Design, synthesis and pharmacophoric model building of new 3-alkoxymethyl/3-phenyl indole-2-carboxamides with potential antiproliferative activity

Article abstract of DOI:10.1111/cbdd.12928

Novel 3-alkoxymethyl/3-phenyl indole-2-carboxamide derivatives were synthesized and evaluated for their anticancer activity. Most of the tested compounds showed moderate to excellent activity against the tested cell lines (MCF7 and HCT116). 3-Phenyl substitution on indole with p-piperidinyl phenethyl 24a and p-dimethylamino phenethyl 24c exhibited anticancer activity against MCF7 with IC₅₀ of 0.13 and 0.14?μm, respectively. Further mechanistic study of the most active compounds through their action on cell cycle showed disturbance in cell cycle progression and cell cycle arrest. For future development of this series of compounds, pharmacophore study was conducted which indicated that the enhancement of the activity could be achieved through the addition of acceptor or donating groups to the already-present indole nucleus.

Full text of DOI:10.1111/cbdd.12928

DR. BAHAA YOUSSIF (Orcid ID : 0000-)0002-6834-6548
Received Date : 24-Aug-2016
Revised Date : 07-Nov-2016
Accepted Date : 10-Dec-2016
Article type : Research Article
Design, Synthesis and Pharmacophoric Model Building of New 3-
Alkoxymethyl / 3-Phenyl indole-2-carboxamides with Potential
Antiproliferative Activity
Mostafa H. Abdelrahman1, Ahmed S. Aboraia2, Bahaa G. M. Youssif3,4*, Bakheet E.
M. Elsadek5
1Department of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Assiut 71524,
Egypt
2Department of Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526,
Egypt
3Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut University,
Assiut 71526, Egypt
4Department of Pharmaceutical Chemistry, College of Pharmacy, Aljouf University, Aljouf,
Sakaka-2014, Kingdom of Saudi Arabia
5Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University, Assiut 71524,
Egypt
*Corresponding author: Bahaa G. M. Youssif
E-mail: bgyoussif@ju.edu.sa
Tel: 00966537240294
Short running title:
Synthesis and anticancer activity of new indole-2-carboxamides
Abstract
Novel 3-alkoxymethyl/3-phenyl indole-2-carboxamide derivatives were synthesized
and evaluated for their anticancer activity. Most of the tested compounds showed
moderate to excellent activity against the tested cell lines (MCF7 and HCT116). 3-
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which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1111/cbdd.12928
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Phenyl substitution on indole with and p-piperidinyl phenethyl 24a and p-
dimethylamino phenethyl 24c exhibited anticancer activity against MCF7 with IC50 of
0.13 and 0.14 µM respectively. Further mechanistic study of the most active
compounds through their action on Cell cycle showed disturbance in cell cycle
progression and cell cycle arrest. For future development of this series of compounds,
Pharmacophore study was conducted which indicated that the enhancement of the
activity could be achieved through addition of acceptor or donating groups to the
already present indole nucleus.
Keywords: Indole, Pharmacophore, Anticancer, Cell cycle, Synthesis
Introduction
Cancers are main leading causes of morbidity and mortality worldwide, with around
14 million new cases and 8.2 million cancer related deaths in 2012. This number is
expected to rise by about 70% over the next 2 decades (1). Improvement of quality of
life and survival of cancer patients will be greatly enhanced by the development of
highly effective drugs to selectively kill malignant cells.
Combating cancer is a topic of global high priority. However, there is much room for
improvement in this aspect given the fact that mortality rates for the most prevalent
cancers have not been significantly reduced despite the recent “spectacular” advances
in molecular medicine, genomics, proteomics, and translational research. Main
strategies currently applied to combat cancer include primary prevention, early
diagnosis and improved therapeutic interventions (2). Numerous anticancer agents
including alkylating agent (3), antimetabolites (4), natural products (5), hormones (6),
etc. are in clinical use against different types of cancer. However, most of those
agents produce side effects like gastrointestinal side effects, hair loss, blood problems,
etc. Therefore, there is an urgent need for the exploration of new chemical entities for
an effective and safer cure of cancer.
The chemistry and biological study of heterocyclic compounds has been an interesting
field for a long time in medicinal chemistry; mainly due to the ability of the
heterocyclic compounds to mimic the structure of endogenous mediators and to bind
to different proteins (7-10).
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Indoles are constantly of interest for the development of newer biologically active
moieties because it is built into proteins in the form of amino acid tryptophan (11) and
because of its wide range of pharmacological activities like anti-inflammatory (12),
antimicrobial (13), antihistaminic (14) and specially anticancer activity (15). Since
indole derivatives have shown good activity as anticancer agents, many structural
changes have been made to enhance their anticancer activity (16-20). For example,
Panobinostat (21) is also a drug developed by Novartis for the treatment of various
cancers. Moreover, the indole nucleus is the core structure of a great number of
tubulin polymerization inhibitors (Figure 1). The indolyl-3-glyoxamide D-24851 was
discovered by Baxter Oncology (22). In view of those observations, we report herein
the synthesis of the new 3-alkoxymethyl/3-phenyl indole-2-carboxamides with their
pharmacophore study and evaluation of the anticancer activity. In addition the cell
cycle activity has been determined for the most active compounds, which gives an
idea about the possible phase in which those new compounds halt the growth of
cancer cells.
Figure 1: a) Structures of anticancer compounds bearing indole-based moiety, b)
General structure of the designed indole-2-carboxamide derivatives.
Methods and Materials
Chemistry
All the chemicals used were of analytical grade and purified by standard methods
prior to use. Silica gel column chromatography was carried out using kieselgel 60
(merck). TLC analysis was performed on aluminium-backed plates coated with silica
gel 60 F254 (Merck). Melting points were determined using a Gallen Kamp melting
point apparatus and are uncorrected. Infrared spectra were recorded with a Nicolet
Avatar 320 FTIR spectrometer. Components were visualized using potassium
permanganate solution and UV light. NMR Spectra were taken using a Varian Unity
INOVA 400 MHz and Bruker AC250 MHz spectrometers for proton and carbon. All
numbers referring to NMR data obtained are in parts per million (ppm). High
resolution mass spectrometric data were obtained using the EPSRC mass
spectrometry centre in Swansea and Thermo Instruments MS system (LTQ XL/LTQ
Orbitrap Discovery) coupled to a Thermo Instruments HPLC system (Accela PDA
detector, Accela PDA autosampler and Pump) at university of Aberdeen.
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General procedure for the synthesis of compound 3-6
To a stirred solution of aminobenzylcyanide (1 equiv) in toluene (0.75 M), 1,5-
dibromopentane, 1,4-dibromobutane or bromoethyl ether (1.1 equiv) and DIPEA (2
equiv) were added. The reaction mixture was refluxed for 20 h in an oil bath. After
cooling, toluene was added and the formed reddish brown precipitate (Br-salt) was
collected by filtration. The filtrate was washed with water, dried over anhydrous
MgSO4, and evaporated under reduced pressure to afford 3-6.
General procedure for the synthesis of compound 7-10
To a suspension of lithium aluminium hydride (LiAlH4) (10 equiv) in Et2O (0.2 M) at
o
0 C in a two-necked rounded bottom flask maintaining anhydrous conditions under
nitrogen atmosphere, benzyl cyanides 3-6 (1 equiv) dissolved in dry Et2O (0.5 M) was
added and the reaction mixture was stirred overnight at rt. A saturated solution of
o
Na2SO4 was slowly added at 0 C with stirring for an additional 30 min followed by
addition of H2O. The reaction mixture was filtered and the residue was washed three
times with Et2O. The organic layer was washed with brine, dried over MgSO4 and
evaporated under reduced pressure to yield 7-10.
General procedure for the synthesis of compound 14
To a stirred suspension of ethyl 5-chloroindole-2-carboxylate 13 (0.3 g, 1.34 mmol, 1
equiv) in acetic acid (3 mL) at 0oC, diethylamine (0.2 mL, 1.5 equiv) and 37%
aqueous formaldehyde (2 mL) were successively added. The resulting reaction
mixture was refluxed for 6 h. After dilution with excess of water and removal of non-
basic material by extraction with Et2O, the aqueous layer was made basic with 5%
NaOH. The formed precipitate was then collected by filtration to afford 14.
General procedure for the synthesis of compound 22
To a stirred suspension of p-chlorophenylhydrazine hydrochloride 20 (0.5 g, 2.793
mmol, 1 equiv) in absolute ethanol (20 mL), phenyl pyruvic acid 21 (0.5 g, 1.1 equiv)
and p-toluenesulfonic acid monohydrate (PTSA) (2.66 g, 5 equiv) were added and the
resulting reaction mixture was refluxed for 20 h. The residue after removal of ethanol
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under reduced pressure was extracted with DCM, washed twice with saturated
NaHCO3 solution, dried over MgSO4, and evaporated under reduced pressure to give
crude product which was recrystallised from light petroleum and EtOAc to yield 22
General procedure for the synthesis of compound 16a-16e, 18 and 23
To a solution of indole ester 14, 13 and 22 (1equiv) in appropriate alcohol, (0.5 M)
o
5% NaOH (5 equiv) was added. The reaction mixture was kept at 40 C with stirring
overnight. The residue after removal of alcohol under reduced pressure was then
taken into water, precipitated out at pH = 1 using 5% HCl. The precipitate was
extracted with EtOAc, dried over MgSO4 and evaporated under reduced pressure to
give the corresponding carboxylic acid 16a-16e, 18 and 23.
General procedure for the synthesis of compound 17a-m, 19, 24a-d, 25 and 26
A mixture of indole-2-carboxylic acids 16a-e, 18 and 23 (1 equiv), BOP (1.5 equiv),
and DIPEA (2 equiv) in DCM (0.05 M) was stirred for 10 min at rt before addition of
the appropriate amine (1.2 equiv) and the resulting reaction mixture was stirred
overnight at rt. After removing of the solvent in vacuo, the residue was extracted with
EtOAc, washed with 5% HCl, saturated NaHCO3 solution, brine, dried over MgSO4,
and evaporated under reduced pressure to give a crude product, which was purified by
flash chromatography on silica gel.
