Synthesis and evaluation of antitumoral activity of ester and amide derivatives of 2-arylamino-6-trifluoromethyl-3-pyridinecarboxylic acids

Article abstract of DOI:10.1016/j.bmc.2007.11.069

The synthesis and antitumoral activity of ester and amide derivatives of 2-arylamino-6-trifluoromethyl-3-pyridinecarboxylic acids 8-58 is described. Trifluoromethylpyridine derivatives 8-58 were evaluated for their anticancer activity toward human tumoral cell lines by the National Cancer Institute (NCI). Most of them possess encouraging anticancer activity, having GI₅₀ values in the low micromolar to nanomolar concentration range. The 3,4,5-trimethoxyphenylamide 44 was the most active, and it is now under review by NCI Biological Evaluation Committee for possible further studies.

Full text of DOI:10.1016/j.bmc.2007.11.069

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2367–2378
Synthesis and evaluation of antitumoral activity of ester
and amide derivatives of 2-arylamino-6-
trifluoromethyl-3-pyridinecarboxylic acids
Valentina Onnis,* Maria T. Cocco, Valentina Lilliu and Cenzo Congiu
`
Dipartimento di Tossicologia, Universita degli Studi di Cagliari, via Ospedale 72, Cagliari I-09124, Italy
Received 31 July 2007; revised 19 November 2007; accepted 23 November 2007
Available online 11 December 2007
Abstract—The synthesis and antitumoral activity of ester and amide derivatives of 2-arylamino-6-trifluoromethyl-3-pyridinecarb-
oxylic acids 8–58 is described. Trifluoromethylpyridine derivatives 8–58 were evaluated for their anticancer activity toward human
tumoral cell lines by the National Cancer Institute (NCI). Most of them possess encouraging anticancer activity, having GI50 values
in the low micromolar to nanomolar concentration range. The 3,4,5-trimethoxyphenylamide 44 was the most active, and it is now
under review by NCI Biological Evaluation Committee for possible further studies.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
mides7 are potent and selective VEGFR-1 and VEG-
FR-3 inhibitors, possessing potent antiangiogenic and
antitumor properties. Furthermore, Nilonitib is a novel
and selective inhibitor of BCR-ABL more potent than
imanitib and also significantly active against imatinib-
resistant BCR-ABL-expressing cells.8 4-Amino-5-(4-((2-
fluoro-5-(trifluoromethyl)phenyl)aminocarbonylamino)
phenyl)furo[2,3-d]pyrimidine is a potent dual VEGFR-2
and Tie-2 inhibitor, and showed marked tumor growth
inhibition and anti-angiogenic activity in mouse HT-29
xenograft model via once-daily oral administration.9
Furthermore, quite a few of trifluoromethylated mole-
cules demonstrated their antiproliferative activity
through modulation of cell cycle proteins. Thus, 1,1-
bis(30-indolyl)-1-(p-trifluoromethylphenyl)methane in-
duced peroxisome proliferator-activated receptor gam-
ma (PPARc)-mediated transactivation in SW480 colon
cancer cells and also it inhibited cell proliferation and
modulated some cell cycle proteins.10 While N-(3,
5-bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxybenza-
mide is an IKKb inhibitor that inhibited NF-KB activity
in HMC-1 cells, resulting in complete repression of
growth factor-independent proliferation of mast cells.
Inhibition of NF-KB activity decreased the expression
of cyclin D3 and the phosphorylation of pRb, leading
to cell cycle arrest and apoptosis.11 Apoptosis induction
in tumoral cell lines has been also reported for molecules
such as 4-(3-methoxyanilino)-2-(2-pyridinyl)-6-(trifluo-
Fluorinated organic molecules are known to perform a
wide range of biological functions and fluorinated anti-
cancer agents have become a focus in the development
of new therapies for cancer. An increasing number of
fluorinated antitumor agents have now become available
for cancer treatment.1,2 Trifluoromethylated drugs such
as nilutamide, flutamide, hydroxyflutamide, and bicalu-
tamide are non-steroidal anti-androgens which are
widely used for the treatment of metastatic prostate can-
cer.3,4 Furthermore, several studies have been devoted to
the antiproliferative activity of trifluoromethyl bearing
molecules. Thus, Panomifene (EGIS-5656, GYKI-
13504), a trifluoromethyl tetra-substituted alkene, is a
follow-up molecule to tamoxifen and is reported to exhi-
bit anti-estrogenic activity superior to that of tamoxifen
in the treatment of breast cancer.5 A number of trifluo-
romethylated compounds demonstrated their antiprolif-
erative activity through inhibition of kinases. For
example, the biaryl urea BAY 43-9006 (sorafenib), that
is now in phase II clinical trials, is known to inhibit a
broad range of kinases including the vascular endothe-
lial growth factor receptor tyrosine kinases (VEGFR-1
and VEGFR-2).6 While trifluoromethyl-anthranila-
Keywords: Trifluoromethylpyridine; Ester derivatives; Amide deriva-
tives; Synthesis; Antitumoral activity; SAR.
romethyl)pyrimidine12
(4-tolyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzene-
and
N-(2-aminoethyl)-4-[5-
*
Corresponding author. Tel.: +39 0706758632; fax: +39
0706758612; e-mail: vonnis@unica.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.069

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V. Onnis et al. / Bioorg. Med. Chem. 16 (2008) 2367–2378
acids. The interest for these analogs is also derived from
COOAr
NH
antitumor activities associated with the structure of 2-
(substituted amino)-3-pyridinecarboxamide deriva-
tives.19–23
2. Results and discussion
2.1. Chemistry
N
CF3
Ar = Aryl, Heteroaryl
Figure
derivatives.
