Synthesis and In Vitro Antitumor Activity of Some New Mannich Bases

Abstract

Two series of Novel Mannich bases has been synthesized from chalcones 3 and 7 and evaluated for their in vitro cytotoxic activity. Out of the newly synthesized compounds, four derivatives 4a, 4b, 4e, 4f were selected by the National Cancer Institute (NCI) to be evaluated for their in-vitro antitumor activity by in-vitro disease-oriented human cells screening panel assay. All the tested compounds exhibited a broad spectrum of antitumor activity against renal cancer UO-31. Keywords: Antitumor activity /cytotoxicity/ Chalcones /Mannich bases / Synthesis .

Full text

International Journal of ChemTech Research
CODEN( USA): IJCRGG ISSN : 0974-4290
Vol.5, No.1, pp 401-408, Jan-Mar 2013
Synthesis and In Vitro Antitumor Activity of Some
New Mannich Bases
Manal M. Kandeel1, Nadia A. Abdou2, Hanan H. Kadry1*, Rana M. El-Masry2
1Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo
University, Cairo, 11562, Egypt.
2Organic Chemistry Department, October University for Modern Sciences and Arts,6
October City, Egypt.
*Corres. author : hanankadry2005@yahoo.com
Tel.: +0020101960964, Fax: 002 02 23635140
Abstract: Two series of Novel Mannich bases has been synthesized from chalcones 3 and 7 and evaluated for
their in vitro cytotoxic activity. Out of the newly synthesized compounds, four derivatives 4a,4b,4e,4f were
selected by the National Cancer Institute (NCI) to be evaluated for their in-vitro antitumor activity by in-vitro
disease-oriented human cells screening panel assay. All the tested compounds exhibited a broad spectrum of
antitumor activity against renal cancer UO-31.
Keywords: Antitumor activity /cytotoxicity/ Chalcones /Mannich bases / Synthesis .
/
Introduction
The principal aim of our work is the discovery of
novel cytotoxic and anticancer agents. Several
publications were reported on Mannich ketones as
potential cytotoxins[1-4]. Some Mannich bases
synthesized from 4-hydroxyacetophenone or its
derivatives containing aromatic or heterocyclic rings
in the B ring proved their effectiveness as cytotoxic
[1, 2, 5]and antitumor agents.[6, 7] Also Mannich
bases, which have been recently synthesized based
on heterocylic chalcones, exhibited very potent
activity against some tumor cell lines.[5, 8] These
studies showed that besides the importance of 4-
hydroxy group in the A ring, heterocyclic rings in
the B ring made significant contribtution to the
bioactivity of Mannich bases. The bioactivity of
Mannich bases have been attributed to the
deamination of the Mannich base group in chalcones
into the corresponding cyclohexadienones that may
generate a further site for nucleophilic attack by
cellular thiols, and to chemical structure of α,β-
unsaturated ketone that can alkylate nucleophiles,
especially toward thiols rather than hydroxyl and
amino groups present in the nucleic acids[9, 10] (fig
1).
O
Ar
NR
HO
R
Deamination
O
Ar
Ocellular Nu
O
Ar
O
Nu
Fig 1: Formation of cyclohexadienone from mannich bases on deamination, which generates an
additional alkylating site for cellular sulfahydryl nucleophiles.

Hanan H. Kadry et al /Int.J.ChemTech Res.2013,5(1)
402
Literature survey demonstrated that there are low
studies concerning the cytotoxic activity of Mannich
bases, in which the A ring possesses a series of
different Mannich bases. Encouraged by these
observations and in our continuous search for new
candidates as cytotoxic agents, we turned our
interest to the synthesis of new Mannich base
derivatives based on the 4-hydroxychalcones with
different substitution groups in the A-ring and in
vitro cytotoxic activity evaluation of Mannich bases
according to the current one-dose protocol of the
National Cancer Institute (NCI) in vitro disease-
oriented human cells screening panel assay.
Experimental
Chemistry
Melting points are uncorrected and determined
in one end open capillary tubes using Gallen Kamp
melting point apparatus MFB-595-010M (Gallen
Kamp, London, England). Microanalysis was carried
out at Micro-analytical Unit, Faculty of Science,
Cairo University and the regional center for
microbiology and biotechnology, Al-Azhar
University. Analyses indicated were within ± 0.4 %
of the theoretical values. Infrared Spectra were
recorded on Schimadzu FT-IR 8400S
spectrophotometer (Shimadzu, Kyoto, Japan) and
expressed in wave number (cm-1) using potassium
bromide discs. The 1HNMR spectra were recorded
on a Varian Gemini 200 MHz and Varian Mercury
VX-300 NMR spectrometer, in chloroform (CDCl3).