The character of all compounds
2-(4-(Piperidin-1-yl)phenyl)acetonitrile (3). Compound 3 was obtained according to
reference (23). 90% yield; pale pink solid; m.p. 71-72 oC; 1HNMR (250 MHz, CDCl3)
δ (ppm): 7.17 (d, J = 8.3 Hz, 2H, Ar-H), 6.91 (d, J = 8.3 Hz, 2H, Ar-H), 3.64 (s, 2H,
CH2CN), 3.15 (t, J = 4.5 Hz, 4H, piperidin-H), 1.69-157 (m, 6H, piperidin-H); 13C-
NMR (62.5 MHz, CDCl3) δ(ppm): 151.9, 128.7, 119.7, 118.5, 116.7, 50.4, 25.7,
24.3, 22.8; HRESI-MS m/z calcd for [M+H]+ C13H17N2: 201.1386, found: 201.1388.
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2-(4-Morpholinophenyl)acetonitrile (4). Compound 4 was obtained according to
reference (23). 92% yield; yellow solid; m.p. 77-78 oC; 1HNMR (250 MHz, CDCl3): δ
(ppm): 7.21 (d, J = 8.3 Hz, 2H, Ar-H), 6.89 (d, J = 8.8 Hz, 2H, Ar-H), 3.85 (t, J = 4.5
Hz, 4H, morphH), 3.66 (s, 2H, CH2CN), 3.14 (t, J = 4.8 Hz, 4H, morph-H); 13CNMR
(62.5 MHz, CDCl3) δ (ppm):151.0, 128.8, 120.8, 118.0 116.0, 66.8, 49.1, 22.8;
HRESI-MS m/z calcd for [M+H]+ C12H15N2O: 203.1179, found: 203.1181.
2-(3-(Piperidin-1-yl)phenyl)acetonitrile (5). 88% yield; reddish brown oil; 1H NMR
(250 MHz, CDCl3) δ (ppm): 7.22 (t, J = 7.5 Hz, 1H, Ar-H), 6.87 (m, 2H, Ar-H), 6.72
(d, J = 7.8 Hz, 1H, Ar-H), 3.66 (s, 2H, CH2CN), 3.18 (t, J = 4.5 Hz, 2H, piperidin-H),
1.80-1.45 (m, 6H, piperidin-H); 13C NMR (62.5 MHz, CDCl3) δ (ppm): 152.7, 130.7,
129.8, 118.2, 115.7, 115.5, 50.3, 25.8, 24.3, 23.9; HRESIMS m/z calcd for [M+H]+
C13H17N2: 201.1386, found: 201.1367.
2-(4-(Pyrrolidin-1-yl)phenyl)acetonitrile (6). 92% yield; pale brown solid; m.p. 82-
83 oC; 1H NMR (400 MHz, CDCl3) δ (ppm): 7.13 (d, J = 8.8 Hz, 2H, Ar-H), 6.52 (d, J
= 8.0 Hz, 2H, Ar-H), 3.62 (s, 2H, CH2CN), 3.26 (t, J = 6.0 Hz, 4H, pyrrolidin-H),
2.10-1.95 (m, 4H, pyrrolidin-H); 13C NMR (62.5 MHz, CDCl3) δ (ppm): 147.6, 128.8,
118.9, 115.8, 112.0, 47.6, 25.5, 22.8; HRESI-MS m/z calcd for [M+H]+ C12H15N2:
187.1230, found: 187.1232.
2-(4-(Piperidin-1-yl)phenyl)ethanamine (7). Compound 7 was obtained according
1
to reference (23). 75% yield; pale yellow oil; H NMR (250 MHz, CDCl3) δ (ppm):
7.05 (d, J = 8. 8 Hz, 2H, Ar-H), 6.86 (d, J = 8. 8 Hz, 2H, Ar-H), 3.09 (t, J = 4. 8 Hz,
4H, piperidin-H), 2.89 (t, J = 6. 8 Hz, 2H, CH2NH2), 2.63 (t, J = 6.5 Hz, 2H,
13
CH2CH2NH2), 1.68-153 (m, 6H, piperidin-H), 1.30 (s br, 2H, NH2); CNMR (62.5
MHz, CDCl3) δ (ppm): 150.8, 130.5, 129.4, 116.8, 51.0, 43.8, 39.3, 26.0, 24.3;
HRESI-MS m/z calcd for [M+H]+ C12H15N2O: 205.1699, found: 205.1701.
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2-(4-Morpholinophenyl)ethanamine (8). Compound 8 was obtained according to
reference (23). 84% yield; pale yellow oil; 1H NMR (250 MHz, CDCl3) δ (ppm):7.09
(d, J = 8.3 Hz, 2H, Ar-H), 6.85 (d, J = 8.5 Hz, 2H, Ar-H), 3.84 (t, J = 4. 8 Hz, 4H,
morph-H), 3.10 (t, J = 4. 8 Hz, 4H, morph-H), 2.90 (t, J = 6. 85 Hz, 2H, CH2NH2),
2.65 (t, J = 6. 8 Hz, 2H, CH2CH2NH2), 1.92 (s br, 2H, NH2); 13CNMR (62.5 MHz,
CDCl3) δ (ppm): 149.7, 131.3, 129.5, 115.9, 66.9, 49.6, 43.6, 39.0. HRESI-MS m/z
calcd for [M+H]+ C12H19N2O: 207.1492, found: 207.1488.
1
2-(3-(Piperidin-1-yl)phenyl)ethylamine (9). 77% yield; pale yellow oil; H-NMR
(250 MHz, CDCl3) δ (ppm): 7.16 (t, J = 8.0 Hz, 1H, Ar-H), 6.78 (m, 2H, Ar-H), 6.65
(d, J = 6.5 Hz, 1H, Ar-H), 3.13 (t, J = 5.3 Hz, 4H, piperidin-H), 2.94 (t, J = 6. 8 Hz,
2H, CH2NH2), 2.69 (t, J = 6. 8 Hz, 2H, CH2CH2NH2), 1.84-1.33 (m, 6H, piperidin-H)
1.30 (s br, 2H, NH2); 13C-NMR (62.5 MHz, CDCl3) δ (ppm): 152.5, 140.5, 129.1,
119.8, 117.3, 114.3, 50.7, 43.5, 40.5, 25.9, 24.3; HRESI-MS m/z calcd for [M+H]+
C13H21N2: 205.1699, found: 205.1693.
1
2-(4-(Pyrrolidin-1-yl)phenyl)ethanamine (10). 78% yield; reddish brown oil; H-
NMR (250 MHz, CDCl3) δ (ppm): 7.05 (d, J = 8.3 Hz, 2H, Ar-H), 6.52 (d, J = 8. 8
Hz, 2H, Ar-H), 3.26 (t, J = 6.5 Hz, 4H, pyrrolidin-H), 2.90 (t, J = 7.0 Hz, 2H,
CH2NH2), 2.64 (t, J = 6. 8 Hz, 2H, CH2CH2NH2), 2.10-1.95 (m, 4H, pyrrolidin-H),
13
1.29 (s br, 2H, NH2); C-NMR (62.5 MHz, CDCl3) δ (ppm): 146.6, 129.6, 126.4,
111.8, 47.8, 44.0, 39.9, 25.5; HRESI-MS m/z calcd for [M+H]+ C12H19N2: 191.1543,
found: 191.1540.
2-(4-(Dimethylamino)phenyl)acetonitrile (11). Compound 11 was obtained
o
1
according to reference (23). 81% yield; pale brown solid; m.p. 48-49 C; HNMR
(250 MHz, CDCl3) δ (ppm): 7.16 (d, J = 8.5 Hz, 2H, Ar-H), 6.71 (d, J = 8. 8 Hz, 2H,
13
Ar-H), 3.63 (s, 2H, CH2CN), 2.95 (s, 6H, N(CH3)2); CNMR (62.5 MHz, CDCl3) δ
(ppm):150.2, 128.8, 118.8, 117.1, 112.8, 40.6, 22.7; HRESI-MS m/z calcd for
[M+H]+ C10H13N2: 161.1073, found: 161.1073.
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4-(2-Aminoethyl)-N,N-dimethylaniline (12). Compound 12 was obtained according
to reference (23). 82% yield; reddish brown oil; 1HNMR (250 MHz, CDCl3) δ (ppm):
7.07 (d, J = 8.3 Hz, 2H, Ar-H), 6.68 (d, J = 8.3 Hz, 2H, Ar-H), 4.65 (s br, 2H, NH2),
13
3.04-2.81 (m, 8H, N(CH3)2, CH2NH2), 2.72 (t, J = 4.5 Hz, 2H, CH2CH2NH2); C
NMR (62.5 MHz, CDCl3) δ (ppm): 149.4, 129.5, 127.2, 113.0, 42.9, 40.9, 37.3;
HRESI-MS m/z calcd for [M+H]+ C10H17N2: 165.1386, found: 165.1382.
Ethyl 5-chloro-3-((diethylamino)methyl)-1H-indole-2-carboxylate (14). 70%
o
yield; white solid; m.p. 110-112 C; IR(KBr): 3327 (NH), 2965, 2934, 2868, 2797,
1
1682 (C=O), 1541, 1457, 1380, 1261, 1012, 802, 772, 679; H-NMR (400 MHz,
DMSO-d6) δ (ppm): 11.73 (s, 1H, indole NH), 7.88 (s, 1H, Ar-H), 7.41 (d, J = 8.8 Hz,
1H, Ar-H), 7.23 (d, J = 8.7 Hz, 1H, Ar-H), 4.33 (q, J = 7.1 Hz, 2H, OCH2CH3), 3.99
(s, 2H, CH2N), 2.46 (q, J = 7.0 Hz, 4H, 2 x NCH2CH3), 1.33 (t, J = 7.1 Hz, 3H,
OCH2CH3), 0.96 (t, J = 7.0 Hz, 6H, 2 x NCH2CH3); 13C-NMR (101 MHz, DMSO-d6)
δ (ppm): 162.75 (C=O), 135.95, 129.79, 126.81, 126.10, 125.03, 122.20, 121.27,
115.30, 61.76, 48.52, 47.37, 15.50, 12.73; HRESI-MS m/z calcd for [M+H]+
C16H22ClN2O2: 309.1364, found: 309.1367.
Ethyl 5-chloro-3-phenyl-1H-indole-2-carboxylate (22). 75% yield; white solid;
m.p. 158-160 oC; 1H-NMR (400 MHz, CDCl3) δ (ppm): 9.11 (s, 1H, indole NH), 7.59
(d, J = 1.9 Hz, 1H, Ar-H), 7.56 – 7.34 (m, 6H, Ar-H), 7.30 (dd, J = 8.7, 2.0 Hz, 1H,
13
Ar-H), 4.30 (q, J = 7.1 Hz, 2H, CH2CH3), 1.23 (t, J = 7.1 Hz, 3H, CH2CH3); C-
NMR (101 MHz, CDCl3) δ(ppm):161.72 (C=O), 133.90, 132.81, 130.49, 128.90,
127.88, 127.47, 126.67, 126.29, 123.95, 123.63, 120.97, 112.84, 61.10, 14.01;
HRESI-MS m/z calcd for [M+H]+ C17H15ClNO2: 300.0786, found: 300.0791.
