, 1026, 807, 757 cm-1; 1H-NMR (DMSO-d6) δ 1.58-1.62 (m, 2H), 2.47 (t, J = 8.00 Hz 2H), 2.60 (t, J = 7.80 Hz 2H), 2.94-2.99 (m, 2H), 3.173.22 (m, 2H), 3.70 (s, 3H), 3.71 (s, 3H), 3.73 (s, 3H), 5.72-5,75 (m, 1H), 5.90-5,93 (m, 1H), 6.68-6.71 (m, 1H), 6.78 (s, 1H), 6.83-6.86 (m, 3H), 7.09-7.11 (m, 2H); 13C-NMR (DMSO-d6) δ 31.59, 32.11, 35.75, 38.78, 41.07, 54.98, 55.39, 55.53, 111.96, 112.64, 113.83, 120.57, 129.36,
ACCEPTED MANUSCRIPT
132.35, 133.81, 147.28, 148.74, 157.54, 158.20; HRMS Calcd for C21H28N2O4 m/z [M+H] 373.2127, found 373.2140. 6.1.1.32.
4-(2-(3-(3-(4-Fluorophenyl)propyl)ureido)ethyl)-N,N-dimethylbenzenesulfonamide
RI PT
(34); Prepared by methylation of compound 30. The methylation procedure was analogous to the preparation of compound 12. Yield 75%; White solid; Rf 0.40 (Ethylacetate); mp: 64-68 °C; IR (neat): 3315, 2934, 2859, 1624, 1560, 1508, 1409, 1334, 1219, 1156, 1089, 952, 822, 704 cm-1;
SC
1H NMR (CDCl3) δ 1.72-1.82 (m, 2H), 2.57-2.66 (m, 2H), 2.69 (s, 6H), 2.88 (t, J = 7.83 Hz, 2H), 3.15 (m, 2H), 3.43 (m, 2H), 4.34-4.48 (m, 2H), 6.96 (m, 2H), 7.12 (m, 2H), 7.35 (d, J =
M AN U
8.40 Hz, 2H), 7.68 (d, J = 8.40 Hz, 2H); 13C NMR (CDCl3) δ 32.12, 32.50, 36.51, 38.17, 40.19, 41.44, 115.40, 115.61, 128.31, 129.91, 130.00, 130.08, 133.78, 137.42, 145.25, 158.55, 162.90; HRMS Calcd for C20H26FN3O3S m/z [M+H] 408.1757, found 408.1785. 6.1.1.33.
N-(4-(N,N-Dimethylsulfamoyl)benzyl)-4-phenylpiperidine-1-carboxamide
(35);
TE D
6.1.1.33.1. Preparation of 4-Phenyl-N-(4-sulfamoylbenzyl)piperidine-1-carboxamide (67); The reaction of 4-(aminomethyl)benzenesulfonamide (64) and 4-phenylpiperidine (66) according to the general procedure (ii) afforded 4-phenyl-N-(4-sulfamoylbenzyl)piperidine-1-carboxamide
EP
(67). 1H-NMR (DMSO-d6) δ 1.47-1.55 (m, 2H), 1.73-1.77 (m, 2H), 2.68-2.70 (m, 1H), 2.762.82 (m, 2H), 4.11-4.14 (m, 2H), 4.31 (d, J = 6.00 Hz, 2H), 7.17-7.20 (m, 4H), 7.23-7.29 (m,
AC C
4H), 7.44 (d, J = 8.40 Hz, 2H), 7.76 (d, J = 8.40 Hz, 2H). 6.1.1.33.2.
N-(4-(N,N-Dimethylsulfamoyl)benzyl)-4-phenylpiperidine-1-carboxamide
(35);
Prepared by the methylation of 67. Methylation procedure is analogous to the preparation of compound 12. Yield 82%; Colourless viscous oil; Rf 0.42 (Ethylacetate); IR (neat): 3325, 1647, 1535, 1321, 1301, 1303, 1152, 1078, 944 cm-1; 1H-NMR (CDCl3) δ 1.65-1.76 (m, 2H), 1.891.92 (m, 2H), 2.67-2.74 (m, 1H), 2.70 (s, 6H), 2.92-2.99 (m, 2H), 4.13-4.16 (m, 2H), 4.56 (d, J =
ACCEPTED MANUSCRIPT
6.00 Hz, 2H), 5.05-5.07 (bs, 1H), 7.21-7.25 (m, 3H), 7.31-7.35 (m, 2H), 7.48 (d, J = 8.40 Hz, 2H), 7.72 (d, J = 8.40 Hz, 2H); 13C-NMR (CDCl3) δ 32.85, 36.33, 37.85, 42.44, 44.61, 126.49, 126.73, 128.01, 128.59, 129.53, 133.38, 145.07, 145.38, 157.40; HRMS Calcd for C21H27N3O3S
6.1.1.34.