1. N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic
acid
The target derivatives of 2-arylamino-6-trifluoromethyl-
3-pyridinecarboxylic acids 8–58 (Table 1) were synthe-
sized as shown in Schemes 1–3.
sulfonamide.13 We have recently reported on some
derivatives of N-(2-(trifluoromethyl)pyridin-4-yl)anthra-
nilic acid (Fig. 1) demonstrating antiproliferative activ-
ity in nanomolar to low micromolar concentrations
against a wide array of human tumor cell lines.14
We have previously reported an easy and convenient
method for the synthesis of 2-arylamino-6-trifluoro-
methyl-3-pyridinecarboxylic acids.24
According to Scheme 1, 3-amino-3-ethoxypropenenitrile
1 was first treated with the appropriate substituted aryl-
amines 2 and 3 in MeCN solution to give the non-isola-
ble 3-amino-3-arylaminopropenenitriles 4, 5 and then
with 1,1,1-trifluoro-4-iso-butoxy-3-buten-2-one. The
temperature was gradually allowed to reach 60 ꢁC. After
3 h, pyridinecarbonitriles 6, 7 are obtained in good
yields. Sodium hydroxide catalyzed hydrolysis of com-
pounds 6, 7 afforded pyridine-3-carboxamides 8, 9. High
Prompted by the above findings, in continuation of our
ongoing research on pyridine derivatives endowed with
anticancer activity,15–18 we became interested in novel
compounds containing the trifluoromethylpyridine scaf-
fold. In this paper, we report the synthesis and evalua-
tion for in vitro antitumoral efficacy against human
cancer cell lines of a series of ester and amide derivatives
of 2-arylamino-6-trifluoromethyl-3-pyridinecarboxylic
Table 1. Ester (15–32) and amide (33–58) derivatives of 2-arylamino-6-trifluoromethylpyridin-3-carboxylic acids
O
COOAr'
CONHR
N
N
R
CF3
N
NHAr
CF3
N
NHAr
CF3
N
NHAr
15-32
51-58
33-50
Compound
Ar
Ar0
Compound
Ar
R
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Cl-phenyl
2-Cl-phenyl
3-Cl-phenyl
3-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
2,4-Cl2-phenyl
2,4-Cl2-phenyl
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
3-Me-phenyl
3-Me-phenyl
3-CF3-phenyl
3-CF3-phenyl
3-MeO-phenyl
3-MeO-phenyl
3,4-(MeO)2-phenyl
3,4-(MeO)2-phenyl
3,5-(MeO)2-phenyl
3,5-(MeO)2-phenyl
3,4,5-(MeO)3-phenyl
3,4,5-(MeO)3-phenyl
3-Cl-phenyl
2,4,6-Cl3-phenyl
2,4,6-Cl3-phenyl
3-MeO-phenyl
3-MeO-phenyl
4-MeO-phenyl
4-MeO-phenyl
3,5-(MeO)2-phenyl
3,5-(MeO)2-phenyl
3-Pyridyl
3-Cl-phenyl
3-Cl-4-OMe-phenyl
3-Cl-4-OMe-phenyl
3-Pyridyl
3-Pyridyl
3-Pyridyl
COOEt
COOEt
3-Cl-phenyl
3-Cl-phenyl
4-F-phenyl
4-F-phenyl
4-MeO-phenyl
4-MeO-phenyl

V. Onnis et al. / Bioorg. Med. Chem. 16 (2008) 2367–2378
2369
noate 12 was sequentially treated with the appropriate
substituted arylamines 2, 3, then with 1,1,1-trifluoro-4-
iso-butoxy-3-buten-2-one in MeCN solution to give pyr-
idine derivatives 13, 14.
NH2
CN
CH3
CN
+
(i)
H2N
OEt
H2N
NHAr
Cl
Aryl esters and amides were prepared as reported in
Scheme 3. Treatment of acids 10, 11 with the appropri-
ate phenol in the presence of 1-(3-dimethylaminopro-
pyl)-3-ethylcarbodiimide hydrochloride (EDCI) and
hydroxybenzotriazole (HOBt) in MeCN solution gave
esters 15–32 (Table 1) in 73–97% yields. Amide deriva-
tives 33–58 (Table 1) were obtained in 73–96% yields
by reaction of acids 10, 11 with the appropriate amine
by EDCI method.