5-chloro-3-(ethoxymethyl)-1H-indole-2-carboxylic acid (16a). 85% yield; white
o
solid; m.p. 200-202 C; IR(KBr): 3100 (br, OH), 1668 (C=O), 1553, 1455, 1331,
1
1258, 1070, 838, 799, 734, 657; H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.82 (s,
1H, indole NH), 7.75 (d, J = 2.1 Hz, 1H, Ar-H), 7.41 (d, J = 8.8 Hz, 1H, Ar-H), 7.24
(dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 4.93 (s, 2H, CH2OCH2CH3), 3.46 (q, J = 7.0 Hz, 2H,
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13
OCH2CH3), 1.11 (t, J = 7.0 Hz, 3H, OCH2CH3); C-NMR (101 MHz, DMSO-d6) δ
(ppm): 163.23 (COOH), 134.82, 128.63, 126.81, 125.11, 124.68, 120.66, 118.37,
114.57, 65.05, 63.04, 15.62; HRESI-MS m/z calcd for [M-H]- C12H11ClNO3:
252.0433, found: 252.0431.
5-chloro-3-(methoxymethyl)-1H-indole-2-carboxylic acid (16b). 90% yield; white
solid; m.p. 203-205 oC; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.85 (s, 1H, indole
NH), 7.74 (d, J = 2.0 Hz, 1H, Ar-H), 7.42 (d, J = 8.7, 0.7 Hz, 1H, Ar-H), 7.24 (dd, J =
8.8, 2.2 Hz, 1H, Ar-H), 4.89 (s, 2H, CH2OCH3), 3.25 (s, 3H, OCH3); 13C-NMR (101
MHz, DMSO-d6) δ (ppm):163.21 (COOH), 134.79, 128.64, 127.05, 125.14, 124.76,
120.59, 117.91, 114.58, 64.88, 57.64; HRESI-MS m/z calcd for [M-H]- C11H9ClNO3:
238.0276, found: 238.0271.
5-chloro-3-(isopropoxymethyl)-1H-indole-2-carboxylic acid (16c). 84% yield;
1
white solid; m.p.198-200 oC; H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.51 (s, 1H,
indole NH), 7.68 (d, J = 2.1 Hz, 1H, Ar-H), 7.23 (d, J = 8.7 Hz, 1H, Ar-H), 7.02 (d, J
= 8.8 Hz, 1H, Ar-H), 4.76 (s, 2H, CH2OCH), 3.50–3.40 (m, 1H, CH(CH3)2), 0.92 (d, J
= 6.5 Hz, 6H, CH(CH3)2); 13C-NMR (101 MHz, DMSO-d6) δ (ppm): 163.40 (COOH),
134.87, 128.64, 126.55, 125.68, 122.31, 120.79, 118.85, 114.55, 70.21, 54.90, 22.53;
HRESI-MS m/z calcd for [M-H]- C13H13ClNO3: 266.0589, found: 266.0584.
5-chloro-3-(isobutoxymethyl)-1H-indole-2-carboxylic acid (16d). 82% yield; white
solid; m.p. 200-202 oC; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.70 (s, 1H, indole
NH), 7.45 (d, J = 2.1 Hz, 1H, Ar-H), 7.35 (d, J = 8.8 Hz, 1H, Ar-H), 7.13 (dd, J = 8.8,
2.2 Hz, 1H, Ar-H), 5.00 (s, 2H, CH2OCH2CH), 3.04 (d, J = 7.0 Hz, 2H,
13
CH2OCH2CH), 2.48 (m, 1H, CH(CH3)2), 1.19 (d, J = 7.2 Hz, 6H, CH(CH3)2); C-
NMR (101 MHz, DMSO-d6) δ (ppm): 163.32 (COOH), 134.24, 127.89, 125.11,
124.35, 123.45, 120.02, 119.58, 113.93, 46.30, 44.19, 19.36, 8.69; HRESI-MS m/z
calcd for [M-H]- C14H15ClNO3: 280.0746, found: 280.0742.
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5-chloro-3-(propoxymethyl)-1H-indole-2-carboxylic acid (16e). 87% yield; white
solid; m.p. 202-204 oC; 1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.83 (s, 1H, indole
NH), 7.76 (d, J = 2.1 Hz, 1H, Ar-H), 7.42 (d, J = 8.8 Hz, 1H, Ar-H), 7.24 (dd, J = 8.7,
2.1 Hz, 1H, Ar-H), 4.94 (s, 2H, CH2OCH2CH2), 3.38 (t, J = 6.5 Hz, 2H,
OCH2CH2CH3), 1.56-1.47 (m, 2H, CH2CH3), 0.85 (t, J = 7.4 Hz, 3H, CH2CH3); 13C-
NMR (101 MHz, DMSO-d6) δ (ppm): 163.25 (COOH), 134.84, 128.63, 126.75,
125.12, 124.66, 120.75, 118.46, 114.57, 71.43, 63.38, 22.96, 11.10; HRESI-MS m/z
calcd for [M-H]- C13H13ClNO3: 266.0589, found: 266.0586.
5-chloro-1H-indole-2-carboxylic acid (18). 96% yield; white solid; m.p. 278 - 280
1
oC; H NMR (400 MHz, DMSO-d6) δ (ppm): 13.09 (s, 1H, COOH), 11.94 (s, 1H,
indole NH), 7.69 (d, J = 2.1 Hz, 1H, Ar-H), 7.42 (d, J = 8.7 Hz, 1H, Ar-H), 7.22 (dd, J
= 8.8, 2.1 Hz, 1H, Ar-H), 7.05 (d, J = 2.2, Hz, 1H, Ar-H); 13C-NMR (101 MHz,
DMSO-d6) δ (ppm): 162.92 (COOH), 136.02, 130.37, 128.29, 124.89, 124.83, 121.39,
114.56, 107.22; HRESI-MS m/z calcd for [M-H]- C9H5ClNO2: 194.0014, found:
194.0011.
5-Chloro-3-phenyl-1H-indole-2-carboxylic acid (25). 92% yield; white solid; m.p.
o
1
220-222 C; H-NMR (400 MHz, DMSO-d6) δ (ppm): 13.02 (s, 1H, COOH), 12.03
(s, 1H, indole NH), 7.52 – 7.31 (m, 7H, Ar-H), 7.28 (dd, J = 8.8, 2.0 Hz, 1H, Ar-H);
13C-NMR (101 MHz, DMSO-d6) δ (ppm): 162.93 (COOH), 134.75, 133.62, 130.80,
128.37, 128.33, 127.35, 125.54, 125.36, 121.76, 119.74, 114.81; HRESI-MS m/z
calcd for [M+H]+ C15H11ClNO2: 272.0473, found: 272.0480.
5-chloro-3-(ethoxymethyl)-N-phenethyl-1H-indole-2-carboxamide (17a). 84%
yield; white solid after purification by flash chromatography on silica gel using a
o
1
mixture of EtOAc, hexanes (1:4) as eluent; m.p.142-144 C; H-NMR (400 MHz,
CDCl3) δ (ppm): 10.55 (s, 1H, indole NH), 8.59 (t, J = 5.7 Hz, 1H, amide NH), 7.58
(d, J = 2.0 Hz, 1H, Ar-H), 7.42 (d, J = 8.7 Hz, 1H, Ar-H), 7.37–7.17 (m, 6H, Ar-H),
4.67 (s, 2H, CH2OCH2CH3), 3.81 (q, J = 7.1 Hz, 2H, OCH2CH3), 3.38 (q, J = 7.0 Hz,
2H, NHCH2CH2), 3.00 (t, J = 7.0 Hz, 2H, NHCH2CH2), 1.08 (t, J = 7.0 Hz, 3H,
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OCH2CH3); 13C-NMR (101 MHz, CDCl3) δ (ppm): 161.85 (C=O), 138.96, 133.27,
131.79, 128.78, 128.69, 128.60, 126.52, 126.08, 124.65, 118.46, 113.42, 111.47,
65.11, 62.23, 41.25, 35.74, 14.98; HRESI-MS m/z calcd for [M+H]+ C20H22ClN2O2:
357.1364, found: 357.1370.
5-chloro-N-(4-(dimethylamino)phenethyl)-3-(ethoxymethyl)-1H-indole-2-
carboxamide (17b). 79% yield; white solid after purification by flash
chromatography on silica gel using a mixture of EtOAc, hexanes (2:3) as eluent; m.p.
1
155-157 oC; H-NMR (400 MHz, CDCl3) δ (ppm): 10.70 (s, 1H, indole NH), 8.56 (t,
J = 5.7 Hz, 1H, amide NH), 7.59 (d, J = 1.9 Hz, 1H, Ar-H), 7.43 (d, J = 8.7 Hz, 1H,
Ar-H), 7.21 (dd, J = 8.7, 2.0 Hz, 1H, Ar-H), 7.15 (d, J = 8.6 Hz, 2H, Ar-H), 6.72 (d, J
= 8.6 Hz, 2H, Ar-H), 4.71 (s, 2H, CH2OCH2CH3), 3.77 (q, J = 7.1 Hz, 2H,
OCH2CH3), 3.42 (q, J = 7.0 Hz, 2H, NHCH2CH2), 2.93-2.88 (m, 8H, NHCH2CH2,
N(CH3)2), 1.12 (t, J = 7.0 Hz, 3H, OCH2CH3); 13C-NMR (101 MHz, CDCl3) δ (ppm):
161.83 (C=O), 149.54, 133.34, 131.97, 129.36, 128.73, 126.86, 126.00, 124.56,
118.41, 113.51, 113.10, 111.37, 65.11, 62.27, 41.62, 40.84, 34.79, 15.04; HRESI-MS
m/z calcd for [M+H]+ C22H27ClN3O2: 400.1786, found: 400.1788.
5-chloro-3-(ethoxymethyl)-N-(4-(pyrrolidin-1-yl)phenethyl)-1H-indole-2-
carboxamide (17c). 81% yield; white solid after purification by flash
chromatography on silica gel using a mixture of EtOAc, hexanes (2:3) as eluent; m.p.