RI PT
m/z [M+H] 402.1851, found 402.1877.
N-(4-(N,N-Dimethylsulfamoyl)phenethyl)-4-phenylpiperidine-1-carboxamide
(36);
6.1.1.33.1. Preparation of 4-phenyl-N-(4-sulfamoylphenethyl)piperidine-1-carboxamide (68);
SC
The reaction of 4-(2-aminoethyl)benzenesulfonamide (65) and 4-phenylpiperidine (66) according to the general procedure (ii) afforded 4-phenyl-N-(4-sulfamoylphenethyl)piperidine-1-
M AN U
carboxamide 68. 1H-NMR (DMSO-d6) δ 1.43-1.47 (m, 2H), 1.70-1.73 (m, 2H), 2.65-2.75 (m, 3H), 2.81 (t, J = 7.20 Hz, 2H), 3.25-3.29 (m, 2H), 4.05-4.08 (m, 2H), 6.60-6.63 (m, 1H), 7.197.30 (m, 7H), 7.39 (d, J = 8.40 Hz, 2H), 7.75 (d, J = 8.40 Hz, 2H). 6.1.1.34.
N-(4-(N,N-Dimethylsulfamoyl)phenethyl)-4-phenylpiperidine-1-carboxamide
(36);
TE D
Prepared by the methylation of 68. Methylation procedure is analogous to the preparation of compound 12. Yield 80%; Colorless viscous oil; Rf 0.44 (Ethylacetate); IR (neat): 3332, 1625, 1538, 1334, 1319, 1160, 1088, 951 cm-1; 1H-NMR (CDCl3) δ 1.62-1.69 (m, 2H), 1.85-1.88 (m,
EP
2H), 2.64-2.68 (m, 1H), 2.70 (s, 6H), 2.84-2.91 (m, 2H), 2.95 (t, J = 7.20 Hz, 2H), 3.51-3.58 (m, 2H), 4.00-4.04 (m, 2H), 4.56 (bs, 1H), 7.19-7.24 (m, 3H), 7.30-7.34 (m, 2H), 7.39 (d, J = 8.40
AC C
Hz, 2H), 7.72 (d, J = 8.40 Hz, 2H). 13C-NMR (CDCl3) δ 32.92, 36.39, 37.86, 41.81, 42.52, 44.83, 128.53, 126.76, 128.07, 128.63, 129.54, 133.64, 145.06, 145.42, 157.48; HRMS Calcd for C22H29N3O3S m/z [M+H] 416.2008, found 416.2029. 6.1.1.35.
N-Benzyl-4-(4-(N,N-dimethylsulfamoyl)phenylpiperidine-1-carboxamide
(37);
6.1.1.35.1. Preparation of N,N-dimethyl-4-(piperidin-4-yl)benzenesulfonamide (69); Prepared by the procedure analogous to the preparation of intermediate 62. 1H-NMR (CDCl3) δ 1.67-1.71 (m,
ACCEPTED MANUSCRIPT
2H), 1.87-1.88 (m, 2H), 2.69-2.71 (m, 1H), 2.71 (s, 6H), 2.78-2.81 (m, 2H), 3.22-3.26 (m, 2H), 7.39 (d, J = 8.40 Hz, 2H), 7.72 (d, J = 8.40 Hz, 2H). 6.1.1.35.2.
N-Benzyl-4-(4-(N,N-dimethylsulfamoyl)phenylpiperidine-1-carboxamide
(37);
RI PT
Prepared from the reaction of 69 and (isocyanatomethyl)benzene (70) according to the general procedure (i). Yield 88%; White solid; Rf 0.40 (Ethylacetate); mp: 158-160 °C; IR (neat): 3330, 1625, 1538, 1379, 1319, 1160, 1088, 951 cm-1; 1H-NMR (CDCl3) δ 1.64-1.75 (m, 2H), 1.87-
SC
1.90 (m, 2H), 2.72 (s, 6H), 2.74-2.81 (m, 1H), 2.89-2.96 (m, 2H), 4.12-4.15 (m, 2H), 4.47 (d, J = 4.80 Hz, 2H), 4.80 (bs, 1H), 7.22-7.26 (m, 3H), 7.31-7.32 (m, 2H), 7.35 (d, J = 8.40 Hz, 2H),
M AN U
7.72 (d, J = 8.40 Hz, 2H); 13C-NMR (CDCl3) δ 32.59, 37.86, 42.53, 44.55, 45.07, 127.41, 127.47, 127.89, 128.17, 128.69, 133.71, 139.47, 150.61, 157.46; HRMS Calcd for C21H27N3O3S m/z [M+H] 402.1851, found 402.1871. 6.1.1.36.