Chemical shifts were quoted in δ and related to that
of the solvents. Mass spectra were recorded using
Hewlett Packard Varian (Varian, Polo, USA) and
Shimadzu Gas Chromatograph Mass spectrometer-
QP 1000 EX (Shimadzu, Kyoto, Japan). TLC was
carried out using Art.DC-Plastikfolien, Kieselgel 60
F254 sheets (Merck, Darmstadt, Germany). The
developing solvents were benzene/acetone (4:1) and
the spots were visualized at 366, 254 nm by UV
Vilber Lourmat 77202 (Vilber, Marne La Vallee,
France). Compounds 3[11] ,7 [12]were obtained
according to the reported procedures.
General procedure for the synthesis of 4a-f
Secondary amine (0.0012 mol) and para-
formaldehyde (0.045 g; 0.0015 mol) were dissolved
in absolute ethanol (10 mL) and refluxed for 1 h. To
this reaction mixture, 1-(4-hydroxyphenyl)-3-(1H-
indol-3-yl)prop-2-en-1-one (3) (0.26 g; 0.001 mol)
was added. The mixture was refluxed for 10-24
hours with stirring then left overnight at room
temperature. The formed crystals were then
crystallized from aqueous ethanol.
1-(4-hydroxy-3-(morpholinomethyl)phenyl)-3-
(1H-indol-3-yl)prop-2-en-1-one (4a)
solid, Yield: 97%; mp: 130-131 oC; IR νmax/cm-1:
3744.12 (OH phenolic), 3435.56 (NH), 3045.05 (CH
aromatic), 2928.38, 2819.42 (CH aliphatic), 1638.23
(C=O); 1H NMR (DMSO-d6) δ ppm: 2.681-3.782
(m, 8H, 4CH2 of morpholine), 4.941 (s, 2H, CH2),
7.271-8.349 (m, 7H, Ar-H and Hs of indole), 7.289
(d, 1H, J=8.1 Hz, olefinic H), 7.450 (d, 1H, J=8.7
Hz, olefinic H), 8.100 (s, 1H, OH, D2O exch.), 9.780
(s, 1H, NH, D2O exch.), 10.071 (s, 1H, H-2 of
indole); Anal. Calcd for C22H22N2O3: C, 72.91; H,
6.12; N, 7.73. Found: C, 72.97; H, 6.15; N, 7.91.
1-(4-hydroxy-3-((4-methylpiperazin-1-yl)methyl)
phenyl)-3-(1H-indol-3-yl)prop-2-en-1-one (4b)
solid, Yield: 49%; mp: 125-126 oC; IR νmax/cm-1:
3620.00-3380.00 (OH, NH), 3044.09 (CH aromatic),
2933.20, 2818.45 (CH aliphatic), 1645.95 (C=O); 1H
NMR (DMSO-d6) δ ppm: 2.461 (s, 3H, CH3), 2.715-
2.800 (m, 8H, 4CH2 of piperazine), 4.823 (s, 2H,
CH2), 7.318-8.306 (m, 7H, Ar-H and Hs of indole),
7.452 (d, 1H, J=7.5 Hz, olefinic H), 8.323 (d, 1H,
J=7.5 Hz, olefinic H), 9.137 (s, 1H, NH, D2O exch.),
10.080 (s, 1H, H-2 of indole); Anal. Calcd for
C23H25N3O2: C, 73.57; H, 6.71; N, 11.19. Found: C,
73.62; H, 6.68; N, 11.28.