180-182 oC; 1H-NMR (400 MHz, CDCl3) δ (ppm): 10.42 (s, 1H, indole NH), 8.53 (t, J
= 5.7 Hz, 1H, amide NH), 7.59 (d, J = 1.9 Hz, 1H, Ar-H), 7.41 (d, J = 8.7 Hz, 1H, Ar-
H), 7.21 (dd, J = 8.7, 2.0 Hz, 1H, Ar-H), 7.13 (d, J = 8.4 Hz, 2H, Ar-H), 6.54 (d, J =
8.4 Hz, 2H, Ar-H), 4.71 (s, 2H, CH2OCH2CH3), 3.74 (q, J = 7.2 Hz, 2H, OCH2CH3),
3.44 (q, J = 7.0 Hz, 2H, NHCH2CH2), 3.31–3.22 (m, 4H, pyrrolidin-H), 2.88 (t, J =
7.3 Hz, 2H, NHCH2CH2), 2.04 – 1.96 (m, 4H, pyrrolidin-H), 1.13 (t, J = 7.0 Hz, 3H,
13
OCH2CH3); C-NMR (101 MHz, CDCl3) δ (ppm): 161.71 (C=O), 133.20, 131.96,
129.41, 128.76, 126.03, 124.58, 118.44, 113.41, 111.96, 111.38, 65.14, 62.27, 47.79,
41.71, 34.89, 25.40, 15.05; HRESI-MS m/z calcd for [M+H]+ C24H29ClN3O2:
426.1943, found: 426.1944.
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5-chloro-3-(ethoxymethyl)-N-(3-(piperidin-1-yl)phenethyl)-1H-indole-2-
carboxamide (17d). 75% yield; white solid after purification by flash
chromatography on silica gel using a mixture of EtOAc, hexanes (1:2) as eluent; m.p.
1
110-112 oC; H-NMR (400 MHz, CDCl3) δ (ppm): 11.00 (s, 1H, indole NH), 8.64 (t,
J = 5.9 Hz, 1H, amide NH), 7.59 (d, J = 1.9 Hz, 1H, Ar-H), 7.46 (d, J = 8.7 Hz, 1H,
Ar-H), 7.25–7.16 (m, 2H, Ar-H), 6.88 – 6.79 (m, 2H, Ar-H), 6.75 (d, J = 7.5 Hz, 1H,
Ar-H), 4.70 (s, 2H, CH2OCH2CH3), 3.82 (q, J = 7.3 Hz, 2H, OCH2CH3), 3.41 (q, J =
7.0 Hz, 2H, NHCH2CH2), 3.17–3.09 (m, 4H, piperidin-H), 2.97 (t, J = 7.1 Hz, 2H,
NHCH2CH2), 1.73–1.62 (m, 4H, piperidin-H), 1.58-1.53 (m, 2H, piperidin-H), 1.12 (t,
13
J = 7.0 Hz, 3H, OCH2CH3); C-NMR (101 MHz, CDCl3) δ (ppm): 161.98 (C=O),
152.58, 139.65, 133.48, 131.87, 129.21, 128.68, 125.99, 124.59, 119.51, 118.38,
117.02, 114.67, 113.60, 113.58, 111.47, 65.08, 62.24, 50.64, 41.40, 36.16, 25.85,
24.30, 15.06; HRESI-MS m/z calcd for [M+H]+ C25H31ClN3O2: 440.2099, found:
440.2101.
5-chloro-3-(ethoxymethyl)-N-(4-morpholinophenethyl)-1H-indole-2-carboxamide
(17e). 80% yield; white solid after purification by flash chromatography on silica gel
o
1
using a mixture of EtOAc, hexanes (1:1) as eluent; m.p. 232-234 C; H-NMR (400
MHz, CDCl3) δ (ppm): 10.33 (s, H, indole NH), 8.55 (t, J = 5.6 Hz, 1H, amide NH),
7.58 (d, J = 1.9 Hz, 1H, Ar-H), 7.39 (d, J = 8.7 Hz, 1H, Ar-H), 7.24–7.14 (m, 3H, Ar-
H), 6.87 (d, J = 8.7 Hz, 2H, Ar-H), 4.69 (s, 2H, CH2OCH2CH3), 3.90– 3.82 (m, 4H,
morph-H), 3.75 (q, J = 6.7 Hz, 2H, OCH2CH3), 3.41 (q, J = 7.0 Hz, 2H, NHCH2CH2),
3.16–3.08 (m, 4H, morph-H), 2.91 (t, J = 7.1 Hz, 2H, NHCH2CH2), 1.10 (t, J = 7.0
13
Hz, 3H, OCH2CH3); C-NMR (101 MHz, CDCl3) δ (ppm): 161.74 (C=O), 150.03,
133.17, 131.84, 130.40, 129.47, 128.72, 126.11, 124.67, 118.46, 116.03, 113.36,
111.42, 66.91, 65.12, 62.24, 49.61, 41.38, 34.84, 15.04; HRESI-MS m/z calcd for
[M+H]+ C24H29ClN3O3: 442.1892, found: 442.1892.
5-chloro-3-(methoxymethyl)-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-
carboxamide (17f). 81% yield; white solid after purification by flash chromatography
on silica gel using a mixture of EtOAc, hexanes (1:2) as eluent; m.p. 158-160 oC; 1H-
NMR (400 MHz, CDCl3) δ (ppm): 10.23 (s, 1H, indole NH), 8.33 (t, J = 5.5 Hz, 1H,
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amide NH), 7.58 (d, J = 1.9 Hz, 1H, Ar-H), 7.40 (d, J = 8.7 Hz, 1H, Ar-H), 7.22 (dd, J
= 8.7, 2.0 Hz, 1H, Ar-H), 7.14 (d, J = 8.6 Hz, 2H, Ar-H), 6.90 (d, J = 8.6 Hz, 2H, Ar-
H), 4.63 (s, 2H, CH2OCH3), 3.76 (q, J = 6.9 Hz, 2H, NHCH2CH2), 3.17 (s, 3H,
OCH3), 3.13 – 3.07 (m, 4H, piperidin-H), 2.89 (t, J = 6.9 Hz, 2H, NHCH2CH2), 1.73-
13
1.68 (m, 4H, piperidin-H), 1.62 – 1.52 (m, 2H, piperidin-H); C-NMR (101 MHz,
CDCl3) δ (ppm): 161.60 (C=O), 151.04, 133.11, 131.95, 129.40, 129.28, 128.89,
126.14, 124.67, 118.46, 116.87, 113.33, 110.98, 64.11, 57.05, 50.97, 41.23, 34.66,
25.83, 24.27; HRESI-MS m/z calcd for [M+H]+ C24H29ClN3O2: 426.1943, found:
426.1944.
5-chloro-3-(methoxymethyl)-N-(4-morpholinophenethyl)-1H-indole-2-
carboxamide (17g). 82% yield; white solid afterpurification by flash chromatography
on silica gel using a mixture of EtOAc, hexanes (1:1) as eluent; m.p. 260-262 oC; 1H-
NMR (400 MHz, CDCl3) δ (ppm): 10.26 (s, 1H, indole NH), 8.34 (t, J = 5.5 Hz, 1H,
amide NH), 7.59 (d, J = 2.0 Hz, 1H, Ar-H), 7.39 (d, J = 8.7 Hz, 1H, Ar-H), 7.22 (dd, J
= 8.7, 2.0 Hz, 1H, Ar-H), 7.17 (d, J = 8.6 Hz, 2H, Ar-H), 6.87 (d, J = 8.6 Hz, 2H, Ar-
H), 4.64 (s, 2H, CH2OCH3), 3.89–3.82 (m, 4H, morph-H), 3.76 (q, J = 7.0 Hz, 2H,
NHCH2CH2), 3.18 (s, 3H, OCH3), 3.16–3.08 (m, 4H, morph-H), 2.90 (t, J = 6.9 Hz,
2H, NHCH2CH2); 13C-NMR (101 MHz, CDCl3) δ (ppm): 161.65 (C=O), 150.03,
133.13, 131.89, 130.32, 129.47, 128.88, 126.18, 124.72, 118.46, 116.02, 113.35,
111.00, 66.89, 64.11, 57.03, 49.61, 41.22, 34.68; HRESI-MS m/z calcd for [M+H]+
C23H27ClN3O3: 428.1735, found: 428.1736.
5-chloro-3-(isopropoxymethyl)-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-
carboxamide (17h). 75% yield; white solid after purification by flash
chromatography on silica gel using a mixture of EtOAc, hexanes (1:2) as eluent; m.p.
190-192 oC; 1H-NMR (400 MHz, CDCl3) δ (ppm): 10.49 (s, 1H, indole NH), 8.93 (s,
1H, amide NH), 7.86 (s, 1H, Ar-H), 7.68 (d, J = 8.6 Hz, 1H, Ar-H), 7.49 (d, J = 8.7
Hz, 1H, Ar-H), 7.42 (d, J = 8.0 Hz, 2H, Ar-H), 7.17 (d, J = 7.9 Hz, 2H, Ar-H), 4.99
(s, 2H, CH2OCH), 4.01 (q, J = 7.0 Hz, 2H, NHCH2CH2), 3.91–3.82 (m, 1H,
CH(CH3)2), 3.41- 3.37 (m, 4H, piperidin-H), 3.17 (t, J = 7.0 Hz, 2H, NHCH2CH2),
2.03–1.91 (m, 4H, piperidin-H), 1.87-1.83 (m, 2H, piperidin-H), 1.40 (d, J = 6.6 Hz,
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6H, CH(CH3)2); 13C-NMR (101 MHz, CDCl3) δ (ppm): 161.67 (C=O), 151.04,
133.13, 131.96, 129.56, 129.37, 128.65, 126.05, 124.62, 118.43, 116.89, 113.34,
111.66, 70.61, 59.60, 50.97, 41.57, 34.99, 25.87, 24.29, 21.92; HRESI-MS m/z calcd
for [M+H]+ C26H33ClN3O2: 454.2256, found: 454.2245.