4-(4-(N,N-Dimethylsulfamoyl)phenyl)-N-phenethylpiperidine-1-carboxamide
(38);
TE D
Prepared from the reaction of 69 and (2-isocyanatoethyl)benzene (71) according to the general procedure (i). Yield 86%; Pale yellow oil; Rf 0.42 (Ethylacetate); IR (neat): 3349, 1624, 1538, 1361, 1322, 1235, 1157, 1089, 950 cm-1; 1H-NMR (CDCl3) δ 1.58-1.69 (m, 2H), 1.83-1.87 (m,
EP
2H), 2.73-2.78 (m, 1H), 2.72 (s, 6H), 2.83-2.90 (m, 4H), 3.51-3.56 (m, 2H), 4.02-4.05 (m, 2H), 4.51 (bs, 1H), 7.22-7.26 (m, 3H), 7.31-7.32 (m, 2H), 7.35 (d, J = 8.40 Hz, 2H), 7.72 (d, J = 8.40
AC C
Hz, 2H); 13C-NMR (CDCl3) δ 32.63, 36.33, 37.92, 42.07, 42.62, 44.46, 126.39, 127.40, 128.11, 128.59, 128.84, 133.67, 139.37, 150.56, 157.41; HRMS Calcd for C22H29N3O3S m/z [M+H] 416.2008, found 416.2032. 6.1.1.37.
4-(4-(N,N-Dimethylsulfamoyl)phenyl)-N-(3-phenylpropyl)piperidine-1-carboxamide
(39); Prepared from the reaction of 69 and (3-isocyanatopropyl)benzene (47) according to the general procedure (i). Yield 86%; Pale white solid; Rf 0.44 (Ethylacetate); mp: 125-127 °C; IR
ACCEPTED MANUSCRIPT
(neat): 3358, 1608, 1538, 1339, 1324, 1160, 1089, 980 cm-1; 1H-NMR (CDCl3) δ 1.60-1.68 (m, 2H), 1.82-1.93 (m, 4H), 2.68-2.74 (m, 3H), 2.71 (s, 6H), 2.79-2.85 (m, 2H), 3.30-3.35 (m, 2H), 3.95-3.99 (m, 2H), 4.40 (bs, 1H), 7.19-7.22 (m, 3H), 7.27-7.31 (m, 2H), 7.35 (d, J = 8.40 Hz,
RI PT
2H), 7.72 (d, J = 8.40 Hz, 2H); 13C-NMR (CDCl3) δ 31.64, 32.64, 33.61, 37.90, 40.84, 42.60, 44.36, 125.90, 127.39, 128.07, 128.38, 128.45, 133.60, 141.84, 150.62, 157.47; HRMS Calcd for C23H31N3O3S m/z [M+H] 430.2164, found 430.2183. HPLC purity 100%
SC
6.1.1.38. Preparation of N,N-Dimethyl-4-(2-(1-methyl-3-(3-phenylpropyl)ureido)ethyl)benzene sulfonamide (40);
M AN U
6.1.1.38.1. Preparation of N-(4-sulfamoylphenethyl)acetamide (72): To a cooled solution of 65 (10 mmol) in methylene chloride at 0 °C, triethylamine (15 mmol) was added and stirred for 5 minutes. Then, acetyl chloride (15 mmol) was added at 0 °C and the whole reaction mixture was slowly allowed to attain ambient temperature and further stirred for 3 h. After the completion of
TE D
the reaction, water was added and the reaction mixture was extracted using methylene chloride. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. Without further purification, the crude product 72 was taken for the next step. Yield 85%
EP
6.1.1.38.2. Preparation of N-(4-(N,N-dimethylsulfamoyl)phenethyl)-N-methylacetamide (73): To a suspension of NaH (16.20 mmol) in DMF at 0 °C, 72 (5.40 mmol) was added and stirred for 45
AC C
minutes at ambient temperature. The reaction mixture was cooled and methyl iodide (16.20 mmol) was added and further stirred at ambient temperature for 3 h. After the completion of the reaction, 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 product 73 was purified by column chromatography. Yield 87%
ACCEPTED MANUSCRIPT
6.1.1.38.3. Preparation of N,N-dimethyl-4-(2-(methylamino)ethyl)benzenesulfonamide (74): The above obtained 73 (3.50 mmol) was dissolved in n-butanol (3 mL) and 3N HCl (100 mL) was added. The reaction mixture was refluxed for 8 h. After the completion of the reaction, the
RI PT
solution was extracted using diethyl ether and the aqueous layer was neutralized with solid K2CO3. Then it was extracted using ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum to get the amine 74. Yield 75%; 1H-NMR
SC
(CDCl3) δ 2.28 (s, 3H), 2.59 (s, 6H), 2.71-2.74 (m, 2H), 2.76-2.81 (m, 2H), 3.32 (bs, 1H), 7.49 (d, J = 8.20 Hz, 2H), 7.65 (d, J = 8.20 Hz, 2H).