1-(3-((4-ethylpiperazin-1-yl)methyl)-4-hydroxy
phenyl)-3-(1H-indol-3-yl)prop-2-en-1-one (4c)
solid, Yield: 42%; mp: 139-140 oC; IR νmax/cm-1:
3760.00 (OH), 3400.00 (NH), 3043.67 (CH
aromatic), 2931.80, 2889.37 (CH aliphatic), 1635.64
(C=O), 1612.49 (C=N); 1H NMR (DMSO-d6) δ
ppm: 1.476 (t, 3H, J=7.2 Hz, CH2CH3), 1.544-1.620
(m, 8H, 4CH2 of piperazine), 3.102 (q, 2H, J= 7.2
Hz, CH2CH3), 4.766 (s, 2H, CH2), 7.323-8.336 (m,
7H, Ar-H and Hs of indole), 7.465 (d, 1H, J=7.5 Hz,
olefinic H), 7.696 (s, 1H, OH, D2O exch.), 8.359 (d,
1H, J=7.5 Hz, olefinic H), 8.985 (s, 1H, NH, D2O
exch.), 10.087 (s, 1H, H-2 of indole); Anal. Calcd
for C24H27N3O2: C, 74.01; H, 6.99; N, 10.79. Found:
C, 73.98; H, 7.02; N, 10.87.
1-(4-hydroxy-3-((4-phenylpiperazin-1-yl)methyl)
phenyl)-3-(1H-indol-3-yl)prop-2-en-1-one (4d)
solid, Yield: 88%; mp: 110-111 oC; IR νmax/cm-1:
3515.00-3293.00 (OH, NH), 3105.39 (CH aromatic),
2943.37, 2823.79 (CH aliphatic), 1658.78 (C=O),
1600.92 (C=N); 1H NMR (DMSO-d6) δ ppm: 1.605-
3.282 (m, 8H, 4CH2 of piperazine), 4.971 (s, 2H,
CH2), 6.945-7.366 (m, 12 H, Ar-H and Hs of
indole), 7.521 (d, 1H, J=6.8 Hz, olefinic H), 7.879
(s, 1H, OH, D2O exch.), 8.320 (d, 1H, J=6.8 Hz,
olefinic H), 9.753 (s, 1H, NH, D2O exch.), 10.053 (s,

Hanan H. Kadry et al /Int.J.ChemTech Res.2013,5(1)
403
1H, H-2 of indole); Anal. Calcd for C28H27N3O2: C,
76.86; H, 6.22; N, 9.60. Found: C, 76.92; H, 6.25; N,
9.71.
1-(3-((4-(4-fluorophenyl)piperazin-1-yl)methyl)-
4-hydroxyphenyl)-3-(1H-indol-3-yl)prop-2-en-1-
one (4e)
solid, Yield: 51%; mp: 130-131 oC; IR νmax/cm-1:
3610.00-3348.00 (OH, NH), 3045.05 (CH aromatic),
2934.16, 2820.38 (CH aliphatic), 1638.23 (C=O); 1H
NMR (DMSO-d6) δ ppm: 2.788-4.747 (m, 8H, 4CH2
of piperazine), 4.949 (s, 2H, CH2), 6.890-7.862 (m,
11H, Ar-H and Hs of indole), 7.340 (d, 1H, J=6.6
Hz, olefinic H), 8.329 (d, 1H, J=6.9 Hz, olefinic H),
9.765 (s, 1H, NH, D2O exch.), 10.080 (s, 1H, H-2 of
indole); Anal. Calcd for C28H26FN3O2: C, 73.83; H,
5.75; N, 9.22. Found: C, 73.86; H, 5.77; N, 9.28.
1-(4-hydroxy-3-((4-(4-methoxyphenyl)piperazin-
1-yl)methyl)phenyl)-3-(1H-indol-3-yl)prop-2-en-
1-one (4f)
solid, Yield: 78%; mp: 129-130 oC; IR νmax/cm-1:
3570.00-3280.00 (OH, NH), 2930.31, 2822.31 (CH
aliphatic), 1652.70 (C=O); 1H NMR (DMSO-d6) δ
ppm: 2.848-3.164 (m, 8H, 4CH2 of piperazine),
3.788 (s, 3H, OCH3), 4.933 (s, 2H, CH2), 6.851 (d,
1H, J=8.7 Hz, olefinic H), 6.879-7.516 (m, 7H, Ar-
H and Hs of indole), 7.330 (d, 2H, J=6.6 Hz, H-2,
H-6 of 4-OCH3 C6H4), 7.846 (d, 2H, J=6.9 Hz, H-3,
H-5 of 4-OCH3 C6H4), 8.336 (d, 1H, J=9.0 Hz,
olefinic H), 9.000 (s, 1H, OH, D2O exch.), 9.752 (s,
1H, NH, D2O exch.), 10.084 (s, 1H, H-2 of indole);
MS m/s 467.30 (M+, 1.92%); Anal. Calcd for
C29H29N3O3: C, 74.50; H, 6.25; N, 8.99. Found: C,
74.48; H, 6.29; N, 9.12.