1
2 : 3-Cl
3 : 4-Cl
4: Ar = 2-CH3-4-ClC6H3
5: Ar = 2CH3-5-ClC6H3
(ii)
CN
CONH2
(iii)
CF3
N
NHAr
CF3
N
NHAr
6: Ar = 2-CH3-4-ClC6H3
7: Ar = 2CH3-5-ClC6H3
8: Ar = 2-CH3-4-ClC6H3
9: Ar = 2CH3-5-ClC6H3
All the newly synthesized compounds gave corrected
analytical data. The IR and NMR spectral data were
consistent with the assigned structure.
(iv)
COOH
2.2. Pharmacology
CF3
N
NHAr
The compounds 8–58 were submitted to the US Na-
tional Cancer Institute (NCI; Bethesda, MD), for
in vitro testing against a panel of approximately 60 tu-
mor cell lines, derived from nine different cancer types:
leukemia, lung, colon, CNS, melanoma, ovarian, renal,
prostate, and breast. The compounds were tested at five
concentrations at 10-fold dilution. A 48-h continuous
drug exposure protocol was used and sulforhodamine
B (SRB) protein assay was used to estimate cell growth.
Details of this system and the information, which is en-
coded by the activity pattern over all cell lines, have
been published.25–27 The antitumoral activity of tested
compounds is given by three parameters for each cell
line: logGI50 value (GI50 = molar concentration of the
compound that inhibits 50% net cell growth), logTGI
value (TGI = molar concentration of the compound
leading to total inhibition), and logLC50 value
(LC50 = molar concentration of the compound leading
to 50% net cell death). Furthermore, a mean graph mid-
point (MG_MID) is calculated for each of the men-
tioned parameters, giving an averaged activity
parameter over all cell lines. For the calculation of the
MG_MID, insensitive cell lines are included with the
highest concentration tested. Selectivity of the com-
pound with respect to one or more cell lines of the screen
is characterized by a high deviation (D) of the particular
cell line parameter compared to the MG_MID value.
From the analysis of the data reported in Tables 2 and
3 we can evince that acids 10, 11 were inactive, while
the primary amide 9 showed cytostatic activity against
36 different tumoral cell lines at micromolar GI50 values
(logGI50 values between ꢀ5.84 and ꢀ5.07). Amide 9 dis-
played ꢀ5.18 MG_MID value with the best results
against HS 578T cell line at GI50 (logGI50 ꢀ6.09) and
TGI levels (logTGI ꢀ5.40). In the ester series, an aryl
substituent appears to favorably modulate antiprolifera-
tive activity. Thus, replacement of phenyl moiety with
an alkyl led to compounds 13 and 14 that had no anti-
proliferative activity. Furthermore, the presence of func-
tionality as 4-chlorophenyl (compound 20) or 3-
methoxyphenyl (compound 26) led to compounds en-
dowed with high antitumoral activity (GI50 MG_MID
10: Ar = 2-CH3-4-ClC6H3
11: Ar = 2CH3-5-ClC6H3
Scheme 1. Reagents and conditions: (i) MeCN, rt; (ii) 1,1,1-trifluoro-4-
iso-butoxy-3-buten-2-one, reflux; (iii) 20% NaOH, 100 ꢁC; (iv) H2SO4,
H2O, 100 ꢁC.
COOEt
COOEt
(ii)
(i)
CF3
N
NHAr
H2N
OEt
13: Ar = 2-Me-4-Cl-Ph
14: Ar = 2-Me-5-Cl-Ph
12
Scheme 2. Reagents and conditions: (i) ArNH2(2, 3),MeCN, rt; (ii)
1,1,1-trifluoro-4-iso-butoxy-3-buten-2-one, reflux.
COOAr'
NHAr
CONHR
NHAr
(ii)
(i)
10,11
CF3
N
CF3
N
(iii)
33-50
15-32
O
N
NHAr
N
R
CF3
N
51-58
Scheme 3. Reagents and conditions: (i) Ar0OH, EDCI, HOBt, MeCN,
rt; (ii) RNH2, EDCI, HOBt, MeCN, rt; (iii) substituted piperazine,
EDCI, HOBt, MeCN, rt.
yields of 2-arylamino-6-trifluoromethyl-3-pyridinecarb-
oxylic acids 10, 11 were achieved by hydrolysis of deriv-
atives 6, 7 in 50% aqueous sulfuric acid. A chemistry
similar to that described in the synthesis of compounds
4, 5 was used for the preparation of ethyl esters 13, 14.
