Synthesis, in vitro antimicrobial, antiproliferative, and QSAR studies of N-(substituted phenyl)-2/4-(1H-indol-3-ylazo)-benzamides

Article abstract of DOI:10.1007/s00044-012-0181-0

In this study, N-(substituted phenyl)-2/4-(1H-indol-3-ylazo)-benzamides (1-26) were synthesized and screened for their in vitro antibacterial (Gram positive; S. aureus, B. subtilis and Gram negative; E. coli) and antifungal (C. albicans and A. niger) activities. The antimicrobial activity results indicated that compound, 4-(1H-indol-3-ylazo)-N-(4-nitro-phenyl)-benzamide (12, pMIC _am = 1.61) was the most potent. In general, it was found that the synthesized compounds were bacteriostatic/fungistatic in action except fungicidal for A. niger. The synthesized compounds were also evaluated for their antiproliferative activity against human colon cancer (HCT116), murine leukemia (P388), and breast cancer (MCF7) cell lines. The antiproliferative study results demonstrated 4-(1H-indol-3-ylazo)-N-p-tolyl-benzamide (2, IC ₅₀ = 0.0003 μM/mL) and 4-(1H-indol-3-ylazo)-N-p-tolyl-benzamide (21, 0.0003 μM/mL) as lead compounds for the development of novel antiproliferative agents. The QSAR studies indicated the importance of topological parameters, Kier's alpha second-order shape indice (κα₂) and Wiener index (W) in describing the antimicrobial activity of the synthesized compounds.

Full text of DOI:10.1007/s00044-012-0181-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1957–1971
DOI 10.1007/s00044-012-0181-0
ORIGINAL RESEARCH
Synthesis, in vitro antimicrobial, antiproliferative, and QSAR
studies of N-(substituted phenyl)-2/4-(1H-indol-3-ylazo)-
benzamides
•
Harsh Kumar Pradeep Kumar Balasubramanian Narasimhan
•
•
•
•
•
Kalavathy Ramasamy Vasudevan Mani Rakesh Kumar Mishra
Abu Bakar Abdul Majeed
Received: 4 February 2012 / Accepted: 21 July 2012 / Published online: 23 August 2012
Ó Springer Science+Business Media, LLC 2012
Abstract In this study, N-(substituted phenyl)-2/4-(1H-
indol-3-ylazo)-benzamides (1–26) were synthesized and
screened for their in vitro antibacterial (Gram positive;
S. aureus, B. subtilis and Gram negative; E. coli) and
antifungal (C. albicans and A. niger) activities. The antimi-
crobial activity results indicated that compound, 4-(1H-indol-
3-ylazo)-N-(4-nitro-phenyl)-benzamide(12, pMICam = 1.61)
was the most potent. In general, it was found that the synthe-
sized compounds were bacteriostatic/fungistatic in action
except fungicidal for A. niger. The synthesized compounds
were also evaluated for their antiproliferative activity against
human colon cancer (HCT116), murine leukemia (P388), and
breast cancer (MCF7) cell lines. The antiproliferative study
results demonstrated 4-(1H-indol-3-ylazo)-N-p-tolyl-benz-
amide (2, IC50 = 0.0003 lM/mL) and 4-(1H-indol-3-ylazo)-
N-p-tolyl-benzamide (21, 0.0003 lM/mL) as lead compounds
for the development of novel antiproliferative agents. The
QSAR studies indicated the importance of topological
parameters, Kier’s alpha second-order shape indice (ja2) and
Wiener index (W) in describing the antimicrobial activity of
the synthesized compounds.
Keywords Benzamides ꢀ QSAR ꢀ Antimicrobial ꢀ
Antiproliferative
Introduction
The microbial resistance toward current antimicrobial
(b-lactum antibiotics, macrolides, quinolones, etc.) drugs
and the development of resistance by cancer cell toward
anticancer drugs remain a serious global health concern.
The advances in the area of chemotherapeutic agents
are occurring at a much slower pace than expected.
Hence, there is an urgency to discover novel molecules to
treat microbial infections (Ozkay et al., 2010) and cancer
(Abdel-Jalil et al., 2010).
The quantitative structure–activity relationship (QSAR)
methodology is a useful tool for analyzing the relation
between the information incorporated in the chemical
structure of compounds and their available biological data
in a systematic way. This methodology has been widely
accepted as it supports the reduction, refinement, and/or
replacement of experimental studies (Giaginis et al., 2009).
Indole derivatives have been reported to possess a wide
spectrum of biological activities like antimicrobial (Laxmi
and Rajitha, 2012), antiproliferative (Jacquemard et al.,
2008), analgesic (Radwan et al., 2007), antasthmatic
(Velankar et al., 2010), antihypertensive (Strappaghetti
et al., 2008), antiAlzheimer (Ivachtchenko et al., 2010),
antidiabetic (Lamotte et al., 2010), and antimalarial
(Chiyanzu et al., 2005) activities. Natural and synthetic
compounds with an indole nucleus have been shown to
induce apoptosis through cell cycle arrest or a cellular stress
activation mechanism. For example, vinca alkaloids and
synthetic indole derivatives (ABT-751 and BPR0L075) were
reported to cause G2/M arrest and apoptosis. Indole-3-carbinol
H. Kumar ꢀ P. Kumar ꢀ B. Narasimhan (&)
Faculty of Pharmaceutical Sciences, Maharshi Dayanand
University, Rohtak 124001, India
e-mail: naru2000us@yahoo.com
K. Ramasamy
Collaborative Drug Discovery Research Group, Faculty
of Pharmacy, Universiti Teknologi MARA (UiTM), Campus
Puncak Alam, 42300 Bandar Puncak Alam, Selangor, Malaysia
V. Mani ꢀ R. K. Mishra ꢀ A. B. A. Majeed
Brain Research Laboratory, Faculty of Pharmacy,
Universiti Teknologi MARA (UiTM), Campus Puncak Alam,
42300 Bandar Puncak Alam, Selangor, Malaysia
123

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Med Chem Res (2013) 22:1957–1971
(I3C) and 3,30-diindolylmethane, phytochemicals com-
monly found in cruciferous vegetables, induce G1 cell
cycle arrest and apoptosis mediated by alterations in stress-
activated protein kinase and activation of a DNA damage
mechanism. However, these natural and semi-synthetic
indole compounds have disadvantages, such as harsh
reaction conditions, long reaction times, and expensive
preparation. This instigated researchers to search for syn-
thetic indole derivatives. In light of the above, recently Lee
et al. (2008) studied the antiproliferative potential of indole
derivatives. The authors found that 3-indole derivative
induced a time-dependent increase in ROS production and
triggered DNA damage. 3-Indole induced G1 cell cycle
arrest at low concentration and apoptosis at high concen-
tration in various human lung cancer cell lines. The in vivo
antitumor activities against murine xenograft model dem-
onstrated 3-indole as a potential lead compound for cancer
treatment (Lee et al., 2008).
3-indole derivatives viz. synthesis of N-(substituted phe-
nyl)-2/4-(1H-indol-3-ylazo)-benzamides and to investigate
their antiproliferative and antimicrobial activities.
Experimental
Melting points were determined in open glass capillary
tubes using Sonar melting point apparatus and were
uncorrected. Reaction progress was monitored by thin layer
chromatography. 1H nuclear magnetic resonance (1H
NMR) spectra were recorded on Bruker Avance II 400
NMR spectrometer (400 MHz) at 298 K, in appropriate
deuterated solvents. Chemical shifts were reported as d
(ppm) relative to tetramethylsilane (TMS) as internal
standard. Infrared (IR) spectra were recorded as KBr pellet
on Varian Resolutions Pro FTIR spectrometer. The wave
number is given in cm-1. Elemental analysis was per-
formed on a Perkin–Elmer 2400 C, H, N analyzer.
In a recent study, Lee et al. (2009) determined the impact
of adding indole to P. aeruginosa on 756 phenotypes using
phenotype arrays. The additionof indole severely suppressed
the growth in the presence of many carbon (e.g., D-saccharic
acid, L-alanine, glucuronamide, M-tartaric acid, and
D-aspartic acid) nitrogen (e.g. xanthosine, tyramine, cyti-
dine, cytosine, guanine, and D-lysine), phosphorus (e.g.,
D-mannose-1-phosphate, D-2-phospho-glyceric acid, and
cytidine-2-monophosphate) and three sulfur (lanthionine,
glutathione, and L-cysteine) sources. The reduced cell
growth in the presence of indole with three sulfur sources
corroborates the microarray data in that indole repressed
genes (sbp, ssuFBCDAE, cysI, and cysND) involved in
sulfur uptake which suggests that indole decreases sul-
fur uptake in P. aeruginosa. Overall, indole alters the carbon,
nitrogen, sulfur, and phosphorus metabolism of P. aeruginosa
and thereby diminishes virulence of P. aeruginosa through
a multidrug efflux pump, mexAB-oprM. Some natural
indole derivatives, such as indole-3-carbinol and 3,
30-diindolylmethane derived from Cruciferous vegetables,
also showed antimicrobial, antiviral, and antiproliferative
activities (Lee et al., 2009). In another study, indole and its
azo dye methyl derivatives like 4-(1-methyl-1H-indol-
3-ylazo)-benzoic acid ester and the (4-fluorophenyl)-(1-methyl-
1H-indol-3-yl)-diazene showed remarkable antimicrobial
activity against B. megaterium, B. subtilis, B. thuringiensis,
and S. aureus (Ozturk and Abdullah, 2006). These studies of
Lee et al. (2008, 2009) and Ozturk and Abdullah (2006)
indicated that the 3-indole azo derivatives have the
potential to be explored as antiproliferative and antimicro-
bial agents.