General procedure for the synthesis of 8a-g
Secondary amine (0.0012mol) and
paraformaldehyde (0.045g; 0.0015mol) were
dissolved in absolute ethanol (10 mL) and refluxed
for 1 h. To this reaction mixture, 1-(4-
bromophenyl)-3-(4-hydroxy-3-methoxyphenyl)
prop-2-en-1-one (7) (0.33 g; 0.001 mol) was added.
The mixture was refluxed for 14-30 hours with
stirring then left overnight at room temperature. The
formed crystals were then crystallized from aqueous
ethanol.
1-(4-bromophenyl)-3-(4-hydroxy-3-methoxy-
5(morpholinomethyl)phenyl)prop-2-en-1-one (8a)
solid, Yield: 86%; mp: 165-166 oC; IR νmax/cm-1:
3502.73 (OH phenolic), 3025.00 (CH aromatic),
2935.66, 2846.93 (CH aliphatic), 1651.07 (C=O); 1H
NMR (DMSO-d6) δ ppm: 2.824 (m, 4H, 2CH2 of
morpholine), 3.760 (s, 1H, OH, D2O exch.), 3.846-
3.967 (m, 4H, 2CH2 of morpholine), 3.976 (s, 3H,
OCH3), 4.019 (s, 2H, CH2), 7.101 (s, 1H, H-6 of 3-
OCH3-4-OH C6H3), 7.214 (s, 1H, H-2 of 3-OCH3-4-
OH C6H3), 7.238 (d, 1H, J=16.8 Hz, olefinic H),
7.668 (d, 1H, J=17.7 Hz, olefinic H), 7.771 (d, 2H,
J=8.1 Hz, H-3, H-5 of 4-Br C6H4), 7.944 (d, 2H,
J=8.4 Hz, H-2, H-6 of 4-Br C6H4); MS m/s 430.95
(M+, 75.84%), 432.95 (M+2, 74.14%); Anal. Calcd
for C21H22BrNO4: C, 58.34; H, 5.13; N, 3.24. Found:
C, 58.39; H, 5.18; N, 3.30.
1-(4-bromophenyl)-3-(4-hydroxy-3-methoxy-5-
((4-methylpiperazin-1-yl)methyl)phenyl)prop-2-
en-1-one (8b)
solid, Yield: 81%; mp: 202-203 oC; IR νmax/cm-1:
3444.87 (OH phenolic), 3032.10 (CH aromatic),
2939.52, 2804.50 (CH aliphatic), 1654.92 (C=O); 1H
NMR (DMSO-d6) δ ppm: 2.392 (s, 3H, CH3), 2.736-
2.892 (m, 8H, 4CH2 of piperazine), 3.801 (s, 2H,
CH2), 3.963 (s, 3H, OCH3), 6.978 (s, 1H, H-6 of 3-
OCH3-4-OH C6H3), 7.089 (s, 1H, H-2 of 3-OCH3-4-
OH C6H3), 7.297 (d, 1H, J=15.3 Hz, olefinic H),
7.642 (d, 2H, J=8.4 Hz, H-3, H-5 of 4-Br C6H4),
7.722 (d, 1H, J=15.6 Hz, olefinic H), 7.881 (d, 2H,
J=8.4 Hz, H-2, H-6 of 4-Br C6H4); MS m/s 443.95
(M+, 61.01%), 445.95 (M+2, 59.13%); Anal. Calcd
for C22H25BrN2O3: C, 59.33; H, 5.66; N, 6.29.
Found: C, 59.32; H, 5.69; N, 6.36.