As reported in Scheme 2, ethyl 3-amino-3-ethoxyprope-

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V. Onnis et al. / Bioorg. Med. Chem. 16 (2008) 2367–2378
values ꢀ5.03 and ꢀ5.10, respectively). Compounds 20
and 26 showed an in vitro chemosensitive profile toward
28 and 32 different cancer cell lines, respectively, with
GI50 values lying in the concentration range between
submicromolar to micromolar. Compound 26 displayed
selectivity on renal cancer RXF 393 cell line (Table 3) at
GI50 (logGI50 value ꢀ6.61) and TGI levels (logTGI va-
lue ꢀ6.01). Simply moving 4-chlorine or 3-methoxy
groups into other position of phenyl ring (esters 15–
18) as well as the introduction of the same group on
more position of phenyl ring (esters 21–24) resulted in
loss of activity. Among amide derivatives, piperazino-
carbonyl compounds 52–54, 56, and 58 as well as aro-
matic amides 35–37, 43, 44, and 47 exhibited moderate
to excellent activity. In the piperazinocarbonyl series
(51–58) the results indicated that inhibition of cell
growth was strongly dependent on the kind of substitu-
ent at N-4 of piperazine ring. A comparison of substitu-
ent effects revealed that the introduction in this position
of the ethoxycarbonyl group led to compound 52 that is
endowed with weak antiproliferative activity (GI50
MG_MID values ꢀ4.79). Replacement of the ethoxy-
carbonyl by a 3-chlorophenyl ring (amides 53 and 54)
resulted in marginal reduction of activity (GI50
MG_MID values ꢀ4.23 and ꢀ4.17, respectively). How-
ever, substitution of the ethoxycarbonyl by a 4-fluoro-
phenyl (amide 56, GI50 MG_MID ꢀ5.01) or by a 4-
methoxyphenyl ring (amide 58, GI50 MG_MID ꢀ5.22)
caused an increase in activity. Amide 58 inhibited the
growth of 38 cell lines with micro and nanomolar GI50
values, showing high selectivity (Table 3) against ovar-
ian cancer IGROV1 cell line (logGI50 ꢀ7.29, D logGI50
2.07). Amide 56 selectively exhibited high potency
against CNS cancer cell line SNB-75 (logGI50 < ꢀ8, D
logGI50 2.99) (Table 3). In the aromatic amide series
(33–50), the results indicated that the 3,4,5-trimethoxy-
phenyl substitution (compounds 43 and 44) was crucial
for potent cell growth inhibition. Compound 44 showed
the best activity, as a matter of fact it inhibited the
growth of all tested cell lines at submicromolar to
micromolar concentrations with MG_MID value
ꢀ5.54, and it is now under review by Biological Evalu-
ation Committee of the NCI for possible further studies.
Amide 44 displayed cytostatic activity at nanomolar
concentrations against four cell lines: HOP-92, HCC-
2998, CCRF-CEM, and MOLT-4 as well as high selec-
tivity on the same cell lines (logGI50 < ꢀ8, D logGI50
2.46). The growth of leukemia MOLT-4 and SR cell
lines was totally inhibited by submicromolar concentra-
tions of compound 44 (logTGI ꢀ7.38 and ꢀ6.52, D
logTGI 2.60 and 1.74, respectively). In addition, 44
was very active against leukemia HL-60 (TB) cell line
at GI50 (logGI50 value ꢀ7.64, D logGI50 2.1), TGI
(logTGI value ꢀ5.79, D logTGI 1.01), and LC50 levels
(logLC50 value ꢀ5.14). Although less potent when com-
pared to 44, compound 43 inhibited the growth of 50
cell lines at micromolar concentrations (GI50MG_MID
ꢀ5.40). Substitution of the 3,4,5-trimethoxyphenyl with
a 3,4-dimethoxyphenyl or 3,5-dimethoxyphenyl moiety
(compounds 39, 40 and 41, 42, respectively) led to the
loss of growth inhibition activity. However, a moderate
loss of activity was observed with methoxy substituents
at C-3 of phenyl ring (amide 37, GI50MG_MID ꢀ5.08).

V. Onnis et al. / Bioorg. Med. Chem. 16 (2008) 2367–2378
2371
Table 3. The in vitro activitya and selectivity toward most sensitive tumor cell lines for compounds 9, 20, 26, 35–37, 43, 44, 47, 52–54, 56, and 58
Compound
Most sensitive tumor cell lines
logGI50 (M)
logTGI (M)
logLC50 (M)
Selectivity toward
tumor cell lines (D)
for logGI50/TGI (M).