General procedure for the synthesis of 2/4-(indol-3-
ylazo)-benzoic acid
o/p-Amino benzoic acid (0.1 mol) was dissolved in a
mixture of water and hydrochloric acid solution (35–38 %)
(40 mL (1:1) and added with sodium nitrite (0.1 mol) at
0 °C and allowed to stand for 15 min. This diazo solution
was added drop by drop to a well-stirred suspension of
indole (0.1 mol) in 50 mL of water containing sodium
hydroxide (0.1 g) and sodium carbonate (1.0 g). The tem-
perature was maintained at 0 °C and the addition was
completed within 1 h. Stirring was continued for another
hour or until coupling was complete. The precipitated solid
obtained was filtered, dried, and recrystallized from
ethanol.
2-(Indol-3-ylazo)-benzoic acid
Mp (°C) 477–479; Yield—74.20 %; 1H NMR (CD3OD): d
7.29–8.22 (m, 9H, ArH of indole and ArCOOH), 11.30 (s,
1H, H of ArCOOH), 9.95 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,607(Ring str., indole), 901 (C–H out of
plane bending, indole), 3,033 (C–H str., Ar), 2,057 (N=N
str., diazo), 1,521(C=C skeletal str., Ar); 1,720 (CO str.,
COOH); Anal. Calculated for C15H11N3O2: C, 67.92; H,
4.18; N, 15.84; Found: C, 68.01; H, 4.11; N, 15.88.
4-(Indol-3-ylazo)-benzoic acid
1
Motivated by these findings and in continuation of our
research efforts in the field of synthesis, antimicrobial, and
QSAR studies (Narang et al., 2012; Judge et al., 2012;
Kumar et al., 2009, 2010), this study is aimed to synthesize
Mp (°C) 456–458; Yield—78.15 %; H NMR (CD3OD):
d 7.24–8.15 (m, 9H, ArH of indole and ArCOOH), 11.23
(s, 1H, H of ArCOOH), 9.87 (s, 1H, NH of indole); IR
(KBr pellets) cm-1: 1,610 (Ring str., indole), 909 (C–H out
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Med Chem Res (2013) 22:1957–1971
1959
N-(2,3-Dimethyl-phenyl)-4-(1H-indol-3-ylazo)-
benzamide (3)
of plane bending, indole), 3,044 (C–H str., Ar), 2,066
(N=N str., diazo), 1,524 (C=C skeletal str., Ar); 1,712 (CO
str., COOH); Anal. Calculated for C15H11N3O2: C, 67.92;
H, 4.18; N, 15.84; Found: C, 67.96; H, 4.21; N, 15.82.
1
Mp (°C) 170–172; Yield—88.50 %; H NMR (CD3OD): d
7.27–7.63 (m, 12H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 2.05 (s, 6H, CH3 of
ArCH3), 8.01(s, 1H, HofNH adjacenttoCO), 9.88(s, 1H, NH
of indole); IR (KBr pellets) cm-1: 1,577 (Ring str., indole),
886 (C–H out of plane bending, indole), 2,058 (N=N str.,
diazo), 1,618 (C=O str., secondary amide), 1,538 (C=C skel-
etal str., Ar), 764 (C–C out of plane bending, 1,2,3-trisubsti-
tuted benzene ring); Anal. Calculated for C23H20N4O: C,
74.98; H, 5.47; N, 15.21; Found: C, 75.00; H, 5.42; N, 15.18.
General procedure for the synthesis of 2/4-(indol-3-
ylazo)-benzoyl chloride
Thionyl chloride (0.3 mol) was added to 2/4-(indol-
3-ylazo) benzoic acid (0.25 mol) and the mixture was
refluxed for 2 h. The excess of thionyl chloride was
removed by distillation.
General procedure for the synthesis of N-(substituted
phenyl)-2/4-(1H-indol-3-ylazo)-benzamides (1–26)
4-(1H-Indol-3-ylazo)-N-(2-nitro-phenyl)-benzamide (4)
Mp (°C) 125–127; Yield—83.35 %; 1H NMR (CD3OD): d
6.89–7.43 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 8.10 (s, 1H, H of NH
adjacent to CO), 9.82 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,615 (Ring str., indole), 876 (C–H out of
plane bending, indole), 1,670 (C=O str., secondary amide),
1,527 (C=C skeletal str., Ar), 1,504 (NO2 str., aromatic
nitro group); Anal. Calculated for C21H15N5O3: C, 65.45;
H, 3.92; N, 18.17; Found: C, 65.40; H, 3.94; N, 18.18.
The solution of corresponding aniline (0.1 mol) in ether
(50 mL) was added dropwise to a solution of correspond-
ing 2/4-(indol-3-ylazo) benzoyl chloride (0.1 mol) in ether
(50 mL) and the reaction mixture was maintained at
0–10 °C temperature. The solution was stirred for 30 min
and the precipitated anilide was separated and treated with
5 % hydrochloric acid, 4 % sodium carbonate, and water
to remove the residual aniline. The resultant anilide was
recrystallized using alcohol.
N-(2-Chloro-phenyl)-4-(1H-indol-3-ylazo)-
benzamide (5)
4-(1H-Indol-3-ylazo)-N-phenyl-benzamide (1)
Mp (°C) 190–192; Yield—85.10 %; 1H NMR (CD3OD): d
7.21–7.56 (m, 14H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 7.73 (s, 1H, H of NH
adjacent to CO), 9.89 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,600 (Ring str., indole), 904 (C–H out of
plane bending, indole), 3,039 (C–H str., Ar), 2,057 (N=N
str., diazo), 1,634 (C=O str., secondary amide), 1,518 (C=C
skeletal str., Ar); Anal. Calculated for C21H16N4O: C,
74.10; H, 4.74; N, 16.46; Found: C, 75.12; H, 4.71; N,
16.42.
Mp (°C) 185–187; Yield—89.40 %; 1H NMR (CD3OD): d
6.96–7.87 (m, 12H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 7.28–7.33 (d, 1H,
ArH adjacent to Cl, J = 25 Hz), 8.02 (s, 1H, H of NH
adjacent to CO), 10.12 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,582 (Ring str., indole), 854 (C–H out of
plane bending, indole), 3,030 (C–H str., Ar), 2,047 (N=N
str., diazo), 1,614 (C=O str., secondary amide), 1,513 (C=C
skeletal str., Ar), 754 (C–Cl str., mono chlorinated benzene
ring); Anal. Calculated for C21H15N4OCl: C, 67.29; H,
4.03; N, 14.95; Found: C, 67.31; H, 4.01; N, 14.98.
4-(1H-Indol-3-ylazo)-N-p-tolyl-benzamide (2)
N-(2,5-Dimethyl-phenyl)-4-(1H-indol-3-ylazo)-
benzamide (6)
Mp (°C) 220–222; Yield—92.45 %; 1H NMR (CD3OD): d
7.18–7.34 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 2.29 (s, 3H, CH3 of
ArCH3), 7.59 (s, 1H, H of NH adjacent to CO), 9.93 (s, 1H,
NH of indole); IR (KBr pellets) cm-1: 1,586 (Ring str.,
indole), 808 (C–H out of plane bending, indole), 1,993
(N=N str., diazo), 1,625 (C=O str., secondary amide), 1,528
(C=C skeletal str., Ar), 808 (C–C out of plane bending, 1,
4-disubstituted benzene ring); Anal. Calculated for
C22H18N4O: C, 74.56; H, 5.12; N, 15.81; Found: C, 74.59;
H, 5.09; N, 15.85.
1
Mp (°C) 158–160; Yield—94.25 %; H NMR (CD3OD): d
7.16–7.30 (m, 12H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 2.35 {s, 3H, CH3 of
ArCH3), 2.38 (s, 3H, CH3 of ArCH3), 7.73 (s, 1H, H of NH
adjacent to CO), 9.96 (s, 1H, NH of indole); IR (KBr pellets)
cm-1: 1,562 (Ring str., indole), 883 (C–H out of plane
bending,indole),2,982(C–Hstr., Ar),2,116(N=Nstr., diazo),
1,581 (C=O str., secondary amide), 1,514 (C=C skeletal str.,
Ar), 863 (C–C out of plane bending, 1,2,5-trisubstituted
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Med Chem Res (2013) 22:1957–1971
benzene ring); Anal. Calculated for C23H20N4O: C, 74.98; H,
5.47; N, 15.21; Found: C, 75.01; H, 5.43; N, 15.26.
secondary amide), 1,498 (C=C skeletal str., Ar), 750 (C–C
out of plane bending, 1,2-disubstituted benzene ring); Anal.