M AN U
6.1.1.38.4. N,N-Dimethyl-4-(2-(1-methyl-3-(3-phenylpropyl)ureido)ethyl)benzene sulfonamide (40); Prepared from the reaction of the above obtained amine 74 and 47 according to the general procedure (i). Yield 78%; White solid; Rf 0.40 (Ethylacetate); mp 72-74 °C; IR (neat): 3442, 2916, 1647, 1538, 1490, 1363, 1158, 1088 cm-1; 1H-NMR (CDCl3) δ 1.81-1.89 (m, 2H), 2.65-
TE D
2.71 (m, 2H), 2.67 (s, 3H), 2.68 (s, 6H), 2.90 (t, J = 7.20 Hz, 2H), 3.25-3.30 (m, 2H), 3.51 (t, J = 7.80 Hz, 2H), 4.22 (bs, 1H), 7.19-7.31 (m, 5H), 7.38 (d, J = 8.20 Hz, 2H), 7.70 (d, J = 8.20 Hz, 2H). 13C-NMR (CDCl3) δ 31.74, 33.44, 34.51, 34.67, 37.86, 40.62, 50.39, 125.96, 128.01,
EP
128.40, 128.50, 129.51, 133.26, 141.82, 144.92, 157.78; HRMS Calcd for C21H29N3O3S m/z [M+H] 404.2008, found 404.2027. HPLC purity 99.01% Preparation
AC C
6.1.1.39.
of
N,N-Dimethyl-4-(2-(3-methyl-3-(3-
phenylpropyl)ureido)ethyl)benzenesulfonamide (41); 6.1.1.39.1. Preparation of N-methyl-3-phenylpropan-1-amine (75); The intermediate 75 was prepared from 59 analogous to the preparation of 74. 1H-NMR (CDCl3) δ 1.80-1.85 (m, 2H), 2.41 (s, 3H), 2.58-2.68 (m, 4H), 7.17-7.21 (m, 3H), 7.27-7.31 (m, 2H).
ACCEPTED MANUSCRIPT
6.1.1.39.2. 4-(2-(3-Methyl-3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (76); Prepared by the reaction of 75 and 65 according to the general procedure (ii). Yield 75%; 1H-NMR (DMSO-d6) δ 1.67-1.75 (m, 2H), 2.50-2.54 (m, 2H), 2.75 (s, 3H), 2.79 (t, J = 7.20 Hz, 2H),
RI PT
3.18-3.27 (m, 4H), 6.33 (bs, 1H), 7.15-7.21 (m, 3H), 7.27-7.30 (m, 4H), 7.37 (d, J = 8.20 Hz, 2H), 7.74 (d, J = 8.20 Hz, 2H). 6.1.1.39.3.
N,N-Dimethyl-4-(2-(3-methyl-3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide
SC
(41); Prepared by the methylation of 76 with 2 equivalents of methyl iodide. Methylation procedure is analogous to the preparation of compound 12. Yield 80%; White solid; Rf 0.40
M AN U
(Ethylacetate); mp 67-69 °C; IR (neat): 3430, 2928, 1632, 1527, 1386, 1185, 1157, 1090 cm-1; 1H-NMR (CDCl3) δ 1.81-1.88 (m, 2H), 2.61 (t, J = 7.60 Hz, 2H), 2.69 (s, 6H), 2.81 (s, 3H), 2.89 (t, J = 7.80 Hz, 2H), 3.26 (t, J = 7.80 Hz, 2H), 3.44-3.49 (m, 2H), 4.26 (bs, 1H), 7.16-7.22 (m, 3H), 7.27-7.31 (m, 2H), 7.36 (d, J = 8.40 Hz, 2H), 7.71 (d, J = 8.40 Hz, 2H). 13C-NMR (CDCl3)
TE D
δ 29.39, 32.82, 34.07, 36.37, 37.89, 41.75, 48.12, 126.03, 128.03, 128.31, 128.50, 129.53, 133.20, 141.47, 145.07, 157.77; HRMS Calcd for C21H29N3O3S m/z [M+H] 404.2008, found 404.2030. HPLC purity 100%
(42);
EP
6.1.1.40. 4-(2-(1,3-Dimethyl-3-(3-phenylpropyl)ureido)ethyl)N,N-dimethylbenzene sulfonamide
AC C