The value is
Mean value
for all tested cell
lines (MG_MID)
for logGI50 (M)
shown if D > 1b,c
9
Non-small cell lung: NCI-H226
Non-small cell lung: NCI-322M
Melanoma: LOX IMVI
Melanoma: SK-MEL-5
Breast: HS 578T
ꢀ5.84
ꢀ5.69
ꢀ5.65
ꢀ5.72
ꢀ6.09
ꢀ5.01
ꢀ5.18
ꢀ5.18
ꢀ5.40
20
26
Non-small cell lung: HOP-92
Ovarian:OVCAR-3
ꢀ5.72
ꢀ5.03
ꢀ5.10
ꢀ5.59
ꢀ5.05
Leukemia: CCRF-CEM
Leukemia: K-562
ꢀ5.67
ꢀ5.51
ꢀ5.51
ꢀ5.79
ꢀ6.61
ꢀ5.91
ꢀ5.29
ꢀ5.03
ꢀ5.10
ꢀ5.16
ꢀ6.01
ꢀ5.32
Leukemia: MOLT-4
Non-small cell lung: HOP-92
Renal: RXF 393
1.51/1.65
Breast: HS 578T
35
Leukemia: HL-60(TB)
Leukemia: SR
ꢀ5.69
ꢀ5.59
ꢀ5.73
ꢀ5.66
ꢀ5.60
ꢀ5.59
ꢀ5.68
ꢀ5.52
ꢀ5.55
ꢀ5.70
ꢀ5.55
ꢀ5.61
ꢀ5.55
ꢀ5.56
ꢀ5.64
ꢀ5.69
ꢀ5.33
ꢀ5.50
ꢀ5.08
Non-small cell lung: EKVX
Non-small cell lung: HOP-92
Colon: COLO205
ꢀ5.09
ꢀ5.21
ꢀ5.13
Colon: HCT-15
Colon: KM12
CNS: U251
ꢀ5.01
ꢀ5.07
ꢀ5.04
ꢀ5.03
Melanoma: LOX IMVI
Melanoma: MALME-3M
Melanoma: SK-MEL-5
Ovarian: SK-OV-3
Renal: A498
ꢀ5.13
ꢀ5.05
ꢀ5.11
ꢀ5.09
Renal: ACHN
Breast: MCF7
Breast: MDA-MB-231/ATCC
36
Leukemia: CCRF-CEM
Leukemia: MOLT-4
Leukemia: RPMI-8226
Non-small cell lung: HOP-92
Non-small cell lung: NCI-H460
Non-small cell lung: NCI-H522
Colon: COLO205
ꢀ5.55
ꢀ5.56
ꢀ5.63
ꢀ5.66
ꢀ5.55
ꢀ5.75
ꢀ5.76
ꢀ5.75
ꢀ5.62
ꢀ5.61
ꢀ5.66
ꢀ5.53
ꢀ5.77
ꢀ5.76
ꢀ5.53
ꢀ5.57
ꢀ5.68
ꢀ5.60
ꢀ5.67
ꢀ5.64
ꢀ5.65
ꢀ5.51
ꢀ5.00
ꢀ5.06
ꢀ5.00
ꢀ5.20
ꢀ5.47
ꢀ5.35
Colon: HCC2998
Colon: HCT-116
Melanoma: SK-MEL-2
Melanoma: SK-MEL-5
Melanoma: UACC-257
Ovarian: IGROV1
ꢀ5.18
ꢀ5.35
ꢀ5.08
Ovarian: SK-OV-3
ꢀ5.26
ꢀ5.07
ꢀ5.10
Renal: A498
Renal: RXF 393
Prostate: PC-3
Breast: MCF7
ꢀ5.02
ꢀ5.12
ꢀ5.02
ꢀ5.05
Breast: NCI/ADR-RES
Breast: MDA-MB-231/ATCC
Breast: T-47D
37
43
Non-small cell lung: NCI-H522
Ovarian:OVCAR-5
Renal: A498
ꢀ5.89
ꢀ5.60
ꢀ5.78
ꢀ5.56
ꢀ5.93
ꢀ5.84
ꢀ5.80
ꢀ5.76
ꢀ5.66
ꢀ5.14
ꢀ5.08
Renal: CAKI-1
Renal: RXF 393
Renal: UO-31
ꢀ5.08
Leukemia: CCRF-CEM
Leukemia: HL-60(TB)
Non-small cell
ꢀ5.40
lung: H23
Non-small cell lung:
NCI-H522
ꢀ5.79
(continued on next page)

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V. Onnis et al. / Bioorg. Med. Chem. 16 (2008) 2367–2378
Table 3 (continued)
Compound
Most sensitive tumor cell lines
logGI50 (M)
logTGI (M)
logLC50 (M)
Selectivity toward
tumor cell lines (D)
for logGI50/TGI (M).
The value is
Mean value
for all tested cell
lines (MG_MID)
for logGI50 (M)
shown if D > 1b,c
43
Colon: HCC2998
Colon: KM12
ꢀ5.72
ꢀ5.81
ꢀ6.02
ꢀ5.62
ꢀ5.73
ꢀ5.62
ꢀ5.61
ꢀ5.25
Ovarian: IGROV1
Ovarian: OVCAR-3
Prostate: PC-3
Breast: MCF7
Breast: T-47D
44
Leukemia: CCRF-CEM
Leukemia: HL-60(TB)
Leukemia: K-562
<ꢀ8.00
ꢀ7.64
ꢀ6.17
<ꢀ8.00
ꢀ6.34
ꢀ7.73
ꢀ5.56
<ꢀ8.00
ꢀ5.66
ꢀ5.64
<ꢀ8.00
ꢀ5.60
ꢀ5.97
ꢀ6.14
ꢀ5.93
ꢀ6.02
ꢀ6.39
ꢀ5.76
ꢀ5.69
ꢀ5.63
ꢀ5.75
ꢀ5.62
ꢀ5.92
ꢀ5.78
ꢀ5.67
2.46
ꢀ5.54
ꢀ5.79
ꢀ7.38
ꢀ6.52
ꢀ5.78
ꢀ5.14
2.10/1.01
Leukemia: MOLT-4
Leukemia: RPMI-8226
Leukemia: SR
2.46/2.60
2.19/1.74
2.46
Non-small cell lung: EKVX
Non-small cell lung: HOP-92
Non-small cell lung: H23
Non-small cell lung: NCI-H522
Colon: HCC2998
ꢀ5.13
ꢀ5.27
2.46
Colon: HCT-116
CNS: SNB-75
Melanoma: LOX IMVI
Melanoma: SK-MEL-5
Melanoma: UACC-62
Ovarian: IGROV1
Ovarian: OVCAR-4
Ovarian: SK-OV-3
Renal: A498
ꢀ5.62
ꢀ5.00
Renal: ACHN
Renal: CAKI-1
Prostate: PC-3
ꢀ5.27
ꢀ5.05
ꢀ5.02
Breast: MDA-MB-231/ATCC
Breast: T-47D
47
Leukemia: HL-60(TB)
Leukemia: K-562
ꢀ5.62
ꢀ5.62
ꢀ5.81
ꢀ5.68
ꢀ5.77
ꢀ5.63
ꢀ5.62
ꢀ5.78
ꢀ5.27
ꢀ5.34
Leukemia: MOLT-4
Leukemia: RPMI-8226
Leukemia: SR
ꢀ5.31
ꢀ5.31
ꢀ5.19
CNS: SF-295
Breast: MCF7
Breast: MDA-MB-231/ATCC