Calculated for C22H18N4O: 74.56; H, 5.12; N, 15.81;
Found: C, 74.54; H, 5.16; N, 15.80.
N-(2,4-Dimethyl-phenyl)-4-(1H-indol-3-ylazo)-
benzamide (7)
N-(2-Fluoro-phenyl)-4-(1H-indol-3-ylazo)-
benzamide (11)
Mp (°C) 200–202; Yield—91.80 %; 1H NMR (CD3OD): d
6.73–7.59 (m, 12H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 2.45 {s, 6H, CH3 of
Ar(CH3)2}, 7.97 (s, 1H, H of NH adjacent to CO), 9.83
(s, 1H, NH of indole}; IR (KBr pellets) cm-1: 1,566 (Ring
str., indole), 870 (C–H out of plane bending, indole), 3,016
(C–H str., Ar), 2,121 (N=N str., diazo), 1,593 (C=O str.,
secondary amide), 1,503 (C=C skeletal str., Ar), 870 (C–C
out of plane bending, 1,2,4-trisubstituted benzene ring);
Anal. Calculated for C23H20N4O: C, 74.98; H, 5.47; N,
15.21; Found: C, 74.96; H, 5.42; N, 15.25.
Mp (°C) 180–182; Yield—80.75 %; 1H NMR (CD3OD): d
7.20–7.73 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 7.92 (s, 1H, H of NH
adjacent to CO), 9.86 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,588 (Ring str., indole), 843 (C–H out of
plane bending, indole), 3,043 (C–H str., Ar), 2,040 (N=N
str., diazo), 1,613 (C=O str., secondary amide), 1,525 (C=C
skeletal str., Ar), 1,072 (C–F str., mono fluorinated benzene
ring); Anal. Calculated for C21H15N4OF: C, 70.38; H, 4.22;
N, 15.63; Found: C, 70.33; H, 4.24; N, 15.65.
N-(3-Chloro-phenyl)-4-(1H-indol-3-ylazo)-benzamide (8)
4-(1H-Indol-3-ylazo)-N-(4-nitro-phenyl)-
benzamide (12)
Mp (°C) 220–222; Yield—80.30 %; 1H NMR (CD3OD): d
7.39–7.56 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 7.57 (s, 1H, H of NH
adjacent to CO), 9.89 (s, 1H, NH of indole); IR (KBr pellets)
cm-1: 1,574 (Ring str., indole), 870 (C–H out of plane
bending, indole), 1,598 (C=O str., secondary amide), 1,513
(C=C skeletal str., Ar), 786 (C–Cl str., mono chlorinated
benzene ring); Anal. Calculated for C21H15N4OCl: C, 67.29;
H, 4.03; N, 14.95; Found: C, 67.30; H, 4.06; N, 14.92.
Mp (°C) 197–199; Yield—88.20 %; 1H NMR (CD3OD): d
6.93–8.01 (m, 12H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 8.26–8.49 (d, 2H,
ArH adjacent to NO2, J = 115 Hz), 8.49 (s, 1H, H of NH
adjacent to CO), 9.98 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,565 (Ring str., indole), 860 (C–H out of
plane bending, indole), 3,034 (C–H str., Ar), 2,035 (N=N
str., diazo), 1,600 (C=O str., secondary amide), 1,522 (C=C
skeletal str., Ar), 1,498 (NO2 str., aromatic nitro group);
Anal. Calculated for C21H15N5O3: C, 65.45; H, 3.92; N,
18.17; Found: C, 65.48; H, 3.90; N, 18.18.
4-(1H-Indol-3-ylazo)-N-(2-methoxy-phenyl)-
benzamide (9)
Mp (°C) 100–102; Yield—75.75 %; 1H NMR (CD3OD): d
6.95–7.65 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 3.88 (s, 3H, CH3 of
ArOCH3), 8.02 (s, 1H, H of NH adjacent to CO), 10.12
(s, 1H, NH of indole); IR (KBr pellets) cm-1: 1,568 (Ring
str., indole), 861 (C–H out of plane bending, indole), 1,630
(C=O str., secondary amide), 1,524 (C=C skeletal str., Ar),
1,265 (C–O-C str., methoxy benzene ring); Anal. Calcu-
lated for C22H18N4O2: C, 74.34; H, 4.90; N, 15.13; Found:
C, 74.33; H, 4.96; N, 15.11.
N-(4-Chloro-phenyl)-4-(1H-indol-3-ylazo)-
benzamide (13)
Mp (°C) 230–232; Yield—82.35 %; 1H NMR (CD3OD): d
7.45–7.88 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 8.02 (s, 1H, H of NH
adjacent to CO), 9.95 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,592 (Ring str., indole), 918 (C–H out of
plane bending, indole), 3,052 (C–H str., Ar), 1,615 (C=O
str., secondary amide), 1,509 (C=C skeletal str., Ar), 753
(C–Cl str., mono chlorinated benzene ring); Anal. Calcu-
lated for C21H15N4OCl: C, 67.29; H, 4.03; N, 14.95;
Found: C, 67.34; H, 4.05; N, 14.98.
4-(1H-Indol-3-ylazo)-N-o-tolyl-benzamide (10)
Mp (°C) 206–208; Yield—90.70 %; 1H NMR (CD3OD): d
7.27–7.50 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 2.40 (s, 3H, CH3 of
ArCH3), 7.57 (s, 1H, H of NH adjacent to CO), 9.94 (s, 1H,
NH of indole); IR (KBr pellets) cm-1: 1,579 (Ring str.,
indole), 808 (C–H out of plane bending, indole), 3,027
(C–H str., Ar), 2,075 (N=N str., diazo), 1,600 (C=O str.,
N-(2,3-Dimethyl-phenyl)-2-(1H-indol-3-ylazo)-
benzamide (14)
Mp (°C) 258–260; Yield—91.35 %; 1H NMR (CD3OD): d
7.15–7.32 (m, 12H, ArH of ArH adjacent to CONH, ArH
123

Med Chem Res (2013) 22:1957–1971
1961
adjacent to N=N and ArH of indole), 2.22 (s, 3H, CH3 of
ArCH3), 2.35 (s, 3H, CH3 of ArCH3), 8.03 (s, 1H, H of NH
adjacent to CO), 9.92 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,588 (Ring str., indole), 886 (C–H out of
plane bending, indole), 3,077 (C–H str., Ar), 2,058 (N=N
str., diazo), 1,614 (C=O str., secondary amide), 1,538 (C=C
skeletal str., Ar), 776 (C–C out of plane bending, 1,2,
3-trisubstituted benzene ring); Anal. Calculated for
C23H20N4O: C, 74.98; H, 5.47; N, 15.21; Found: C, 75.00;
H, 5.45; N, 15.24.
ring); Anal. Calculated for C21H15N4OCl: C, 67.29; H,
4.03; N, 14.95; Found: C, 67.25; H, 4.06; N, 14.92.
N-(3-Chloro-phenyl)-2-(1H-indol-3-ylazo)-
benzamide (18)
Mp (°C) 210–212; Yield—92.20 %; 1H NMR (CD3OD): d
7.39–7.50 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 7.54 (s, 1H, H of NH
adjacent to CO), 9.98 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,574 (Ring str., indole), 870 (C–H out of
plane bending, indole), 3,016 (C–H str., Ar), 2,074 (N=N
str., diazo), 1,598 (C=O str., secondary amide), 1,513 (C=C
skeletal str., Ar), 786 (C–Cl str., mono chlorinated benzene
ring); Anal. Calculated for C21H15N4OCl: C, 67.29; H,
4.03; N, 14.95; Found: C, 67.26; H, 4.03; N, 14.92.
N-(2,5-Dimethyl-phenyl)-2-(1H-indol-3-ylazo)-
benzamide (15)
Mp (°C) 206–208; Yield—78.75 %; 1H NMR (CD3OD): d
7.12–7.28 (m, 12H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 2.32 (s, 3H, CH3 of
ArCH3), 2.38 (s, 3H, CH3 of ArCH3), 7.96 (s, 1H, H of NH
adjacent to CO), 10.12 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,562 (Ring str., indole), 883 (C–H out of
plane bending, indole), 2,980 (C–H str., Ar), 2,074 (N=N
str., diazo), 1,581 (C=O str., secondary amide), 1,513 (C=C
skeletal str., Ar), 883 (C–C out of plane bending, 1,2,
5-trisubstituted benzene ring); Anal. Calculated for
C23H20N4O: C, 74.98; H, 5.47; N, 15.21; Found: C, 75.02;
H, 5.42; N, 15.22.