5-chloro-3-(isopropoxymethyl)-N-(4-morpholinophenethyl)-1H-indole-2-
carboxamide (17i). 77% yield; white solid after purification by flash chromatography
on silica gel using a mixture of EtOAc, hexanes (1:1) as eluent; m.p. 282-284 oC; 1H-
NMR (400 MHz, CDCl3) δ (ppm): 9.95 (s, 1H, indole NH), 8.64 (t, J = 5.7 Hz, 1H,
amide NH), 7.58 (d, J = 2.0 Hz, 1H, Ar-H), 7.38 (d, J = 8.7 Hz, 1H, Ar-H), 7.22 (dd, J
= 8.7, 2.0 Hz, 1H, Ar-H), 7.17 (d, J = 8.6 Hz, 2H, Ar-H), 6.87 (d, J = 8.6 Hz, 2H, Ar-
H), 4.71 (s, 2H, CH2OCH), 3.90–3.82 (m, 4H, morph-H), 3.72 (q, J = 7.2 Hz, 2H,
NHCH2CH2), 3.60 (p, J = 6.1 Hz, 1H, CH(CH3)2), 3.16–3.08 (m, 4H, morph-H), 2.90
(t, J = 7.2 Hz, 2H, NHCH2CH2), 1.12 (d, J = 6.1 Hz, 6H, CH(CH3)2); 13C-NMR (101
MHz, CDCl3) δ (ppm): 161.59 (C=O), 149.98, 133.00, 131.88, 130.52, 129.53,
128.65, 126.13, 124.70, 118.47, 116.04, 113.24, 111.69, 70.64, 66.91, 59.61, 49.64,
41.48, 34.98, 21.92; HRESI-MS m/z calcd for [M+H]+ C25H31ClN3O3: 456.2048,
found: 456.2051.
5-chloro-3-(isobutoxymethyl)-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-
carboxamide (17j). 74% yield; white solid after purification by flash chromatography
on silica gel using a mixture of EtOAc, hexanes (1:2) as eluent; m.p. 201-203 oC; 1H-
NMR (400 MHz, CDCl3) δ (ppm): 10.51 (s, 1H, indole NH), 8.49 (t, J = 5.8 Hz, 1H,
amide NH), 7.59 (d, J = 2.1 Hz, 1H, Ar-H), 7.42 (d, J = 8.7 Hz, 1H, Ar-H), 7.22 (dd, J
= 8.8, 2.0 Hz, 1H, Ar-H), 7.15 (d, J = 8.6 Hz, 2H, Ar-H), 6.90 (d, J = 8.6 Hz, 2H, Ar-
H), 4.67 (s, 2H, CH2OCH2CH), 3.74 (q, J = 7.2 Hz, 2H, NHCH2CH2), 3.16–3.08 (m,
6H, piperidin-H, CH2OCH2CH ), 2.90 (t, J = 7.1 Hz, 2H, NHCH2CH2), 1.83 – 1.66
(m, 5H, CH(CH3)2, piperidin-H), 1.60 - 1.55 (m, 2H, piperidin-H), 0.86 (d, J = 6.6
Hz, 6H, CH(CH3)2); 13C-NMR (101 MHz, CDCl3) δ (ppm): 161.76 (C=O), 151.02,
133.27, 131.96, 129.46, 129.39, 128.80, 126.05, 124.60, 118.46, 116.83, 113.44,
111.54, 76.57, 62.61, 50.91, 41.59, 34.95, 28.38, 25.87, 24.29, 19.36; HRESI-MS m/z
calcd for [M+H]+ C27H35ClN3O2: 468.2412, found: 468.2414.
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5-chloro-3-(isobutoxymethyl)-N-(4-morpholinophenethyl)-1H-indole-2-
carboxamide (17k). 75% yield; white solid after purification by flash
chromatography on silica gel using a mixture of EtOAc, hexanes (1:1) as eluent; m.p.
165-167 oC; 1H-NMR (400 MHz, CDCl3) δ (ppm): 10.09 (s, 1H, indole NH), 8.47 (s,
1H, amide NH), 7.59 (s, 1H, Ar-H), 7.39 (d, J = 8.7 Hz, 1H, Ar-H), 7.26 – 7.14 (m,
3H, Ar-H), 6.89 (d, J = 8.4 Hz, 2H, Ar-H), 4.67 (s, 2H, CH2OCH2CH), 3.91 – 3.84
(m, 4H, morph-H), 3.72 (q, J = 6.7 Hz, 2H, NHCH2CH2), 3.18–3.06 (m, 6H,
CH2OCH2CH, morph-H), 2.90 (t, J = 7.2 Hz, 2H, NHCH2CH2), 1.80-1.73 (m, 1H,
13
CH(CH3)2), 0.85 (d, J = 6.6 Hz, 6H, CH(CH3)2); C-NMR (101 MHz, CDCl3) δ
(ppm): 161.63 (C=O), 149.77, 133.07, 131.87, 130.64, 129.57, 128.82, 126.16,
124.70, 118.51, 116.08, 113.29, 111.55, 76.60, 66.86, 62.61, 49.69, 41.47, 34.96,
28.37, 19.36; HRESI-MS m/z calcd for [M+H]+ C26H33ClN3O3: 470.2205, found:
470.2205.
5-chloro-N-(4-(piperidin-1-yl)phenethyl)-3-(propoxymethyl)-1H-indole-2-
carboxamide (17l). 73% yield; white solid after purification by flash chromatography
on silica gel using a mixture of EtOAc, hexanes (1:2) as eluent; m.p. 181-182 oC; 1H-
NMR (400 MHz, CDCl3) δ (ppm): 10.36 (s, 1H, indole NH), 8.52 (t, J = 5.7 Hz, 1H,
amide NH), 7.59 (d, J = 1.9 Hz, 1H, Ar-H), 7.41 (d, J = 8.7 Hz, 1H, Ar-H), 7.21 (dd, J
= 8.7, 1.9 Hz, 1H, Ar-H), 7.14 (d, J = 8.5 Hz, 2H, Ar-H), 6.90 (d, J = 8.6 Hz, 2H, Ar-
H), 4.68 (s, 2H, CH2OCH2CH2), 3.74 (q, J = 7.1 Hz, 2H, NHCH2CH2), 3.30 (t, J = 6.7
Hz, 2H, OCH2CH2CH3), 3.16–3.08 (m, 4H, piperidin-H), 2.89 (t, J = 7.1 Hz, 2H,
NHCH2CH2), 1.75-1.88 (m, 4H, piperidin-H), 1.62–1.47 (m, 4H, piperidin-H,
CH2CH3), 0.86 (t, J = 7.4 Hz, 3H, CH2CH3); 13C-NMR (101 MHz, CDCl3) δ (ppm):
161.72 (C=O), 151.00, 133.19, 131.93, 129.49, 129.32, 128.77, 126.06, 124.62,
118.47, 116.85, 113.38, 111.48, 71.44, 62.42, 50.95, 41.50, 34.88, 25.85, 24.27,
22.76, 10.59; HRESI-MS m/z calcd for [M+H]+ C26H33ClN3O2: 454.2256, found:
454.2255.
5-chloro-N-(4-morpholinophenethyl)-3-(propoxymethyl)-1H-indole-2-
carboxamide (17m). 72% yield; white solid after purification by flash
chromatography on silica gel using a mixture of EtOAc, hexanes (1:1) as eluent; m.p.
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1
185-187 oC; H-NMR (400 MHz, CDCl3) δ (ppm): 10.11 (s, 1H, indole NH), 8.51 (t,
J = 5.6 Hz, 1H, amide NH), 7.58 (d, J = 1.9 Hz, 1H, Ar-H), 7.39 (d, J = 8.8 Hz, 1H,
Ar-H), 7.25–7.13 (m, 3H, Ar-H), 6.87 (d, J = 8.4 Hz, 2H, Ar-H), 4.68 (s, 2H,
CH2OCH2CH2), 3.86 (t, J = 5.9 Hz, 4H, morph-H), 3.72 (q, J = 7.2 Hz, 2H,
NHCH2CH2), 3.31 (t, J = 6.7 Hz, 2H, OCH2CH2CH3), 3.13 (t, J = 5.8 Hz, 4H, morph-
H), 2.90 (t, J = 7.1 Hz, 2H, NHCH2CH2), 1.52-1.47 (m, 2H, CH2CH3), 0.86 (t, J = 7.4,
1.0 Hz, 3H, CH2CH3); 13C-NMR (101 MHz, CDCl3) δ (ppm): 161.65 (C=O), 149.99,
133.08, 131.86, 130.39, 129.50, 128.78, 126.14, 124.69, 118.50, 115.97, 113.29,
111.49, 71.45, 66.91, 62.42, 49.58, 41.43, 34.87, 22.75, 10.60; HRESI-MS m/z calcd
for [M+H]+ C25H31ClN3O3: 456.2048, found: 456.2048.
5-chloro-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (19). 90%
yield; white solid after purification by flash chromatography on silica gel using a
o
1
mixture of EtOAc, hexanes (1:4) as eluent; m.p. 210 - 212 C; H-NMR (250 MHz,
CDCl3) δ (ppm): 11.77 (s, 1H, indole NH), 8.64 (t, J = 5.5 Hz, 1H, amide NH), 7.70
(s, 1H, Ar-H), 7.42 (d, J = 8.25 Hz, 1H, Ar-H), 7.17 (d, J = 8.25 Hz, 1H, Ar-H), 7.07
(d, J = 8.0 Hz, 2H, Ar-H), 6.85 (d, J = 8.0 Hz, 2H, Ar-H), 3.54 (q, J = 7.25 Hz, 2H,
NHCH2CH2), 3.14–3.02 (m, 4H, piperidin-H), 2.75 (t, J = 7.25 Hz, 2H, NHCH2CH2),
1.65 - 1.50 (m, 6H, piperidin-H); 13C-NMR (62.5 MHz, CDCl3) δ (ppm):161.62
(C=O), 151.14, 133.21, 131.95, 129.40, 129.28, 128.89, 126.14, 124.67, 118.46,
116.87, 113.33, 110.98, 50.97, 41.23, 34.66, 25.83, 24.27; HRESI-MS m/z calcd for
[M+H]+ C22H25ClN3O: 382.1681, found: 382.1678.
5-Chloro-3-phenyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-
carboxamide(24a). 77% yield; white solid after purification by flash chromatography
on silica gel using a mixture of EtOAc, hexanes (1:2) as eluent; m.p. 178-180 oC; 1H-
NMR (400 MHz, CDCl3) δ (ppm): 10.25 (s, 1H, indole NH), 7.51–7.32 (m, 7H, Ar-
H), 7.28–7.20 (m, 1H, Ar-H), 6.88 (d, J = 8.6 Hz, 2H, Ar-H), 6.82 (d, J = 8.6 Hz, 2H,
Ar-H), 6.07 (t, J = 5.7 Hz, 1H, amide NH), 3.58 (q, J = 6.8 Hz, 2H, NHCH2CH2),
3.17 – 3.09 (m, 4H, piperidin-H), 2.65 (t, J = 6.8 Hz, 2H, NHCH2CH2), 1.76 - 1.68
(m, 4H, piperidin-H), 1.62-1.55 (m, 2H, piperidin-H); 13C-NMR (101 MHz, CDCl3) δ
(ppm): 161.52 (C=O), 150.94, 133.45, 132.88, 130.36, 129.33, 129.27, 129.02,
128.77, 128.28, 127.68, 126.23, 125.14, 119.93, 117.53, 116.81, 113.13, 50.84, 41.06,
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34.17, 25.90, 24.29; HRESI-MS m/z calcd for [M+H]+ C28H29ClN3O: 458.1994,
found: 458.1993.