1-(4-bromophenyl)-3-(3-((4-ethylpiperazin-1-
yl)methyl)-4-hydroxy-5-methoxyphenyl)prop-2-
en-1-one (8c)
solid, Yield: 92%; mp: 182-183 oC; IR νmax/cm-1:
3444.87 (OH phenolic), 3024.00 (CH aromatic),
2939.52, 2819.93 (CH aliphatic), 1658.78 (C=O); 1H
NMR (DMSO-d6) δ ppm: 1.139 (t, 3H, J=7.2 Hz,
CH2CH3), 2.509 (q, 2H, J=7.2 Hz, CH2CH3), 2.699-
2.732 (m, 8H, 4CH2 of piperazine), 3.752 (s, 1H,
OH, D2O exch.), 3.799 (s, 2H, CH2), 3.950 (s, 3H,
OCH3), 6.971 (s, 1H, H-6 of 3-OCH3-4-OH C6H3),
7.089 (s, 1H, H-2 of 3-OCH3-4-OH C6H3), 7.294 (d,
1H, J=15.3 Hz, olefinic H), 7.643 (d, 2H, J=8.4 Hz,
H-3, H-5 of 4-Br C6H4), 7.726 (d, 1H, J=15.6 Hz,
olefinic H), 7.881 (d, 2H, J=8.7 Hz, H-2, H-6 of 4-
Br C6H4); Anal. Calcd for C23H27BrN2O3: C, 60.14;
H, 5.92; N, 6.10. Found: C, 60.22; H, 5.98; N, 6.14.
1-(4-bromophenyl)-3-(4-hydroxy-3-methoxy-5-
((4-phenylpiperazin-1-yl)methyl)phenyl)prop-2-
en-1-one (8d)
solid, Yield: 60%; mp: 208-209 oC; IR νmax/cm-1:
3420.00 (OH phenolic), 3040.00 (CH aromatic),
2956.87, 2814.14 (CH aliphatic), 1651.07 (C=O); 1H
NMR (DMSO-d6) δ ppm: 2.838-2.984 (m, 4H, 2CH2
of piprezaine), 3.308 (m, 4H, 2CH2 of piperazine),
3.885 (s, 2H, CH2), 3.963 (s, 3H, OCH3), 6.916 (s,

Hanan H. Kadry et al /Int.J.ChemTech Res.2013,5(1)
404
1H, H-6 of 3-OCH3-4-OH C6H3), 6.936 (s, 1H, H-2
of 3-OCH3-4-OH C6H3), 6.943-7.311 (m, 5H, Ar-H),
7.347 (d, 1H, J=15.6 Hz, olefinic H), 7.647 (d, 2H,
J=7.8 Hz, H-3, H-5 of 4-Br C6H4), 7.743 (d, 1H,
J=15.6, olefinic H), 7.902 (d, 2H, J=7.8, H-2, H-6 of
4-Br C6H4); MS m/s 506.00 (M+, 13.46%), 508.00
(M+2, 12.04%); Anal. Calcd for C27H27BrN2O3: C,
63.91; H, 5.36; N, 5.52. Found: C, 63.98; H, 5.41; N,
5.63.
1-(4-bromophenyl)-3-(3-((4-(4-fluorophenyl)
piperazin-1-yl)methyl)-4-hydroxy-5-methoxy
phenyl)prop-2-en-1-one (8e)
solid, Yield: 43%; mp: 143-144 oC; IR νmax/cm-1:
3441.01 (OH phenolic), 3060.00 (CH aromatic),
2962.66, 2812.21 (CH aliphatic), 1651.07 (C=O); 1H
NMR (DMSO-d6) δ ppm: 2.877-2.983 (m, 4H, 2CH2
of piprezaine), 3.248 (m, 4H, 2CH2 of piperazine),
3.381 (s, 1H, OH, D2O exch.), 3.922 (s, 2H, CH2),
3.966 (s, 3H, OCH3), 6.861 (s, 1H, H-6 of 3-OCH3-
4-OH C6H3), 6.890 (d, 2H, J=8.1 Hz, H-3, H-5 of 4-
Br C6H4), 6.892 (s, 1H, H-2 of 3-OCH3-4-OH C6H3),
6.968 (d, 2H, J=8.4 Hz, H-2, H-6 of 4-F C6H4),
7.377 (d, 1H, J=15.6 Hz, olefinic H), 7.650 (d, 2H,
J=8.1 Hz, H-3, H-5 of 4-Br C6H4), 7.742 (d, 1H,
J=15.3 Hz, olefinic H), 7.913 (d, 2H, J=8.4 Hz, H-2,
H-6 of 4-F C6H4); Anal. Calcd for C27H26BrFN2O3:
C, 61.72; H, 4.99; N, 5.33. Found: C, 61.79; H, 5.05;
N, 5.38.