Prepared by the methylation of compound 13. Methylation procedure is analogous to the preparation of compound 12. Yield 59%; Pale yellow oil; Rf 0.42 (Ethylacetate); IR (neat): 3105, 3020, 2987, 1655, 1617, 1554, 1488 cm-1; 1H-NMR (CDCl3) δ 1.83-1.90 (m, 2H), 2.59 (t, J = 8.00 Hz, 2H), 2.69 (s, 6H), 2.72 (s, 3H), 2.77 (s, 3H), 2.92 (t, J = 8.00 Hz, 2H), 3.14 (t, J = 8.00 Hz, 2H), 3.39 (t, J = 8.00 Hz, 2H), 7.16-7.21 (m, 3H), 7.26-7.30 (m, 2H), 7.36 (d, J = 8.20 Hz, 2H), 7.69 (d, J = 8.20 Hz, 2H); 13C-NMR (CDCl3) δ 29.09, 33.08, 33.81, 36.43, 37.34, 37.86,
ACCEPTED MANUSCRIPT
49.90, 51.32, 125.97, 127.98, 128.35, 128.45, 129.39, 133.37, 141.64, 145.07, 165.08; HRMS Calcd for C22H31N3O3S m/z [M+H] 418.2164, found 418.2186. HPLC purity 99.21% 6.1.1.41. Preparation of 4-(2-(1-Ethyl-3-(3-phenylpropyl)ureido)ethyl)-N,N-dimethylbenzene
RI PT
sulfonamide (43):
6.1.1.41.1. Preparation of 4-(2-ethylamino)ethyl)benzenesulfonamide (77a): To a solution of 65 (0.5 mmol) in DMF, K2CO3 (0.5 mmol) was added and stirred for 15 minutes. After that
SC
ethyliodide (0.5 mmol) was added and allowed to stir for 12 h at ambient temperature. After the formation of the product by TLC, water was added to the reaction mixture and extracted using
M AN U
ethyl acetate. The intermediate 77a was separated by column chromatography. Yield 55%. (The other product was the dialkylated derivative).
6.1.1.41.2. Preparation of 4-(2-(1-ethyl-3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide (78a); Prepared by the reaction of 77a and 47 according to the general procedure (i). Yield 40%;
TE D
1H-NMR (CDCl3) δ 1.07 (t, J = 7.20 Hz, 3H), 1.81-1.89 (m, 2H), 2.65-2.70 (m, 2H), 2.94 (t, J = 7.60 Hz, 2H), 3.24-3.35 (m, 4H), 3.40-3.45 (m, 2H), 4.30 (bs, 1H), 4.94 (s, 2H), 7.18-7.34 (m, 5H), 7.33 (d, J = 8.20 Hz, 2H), 7.79 (d, J = 8.20 Hz, 2H).
(43);
4-(2-(1-Ethyl-3-(3-phenylpropyl)ureido)ethyl)-N,N-dimethylbenzene
sulfonamide
EP
6.1.1.41.3.
Prepared by the methylation of 78a by 2 equivalents of methyl iodide. Methylation
AC C
procedure is analogous to the preparation of compound 12. Yield 30%; Colourless oil; Rf 0.42 (2 : 1 Ethylacetate : Hexane); IR (neat): 3334, 2923, 1642, 1540, 1495, 1339, 1213, 1189 cm-1; 1HNMR (CDCl3) δ 1.05 (t, J = 7.20 Hz, 3H), 1.83-1.90 (m, 2H), 2.65-2.72 (m, 2H), 2.68 (s, 6H), 2.92 (t, J = 7.60 Hz, 2H), 2.99-3.04 (m, 2H), 3.26-3.31 (m, 2H), 3.44 (t, J = 7.80 Hz, 2H), 4.23 (bs, 1H), 7.17-7.21 (m, 2H), 7.27-7.34 (m, 3H), 7.38 (d, J = 8.40 Hz, 2H), 7.70 (d, J = 8.40 Hz, 2H). 13C-NMR (CDCl3) δ 13.62, 31.98, 33.62, 35.19, 37.99, 40.70, 42.36, 48.64, 126.11,
ACCEPTED MANUSCRIPT
128.15, 128.54, 128.64, 129.64, 133.52, 141.95, 145.12, 157.43; HRMS Calcd for C23H33N3O3S m/z [M+H] 432.2321, found 432.2341. 6.1.1.42. 4-(2-(1-Ethyl-3-(3-phenylpropyl)ureido)ethyl)-N,N-dimethylbenzene sulfonamide (44);
RI PT
To a cooled suspension of NaH (3.08 mmol) in DMF, 13 (0.51 mmol) was added and stirred at ambient temperature for 45 minutes. Then the reaction mixture was cooled and ethyliodide (2.06 mmol) was added. The reaction mixture was further stirred at ambient temperature for 15 h.