N-(4-Chloro-phenyl)-2-(1H-indol-3-ylazo)-
benzamide (19)
Mp (°C) 233–235; Yield—96.50 %; 1H NMR (CD3OD): d
7.42–7.59 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 8.22 (s, 1H, H of NH
adjacent to CO), 9.96 (s, 1H, NH of indole); IR (KBr pellets)
cm-1: 1,566 (Ring str., indole), 870 (C–H out of plane
bending, indole), 1,615 (C=O str., secondary amide), 1,508
(C=C skeletal str., Ar), 752 (C–Cl str., mono chlorinated
benzene ring); Anal. Calculated for C21H15N4OCl: C, 67.29;
H, 4.03; N, 14.95; Found: C, 67.28; H, 4.04; N, 14.93.
N-(2-Fluoro-phenyl)-2-(1H-indol-3-ylazo)-
benzamide (16)
Mp (°C) 226–228; Yield—86.90 %; 1H NMR (CD3OD): d
7.25–7.56 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 7.77 (s, 1H, H of NH
adjacent to CO), 9.78 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,587 (Ring str., indole), 843 (C–H out of
plane bending, indole), 3,043 (C–H str., Ar), 1,997 (N=N
str., diazo), 1,613 (C=O str., secondary amide), 1,523 (C=C
skeletal str., Ar), 1,071 (C–F str., mono fluorinated benzene
ring); Anal. Calculated for C21H15N4OF: C, 70.38; H, 4.22;
N, 15.63; Found: C, 70.40; H, 4.20; N, 15.65.
2-(1H-Indol-3-ylazo)-N-o-tolyl-benzamide (20)
Mp (°C) 158–160; Yield—85.25 %; 1H NMR (CD3OD): d
7.25–7.41 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 2.34 (s, 3H, CH3 of
ArCH3), 7.59 (s, 1H, H of NH adjacent to CO), 9.92 (s, 1H,
NH of indole); IR (KBr pellets) cm-1: 1,579 (Ring str.,
indole), 867 (C–H out of plane bending, indole), 3,027
(C–H str., Ar), 2,075 (N=N str., diazo), 1,600 (C=O str.,
secondary amide), 1,498 (C=C skeletal str., Ar), 750 (C–C
out of plane bending, 1,2-disubstituted benzene ring); Anal.
Calculated for C22H18N4O: C, 74.56; H, 5.12; N, 15.81;
Found: C, 74.60; H, 5.13; N, 15.78.
N-(2-Chloro-phenyl)-2-(1H-indol-3-ylazo)-
benzamide (17)
4-(1H-Indol-3-ylazo)-N-m-tolyl-benzamide (21)
Mp (°C) 218–220; Yield—97.15 %; 1H NMR (CD3OD): d
7.23–7.50 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 7.65 (s, 1H, H of NH
adjacent to CO), 9.89 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,582 (Ring str., indole), 855 (C–H out of
plane bending, indole), 3,030 (C–H str., Ar), 2,047 (N=N
str., diazo), 1,614 (C=O str., secondary amide), 1,513 (C=C
skeletal str., Ar), 754 (C–Cl str., mono chlorinated benzene
1
Mp (°C) 180–182; Yield—78.65 %; H NMR (CD3OD): d
7.19–7.76 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 2.40 (s, 3H, CH3 of
ArCH3), 7.92 (s, 1H, H of NH adjacent to CO), 10.52 (s, 1H,
NH of indole); IR (KBr pellets) cm-1: 1,573 (Ring str., indole),
868 (C–H out of plane bending, indole), 2,059 (N=N str.,
diazo),1,602(C=Ostr.,secondaryamide),1,541(C=Cskeletal
123

1962
Med Chem Res (2013) 22:1957–1971
str., Ar), 679 (C–C out of plane bending, 1,3-disubstituted
benzene ring); Anal. Calculated for C22H18N4O: C, 74.56; H,
5.12; N, 15.81; Found: C, 74.61; H, 5.11; N, 15.84.
indole), 868 (C–H out of plane bending, indole), 2,056 (N=N
str., diazo), 1,602 (C=O str., secondary amide), 1,489 (C=C
skeletal str., Ar), 679 (C–C out of plane bending, 1,3-
disubstituted benzene ring); Anal. Calculated for C22H18
N4O: C, 74.56; H, 5.12; N, 15.81; Found: C, 74.55; H, 5.16;
N, 15.84.
4-(1H-Indol-3-ylazo)-N-(3-nitro-phenyl)-benzamide (22)
Mp (°C) 218–220; Yield—76.10 %; 1H NMR (CD3OD): d
7.00–7.86 (m, 12H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 8.31–8.40 (d, 1H,
ArH adjacent to NO2, J = 45 Hz), 8.01 (s, 1H, H of NH
adjacent to CO), 10.51 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,579 (Ring str., indole), 880 (C–H out of
plane bending, indole), 3,092 (C–H str., Ar), 1,980 (N=N
str., diazo), 1,617 (C=O str., secondary amide), 1,527 (C=C
skeletal str., Ar), 1,484 (NO2 str., aromatic nitro group);
Anal. Calculated for C21H15N5O3: C, 65.45; H, 3.92; N,
18.17; Found: C, 65.48; H, 3.88; N, 18.15.
2-(1H-Indol-3-ylazo)-N-p-tolyl-benzamide (26)
Mp (°C) 202–204; Yield—87.90 %; 1H NMR (CD3OD): d
6.98–7.36 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 2.38 (s, 3H, CH3 of
ArCH3), 7.50 (s, 1H, H of NH adjacent to CO), 10.14 (s,
1H, NH of indole); IR (KBr pellets) cm-1: 1,565 (Ring str.,
indole), 808 (C–H out of plane bending, indole), 1,617
(C=O str., secondary amide), 1,508 (C=C skeletal str., Ar),
808 (C–C out of plane bending, 1,4-disubstituted benzene
ring); Anal. Calculated for C22H18N4O: C, 74.56; H, 5.12;
N, 15.81; Found: C, 74.59; H, 5.16; N, 15.83.
2-(1H-Indol-3-ylazo)-N-phenyl-benzamide (23)
Mp (°C) 185–187; Yield—97.95 %; 1H NMR (CD3OD): d
7.23–7.61 (m, 14H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and H of indole), 9.97 (s, 1H, NH of
indole), 7.88 (s, 1H, H of NH adjacent to CO); IR (KBr
pellets) cm-1: 1,600 (Ring str., indole), 904 (C–H out of
plane bending, indole), 3,039 (C–H str., Ar), 2,057 (N=N
str., diazo), 1,620 (C=O str., secondary amide), 1,519 (C=C
skeletal str., Ar); Anal. Calculated for C21H16N4O: C,
74.10; H, 4.74; N, 16.46; Found: C, 74.13; H, 4.73; N,
16.43.
Evaluation of antimicrobial activity (determination
of minimum inhibitory concentration, MIC)
The antimicrobial activity against Gram-positive bacteria: S.
aureus, B. subtilis, Gram-negative bacterium: E. coli, and
fungal strains: Candida albicans and Aspergillus niger was
determined using the tube dilution method (Cappucino and
Sherman, 1999). The standard and test samples were dis-
solved in DMSO to give a concentration of 100 lg/mL.
Dilutions of test and standard compounds were prepared in
double strength nutrient broth—I.P. (bacteria) or Sabouraud
dextrose broth I.P. (fungi) (Pharmacopoeia of India, 2007).
The samples were incubated at 37 1 °C for 24 h (bacte-
ria), at 25 1 °C for 7 days (A. niger) and at 37 1 °C for
48 h (C. albicans), respectively, and the results were recor-
ded in terms of MIC (the lowest concentration of test sub-
stance which inhibited the growth of microorganisms).
N-(2,4-Dimethyl-phenyl)-2-(1H-indol-3-ylazo)-
benzamide (24)
Mp (°C) 222–224; Yield—89.90 %; 1H NMR (CD3OD): d
7.23–7.59 (m, 12H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 2.33 (s, 3H, CH3 of
ArCH3), 2.44 (s, 3H, CH3 of ArCH3), 7.95 (s, 1H, H of NH
adjacent to CO), 9.84 (s, 1H, NH of indole); IR (KBr
pellets) cm-1: 1,566 (Ring str., indole), 870 (C–H out of
plane bending, indole), 2,121 (N=N str., diazo), 1,620
(C=O str., secondary amide), 1,503 (C=C skeletal str., Ar),
870 (C–C out of plane bending, 1,2,4-trisubstituted ben-
zene ring); Anal. Calculated for C23H20N4O: C, 74.98; H,
5.47; N, 15.21; Found: C, 75.00; H, 5.50; N, 15.20.
Determination of MBC/MFC
The minimum bactericidal concentration (MBC) and fun-
gicidal concentration (MFC) were determined by subcul-
turing 100 lL of culture from each tube (which remained
clear in the MIC determination) on fresh medium. MBC
and MFC values represent the lowest concentration of
compound that produces a 99.9 % end point reduction
(Rodriguez-Arguelles et al., 2005).
2-(1H-Indol-3-ylazo)-N-m-tolyl-benzamide (25)
Mp (°C) 216–218; Yield—70.15 %; 1H NMR (CD3OD): d
7.24–7.43 (m, 13H, ArH of ArH adjacent to CONH, ArH
adjacent to N=N and ArH of indole), 2.42 (s, 3H, CH3 of
ArCH3), 7.73 (s, 1H, H of NH adjacent to CO), 9.98 (s, 1H,
NH of indole); IR (KBr pellets) cm-1: 1,572 (Ring str.,
QSAR studies
The calculations of molecular descriptors as well as
regression analysis were conducted using molecular pack-
age TSAR 3D version 3.3 (TSAR 3D Version 3.3, 2000).