5-chloro-N-phenethyl-3-phenyl-1H-indole-2-carboxamide (24b). 80% yield; white
solid after purification by flash chromatography on silica gel using a mixture of
o
1
EtOAc, hexanes (1:4) as eluent; m.p.158-160 C; H-NMR (400 MHz, CDCl3) δ
(ppm): 10.66 (s, 1H, indole NH), 7.50–7.33 (m, 7H, Ar-H), 7.30–7.22 (m, 4H, Ar-H),
7.03 (d, J = 7.5 Hz, 2H, Ar-H), 6.10 (t, J = 5.8 Hz, 1H, amide NH), 3.66 (q, J = 6.6
13
Hz, 2H, NHCH2CH2), 2.77 (t, J = 6.8 Hz, 2H, NHCH2CH2); C-NMR (101 MHz,
CDCl3) δ (ppm):161.74 (C=O), 138.41, 133.67, 132.91, 130.33, 129.35, 129.23,
128.68, 128.52, 128.33, 127.57, 126.52, 126.23, 125.17, 119.93, 117.69, 113.26,
40.91, 35.15; HRESI-MS m/z calcd for [M+H]+ C23H20ClN2O: 375.1259, found:
375.1259.
5-chloro-N-(4-(dimethylamino)phenethyl)-3-phenyl-1H-indole-2-carboxamide
(24c). 81% yield; white solid after purification by flash chromatography on silica gel
o
1
using a mixture of EtOAc, hexanes (1:2) as eluent; m.p. 178-180 C; H-NMR (400
MHz, CDCl3) δ (ppm): 10.60 (s, 1H, indole NH), 7.50–7.36 (m, 7H, Ar-H), 7.29–7.21
(m, 1H, Ar-H), 6.89 (d, J = 8.6 Hz, 2H, Ar-H), 6.65 (d, J = 8.6 Hz, 2H, Ar-H), 6.11 (t,
J = 5.6 Hz, 1H, amide NH), 3.61 (q, J = 7.0 Hz, 2H, NHCH2CH2), 2.95 (s, 6H,
13
N(CH3)2), 2.67 (t, J = 6.9 Hz, 2H, NHCH2CH2); C-NMR (101 MHz, CDCl3) δ
(ppm): 161.66 (C=O), 149.43, 133.63, 132.96, 130.40, 129.33, 129.25, 129.11,
128.25, 127.74, 126.19, 126.17, 125.09, 119.89, 117.55, 113.27, 113.07, 41.26, 40.79,
34.12; HRESI-MS m/z calcd for [M+H]+ C25H25ClN3O: 418.1681, found: 418.1673.
5-chloro-N-(4-morpholinophenethyl)-3-phenyl-1H-indole-2-carboxamide (24d).
78% yield; white solid after purification by flash chromatography on silica gel using a
o
1
mixture of EtOAc, hexanes (1:1) as eluent; m.p. 198-200 C; H-NMR (400 MHz,
CDCl3) δ (ppm): 10.32 (s, 1H, indole NH), 7.48–7.34 (m, 7H, Ar-H), 7.28–7.20 (m,
1H, Ar-H), 6.92 (d, J = 8.5 Hz, 2H, Ar-H), 6.80 (d, J = 8.6 Hz, 2H, Ar-H), 6.07 (t, J =
5.6 Hz, 1H, amide NH), 3.91–3.84 (m, 4H, morph-H), 3.59 (q, J = 6.9, 5.7 Hz, 2H,
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NHCH2CH2), 3.18–3.10 (m, 4H, morph-H), 2.67 (t, J = 6.9 Hz, 2H, NHCH2CH2);
13C-NMR (101 MHz, CDCl3) δ (ppm): 161.56 (C=O), 149.92, 133.49, 132.92,
130.37, 129.76, 129.33, 129.25, 129.22, 128.29, 127.63, 126.26, 125.18, 119.94,
117.55, 115.99, 113.15, 66.93, 49.53, 41.02, 34.21; HRESI-MS m/z calcd for [M+H]+
C27H27ClN3O2: 460.1786, found: 460.1781.
(5-chloro-3-phenyl-1H-indol-2-yl)(4-phenylpiperazin-1-yl)methanone (25). 74%
yield; white solid after purification by flash chromatography on silica gel using a
o
1
mixture of EtOAc, hexanes (1:2) as eluent; m.p. 220-222 C; H-NMR (400 MHz,
CDCl3) δ (ppm): 10.41 (s, 1H, indole NH), 7.72 (d, J = 2.0 Hz, 1H, Ar-H), 7.55–7.32
(m, 6H, Ar-H), 7.28–7.18 (m, 3H, Ar-H), 6.87 (t, J = 7.3 Hz, 1H, Ar-H), 6.75 (d, J =
8.4 Hz, 2H, Ar-H), 3.86 (s, 2H, piperazin-H), 3.28 (s, 2H, piperazin-H), 3.10 (s, 2H,
piperazin-H), 2.38 (s, 2H, piperazin-H); 13C-NMR (101 MHz, CDCl3) δ (ppm):
164.06 (C=O), 150.67, 134.55, 133.68, 129.42, 129.19, 129.17, 127.56, 127.06,
126.45, 124.50, 120.57, 119.56, 117.56, 116.68, 113.10, 48.82, 42.54; HRESI-MS
m/z calcd for [M+H]+ C25H23ClN3O: 416.1524, found: 416.1525.
(4-benzylpiperidin-1-yl) (5-chloro-3-phenyl-1H-indol-2-yl)methanone (26). 75%
yield; white solid after purification by flash chromatography on silica gel using a
o
1
mixture of EtOAc, hexanes (1:2) as eluent; m.p. 222-224 C; H-NMR (400 MHz,
DMSO-d6) δ (ppm): 11.98 (s, 1H, indole NH), 7.62 (d, J = 1.9 Hz, 1H, Ar-H), 7.47–
7.34 (m, 5H, Ar-H), 7.32 (t, J = 6.9 Hz, 1H, Ar-H), 7.21-7.14 (m, 3H, Ar-H), 7.09 (t,
J = 7.3 Hz, 1H, Ar-H), 6.96 (d, J = 7.4 Hz, 2H, Ar-H), 4.45 (d, J = 13.1 Hz, 1H,
piperidin-H), 3.38 (s, 1H, piperidin-H), 2.56–2.44 (m, 2H, piperidin-H), 2.20 (d, J =
6.5 Hz, 2H, PhCH2), 1.56–1.42 (m, 2H, piperidin-H), 0.93 (t, J = 15.0 Hz, 2H,
piperidin-H), 0.05 (s, 1H, piperidin-H); 13C-NMR (101 MHz, DMSO-d6) δ (ppm):
162.57 (C=O), 140.09, 134.61, 133.94, 130.57, 129.32, 129.26, 128.99, 128.50,
127.12, 126.64, 126.18, 125.27, 123.27, 118.73, 114.55, 114.10, 46.90, 42.45, 37.32,
31.14; HRESI-MS m/z calcd for [M+H]+ C27H26ClN2O: 429.1728, found: 429.1728.
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Biological evaluation
Chemicals
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) was purchased
from sigma-Aldrich chemical company (St Lewis, MO, USA). BD Cycle test™ Plus
DNA Reagent Kit was purchased from BD Biosciences (San Jose, CA, USA). All
other chemicals, reagents, and solvents were of analytical grade that obtained from
standard suppliers and were used without further purification.
Cells and cell culture
MCF7, a human breast adenocarcinoma cell line, and HCT116, a colon cancer cell
line, were purchased from American type Culture Collection (ATCC, Manassas,
USA) and grown on Roswell Park Memorial Institute medium (RPMI 1640)
supplemented with 100 µg/mL of streptomycin, 100 units/mL of penicillin and 10%
of heat-inactivated fetal bovine serum in a humidified, 5% (v/v) CO2 atmosphere at 37
ºC. The cells were grown confluence, which could be observed under an inverted
microscope and sub–cultured at three to four days interval.
MTT cytotoxicity assay
Exponentially growing cells from different cancer cell lines were trypsinized and
counted using hemocytometer then were seeded at the appropriate densities (5000
cells/0.33 cm2 well) into 96-well micro plates. Subsequently, cells were incubated at
37oC in 5% CO2 for 24 h to allow cells attachment. The medium was removed and
replaced with fresh medium containing various concentrations of the tested
compounds (0.01, 0.1, 1.0, 10, 100, and 1000 µM) in triplicates (n=3). Cells were
incubated at 37oC, 5% CO2. Seventy-two h later, the viability of treated cells was
determined using MTT technique. Briefly, media were removed and cells were
incubated with 200 μl of 5% MTT solution/well and were allowed to metabolize the
dye into colored-insoluble formazan crystals for 2 h. The remaining MTT solution
were discarded from the wells and the formazan crystals were dissolved in 200
µl/well acidified isopropanol for 30 minutes, covered with aluminum foil and with
continuous shaking using a MaxQ 2000 plate shaker (Thermo Fisher Scientific Inc,
MI) at room temperature. Absorbance was measured at 570 nm using a Stat FaxR
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4200 plate reader (Awareness Technology, Inc., FL). The cell viability were
expressed as percentage of control and the concentration that induces 50% of
maximum inhibition of cell proliferation (IC50) were determined using Graph Pad
Prism version 5 software (Graph Pad software Inc, CA) (24-25) .
DNA-Flow Cytometry Analysis
Cells at a concentration of 1x106 cells/ml were seeded into 6-well plate in 2 ml culture
medium with the compound with highest activity at its IC50 and were incubated at
37ºC in an atmosphere of 5% CO2 for 24 h. Some wells were left with no treatment to
be used as a control. After the incubation period, the cultured cells were collected by
trypsinization, washed in PBS and fixed in ice-cold absolute alcohol. Thereafter, cells
were stained using Cycle test™ Plus DNA Reagent Kit (BD Biosciences, San Jose,
CA, USA) according to the manufacturer’s instructions. Cell-cycle distribution was
determined using a FACS Calibur flow cytometry system (BD Biosciences, San Jose,
CA, USA).