SC
After the formation of product, saturated NH4Cl solution was added to neutralize excess NaH and the reaction mixture was extracted with ethyl acetate. The organic layer was dried over
M AN U
anhydrous sodium sulfate and concentrated under vacuum to get 44. The product was purified by column chromatography. Yield 30%; Colourless oil; Rf 0.50 (2 : 1 Ethylacetate : Hexane); IR (neat): 3340, 2923, 1642, 1555, 1488, 1341, 1211, 1187 cm-1; 1H-NMR (CDCl3) δ 1.02-1.08 (m, 6H), 1.82-1.89 (m, 2H), 2.59 (t, J = 7.80 Hz, 2H), 2.69 (s, 6H), 2.88 (t, J = 7.20 Hz, 2H), 3.08-
TE D
3.16 (m, 6H), 3.34 (t, J = 7.20 Hz, 2H), 7.16-7.20 (m, 3H), 7.26-7.30 (m, 2H), 7.35 (d, J = 8.40 Hz, 2H), 7.69 (d, J = 8.40 Hz, 2H); 13C-NMR (CDCl3) δ 13.14, 13.25, 29.68, 33.40, 34.42, 37.99, 43.11, 44.40, 47.12, 48.59, 126.09, 128.05, 128.47, 128.57, 129.58, 133.37, 141.80,
purity 99.64%
EP
145.58, 164.98; HRMS Calcd for C24H35N3O3S m/z [M+H] 446.2477, found 446.2499. HPLC
AC C
6.1.1.43. Preparation of 4-(2(1-isopropyl-3-(3-phenylpropyl)ureido)ethyl)-N,N-dimethylbenzene sulfonamide (45);
6.1.1.43.1. Preparation of 4-(2-(isopropylamino)ethyl)benzenesulfonamide (77b): To a solution of 65 (0.5 mmol) in DMF, K2CO3 (0.5 mmol) was added and stirred for 15 minutes. After that isopropyl iodide (0.5 mmol) was added and allowed to stir for 12 h at ambient temperature. After the formation of the product by TLC, water was added to the reaction mixture and extracted
ACCEPTED MANUSCRIPT
using ethyl acetate. The intermediate 77b was separated by column chromatography. Yield 50%. (The other product was the dialkylated derivative). 6.1.1.43.2. Preparation of 4-(2-(1-isopropyl-3-(3-phenylpropyl)ureido)ethyl)benzenesulfonamide
RI PT
(78b): Prepared by the reaction of 77b and 47 according to the general procedure (i). Yield 40%; 1H-NMR (CDCl3) δ 1.12 (d, J = 6.80 Hz, 6H), 1.80-1.86 (m, 2H), 2.66 (t, J = 7.60 Hz, 2H), 2.90 (t, J = 7.60 Hz, 2H), 3.24-3.31 (m, 4H), 3.84-3.90 (m, 1H), 4.24 (bs, 1H), 4.91 (s, 2H), 7.19-7.31
SC
(m, 5H), 7.35 (d, J = 8.20 Hz, 2H), 7.85 (d, J = 8.20 Hz, 2H).
6.1.1.43.3. 4-(2(1-Isopropyl-3-(3-phenylpropyl)ureido)ethyl)-N,N-dimethylbenzene sulfonamide
M AN U
(45); Prepared by the methylation of 78b by 2 equivalents of methyl iodide. Methylation procedure is analogous to the preparation of compound 12. Yield 70%; Colourless oil; Rf 0.32 (2 : 1 Ethylacetate : Hexane); IR (neat): 3330, 2984, 1638, 1540, 1490, 1337, 1210, 1188 cm-1; 1HNMR (CDCl3) δ 1.10 (d, J = 6.80 Hz, 6H), 1.84-1.91 (m, 2H), 2.64-2.72 (m, 2H), 2.69 (s, 6H),
TE D
2.90-2.96 (m, 2H), 3.26-3.32 (m, 4H), 3.87-3.90 (m, 1H), 4.28 (bs, 1H), 7.17-7.21 (m, 3H), 7.277.31 (m, 2H), 7.38 (d, J = 8.40 Hz, 2H), 7.70 (d, J = 8.40 Hz, 2H); 13C-NMR (CDCl3) δ 20.91, 31.80, 33.57, 36.72, 37.87, 40.71, 43.21, 46.85, 126.01, 128.09, 128.44, 128.54, 129.48, 133.57,
6.1.1.44.