123

Med Chem Res (2013) 22:1957–1971
1963
Evaluation of antiproliferative activity
mean IC50 of three independent experiments (Mosmann,
1983).
The antiproliferative activity of the synthesized N-(substi-
tuted phenyl)-2/4-(1H-indol-3-ylazo)-benzamides were
determined against human colon (HCT116), murine leu-
kemia (P388), and breast cancer (MCF7) cell lines. All cell
lines were cultured in RPMI 1640 (Sigma) supplemented
with 10 % heat inactivated fetal bovine serum (FBS)
(PAA Laboratories) and 1 % penicillin/streptomycin (PAA
Laboratories). Cultures were maintained in a humidified
incubator at 37 °C in an atmosphere of 5 % CO2. Cytotox-
icity of the synthesized compounds at various concentrations
was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyl tetrazolium bromide (MTT) (Sigma) assay, as described
by Mosmann but with minor modification, following 72 h of
incubation. Assay plates were read using a spectrophotom-
eter at 520 nm. Data generated were used to plot a dose–
response curve from which the concentration of test
compounds required to kill 50 % of cell population (IC50)
was determined. Cytotoxic activity was expressed as the
Results and discussion
Chemistry
The title compounds, N-(Substituted phenyl)-2/4-(1H-in-
dol-3-ylazo)-benzamides and the intermediates 2/4-(indol-
3-ylazo)-benzoic acids, were synthesized as outlined in
Scheme 1 and their physicochemical characteristics are
presented in Table 1. The spectral and elemental analysis
data of the synthesized compounds were found to be in
agreement with the assigned molecular structures.
The formation of target compounds was ascertained
based on their consistent NMR and IR spectral data. The
presence of IR stretching bands around 1,615–1,562 cm-1
indicated the presence of indole nucleus in the synthesized
compounds. The stretching IR bands ranging from 2,121 to
H
H
N
N
Scheme 1 Synthetic route
followed for the synthesis of
N-(substituted phenyl)-2/4-
(1H-indol-3-ylazo)-benzamides
COOH
NaNO2/HCl
H
N
SOCl2
+
COCl
COOH
N
N
NH2
N
N
R1
R2
H2N
R3
H
N
R5
R1
R4
R1
CONH
R5
R2
R4
R2
N
N
R3
H
N
R3
CONH
(1-13, 21-22 )
R5
R4
N
N
(14-20, 23-26)
Comp.
R1
H
R2
R3
H
R4
H
H
H
H
H
R5
Comp.
14
R1
CH3
CH3
F
R2
CH3
H
R3
R4
H
R5
H
H
H
H
H
H
H
H
H
H
H
H
H
1
2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
15
CH3
H
3
CH3
NO2
Cl
CH3
H
16
H
H
4
H
17
Cl
H
H
H
5
H
H
18
H
Cl
H
H
6
CH3
CH3
H
H
H
CH3
H
CH3
H
19
H
H
Cl
H
H
7
H
20
CH3
H
H
H
8
Cl
H
H
21
CH3
NO2
H
H
H
9
OCH3
CH3
F
H
H
22
H
H
H
10
11
12
13
H
H
H
23
H
H
H
H
H
H
24
CH3
H
H
CH3
H
H
H
H
NO2
Cl
H
25
CH3
H
H
H
H
H
26
H
CH3
H
123

1964
Med Chem Res (2013) 22:1957–1971
1,071 cm-1, respectively. The presence of IR out of
plane bending at 808 cm-1 indicated the presence of 1,
4-disubstituted benzene ring in compounds 2 and 26.
Similarly in compounds 10 and 20, out of plane bending at
750 cm-1 indicated the presence of 1,2-disubstituted ben-
zene ring. The presence of 1,3-disubstituted benzene ring
in compounds 21 and 25 was confirmed by IR out of plane
bending at 679 cm-1. The presence of 1,2,3-trisubstituted
benzene ring in compounds 3 and 14 was evidenced by IR
out of plane bending at 764 and 776 cm-1, respectively. IR
out of plane bending at 870 cm-1 in compounds 7 and 24
confirmed the presence of 1,2,4-trisubstituted benzene ring.
The presence of 1,2,5-trisubstituted benzene ring in com-
pounds 6 and 15 was confirmed by IR out of plane bending
Table 1 Physicochemical properties of synthesized N-(substituted
phenyl)-2/4-(1H-indol-3-ylazo)-benzamides
Comp. M. formula
M. wt. Mp (°C) Rf valuea % yield
Training set
1
2
C21H16N4O
340
354
368
385
190–192 0.33
220–222 0.56
170–172 0.50
125–127 0.63
185–187 0.36
158–160 0.58
200–202 0.37
220–222 0.45
100–102 0.44
206–208 0.77
180–182 0.64
197–199 0.43
230–232 0.53
258–260 0.59
206–208 0.48
226–228 0.64
218–220 0.73
210–212 0.54
233–235 0.64
158–160 0.37
85.10
92.45
88.50
83.35
89.40
94.25
91.80
80.30
75.75
90.70
80.75
88.20
82.35
91.35
78.75
86.90
97.15
92.20
96.50
85.25
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
22H18N4O
23H20N4O
21H15N5O3
3
4
5
21H15N4OCl 375
6
23H20N4O
23H20N4O
368
368
7
8
21H15N4OCl 375
9
22H18N4O2
22H18N4O
21H15N4OF
21H15N5O3
370
354
358
385
at 883 cm-1
.
10
11
12
13
14
15
16
17
18
19
20
Test set
21
22
23
24
25
26
The presence of indole nucleus and aromatic moieties in
the synthesized compounds were confirmed by the
appearance of d at 9.6–10.2 and 6.9–7.8 ppm, respectively.
Furthermore, the formation of amide linkage was con-
firmed by appearance of singlet signal at d 7.4–8.2 ppm.
Appearance of singlet signal around d 2.0–2.4 ppm elicited
the presence of aromatic methyl groups in compounds 2, 3,
6, 7, 10, 14, 15, 20, 21, 24, 25, and 26. The presence of
methoxy group in compound 9 was indicated by the
appearance of singlet signal at d 3.8 ppm. Furthermore,
the elemental analysis results were within 0.4 % of the
theoretical values.
21H15N4OCl 375
23H20N4O
23H20N4O
21H15N4OF
368
368
358
21H15N4OCl 375
21H15N4OCl 375
21H15N4OCl 375
22H18N4O
354
C22H18N4O
C21H15N5O3
C21H16N4O
C23H20N4O
C22H18N4O
C22H18N4O
354
385
340
368
354
354
180–182 0.29
218–220 0.44
185–187 0.67
222–224 0.46
216–218 0.36
202–204 0.52
78.65
76.10
97.95
89.90
70.15
87.90
In this study, it was observed that in general, the %
yield of target compounds is very high (almost above
80–90 %) which suggested that none of the problems that
decrease the yield of organic compounds occurred in the
present synthetic procedure. The low yields of synthetic
compounds are often attributed to one or more of the fol-
lowing reasons (Furniss et al., 1998): (a) the reaction may
be reversible and position of equilibrium is unfavorable to
the product; (b) the incursion of side reactions leading to
the formation of by-products; (c) the premature work-up of
the reaction before its completion; (d) the volatilization of
products during reaction or work-up; (e) the loss of product
due to incomplete extraction, inefficient crystallization, or
other work-up procedures; and (f) the presence of con-
taminants in the reactants or reagents leading to a less
efficient reaction.
% yield of final compounds
a
TLC mobile phase = benzene
1,980 cm-1 confirmed the formation of diazo linkage in
the target compounds. The appearance of IR bands in the
range 1,670–1,581 cm-1 (C=O stretching, secondary
amide) further confirmed the formation of title compounds.
The presence of phenyl group in the synthesized com-
pounds was demonstrated by C=C skeletal stretching of
phenyl nucleus at wave number 1,541–1,489 cm-1. The
presence of IR stretching at wave number 3,092–2,980
cm-1 indicated C–H stretching of phenyl nucleus. The
presence of IR bands in the region of 1,504–1,484 cm-1
indicated the presence of aromatic nitro group in com-
pounds 4, 12, and 22. The presence of chloro group in
compounds 5, 8, 13, 17, 18, and 19 was evidenced by IR
stretching bands ranging from 786 to 752 cm-1. IR band at
1,265 cm-1 indicated the presence of methoxy group in
compound 9. The presence of fluoro group in compounds
11 and 16 was confirmed by IR bands at 1,072 and
Antimicrobial activity
The synthesized indole derivatives (1–26) were screened
for their in vitro antimicrobial potential by tube dilution
method (Cappucino and Sherman, 1999) using norfloxacin
(antibacterial) and fluconazole (antifungal) as reference
drugs. Double strength nutrient broth I.P. and Sabouraud
dextrose broth I.P. was employed as media for growth of
bacterial and fungal strains, respectively. The results of
123

Med Chem Res (2013) 22:1957–1971
1965
antimicrobial activity are presented in Table 2. Since the
synthesized compounds were not as sensitive as the standard
drugs, norfloxacin and fluconazole (pMIC[ 2.6 lM/mL)
against the tested microorganisms, these drugs were not
further diluted to determine their absolute MIC values.