1-(4-bromophenyl)-3-(4-hydroxy-3-methoxy-5-
((4-(4-methoxyphenyl)piperazin-1-
yl)methyl)phenyl)prop-2-en-1-one (8f)
solid, Yield: 37%; mp: 190-191 oC; IR νmax/cm-1:
3583.74 (OH phenolic), 3055.24 (CH aromatic),
2943.37, 2812.21 (CH aliphatic), 1658.78 (C=O); 1H
NMR (DMSO-d6) δ ppm: 2.826 (m, 4H, 2CH2 of
piprezaine), 3.183 (m, 4H, 2CH2 of piperazine),
3.301 (s, 1H, OH, D2O exch.), 3.775 (s, 3H, OCH3),
3.871 (s, 2H, CH2), 3.957 (s, 3H, OCH3), 6.847 (d,
2H, J=9.3 Hz, H-2, H-6 of 4-OCH3 C6H4), 6.904 (d,
2H, J=9.3 Hz, H-3, H-5 of 4-OCH3 C6H4), 7.108 (s,
1H, H-6 of 3-OCH3-4-OH C6H3), 7.269 (s, 1H, H-2
of 3-OCH3-4-OH C6H3), 7.335 (d, 1H, J=15.4 Hz,
olefinic H), 7.642 (d, 2H, J=8.4 Hz, H-3, H-5 of 4-
Br C6H4), 7.739 (d, 1H, J=15.4 Hz, olefinic H),
7.896 (d, 2H, J=8.7 Hz, H-2, H-6 of 4-Br C6H4);
Anal. Calcd for C28H29BrN2O4: C, 62.57; H, 5.44; N,
5.21. Found: C, 62.53; H, 5.46; N, 5.30.
1-(4-bromophenyl)-3-(3-((4-(2-ethoxyphenyl)
piperazin-1-yl)methyl)-4-hydroxy-5-methoxy
phenyl)prop-2-en-1-one (8g)
solid, Yield: 54%; mp: 170-171 oC; IR νmax/cm-1:
3433.29 (OH phenolic), 3020.00 (CH aromatic),
2903.32, 2819.93 (CH aliphatic), 1681.23 (C=O); 1H
NMR (DMSO-d6) δ ppm: 1.431 (t, 3H, J=6.9 Hz,
CH2CH3), 2.949-3.276 (m, 8H, 4CH2 of piperazine),
3.459 (s, 1H, OH, D2O exch.), 3.969 (s, 3H, OCH3),
4.004 (s, 2H, CH2), 4.070 (q, 2H, J=6.9 Hz,
CH2CH3), 6.867 (d, 2H, J=7.8 Hz, H-3, H-5 of 4-Br
C6H4), 6.911-7.010 (m, 4H, Hs of 2-ethoxyphenyl),
7.023 (s, 1H, H-6 of 3-OCH3-4-OH C6H3), 7.186 (s,
1H, H-2 of 3-OCH3-4-OH C6H3), 7.664 (d, 1H,
J=15.6 Hz, olefinic H), 7.746 (d, 1H, J=15.3 Hz,
olefinic H), 7.930 (d, 2H, J=8.4 Hz, H-2, H-6 of 4-
Br C6H4); Anal. Calcd for C29H31BrN2O4: C, 63.16;
H, 5.67; N, 5.08. Found: C, 63.22; H, 5.72; N, 5.17.
Scheme I:
HO C
OCH3+N
H
OHC
1
HO C
OC
HC
HNH
3
HO C
OC
HC
HNH
4a-f
H2CNR1R2
N O
N NCH3
N N
N N
N NC2H5
F
OCH3
NR1R2
a=
b=
e=
f=
N N
d=
(i)
(ii)
2
c=
Scheme 1:Reagents and conditions: (i) EtOH, 40% KOH, stir, rt, 24 h; (ii) Secondary amine, Paraformaldehyde,
EtOH, reflux, 10-17 h.

Hanan H. Kadry et al /Int.J.ChemTech Res.2013,5(1)
405
Scheme II:
Br C CH3
O
+OHC
OCH3
OH
Br C
OC
HC
OCH3
OH
5
Br C
OC
HC
OCH3
OH
8a-g H2C NR1R2
N O
N NCH3
N N
N N
N N
F
OCH3
C2H5O
NR1R2
8a=
8b=
8e=
8f=
8 g=
N N
8d=
(i)
(ii)
7
N NCH3
8c=
6
H
H
Scheme 2: Reagents and conditions: (i) EtOH, 20% Alcoholic KOH, reflux, 6 h; (ii) Secondary amine,
Paraformaldehyde, EtOH, reflux, 14-30 h.