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141.87, 145.11, 157.61; HRMS Calcd for C23H33N3O3S m/z [M+H] 432.2321, found 432.2340. 4-(2-(1-Isopropyl-3-methyl-3-(3-phenylpropyl)ureido)ethyl)-N,N-dimethylbenzene
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sulfonamide (46); Prepared by the methylation of compound 45 with 1 equivalent of methyliodide and NaH. Methylation procedure is analogous to the preparation of compound 12. Yield 60%; White solid; Rf 0.35 (2 : 1 Ethylacetate : Hexane); mp 57-59 °C; IR (neat): 3010, 2989, 1641, 1550, 1496, 1335, 1211, 1180 cm-1; 1H-NMR (CDCl3) δ 0.95 (d, J = 6.80 Hz, 6H), 1.87-1.90 (m, 2H), 2.60 (t, J = 7.60 Hz, 2H), 2.68 (s, 6H), 2.76 (s, 3H), 2.82 (t, J = 7.20 Hz, 2H), 3.19-3.24 (m, 4H), 3.57-3.64 (m, 1H), 7.17-7.21 (m, 3H), 7.27-7.31 (m, 2H), 7.33 (d, J = 8.20
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Hz, 2H), 7.68 (d, J = 8.20 Hz, 2H); 13C-NMR (CDCl3) δ 19.98, 29.23, 33.16, 35.29, 36.36, 37.89, 42.44, 49.71, 50.60, 126.02, 127.77, 128.37, 128.50, 129.65, 133.04, 141.67, 146.13, 164.98; HRMS Calcd for C24H35N3O3S m/z [M+H] 446.2477, found 446.2498.
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6.2. Pharmacology
6.2.1. Sarcomere assay procedure for the measurement of myosin ATPase activity:
In the sarcomere, force generation is directly coupled to ATP hydrolysis. Compounds that
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activate the sarcomere were identified by measuring the increase in myosin ATPase activity in a sarcomere assay at 10 µM.
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Actin stimulated ATPase activity was assayed spectrophotometrically as reported previously [28] with modifications. The standard reaction mixture contained 20 mM Tris HCl (pH 7.5), 15 mM KCl, 6 mM MgCl2, 1 mM ATP, S1 myosin (CS-MYS03) and actin thin filament complex (CSTFC01) with pCa = 6.5 [29]. The reaction was stopped by addition of Cytophos reagent
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(Cytoskeleton BK054 kit) after 10 min incubation at room temperature, samples were analyzed for inorganic phosphate liberated by taking the absorbance at 650 nm on TECAN Infinite. Omecamtiv was used as a positive control for the selectivity assay. Blank had buffer, ATP and
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Cytophos mixture while DMSO was used as a negative control. % Activity = (Mean A - B)-(NC -B)/ (NC-B) x100
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Where, NC=Negative control; B=Blank, A=Absorbance 6.2.2. Selectivity Studies
Compound specificity with respect to muscle type were evaluated by comparing the effect of the compound on actin stimulated ATPase activity of a panel of myosin isoforms including cardiac (bovine), skeletal (rabbit) and smooth muscle (chicken gizzard) at a single higher dose (100 uM ) of the compound.
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6.2.2.1. For Skeletal muscle: Actin stimulated ATPase activity was assayed spectrophotometrically as reported previously [2831] with modifications. The standard reaction mixture contained 20 mM Tris HCl (pH 7.5), 15
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mM KCl, 6 mM MgCl2, 1 mM ATP, S1 myosin (CS-MYS04) and actin thin filament complex (CS-TFC01) with pCa = 6.5. The reaction was stopped by addition of Cytophos reagent (Cytoskeleton BK054 kit) After 10 min incubation at room temperature, samples were analyzed
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for inorganic phosphate liberated by taking absorbance at 650 nm on TECAN Infinite.
6.2.2.2. For Smooth muscle:
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Blebbistatin was used as a negative control for selectivity assay [34].
Actin stimulated ATPase activity was assayed spectrophotometrically as reported previously [28,29,32,33] with modifications. The standard reaction mixture contained 20 mM Tris HCl (pH 7.5), 15 mM KCl, 6 mM MgCl2, 1 mM ATP, S1 myosin (CS-MYS05), actin (AD 99) and
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Tropomyosin (T2400) with pCa = 6.5. The reaction was stopped by addition of Cytophos reagent (Cytoskeleton BK054 kit) After 10 min incubation at room temperature, samples were analyzed for inorganic phosphate liberated by taking absorbance at 650 nm on TECAN Infinite.
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Blebbistatin was used as a negative control for selectivity assay [34]. 6.2.3. Animal study (in vivo)
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6.2.3.1. The formulation of sample
The stock solution was prepared by dissolving 8 mg of respective compound in 2 mL of DMSO (i.e. 4 mg/mL or 4000 µg/mL of DMSO). It was diluted 100 times with saline solution to give 40 µg/mL final solution (% of DMSO is 1 %). The maximum % of DMSO in the final solution was limited to 10 %. The concentration of the final solution (unknown concentration) was measured by HPLC along with three standards (known concentration).