Antimicrobial screening results indicated that com-
pound 12 demonstrated potent antibacterial activity against
S. aureus (pMICsa = 1.79). In the case of B. subtilis,
compounds 4 and 22 emerged as the most effective anti-
bacterial agents with pMICbs values of 1.79 each. Against
Gram-negative bacterium (E. coli), compounds 5, 12, and
18 (pMICec values 1.78, 1.79, and 1.78, respectively)
emerged as most active candidates among the synthe-
sized compounds. In the case of antifungal activity against
C. albicans and A. niger, compounds 4, 12, and 22
(pMICca and pMICan = 1.49) demonstrated better effects
when compared to the other synthesized compounds.
In general, according to the results of MBC/MFC
(Table 3), the synthesized compounds were bacteriostatic
and fungistatic (in case of C. albicans) in action as their
MFC and MBC values were 3-fold higher than their MIC
values. A drug is considered to be bacteriostatic/fungistatic
when its MFC and MBC values are 3-fold higher than its
MIC values) and fungicidal against A. niger (Emami et al.,
2004).
Data set and molecular descriptors
The different molecular descriptors (independent variables)
like log of octanol–water partition coefficient (log P),
molar refractivity (MR), Kier’s molecular connectivity (0v,
1
1
2
2
0vv, v, vv, v, vv) and shape (j1, ja1, ja2, ja3) topo-
logical indices, Randic topological index (R), Balaban
topological index (J), Wiener topological index (W), Total
energy (Te), energies of highest occupied molecular orbital
(HOMO) and lowest unoccupied molecular orbital
(LUMO), dipole moment (l), and electronic energy (Ele.E)
(Hansch and Fujita, 1964; Kier and Hall, 1976; Randic,
Table 2 Antimicrobial activity
Comp.
pMICsa
pMICbs
pMICec
pMICca
pMICan
pMICab
pMICaf
pMICam
(lM/mL) of synthesized N-
(substituted phenyl)-2/4-(1H-
indol-3-ylazo)-benzamides
Training set
1
2
1.44
1.44
1.45
1.47
1.79
1.48
1.47
1.47
1.48
1.47
1.45
1.46
1.49
1.48
1.47
1.77
1.46
1.48
1.48
1.48
1.45
1.44
1.45
1.47
1.49
1.78
1.77
1.47
1.48
1.47
1.45
1.46
1.79
1.48
1.77
1.47
1.76
1.48
1.78
1.48
1.45
1.44
1.45
1.47
1.49
1.48
1.47
1.47
1.48
1.47
1.45
1.46
1.49
1.48
1.47
1.47
1.46
1.48
1.48
1.48
1.45
1.44
1.45
1.47
1.49
1.48
1.47
1.47
1.48
1.47
1.45
1.46
1.49
1.48
1.47
1.47
1.46
1.48
1.48
1.48
1.45
1.44
1.45
1.57
1.59
1.48
1.57
1.47
1.48
1.47
1.35
1.46
1.69
1.48
1.47
1.47
1.46
1.38
1.48
1.38
1.35
1.44
1.45
1.47
1.49
1.48
1.47
1.47
1.48
1.47
1.45
1.46
1.49
1.48
1.47
1.47
1.46
1.48
1.48
1.48
1.45
1.44
1.45
1.53
1.55
1.48
1.53
1.47
1.48
1.47
1.39
1.46
1.61
1.48
1.47
1.47
1.46
1.42
1.48
1.42
1.39
1.45
1.77
1.49
1.18
1.47
1.47
1.48
1.47
1.15
1.46
1.79
1.48
1.17
1.17
1.16
1.18
1.18
1.18
1.15
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Test set
21
22
23
24
25
26
SD
Std.
1.45
1.19
1.13
1.17
1.45
1.15
0.2
1.75
1.79
1.74
1.47
1.75
1.45
0.13
2.61a
1.75
1.49
1.74
1.47
1.75
1.45
0.15
2.61a
1.45
1.49
1.44
1.47
1.45
1.45
0.02
2.64b
1.45
1.49
1.44
1.47
1.45
1.45
0.02
2.64b
1.65
1.49
1.54
1.37
1.65
1.35
0.09
–
1.45
1.49
1.44
1.47
1.45
1.45
0.02
–
1.57
1.49
1.5
1.41
1.57
1.39
0.06
–
SD standard deviation, Std.
standard
a
Norfloxacin
2.61a
b
Fluconazole
123

1966
Med Chem Res (2013) 22:1957–1971
Hansch et al. (1973). According to ot-QSAR models, one
should use five different equations to predict the activity of
a new compound against the five microbial species. The
ot-QSAR models, which are almost in the whole literature,
become impractical when we have to predict results of
each compound for more than one target. In these cases, we
have to develop one ot-QSAR model for each target. In
opposition to one-target-QSAR (ot-QSAR), the multitar-
get-QSAR (mt-QSAR) model is a single equation that
considers the nature of molecular descriptors which are
common and essential for describing the antibacterial and
antifungal activities (Prado-Prado et al., 2008; Gonzalez-
Diaz and Prado-Prado, 2008; Cruz-Monteagudo et al.,
2007; Gonzalez-Diaz et al., 2007).
Table 3 MBF/MFC of synthesized N-(substituted phenyl)-2/4-(1H-
indol-3-ylazo)-benzamides
Comp. Minimum bactericidal/fungicidal concentration (lM/mL)
S. aureus B. subtilis E. coli
C. albicans A. niger
Training set
1
2
[0.15
[0.14
[0.14
[0.13
[0.13
[0.14
[0.14
[0.13
[0.13
[0.14
[0.14
[0.13
[0.13
[0.14
[0.14
[0.14
[0.13
[0.13
[0.13
[0.14
[0.15
[0.14
[0.14
[0.13
[0.13
[0.14
[0.14
[0.13
[0.13
[0.14
[0.14
[0.13
[0.13
[0.14
[0.14
[0.14
[0.13
[0.13
[0.13
[0.14
[0.15
[0.14
[0.14
[0.13
[0.13
[0.14
[0.14
[0.13
[0.13
[0.14
[0.14
[0.13
[0.13
[0.14
[0.14
[0.14
[0.13
[0.13
[0.13
[0.14
[0.15
[0.14
[0.14
[0.13
[0.13
0.07
0.07
0.04
0.03
0.03
0.03
0.03
0.03
0.03
0.07
0.04
0.03
0.03
0.13
0.03
0.03
0.03
0.03
0.03
0.03
0.04
3
4
5
6
7
0.14
8
[0.13
0.03
In view of the above and based on the QSAR results of
our previous studies (Narang et al., 2012; Judge et al.,
2012; Kumar et al., 2009, 2010), we observed that the
mt-QSAR models gave better results than ot-QSAR models
in describing the antimicrobial activity. In this study, there-
fore, we decided to develop mt-QSAR models to describe
the antimicrobial activity of the synthesized N-(substituted
phenyl)-2/4-(1H-indol-3-ylazo)-benzamides.
9
10
11
12
13
14
15
16
17
18
19
20
Test set
21
22
23
24
25
26
Std.
[0.14
0.07
0.13
[0.13
[0.14
[0.14
[0.14
[0.13
[0.13
[0.13
[0.14
In order to develop the mt-QSAR models, initially we
calculated the average antibacterial [pMICab = (pMICsa
? pMICbs ? pMICec)/3], antifungal [pMICaf = (pMIC-
ca ? pMICan)/2], and antimicrobial [pMICam = (pMICsa
? pMICbs ? pMICec ? pMICca ? pMICan)/5] activity
values of substituted indole derivatives which are presented
in Table 2.
[0.14
[0.13
[0.15
[0.14
[0.14
[0.14
[0.14
[0.13
[0.15
[0.14
[0.14
[0.14
[0.14
[0.13
[0.15
[0.14
[0.14
[0.14
0.04
0.06
0.04
0.03
0.04
0.03
0.04
0.04
0.040b
Preliminary analysis was carried out in terms of correla-
tion analysis (Table 5) to identify the parameter responsible
for the antibacterial, antifungal, and antimicrobial activities.
In general, high colinearity (r [ 0.5) was observed between
different parameters. The high interrelationship was
observed between Kier’s alpha first-order shape indice (ja1)
and R (r = 0.956), and low interrelationship was observed
between energy of lowest unoccupied molecular orbital
(LUMO) and molar refractivity (MR) (r = 0.018).