ꢀ5.17
52
53
CNS: SNB-75
ꢀ5.85
1.06
1.07
ꢀ4.79
ꢀ4.23
Ovarian: IGROV1
ꢀ5.86
CNS: SNB-75
ꢀ5.95
ꢀ5.75
1.72
1.52
Ovarian: IGROV1
54
56
Leukemia: SR
ꢀ5.56
<ꢀ8.00
ꢀ6.88
ꢀ5.82
1.34
ꢀ4.17
ꢀ5.01
CNS: SNB-75
2.99
1.87
Ovarian: IGROV1
Renal: CAKI-1
58
Non-small cell lung: HOP-92
Melanoma: SK-MEL-5
Ovarian: IGROV1
Renal: CAKI-1
ꢀ5.83
ꢀ6.02
ꢀ7.29
ꢀ5.86
ꢀ5.77
ꢀ5.10
ꢀ5.22
2.07
Renal: UO-31
a Data obtained from the NCI’s in vitro disease-oriented human tumor cells screen (see Refs. 25–27 for details).
b The reported data represent the logarithmic difference between the parametric value referred to the most sensitive cell line and the same mean
parameter, D is considered low if <1, moderate >1 and <3, high if >3.
c The value is shown if D > 1.
Amide 37 totally inhibited the growth of lung cancer
NCI-H522 cell line (logGI50 ꢀ5.89, logTGI ꢀ5.14)
and renal cancer UO-31 cell line (logGI50 ꢀ5.84, logT-
GI ꢀ5.08). The replacement of C-3 methoxy with a C-3
methyl (amides 33 and 34) and C-3 chlorine (amides 45
and 46) gave inactive compounds. We observed the

V. Onnis et al. / Bioorg. Med. Chem. 16 (2008) 2367–2378
2373
opposite effect by substitution of C-3 methoxy with a C-
3 trifluoromethyl (amides 35 and 36, GI50MG_MID
ꢀ5.5) that caused an increase in activity with respect
to amide 37 but a decrease in activity with respect to
amides 43 and 44. Activity intermediate between the
activities of 44 and 37 was observed with 47, bearing a
3-chloro-4-methoxyphenyl, that inhibited the growth
of 55 cell lines at micromolar concentrations (GI50MG_-
MID ꢀ5.34). As a general trend, esters and amides bear-
ing the 2-(5-chloro-2-methylphenylamino) substituent
on pyridine ring are significantly more potent than the
isomeric 2-(4-chloro-2-methylphenylamino) analogs.
showed correlation with the inhibitor of protein synthe-
sis anguidin, while using those of compounds 56 and 44
a correlation was found with proapoptotic agents such
as didemnin (caspase activator), triciribine (inhibitor
of the Akt signaling pathway), and L-buthionine sulfox-
imine (irreversible inhibitor of c-glutamylcysteine
synthetase).
All in all COMPARE analysis indicates that the tested
compounds might exert their antiproliferative activity
through alteration and/or inhibition of processes crucial
for the cell cycle progression.
A COMPARE28 analysis was performed with the more
active compounds to investigate whether they resemble
anticancer drugs of the NCI standard agent database
and to probably predict its mechanism of action. The
COMPARE algorithm was developed to determine the
degree of similarity of mean graph fingerprints obtained
from the in vitro anticancer screen with patterns of
activity of standard agents. The hypothesis is that, if
the data pattern of a compound correlates well with
the data pattern of compounds belonging to the stan-
dard agent database, the compound of interest may have
the same mechanism of action as those agents with
known mechanism. A Pearson’s correlation coefficient
(PCC) of 0.55–0.6 is considered the lowest correlation
that suggests a relationship with another compound.27
When tested as seeds against the NCI ‘Standard Agents’
Database (Table 4), compounds 37, 43, 52, and 56
showed a response pattern that correlated their activity
to those of DNA antimetabolite agents, including
6-methoxyguanosine and 2a-deoxy-6-thioguanosine
(PCC > 0.60 at GI50 level).