Antitumor screening
Under sterile conditions, cell lines were grown in
RPMI 1640 media (Gibco, NY, USA) supplemented
with 10% fetal bovine serum (Biocell, CA, USA),
5Х105cell/ml was used to test the growth inhibition
activity of the synthesized compounds. The
concentrations of the compounds ranging from 0.01
to 100 µM were prepared in phosphate buffer saline.
Each compound was initially solubilized in dimethyl
sulfoxide (DMSO), however, each final dilution
contained less than 1% DMSO. Solutions of
different concentrations (0.2 ml) were pipetted into
separate well of a microtiter tray in duplicate. Cell
culture (1.8 ml) containing a cell population of 6 Х
104 cells/ml was pippeted into each well. Controls,
containing only phosphate buffer saline and DMSO
at identical dilutions, were also prepared in the same
manner. These cultures were incubated in a
humidified incubator at 37oC. The incubator was
supplied with 5% CO2 atmosphere. After 48 h, cells
in each well were diluted 10 times with saline and
counted by using a coulter counter. The counts were
corrected for the dilution [13-16].
Result and Discussion
The synthesis of the target compounds was
accomplished according to the reaction sequences
illustrated in Schemes 1and 2. Chalcones 3[11] 7
[12] were synthesized by reacting 4-substituted
acetophenone with indolyl-3-carboxaldehyde or
vaniline respectively in the presence of potassium
hydroxide by conventional Claisen-Schmidt
condensation. Mannich bases 4a-f were prepared in
42-97% yield by refluxing a mixture of chalcone 3,
paraformaldehyde and different secondary amines
dissolved in ethanol according to the previously
described procedure for the preparation of analogue
compounds.[3, 4] The synthesized compounds were
characterized by their physical and spectral data (IR,
1H-NMR, MS) that confirmed the structures of the
novel compounds. The case of compound 4a was an
example, the 1H NMR spectrum showed multiplet
signal at δ 2.681-3.782 ppm for 8 protons of 4CH2
groups of morpholine ring. While the spectra of
compounds 4b-f demonstrate multiplet signals in
range of δ 1.605-3.282 ppm for 8 protons of 4CH2
groups of piperazine rings. However, the spectra of
all compounds showed the presence of singlet
signals at δ 7.696-9.000 ppm for the phenolic OH
protons, they also showed the presence of singlet
signals at δ 4.933-4.971 ppm for the 2 protons of the
methylene groups. Protons of α,β-unsaturated ketone
of Mannich bases derived from indolylchalcone 4a-f
were observed as doublets with J =6.8-8.1 Hz at δ
6.8-7.3 ppm for Hβ and δ 8.3 ppm for Hα. The J
characteristic values were the evidence that Mannich
bases derived from indolylchalcone 4a-f appeared as
z isomers. Further elucidation of the structure of

Hanan H. Kadry et al /Int.J.ChemTech Res.2013,5(1)
406
compound 4f came from MS spectrum which gave
molecular ion peak at 467.30. On the other hand,
Bamnela and Shrivastava[12] prepared 1-(4-
bromophenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-
2-en-1-one 7. Unfortunately, no physical or spectral
data were reported for the compound. So, we
elucidate their structure by spectral data. The IR
spectrum showed the presence of the characteristic
band for conjugated C=O of chalcones at 1639 cm-1.
The structure was further supported by its 1H NMR
spectrum which showed the presence of two
characteristic doublet signals for the olefinic protons
at δ 7.316 and 7.760 ppm with a coupling constant
J=15.6 Hz confirming the E configuration. It also
showed a singlet signal at δ 5.940 ppm for the
phenolic OH proton, where the 3 protons of the
methoxy group were seen as a singlet signal at δ
3.976 ppm.
The designed target compounds 8a-g were prepared
by the reaction of 7 with various secondary amines
and paraformaldehyde in ethanol under reflux for
14-30 h, [4] (Scheme 2) to give Mannich bases 8a-g
in 37-92% yield. The structure conformations of
compounds 8a-g were based on the spectral data.
The1H NMR spectrum of compounds 8b-f showed
methylene protons as a singlet signal at δ 3.799-
4.019 ppm, and protons of piperazine nucleus were
showed as multiplet signal at δ 2.77 -3.26 ppm.