[0.15
[0.14
[0.14
[0.14
0.019a
0.019a
0.019a
0.040b
Std. standard
a
Norfloxacin
b
Fluconazole
The QSAR models developed using the training set
(1–20) are presented in Table 6. Equation 1 (E1) of Table 6
indicated that the Wiener index (W) has positive effects on
antibacterial activity of the compounds. This is evidenced
by the high antibacterial activities of compounds 12 and 21
(pMICab = 1.69 and 1.65, respectively) which have high
value of Wiener index (W = 2787 and 2227, respectively).
Wiener index W = W(G) of G is defined as the half sum
of the elements of the distance matrix
1975, 1993; Balaban, 1982; Wiener, 1947) were calculated
for indole derivatives and the values are presented in
Table 4. The antimicrobial activity (in terms of pMIC) of
synthesized indole derivatives (1–26) was used as depen-
dent variable in QSAR study. The data set was classified
into training (1–20) and test set (21–26).
QSAR studies
1
W ¼ WðGÞ ¼ = RðDÞij i ¼ 1; j ¼ 1;
2
Linear regression (LR) analysis
where (D)ij is the ijth element of the distance matrix which
denotes the shortest graph–theoretical distance between
sites i and j of G (Wiener, 1947).
In this study, the QSAR study was performed by linear free
energy relationship model (LFER) as that described by
123

Med Chem Res (2013) 22:1957–1971
1967
Table 4 Values of selected parameters used in QSAR studies of synthesized N-(substituted phenyl)-2/4-(1H-indol-3-ylazo)-benzamides
Comp.
log P
MR
ja1
ja2
ja3
R
J
W
LUMO
HOMO
Training set
1
2
4.88
5.35
5.82
4.84
5.40
5.82
5.82
5.40
4.63
5.35
5.02
4.84
5.40
5.82
5.82
5.02
5.40
5.40
5.40
5.35
104.72
109.76
114.80
112.04
109.52
114.80
114.80
109.52
111.18
109.76
104.94
112.04
109.52
114.80
114.80
104.94
109.52
109.52
109.52
109.76
16.84
17.79
18.74
19.27
18.06
18.74
18.74
18.06
18.70
17.79
17.72
19.27
18.06
18.74
18.74
17.72
18.06
18.06
18.06
17.79
7.74
7.96
8.18
8.53
8.14
8.18
8.18
8.14
8.57
7.96
7.91
8.53
8.14
8.18
8.18
7.91
8.14
8.14
8.14
7.96
3.98
4.21
4.14
4.35
4.17
4.30
4.30
4.33
4.28
4.05
4.02
4.51
4.33
3.99
4.14
3.87
4.01
4.17
4.17
3.90
12.79
13.19
13.61
14.11
13.20
13.60
13.60
13.19
13.74
13.20
13.20
14.10
13.19
13.63
13.61
13.22
13.22
13.20
13.20
13.22
1.07
1.07
1.08
1.11
1.09
1.09
1.09
1.07
1.09
1.09
1.09
1.06
1.07
1.21
1.21
1.21
1.21
1.19
1.19
1.21
2005
2247
2432
2667
2207
2434
2453
2227
2436
2207
2207
2787
2247
2190
2192
1987
1987
2007
2027
1987
-0.97
-0.96
-0.96
-1.14
-0.99
-0.96
-0.96
-1.03
-0.91
-0.96
-0.95
-1.26
-1.04
-0.68
-0.69
-0.78
-0.76
-0.79
-0.78
-0.70
-8.51
-8.49
-8.50
-8.52
-8.51
-8.49
-8.46
-8.55
-8.43
-8.50
-8.53
-8.67
-8.55
-8.38
-8.39
-8.44
-8.44
-8.46
-8.46
-8.39
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Test set
21
22
23
24
25
26
5.35
4.84
4.88
5.82
5.35
5.35
109.76
112.04
104.72
114.80
109.76
109.76
17.79
19.27
16.84
18.74
17.79
17.79
7.96
8.53
7.74
8.18
7.96
7.96
4.21
4.51
3.82
4.14
4.05
4.05
13.19
14.10
12.81
13.61
13.20
13.20
1.07
1.06
1.19
1.21
1.19
1.19
2227
2727
1807
2211
2007
2027
-0.96
-1.17
-0.71
-0.66
-0.70
-0.70
-8.50
-8.62
-8.40
-8.39
-8.39
-8.40
Table 5 Correlation matrix for the antibacterial, antifungal, and antimicrobial activities of synthesized N-(substituted phenyl)-2/4-(1H-indol-3-
ylazo)-benzamides
pMICab pMICaf pMICam log P
MR
ja1
ja2
ja3
R
J
W
LUMO HOMO
pMICab
pMICaf
pMICam
log P
MR
1.000
0.537
0.996
1.000
0.610
1.000
-0.115
0.392
0.030 -0.105
1.000
0.386
0.691
0.766
0.647
0.564
0.409
0.715
0.656
0.714
0.734
0.616 1.000
0.169 0.811
ja1
0.683
1.000
ja2
0.613
-0.190 0.576
-0.088 0.443
-0.056 0.685
0.881
0.634
0.956
1.000
ja3
0.687
0.721
0.867
1.000
0.580
R
0.718
1.000
J
-0.423
0.817
0.022 -0.395
0.456 0.815
0.241 0.028 -0.034 -0.119 -0.598 -0.080 1.000
-0.150 0.543 0.795 0.749 0.795 0.851 -0.585
W
1.000
LUMO
HOMO
-0.656 -0.297 -0.647
-0.606 -0.313 -0.602
0.412 0.018 -0.263 -0.366 -0.730 -0.345
0.391 0.178 -0.120 -0.217 -0.630 -0.181
0.867 -0.747 1.000
0.753 -0.568 0.920
1.000
123

1968
Med Chem Res (2013) 22:1957–1971
The developed QSAR model (E1) was cross validated by
well by the plot of predicted pMICab against observed
pMICab (Fig. 1). Further, the plot of observed pMICab
versus residual pMICab (Fig. 2) indicated that there was no
systemic error in development of linear regression model
as the propagation of residuals was observed on both sides
of zero (Kumar et al., 2007).
its q2 value (q2 = 0.574) obtained by leave one out (LOO)
method. The value of q2 more than 0.5 is the basic
requirement for the validity of QSAR model (Golbraikh
and Tropsha, 2002). The predictive ability of E1 was evi-
denced by the low residual values observed in Table 7 as
The second equation (E2) of Table 6 explains the posi-
tive effect of Kier’s alpha second order shape indice (ja2)
on the antifungal activity of synthesized compounds.
According to Kier, the shape of a molecule may be parti-
tioned into attributes, each describable by the count of
bonds of various path lengths. The basis for devising a
relative index of shape is given by the relationship of the
Table 6 The results of LR analysis for antimicrobial activity of syn-
thesized N-(substituted phenyl)-2/4-(1H-indol-3-ylazo)-benzamides
No. Equation
n
r
q2
s
F
E1 pMICab = 0.0003 W ? 0.813 20 0.817 0.574 0.048 36.11
E2 pMICaf = 0.049 ja2 ? 0.738 20 0.766 0.430 0.009 25.55
E3 pMICam = 0.0002 W ? 1.050 20 0.815 0.574 0.031 35.73
l
number of path of length l in the molecule i, Pi, to some
reference values based on molecules with a given number
n number of data points, r correlation coefficient, q2 cross validated,
r2 obtained by LOO method, s standard error of the estimate,
F Fischer statistics
1.7
Table 7 Comparison of observed and predicted antimicrobial activ-
ity obtained by mt-QSAR models
1.6
1.5
1.4
Comp. pMICab
Obs. Pre. Res.
Training set
pMICaf
pMICam
Obs. Pre. Res.
Obs. Pre. Res.