Pharmacophore Generation
The training set was built from 50 compounds using the Molecular Operating
Environment (MOE) package version 2011.10 (Chemical Computing Group, Inc.
Molecular Operating Environment (MOE). CCG, Montreal, Canada. 2011) and saved
in the Sdf format database. A common-features pharmacophore model was derived
with the HipHop module of Catalyst using the Accelrys Discovery Studio 2.5 (DS)
software.
For all compounds in the training set, energy minimization process was performed
with CHARMM force field. Poling algorithm was applied to generate a maximum of
255 diverse conformations with the energy threshold of 20 kcal mol-1 above the
calculated energy minimum for every compound in the dataset. These conformers
were generated using Diverse Conformer Generation protocol running with
Best/Flexible conformer generation option as available in DS. The most active
compound (24a) was considered as ‘reference compound’ specifying a ‘Principal’
value of 2 and a ‘MaxOmitFeat’ value of 0, meaning its structure and conformation
would have the strongest influence in the model building phase. The ‘Principal’ value
and ‘MaxOmitFeat’ value for the remaining active compounds were set to 1 and 1,
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respectively. The inactive compounds were given a ‘Principal’ value and
‘MaxOmitFeat’ value of 0 and 1, respectively. Feature mapping protocol was used to
identify the common chemical groups present in the training set compounds. As
predicted, hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), hydrophobic
(HY) features were selected during the pharmacophore generation. Misses specifies
the number of molecules that do not have to map to all features in the hypothesis.
Feature Misses, the number of maximal molecules which do not have to map to each
feature in generated hypothesis. Complete Misses specify the number of molecules
that do not have to map to any features in the hypothesis. The Misses, Feature Misses
and Complete Misses were set to 2, 2 and 2, respectively. The minimum inter-feature
distance was chosen to be not less than 3.0 Å. Finally, exclusion volume was added in
order to tuning of the pharmacophore using the steric refinement with exclusion
volume protocol within the Discovery studio Software package
Results and Discussion
Chemistry
The synthetic pathway of the required phenethylamine intermediates 7-10 and 12 was
illustrated in scheme 1. Aminobenzyl cyanide is subjected to alkylation using 1,5-
dibromopentane, 1,4-dibromobutane and bromoethyl ether to yield piperidinyl benzyl
cyanide, pyrrolidinyl benzyl cyanide and morpholinyl benzyl cyanide 3-6 respectively
1
(23). The H-NMR spectrum of 6 as an example of this series showed singlet
equivalent to two protons at δ 3.62 ppm which assigned to CH2CN group in addition
topyrrolidinyl peaks. Benzyl cyanides 3-6 are reduced to corresponding amine
1
derivatives 7-10 using LiAlH4. The H-NMR spectrum of 10 as an example of this
series showed two triplet signals at δ 2.90 and 2.64 ppm, which were assigned to the
two methylene protons of NHCH2CH2 and NHCH2CH2 respectively and a broad
singlet at δ 1.29 ppm ascribed to NH2. The structure of 10 was also confirmed by
HRESI-MS which gave a molecular ion m/z 191.1540 [M+H]+, consistent with the
molecular formula C12H19N2 of the desired product. Compound 12 was prepared by
alkylation of p-aminobenzylcyanide followed by reduction by LiAlH4 to the
corresponding amine. Structural confirmation of this amine was provided through 1H-
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NMR, 13C-NMR, and HRESI-MS, which are in comparable with the calculated
values.
Scheme 1: Synthetic route of phenethylamine intermediates 7-10, 12
Reagents and conditions: (a) 1,5-dibromopentane, bromoethyl ether, or 1,4-
dibromobutane, DIPEA, toluene, reflux, 20 h. (b) LiAlH4, Et2O, 0 oC to rt, overnight.
(c) 37 % aqueous formaldehyde, NaBH3CN, acetic acid, CH3CN, rt, 3h. (d) LiAlH4,
Et2O, 0 oC to rt, overnight.
The synthesis of indole-2-carboxamides 17a-m, 19 is described in Scheme 2.
Compound 14 was prepared using the Mannich reaction conditions. Ethyl indole-2-
carboxylate 13 was treated with diethylamine and aqueous formaldehyde in refluxing
acetic acid for 4 h. After dilution of the reaction mixture with excess water and
removal of non-polar compounds by extraction with ether, the solution was made
basic by addition of an excess 5% NaOH. The precipitate formed was collected by
filtration to provide 70% of 14. The structure of compounds 14 was confirmed by 1H-
NMR, 13C-NMR as well as HRESI-MS. Analysis of the 1H-NMR spectrum showed a
triplet at δ 1.33 ppm and quartet at 4.33 ppm corresponding to OCH2CH3 moiety,
singlet signal at 3.99 (-CH2N), quartet at δ 2.46 ppm and triplet at 0.96 ppm (2 x
NCH2CH3). The structure of compound 14 was also confirmed by HRESI-MS which
gave a molecular ion m/z 309.1367[M+H]+, consistent with the molecular formula
C16H22ClN2O2 of the product.
The indole ester 14 was then hydrolysed using an aqueous solution of 5% NaOH in
o
appropriate alcohol. After stirring overnight at 40 C and acidification, the reaction
mixture did not yield the desired 3-diethylaminomethylindole-2-carboxylic acid 15
but instead 3-alkoxymethylindole-2-carboxylic acids 16a-e were obtained. Analysis
1
of the H-NMR spectrum of 16a as an example of this series showed the
disappearance of diethylamino protons and the presence of only one ethyl group with
a quartet at 3.46 ppm and a triplet at 1.11 ppm. The 13C-NMR spectrum of 16a
showed the disappearance of diethylamino carbons and appearance of peaks at 63.0
and 15.6 which were assignedto ethoxy carbons. The structure of 16a was also
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confirmed by HRESI-MS which gave a molecular ion m/z 252.0431 [M-H]-,
consistent with the molecular formula C12H11ClNO3 of the product.
The indole-2-carboxylic acids 16a-m were coupled with appropriate amine using the
BOP coupling reagent in the presence of DIPEA to yield corresponding 5-chloro-3-
1
ethoxymethylindole-2-carboxamides 17a-m. The H-NMR spectrum of 17c as a
representative example of this series revealed the appearance of a signal at 8.53 ppm
assigned to amide NH, two sets of doublets at 7.13 and 6.54 ppm with J = 8.4 Hz each
which is indicative of aromatic para-disubstitution, two signals with two protons
integration each at 3.44 (q) and 2.88 (t) ppm attributed to NHCH2CH2 group and
appearance of a singlet with two proton integration at 4.71 ppm assigned for
methylene protons of CH2OCH2CH3 and ethyl group signals at δ 3.74 (q, J = 7.2 Hz,
2H, OCH2CH3) and 1.13 (t, J = 7.0 Hz, 3H, OCH2CH3) as well as pyrrolidinyl
protons. The structure of 17c was also confirmed by HRESI-MS which gave a
molecular ion m/z 426.1944 [M+H]+, consistent with the molecular formula
C24H29ClN3O2 of the desired product.
The synthesis of indole-2-carboxamide 19 was accomplished by coupling of 2-(4-
(piperidin-1-yl)phenyl)ethanamine7 with 5-chloroindole-2-carboxylic acid 18 which
obtained from alkaline hydrolysis of indole-2-carboxylate 13. The structure of
compound 19 was confirmed by NMR and HRESI-MS
Scheme 2: Synthetic route of indole-2-carboxamides 17a-m and 19
o
Reagents and conditions: (a) diethylamine, 37% HCHO, acetic acid, 100 C,
o
overnight, 70%. (b) Appropriate alcohol, 5% NaOH, 40 C, overnight, then 5% HCl
o
workup. (c) Appropriate amine, BOP, DIPEA, DCM. (d) 5% NaOH, EtOH, 40 C,
overnight, then 5% HCl workup. (e) 2-(4-(piperidin-1-yl)phenyl)ethanamine, BOP,
DIPEA, DCM
Compound
17a
R
R1
Compound
17h
R
R1
H
p-piperidyl
p-dimethylamine
p-pyrrolidinyl
p-morpholinyl
p-piperidyl
17b
17c
17i
17j
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m-piperidyl
p-morpholinyl
p-piperidyl
17d
17e
17f
17g
17k
17l
p-morpholinyl
p-piperidyl
p-morpholinyl
17m
p-morpholinyl
The synthesis of indole-2-carboxamide derivatives 24a-d, 25-26 is demonstrated in
Scheme 3. The preparation of ethyl 5-chloro-3-phenyl-1H-indole-2-carboxylate 22
was achieved by reaction of p-chlorophenyl hydrazine 20 with phenyl pyruvic acid 21
in presence of p-toluenesulfonic acid (PTSA) using modified Fisher indole cyclization
to afford 3-alkylindole-2-carboxylate esters 22. This ester was verified by NMR and
HRESI-MS. Conversion of ester 22 to corresponding carboxylic acid 23 was
performed by hydrolysis using 5% NaOH. The structure of 23 was confirmed by
1
NMR and HRESI-MS. Analysis of the HNMR spectrum of 23 revealed the
disappearance of ethoxy protons in its ester starting material. Furthermore, the ethoxy
carbon signals were also disappeared in the 13C-NMR spectrum of the product,
indicating successful deprotection of the carboxylic acid group. The appropriate
primary or secondary amines were coupled with 5-chloro-3-phenyl-1H-indole-2-
carboxylic acid 23 using BOP as a coupling reagent in the presence of DIPEA to yield
5-chloro-3-phenylindole-2-carboxamides 24a-d, 25-26. All the final 3-phenylindole-
1
2-carboxamides structures 24a-d were verified using H-NMR, 13C-NMR and
HRESI-MS
Scheme 3: Synthetic route of 5-chloro-3-phenylindole-2-carboxamide derivatives
24a-d, 25-26
Reagents and conditions: (a) PTSA, EtOH, reflux, 20 h, 75%. (b) 5% NaOH, EtOH,
40 oC, 92%. (c) Appropriate amine, BOP, DIPEA, DCM, rt, overnight.
Biological evaluation
Cell cytotoxicity studies of the tested compounds 17a-m, 19, 24a-d, 25 and 26 were
evaluated using MTT assay in comparison to 5-Fluorouracil (5-FU) as a reference
standard. The IC50 values of these compounds are shown in Table 1.
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Table 1: IC50 values of tested compound against MCF7 and HCT116 cell lines.
Data are presented as mean SD (n = 3).