In summary, a series of ester and amide derivatives of 2-
arylamino-6-trifluoromethyl-3-pyridinecarboxylic acids
have been successfully synthesized in good yield. They
were screened for their activity against a panel of about
60 human tumor cell lines. Most of them possess encour-
aging anticancer activity having GI50 values in the low
micromolar to nanomolar concentration range. The cell
lines derived from leukemia, renal, lung, and ovarian
cancers appeared to be the most sensitive to the new
agents herein described.
3. Experimental
3.1. Chemistry
Melting points were determined on a Stuart Scientific
Melting point SMP1 and are uncorrected. Proton
NMR spectra were recorded on a Varian Unity 300
spectrometer. The chemical shift are reported in parts
per million (d, ppm) downfield from tetramethylsilane
(TMS), which was used as internal standard. Infrared
spectra were obtained with a Bruker Vector 22 spectro-
photometer. Elemental analyses were carried out with a
Carlo Erba model 1106 Elemental Analyzer and the val-
ues found were within 0.4% of theoretical values. The
compounds 1,29 6, 7, 9–11, 14,24 12,30 and 1,1,1-tri-
COMPARE analysis also indicates that compounds 43,
44, 52, and 47 shared a response pattern with topoiso-
merase II inhibitors, including menogaril, chloroquinox-
aline sulfonamide, and dactinomycin. Using GI50 values
of compounds 26 and 43 as seed, COMPARE analysis
Table 4. COMPARE correlation coefficients (PCC) using GI50 values of compounds 9, 26, 31, 37, 43, 44, 47, 52, 56, and 58 as seeds, tested in the US
NCI 60 Cell lines in vitro screen
Compound
Standard agent
Endpoint level
PCC
No. of common cell lines
9
Rifamicin
GI50
GI50
GI50
GI50
0.656
0.855
0.589
0.645
52
45
27
42
26
31
37
Anguidin
Flavone acetic acid
6-Methoxyguanine
43
Anguidin
GI50
GI50
GI50
0.491
0.810
0.780
57
11
11
Chloroquinoxaline sulfonamide
6-Methoxyguanine
44
Dihydrolenperone
Dactinomycin
GI50
TGI
TGI
GI50
TGI
GI50
0.730
0.811
0.761
0.631
0.731
0.578
23
19
23
23
23
21
Triciribine
L-Buthionine sulfoximine
L-Buthionine sulfoximine
Menogaril
47
52
a-20-Deoxythioguanosine
Chloroquinoxaline sulfonamide
GI50
GI50
0.610
0.930
25
11
56
58
a-20-Deoxythioguanosine
Didemnin B
GI50
GI50
GI50
0.810
0.665
0.616
50
59
37
Chloroquinoxaline sulfonamide

2374
V. Onnis et al. / Bioorg. Med. Chem. 16 (2008) 2367–2378
fluoro-4-iso-butoxy-3-buten-2-one31 were obtained with
previously described procedures.
3.1.3.2. 2-Chlorophenyl 2-(5-chloro-2-methylphenyla-
mino)-6-(trifluoromethyl)nicotinate (16). Yield 94%. Mp
159–160 ꢁC (cyclohexane). 1H NMR (CDCl3): d 2.31
(s, 3H, CH3), 7.04–8.72 (m, 9H, aryl and pyridyl), 9.92
(s, 1H, NH). IR (Nujol) 3313, 1712, 1667, 1618,
1591 cmꢀ1. Anal. Calcd for C20H13Cl2F3N2O2: C,
54.44; H, 2.97; N, 6.35. Found: C, 54.48; H, 2.99; N,
6.33.
3.1.1. 2-((4-Chloro-2-methylphenyl)amino)-6-trifluorom-
ethylpyridine-3-carboxamide (8). Pyridine-3-carbonitrile
6 (3.11 g, 10 mmol) was added 20% aqueous sodium
hydroxide solution (10 mL). The resulting mixture was
heated in boiling water bath for 4 h. After cooling the
reaction mixture was diluted with water (50 mL) and
the formed precipitate was separated by filtration, dried,
and crystallized from toluene. Yield 85%. Mp 234–
235 ꢁC. 1H NMR (DMSO-d6): d 2.28 (s, 3H, CH3),
7.13–8.45 (m, 7H, aryl and NH2), 10.43 (s, 1H, NH).
3.1.3.3. 3-Chlorophenyl 2-(4-chloro-2-methylphenyla-
mino)-6-(trifluoromethyl)nicotinate (17). Yield 86%. Mp
177–180 ꢁC (cyclohexane). 1H NMR (CDCl3): d 2.29
(s, 3H, CH3), 7.11–7.40 (m, 7H, aryl), 8.22 (d, 1H,
J = 6.7 Hz, pyridyl), 8.59 (d, 1H, J = 6.7 Hz, pyridyl),
9.93 (s, 1H, NH). IR (Nujol) 3340, 3310, 1712, 1678,
1618, 1590 cmꢀ1. Anal. Calcd for C20H13Cl2F3N2O2:
C, 54.44; H, 2.97; N, 6.35. Found: C, 54.41; H, 2.98;
N, 6.37.
IR (Nujol) 3488, 3165, 3124, 1663, 1619, 1592 cmꢀ1
.