1
2
1.44 1.40
0.04 1.44 1.45 -0.01 1.44 1.43
0.01
1.45 1.47 -0.02 1.45 1.46 -0.01 1.45 1.47 -0.02
3
1.57 1.53
0.04 1.47 1.47
0.00 1.53 1.51
0.00 1.55 1.55
0.01 1.48 1.47
0.00 1.53 1.51
0.02
0.00
0.01
0.02
4
1.59 1.60 -0.01 1.49 1.49
1.3
5
1.48 1.46
1.57 1.53
0.02 1.48 1.47
0.04 1.47 1.47
1.3
1.4
1.5
1.6
1.7
6
observed pMICab
7
1.47 1.54 -0.07 1.47 1.47
1.48 1.47 0.01 1.48 1.47
0.00 1.47 1.51 -0.04
0.01 1.48 1.47 0.01
8
Fig. 1 Plot of predicted pMICab values against observed pMICab
values for linear regression developed model by Eq. E1
9
1.47 1.53 -0.06 1.47 1.49 -0.02 1.47 1.51 -0.04
1.35 1.46 -0.11 1.45 1.46 -0.01 1.39 1.47 -0.08
10
11
12
13
14
15
16
17
18
19
20
Test set
21
22
23
24
25
26
1.46 1.46
1.69 1.63
1.48 1.47
1.47 1.46
1.47 1.46
1.46 1.40
0.00 1.46 1.46
0.06 1.49 1.49
0.01 1.48 1.47
0.01 1.47 1.47
0.01 1.47 1.47
0.06 1.46 1.46
0.00 1.46 1.47 -0.01
.2
.1
0.00 1.61 1.57
0.01 1.48 1.47
0.00 1.47 1.46
0.00 1.47 1.46
0.00 1.46 1.42
0.01 1.42 1.42
0.01 1.48 1.43
0.04
0.01
0.01
0.01
0.04
0.00
0.05
1.38 1.40 -0.02 1.48 1.47
1.48 1.40 0.08 1.48 1.47
1.38 1.41 -0.03 1.48 1.47
1.35 1.40 -0.05 1.45 1.46 -0.01 1.39 1.42 -0.03
0.0
-.1
-.2
0.01 1.42 1.43 -0.01
1.65 1.48
0.17 1.45 1.45
0.00 1.57 1.50
0.08
1.49 1.63 -0.14 1.49 1.48
0.01 1.49 1.60 -0.11
0.18 1.44 1.44 -0.01 1.50 1.41 0.08
1.54 1.36
1.3
1.4
1.5
1.6
1.7
1.37 1.48 -0.11 1.47 1.46
0.00 1.41 1.49 -0.08
Observed pMICab
1.65 1.42
0.24 1.45 1.45
0.00 1.57 1.45
0.12
Fig. 2 Plot of predicted pMICab values against residual pMICab
values for linear regression developed model by Eq. E2
1.35 1.42 -0.07 1.45 1.45
0.00 1.39 1.46 -0.06
123

Med Chem Res (2013) 22:1957–1971
1969
l
of atoms, n, in which the values of P are maximum and
activity data is in the narrow range. When biological
activity data lies in the narrow range, the presence of
minimum standard deviation of the biological activity
justifies its use in QSAR studies (Narasimhan et al., 2007).
The minimum standard deviation (Table 2) observed in the
antimicrobial activity data justifies its use in QSAR studies.
l
minimum, Pmax and Pmin (Kier and Hall, 1999).
l
The modified kappa shape indices are given by
.
ꢀ
ꢁ
2
2
ja1 ¼ ðn þ aÞ ðn þ a ꢁ 1Þ
1Pi þ a
.
ꢀ
ꢁ
2
2
ja2 ¼ ðn þ a ꢁ 1Þ ðn þ a ꢁ 2Þ
2Pi þ a
.
ꢀ
ꢁ
2
2
3Pi þ a
3Pi þ a
n is odd
The antiproliferative activity
ja3 ¼ ðn þ a ꢁ 1Þ ðn þ a ꢁ 3Þ
ja3 ¼ ðn þ a ꢁ 3Þ ðn þ a ꢁ 2Þ
.
ꢀ
ꢁ
2
2
n is even:
The antiproliferative activity of the synthesized N-
(substituted phenyl)-2/4-(1H-indol-3-ylazo)-benzamides were
determined against human colon cancer (HCT116), murine
leukemia (P388), and breast cancer (MCF7) cell lines using
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay (Mosmann, 1983) and the results are
depicted in Table 8. In general, the synthesized compounds
had poor antiproliferative potential against MCF7 cancer
cell line but were highly potent against P388 cell line.
4-(1H-indol-3-ylazo)-N-p-tolyl-benzamide (2) was found
most effective against HCT116 cancer cell line with an
IC50 value of 0.0254 lM/mL (Table 8). Against MCF7
cancer cell line, 4-(1H-indol-3-ylazo)-N-p-tolyl-benzamide
(2) and N-(2,5-dimethyl-phenyl)-4-(1H-indol-3-ylazo)-
benzamide (6) emerged as the most effective ones with
IC50 values of 0.0424 and 0.0299 lM/mL, respectively.
Against HCT116 cancer cell line, compound 2 was found
to be more active than the standard drug 5-fluorouracil
(5-FU) (IC50 = 0.0462 lM/mL, Table 8).
In the case of P388 cancer cell line, 4-(1H-indol-3-
ylazo)-N-p-tolyl-benzamide (2) and 4-(1H-indol-3-ylazo)-
N-m-tolyl-benzamide (21) (IC50 = 0.0003 lM/mL) were
more potent than the standard drug, 5-FU (IC50 = 0.0219
lM/mL). In general, all the synthesized compounds were
less active as compared to the standard drug 5-FU, but
compound 2 was more effective with an IC50 of about 1.8
times lower than 5-FU against HCT116 cell line. Com-
pounds 2 and 21 which were more potent (73 times) as
compared to standard drug 5-FU against P388, indicates
that these compounds have potential to be taken as lead
compounds for the development of novel antiproliferative
agents against leukemia.
The equation E3 of Table 6 demonstrated the effect of
topological descriptor, Wiener index (W) on the
antimicrobial activity of these compounds. Stepwise
selection and elimination of variables produced a QSAR
equation (E3). This equation, which has a good statistical
quality (q2 = 0.574), demonstrates the positive effect of
Wiener index (W) on the antimicrobial activity of the
synthesized indole derivatives.
The validity and predictability of the QSAR models, i.e.,
E1 and E3 are evidenced by high q2 value (q2 [ 0.5)
obtained by LOO method as well as the low residual
activity values (Tables 6, 7). True predictive power of a
QSAR model is to test their ability to predict accurately the
biological activities of compounds from an external test set
(compounds which were not used in the model develop-
ment). The low residual activity values observed in case of
test set (21–26) justify the selection of the linear regression
models expressed by E1, E2, and E3.
It was observed from mt-QSAR models [E1–E3] that the
antibacterial activity, antifungal activity, and overall anti-
microbial activity of the synthesized indole derivatives are
governed by the topological parameters Kier’s alpha sec-
ond order shape indice (ja2) and Wiener index (W).
It is important to note a fact here that in case the linear
regression model derived for antifungal activity (E2,
Table 6) has q2 value that is \0.5 (q2 = 0.430) it shows
that the developed models are invalid. However, one
should not forget the recommendations of Golbraikh and
Tropsha (2002) who reported that the only way to estimate
the true predictive power of a QSAR model is to test their
ability to predict accurately the biological activities of
compounds. As the observed and predicted values are close
to each other (Table 7), the QSAR model for antifungal
activity (E2) is a valid one.
Structure–activity relationship
1. Antimicrobial activity of indole derivatives synthesized
using p-aminobenzoic acid were more potent than the
ones synthesized using o-aminobenzoic acid. This result
indicated the fact that o-amino group may cause steric
problem at the target site and may prevent the binding of
the indole derivatives with the target site.
In generally, for QSAR studies, the biological activities
of compounds should span 2–3 orders of magnitude.
Nevertheless, in this study the range of antimicrobial
activities of the synthesized compounds is within one order
of magnitude. This is similar to the results obtained by
Bajaj et al. (2005) who stated that the reliability of the
QSAR model lies in its predictive ability even though the
2. The presence of electron-withdrawing groups (nitro or
chloro) on aniline portion increased the antimicrobial
123

1970
Med Chem Res (2013) 22:1957–1971
improved the activity against HCT116 and MCF7 cell
lines. The role of electron-releasing groups in improv-
ing the antiproliferative activity of indole derivatives is
supported by findings of Jacquemard et al. (2008).
Table 8 Cytotoxicity (IC50) of synthesized N-(substituted phenyl)-2/
4-(1H-indol-3-ylazo)-benzamides against human colon (HCT116),
murine leukemia (P388), and breast (MCF7) cancer cell lines
Comp.
IC50(lM/mL)
MCF7
HCT116
P388
Training set
Conclusion
1
2
0.1471
0.0424
0.1060
0.1013
0.1120
0.0299
0.0870
0.0827
0.0865
0.0734
0.0838
0.0545
0.1067
0.1087
0.1087
0.1397
0.1067
0.1067
0.1067
0.1412
0.1029
0.0254
0.2717
0.2078
0.1200
0.5435
0.0761
0.0800
0.1216
0.5085
0.5587
0.1558
0.1867
0.3533
0.1630
0.2235
0.2400
0.2933
0.3467
1.1299
NA
0.0003
0.0516
0.0779
NA
In this study, N-(substituted phenyl)-2/4-(1H-indol-3-ylazo)-
benzamides (1–26) were synthesized and screened for their
in vitro antimicrobial and antiproliferative capacities.
Results of the antimicrobial studies indicated that 4-(1H-
indol-3-ylazo)-N-(4-nitro-phenyl)-benzamide (12) was the
most effective antimicrobial agent. QSAR studies demon-
strated the importance of topological parameters Kier’s
alpha second-order shape indice (ja2) and Wiener index
(W) in describing the antimicrobial activity. In general, the
synthesized compounds had low antimicrobial potential
compared to their antiproliferative potential as evidenced by
the high antiproliferative potential of compounds 2 and 21,
which may be taken as lead compounds to develop novel
antiproliferative agents.
3
4
5
6
NA
7
0.1902
0.2667
NA
8
9
10
11
12
13
14
15
16
17
18
19
20
Test set
21
22
23
24
25
26
5-FU
NA
0.1061
0.0112
0.0800
ND
ND
ND
ND
ND
ND
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