Protons of α,β-unsaturated ketone of Mannich bases
derived from vaniline chalcone 8a-g were observed
as doublets with J =15.3-16.8 Hz at 7.7 ppm for Hβ
and 7.2 ppm for Hα. The J characteristic values were
the evidence that mannich derivatives of 4-hydroxy-
3-methoxyphenylchalcone appeared as E isomers.
Further evidence of the structures of compounds 8a,
8b, and 8d came from MS spectra which showed the
molecular ion peak and M+2 in ratio 1:1.
Preliminary in-vitro anticancer screening
Table 1: Percentage growth inhibition (GI %) of in-vitro subpanel tumor cell lines at 10 µM
concentration of tested compounds .
Cell Line
Compound
4a
4b
4e
4f
Leukemia
HL-60(TB)
15.52
L
-
-
K-562
12.84
L
L
L
MOLT-4
15.39
11.86
-
-
RPMI-8226
-
-
-
-
SR
Nt
Nt
nt
Nt
Non-Small Cell
Lung Cancer
A549/ATCC
L
L
-
L
HOP-62
12.04
-
-
11.04
HOP-92
15.45
-
-
10.13
NCI-H226
-
L
L
L
NCI-H23
-
L
-
L
NCI-H322M
-
L
L
L
NCI-H460
L
L
L
L
NCI-H522
-
-
L
-
Colon Cancer
COLO 205
L
L
L
L
HCC-2998
30.43
Nt
nt
Nt
HCT-116
L
L
-
L
HCT-15
L
-
-
HT29
-
L
-
L
KM12
-
-
-
L
SW-620
L
L
L
L
CNS Cancer
SF-268
L
L
-
L
SF-295
nt
Nt
nt
Nt
SNB-19
L
L
-
L
SNB-75
nt
Nt
nt
Nt

Hanan H. Kadry et al /Int.J.ChemTech Res.2013,5(1)
407
Melanoma
LOX IMVI
-
L
-
-
MALME-3M
-
-
L
-
M14
L
-
-
L
MDA-MB-435
L
L
L
L
SK-MEL-2
L
L
L
L
SK-MEL-28
L
L
L
L
SK-MEL-5
-
-
L
-
UACC-257
11.71
L
L
L
UACC-62
L
L
L
L
Ovarian Cancer
IGROV1
-
-
L
L
OVCAR-3
L
L
L
L
OVCAR-4
L
-
-
-
OVCAR-5
L
L
-
L
NCI/ADR-RES
L
L
L
L
SK-OV-3
L
L
L
-
Renal Cancer
786-0
L
L
L
L
A498
L
L
-
L
ACHN
L
-
-
-
RXF 393
L
L
L
L
TK-10
L
L
L
L
UO-31
40.88
39.48
40.57
44.13
Prostate Cancer
PC-3
18.37
18.55
15.29
11.69
DU-145
L
L
L
L
Breast Cancer
MCF7
L
L
-
-
MDA-MB-
231/ATCC
L
L
-
L
HS 578T
L
L
L
L
BT-549
L
L
-
L
MDA-MB-468
L
L
-
-
a -, GI <10%; nt , not tested; L, compound proved lethal to the intact cell .
All of the newly synthesized compounds were sent
to the National Cancer Institute (NCI) USA to be
tested for their anticancer activity. Unfortunately
four derivatives 4a,4b,4e,4f only were selected to
be tested by in-vitro disease-oriented human cells
screening panel assay to be evaluated for their in-
vitro antitumor activity. A single dose (10 µM) of
the test compounds were used in the full NCI 60 cell
lines panel assay which includes nine tumor
subpanels namely; leukemia, non-small cell lung,
colon, CNS, melanoma, ovarian, renal, prostate, and
breast cancer cells [13-16]. The data were reported
as mean-graph of the percent growth of the treated
cells, and presented as percentage growth inhibition
(GI %). The obtained data revealed that all of the
tested compounds exhibit sensitivity profiles against
the renal cancer UO-31cell line and do not show any
inhibition activity against the other cell lines except
4a ,which demonstrates moderate inhibition activity
against colon cancer HCC-2998 cell line.
Acknowledgements
We are grateful to the NCI, Bethesda, MD, USA for
performing the antitumor testing of the synthesized
compounds.

Hanan H. Kadry et al /Int.J.ChemTech Res.2013,5(1)
408
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