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6.2.3.2. Measurement of fractional shortening (FS) and ejection fraction (EF) by echocardiography 6.2.3.2.1. Animals
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Seven-week-old Sprague-Dawley male Rats were purchased from the Orient Bio (South Korea). The protocols used in this study [35] conformed to the Guide for the Care and use of Laboratory Animals published by National Institutes of Health (NIH Publication 85-23, revised 1996). All
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animal experiments were approved by the Institutional Animal Care and Use Committee of Samsung Biomedical Research Institute (SBRI). SBRI is accredited by the Association for
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Assessment and Accreditation of Laboratory Animal Care International and abided by the Institute of Laboratory Animal Resources guide. 6.2.3.2.2. Echocardiography
The rats were anesthetized by 1.5 % isoflurane inhalation method with nosecone. The right
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internal jugular vein was used as a central venous line for drug administration. Rats were infused with samples at 0, 2, 4, 8, and 16 µg/kg/min for 3 minutes using 40 µg/mL final solution. Mmode echocardiograms were performed at baseline and 16 µg/kg/min. Images were acquired
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with a MS250 transducer operated at 25 MHz connected to a VisualSonics Vevo2100 (VisualSonics Inc., Toronto, Ontario, Canada). LV end-diastolic interventricular septum,
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posterior wall thickness, LV dimension, LV fractional shortening (FS) and ejection fraction (EF) were measured. % increase value was calculated according to following formula: % Increase FS(EF) = (FS(EF) data of 16 µg/kg/min - FS(EF) data of 0 µg/kg/min) / baseline data of FS(EF) x 100 %. Three rats per drug were used. A single sonographer who was blinded to treatment information performed all echacardiograms for data acquisition.
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6.2.4. Ventricular cell contractility 6.2.4.1. Single-cell Isolation Rat ventricular myocytes were enzymatically isolated from male Sprague-Dawley rats (200–300
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g) as described previously [36]. Briefly, rats were deeply anesthetized with sodium pentobarbital (150 mg/kg, intraperitoneally), chest cavities were opened, and hearts were excised. This surgical procedure was carried out in accordance with university ethical guidelines. The excised hearts
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were retrogradely perfused at 7 mL/min through the aorta (at 36.5°C), first for 3 minutes with Ca2+-free Tyrode solution composed of (in millimolar) 137 NaCl, 5.4 KCl, 10 HEPES, 1 MgCl2,
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and 10 glucose (pH 7.3), and then with Ca2+ free Tyrode solution containing collagenase (1.4 mg/mL, Type I; Roche, Indianapolis, IN) and protease (0.14 mg/mL, Type XIV; Sigma-Aldrich, St. Louis, MO) for 12 minutes, and finally with Tyrode solution containing 0.2 mM CaCl2 for 8 minutes. The ventricles of the digested heart were then cut into several sections and subjected to
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gentle agitation to dissociate the cells.
6.2.4.2. Measurement of Cell Shortenings
Isolated myocytes were continuously superfused with normal Tyrode solution (see above)
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containing 2 mM Ca2+. Cells were field stimulated with 2 paralleled platinum wires connected with an electrical stimulator (Stimulator I; Hugo Sach Elektronik, March-Hugstetten, Germany)
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at 1 Hz. Single-cell shortenings were detected with a video edge detector (Model VED-105; Crescent Electronics, Sandy, UT) connected with a CCD camera (LCL902C; Till Photonics, Graefelting, Germany) and video monitor (ViewFinder III, Polychrome V system; Till Photonics). Analog signals from the edge detector were converted into digital signals by an A/D converter (Digidata 1322A; Molecular Devices). The digitized cell shortening signals were recorded with a PC program, pClamp 9 (Molecular Devices, Sunnyvale, CA).
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A Stock solution of the compounds were made in dimethyl sulfoxide (DMSO), which was diluted in the external normal Tyrode solution to make the final testing solutions. The drug solutions were applied to the cells by super fusion using custom-made solution switching
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apparatus. The experiments were all performed at room temperature (22-25 oC). ACKNOWLEDGMENT
This work was supported by Priority Research Centers Program through the National Research
(2009-0093815) and KDDF (201202-09).
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Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology
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Highlights SAR study of sulfonamidophenylethylureas discovered highly potent inotrope.
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These urea analogs are selective cardiac myosin ATPase activators.
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Compound 13, 40 and 41 shows 17.6, 38.9 and 23.2% fractional shortening in the echocardiographic study with rat, respectively.
The cell contractility of 13, 40, and 41 shows 47.9, 45.5 and 63.5% at 5 µM in rat
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ventricle cells, respectively.
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