Analysis of Cellular DNA Cytometry
The most active compounds were selected to be further studied regarding their effects
on the cell cycle progression against MCF7 and HCT116 cell lines. The cells were
incubated with IC50 of compounds 17e, 17i and 24a for 24 h and their effects on the
normal cell cycle profile analyzed using Cycle TESTTM PLUS DNA Reagent Kit (BD
Biosciences, San Jose, CA) according to the manufacturer’s instructions and
employing a FACS Caliber flow cytometer (BD Biosciences, San Jose, CA).
As summarized in Table 2, exposure of MCF7 cells to compound 24a resulted in
drastic changes in the population of different phases in the cell cycle. Compound 24a
induced a significant increase in the percentage of cells at S phase to 180% with
subsequent decrease in G2/M, G0/G1 and Pre-G phases by 73, 12 and 67%;
respectively, compared to control (Table 2, Figure 2).
Similarly, exposure of HCT116 cells to compounds 17e and 17i resulted in an
interference with the normal cell cycle distribution of this cell line. Compound 17e
induced a significant increase in the percentage of cells at pre-G (2 fold) and G0/G1
to 113% with subsequent decrease in G2/M and S phases by 42 and 60%;
respectively, compared to control. Meanwhile 17i induced a significant increase in the
percentage of G0/G1 to 108% with subsequent decrease in Pre-G, G2/M and S phases
by 98, 16 and 30%; compared to control (Table 3, Figure 3). Collectively exposure
of HCT116 and MCF7 to the most active compounds in this series resulted in
disturbance in cell cycle progression and cell cycle arrest.
Table 2: Cell cycle analysis of MCF7 Cells treated with 24a at IC50 for 24 h in
comparison to untreated cells.
Table 3: Cell cycle analysis of HCT116 Cells treated with 17e and 17i at the
corresponding IC50 for 24 h in comparison to untreated cells.
Figure 2: Cell Cycle Analysis of MCF7 treated with 24a at IC50 for 24 h. (A)
Untreated cells, (B) Treatment after 24 h, (C) Bar chart shows percentage of MCF7
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cells at each phase of cell cycle in control cells and cells treated with the IC50 of 24a
after 24 h.
Figure 3: Cell Cycle Analysis of HCT116 treated with 17e and 17i at the
corresponding IC50 for 24 h. (A) Untreated cells, (B) Treatment with IC50 of 17e after
24 h, (C) Treatment with IC50 of 17i after 24 h, (D) Bar chart shows percentage of
HCT116 cells at each phase of cell cycle in control cells and cells treated with the
IC50 of 17e and 17i after 24 h.
Pharmacophore Generation
HipHop Pharmacophore:
Accelrys Discovery Studio Software package (DS) was used for automated ligand-
based pharmacophore generation. The algorithms within Catalyst are used to support
most of the pharmacophore related tasks in Discovery Studio. These tasks include
automatic creation of pharmacophores, creation of ligand conformations, creation of
compact, indexed ligand databases that can be searched by pharmacophores, and
many others (Catalyst 4.11 User Guide, Accelrys Inc., San Diego, CA 92121, USA,
2005). Accelrys Catalyst can generate two types of chemical feature based models or
hypothesis, depending on whether there is activity data or no activity data (26). The
two main algorithms used in Catalyst Pharmacophore generation are, HypoGen (27)
and HipHop (28). HypoGen attempts to generate pharmacophore models from a set of
molecules for which activity values on a given biological target have been measured
and relate this model to the activity. HipHopis intended to derive common feature
pharmacophore models using information from a set of active compounds. The
retrieved pharmacophore models are expected to discriminate between active and
inactive compounds. HipHop identifies configurations or three-dimensional spatial
arrangements of chemical features that are common to molecules in a training set. The
configurations are identified by a pruned exhaustive search, starting with small sets of
features and extending them until no larger common configuration is found. The
resultant pharmacophores are ranked as they are built. The ranking is a measure of
how well the molecules map onto the proposed pharmacophores as well as the rarity
of the pharmacophore model. If a pharmacophore model is less likely to map to an
inactive compound, it will be given a higher rank.
In order to generate the best model, a lot of HipHop runs were carried out by varying
the control parameters. In the model generation methodology, the highest weighting
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was assigned to the most active compound in the training set (24a; IC50 0.13 μM), this
was achieved by putting 2 (which ensures that all of the chemical features in the
compound will be considered in building hypotheses space) and 0 (which forces
mapping of all features of compound) in principle and maximum omitting features
columns, respectively. For the other active compounds (17a, 17b-17f, 17h-17m and
24b-24d), the value of 1 (ensures that at least one mapping for each of generated
hypotheses will be found) and 1 (all but one feature must map) were put in principle
and maximum omitting feature column respectively. For the low active compounds
(17g and 19) the value of 0 (indicates that the molecule is inactive and will not be
used in the creation of the pharmacophore) and 1 were put in principle and maximum
omitting feature column respectively. Feature mapping protocol was used to identify
the common chemical groups present in the training set compounds. As predicted,
hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), hydrophobic (HY)
features were selected during the pharmacophore generation.
The successful HipHop run resulted in the generation of 10 hypotheses, each
composed of five features (Table 4). The 10 hypotheses (Hypos) generated had scores
from 214.656 to 193.712
Table 4: Summery of HipHop generated common feature hypothesis:
This small range of ranking score suggests that the features are spatially arranged in a
similar fashion in all 10 models. To determine the similarity between the ten
hypotheses, a hierarchical cluster analysis was performed. The results of the cluster
analysis indicate that hypos 1 and 3, 4, 7 and 8 belong to the same cluster, whereas
hypos 2, 5, 6, 9 and 10 are slightly different. All ten hypotheses contain five features,
of which four hydrophobic (HY) features and one hydrogen bond acceptor (HBA) are
common for all except Hypo-2 which contains in exchange of (HBA) a hydrogen
bond donor feature (HBD). The first two hypotheses show close statistics in terms of
their rank score which is quite significantly better than the rest hypotheses.
Consequently, both hypotheses (Hypo-1 and Hypo-2) were considered for further
analysis.
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Analysis of Hypo-1 and Hypo-2
Examination of Hypo-1 (Figure 4) showed the distribution of the five features on two
sides, four hydrophobic features (HY-1, HY-2, HY-3 and HY-4) on one side and a
hydrogen bond acceptor (HBA) on the other side. As for Hypo-2 (Figure 5), the only
difference from Hypo-1 is the presence of a hydrogen bond donor (HBD) feature
instead of the (HBA). The two sides are apart from each other by inter-feature
distance of more than 6.5 Å. As for the Hypo-2 (Figure 5), the four features seem to
be more evenly distributed.
The tested compounds were mapped onto Hypo-1 and Hypo-2 using the "best fit"
option where HipHop scores the orientation of a mapped compound within the
hypothesis features using a "fit value" score. For models that show five features, as in
this case, the maximum fit value score is 4.9.
Figure 4. Hypo-1 showing inter-feature distances (Å). Features are colour coded as:
Hydrophobic, cyan; Hydrogen bond acceptor, green.
Figure 5. Hypo-2 showing inter-feature distances (Å). Features are colour coded as:
Hydrophobic, cyan; Hydrogen bond donor, magenta.
Results from the mapped compounds (Table 5) showed a rough correlation between
the fit values and their biological activities. Both models (Hypo-1 and Hypo-2) can
easily identify some low active compounds (17g and 19) giving them poor fit value
scores.
The most active compound (24a) was assigned the highest fit value by both models.
However both models can not differentiate between the small differences in biological
activity exhibited by the rest of the active compounds. Mapping of compound (24a) to
both models (Hypo-1 and Hypo-2) is shown in (Figure 6).
The mapping of all compounds reveals the importance of presence of certain groups
to fit the produced models. The hydrophobic features (HY-1, HY-2 and HY-4) can
accommodate either an aromatic ring, heteroaryl, alkyl or cycloalkyl group. The
hydrophobic feature (HY-3) in both hypotheses can accommodate an aliphatic side
chain with maximum of two carbon atoms. The hydrogen bond acceptor feature
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(HBA) is occupied by the carbonyl next to the pyrrole ring of the indole nucleus in all
of the compounds, while the hydrogen bond donor (HBD) is occupied by the pyrrole
NH group of the indole nucleus with the exception of the compounds (17a and 24b)
where this feature is occupied by the amidic NH.
Table 5: Output for Hypo-1 and Hypo-2 mapping
Figure 6. Mapping of compound (24a) (stick) to (a) Hypo-1 and (b) Hypo-2.
The overall look on both hypotheses shows the predominance of the hydrophobic
features. The hydrogen bond acceptor or donor is an essential part of the compounds,
with an opportunity to extend the groups responsible for fulfilling those features by
adding acceptor or donating groups to the already present indole nucleus.
In order to further tuning of the pharmacophore Hyp-1, exclusion volumes have been
added. The resulting model showed 27 exclusion volumes (Figure 7). The mapping of
the most active compound to the exclusion volumes showed good fit which can help
in future developing of more potent analogues.
Figure 7. Hyp-1 showing 27 exclusion volumes.
Conclusion
The present study reports design and synthesis of novel series of 3-alkoxymethyl/3-
phenyl indole-2-carboxamides 17a-m, 19, 24a-d, 25 and 26 as potential anticancer
1
agents. All the compounds were confirmed by H-NMR, 13C-NMR, an HRESI-MS.
The MTT cytotoxicity activities of the compounds were carried out using 5-
fluorouracil as reference drugs. Mechanistic study of the most active compounds
through cell cycle testing has been conducted. Furthermore, HipHop Pharmacophore
generation for the targeted series has been done for further development of the
synthesized compounds. The target compounds were evaluated for their anticancer
activity. The results revealed that the 3-phenyl-N-(4-(piperidin-1-yl)phenethyl)-1H-
indole-2-carboxamide (24a) and the 5-chloro-N-(4-(dimethylamino) phenethyl)-3-
phenyl-1H-indole-2-carboxamide (24c) proved to be potent anticancer against MCF7
cell line with IC50 of sub-micromolar levels.
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Acknowledgment
We are grateful to Dr. Tamer M. Abdelghany, Dr. Ahmed M. I. Mansour, Department
of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Cairo,
Egypt for cell culture facilities. Great thanks are also expressed to Prof. Laurent
Trembleau and Prof. Marcel Jaspars School of Natural and Computing Sciences,
University of Aberdeen, UK for spectroscopy and HRESI-MS analysis.
Conflict of interest: Authors declare no conflict of interest.
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