Anal. Calcd for C14H11ClF3N3O: C, 51.00; H, 3.36; N,
12.74. Found: C, 50.97; H, 3.35; N, 12.77.
3.1.2. Ethyl 2-((4-chloro-2-methylphenyl)amino)-6-trifluo-
romethylpyridine-3-carboxylate (13). 4-Chloro-2-methy-
laniline (1.41 g, 10 mmol) was added to a solution of
ethyl 3-amino-3-ethoxypropenoate 12 (1.60 g, 10 mmol)
in anhydrous acetonitrile (20 mL). The resulting solution
was kept at room temperature for 6 days and then 1,1,1-
trifluoro-4-iso-butoxy-3-buten-2-one (1.96 g, 10 mmol)
was added. The resulting mixture was stirred at room tem-
perature for 0.5 h and then refluxed for 3 h. Then solvent
was removed to dryness andthe resulting residue wastrea-
ted with isopropyl ether, separated by filtration, and crys-
3.1.3.4. 3-Chlorophenyl 2-(5-chloro-2-methylphenyla-
mino)-6-(trifluoromethyl)nicotinate (18). Yield 94%. Mp
179–180 ꢁC (cyclohexane). 1H NMR (CDCl3): d 2.33
(s, 3H, CH3), 7.15–7.44 (m, 7H, aryl), 8.26 (d, 1H,
J = 6.7 Hz, pyridyl), 8.63 (d, 1H, J = 6.7 Hz, pyridyl),
9.97 (s, 1H, NH). IR (Nujol) 3341, 3306, 1713, 1619,
1590 cmꢀ1. Anal. Calcd for C20H13Cl2F3N2O2: C,
54.44; H, 2.97; N, 6.35. Found: C, 54.49; H, 2.99; N,
6.32.
1
tallized from n-hexane. Yield 80%. Mp 99–110 ꢁC. H
3.1.3.5. 4-Chlorophenyl 2-(4-chloro-2-methylphenyla-
mino)-6-(trifluoromethyl)nicotinate (19). Yield 97%. Mp
NMR (DMSO-d6): d 1.30 (t, J = 7.0 Hz, 3H, CH3), 2.23
(s, 3H, CH3), 4.33 (q, J = 7.0 Hz, 2H, CH2), 7.26 (m,
3H, aryl), 8.06 (d, 1H, J = 8.8 Hz, pyridyl), 8.46 (d, 1H,
J = 8.8 Hz, pyridyl), 10.04 (s, 1H, NH). IR (Nujol)
1
164–165 ꢁC (2-propanol). H NMR (DMSO-d6): d 2.19
(s, 3H, CH3), 7.22–7.52 (m, 7H, aryl), 8.20 (d, 1H,
J = 8.1 Hz, pyridyl), 8.70 (d, 1H, J = 8.1 Hz, pyridyl),
9.77 (s, 1H, NH). IR (Nujol) 3343, 3301, 1717, 1620,
1590 cmꢀ1. Anal. Calcd for C20H13Cl2F3N2O2: C,
54.44; H, 2.97; N, 6.35. Found: C, 54.49; H, 2.96; N,
6.31.
3280,
1696,
1620 cmꢀ1
.
Anal.
Calcd
for
C16H14ClF3N2O2: C, 53.57; H, 3.93; N, 7.81. Found: C,
53.61; H, 3.94; N, 7.78.
3.1.3. General procedure for the synthesis of esters
(15–32). A mixture of acids 10 or 11 (0.33 g, 1 mmol),
EDCI (1.92 g, 1.1 mmol), and HOBt (0.13 g, 1 mmol)
in dry MeCN (10 mL) was stirred at room temperature
for 30 min and then treated with the appropriate phenol
(1 mmol). The mixture was stirred at room temperature
for an additional 24 h. Then the solution was evapo-
rated to dryness in vacuo. The residue was dissolved in
ethyl acetate (20 mL) and washed with brine (2·
5 mL), 5% aqueous sodium hydroxide (2· 5 mL), and
water (2· 5 mL). The organic layer was dried over anhy-
drous magnesium sulfate. Concentration of the dried ex-
tract yielded a residue which was triturated with
isopropyl ether. The formed precipitate was filtered off
and purified by crystallization from the adequate solvent
to give the ester derivatives 15–32.
3.1.3.6. 4-Chlorophenyl 2-(5-chloro-2-methylphenyla-
mino)-6-(trifluoromethyl)nicotinate (20). Yield 91%. Mp
168–170 ꢁC (cyclohexane). 1H NMR (CDCl3): d 2.31
(s, 3H, CH3), 7.04–7.19 (m, 7H, aryl), 7.87 (d, 1H,
J = 6.1 Hz, pyridyl), 8.08 (d, 1H, J = 6.1 Hz, pyridyl),
8.68 (s, 1H, NH). IR (Nujol) 3550, 3306, 1667, 1614,
1597 cmꢀ1. Anal. Calcd for C20H13Cl2F3N2O2: C,
54.44; H, 2.97; N, 6.35. Found: C, 54.39; H, 2.98; N,
6.38.
3.1.3.7. 2,4-Dichlorophenyl 2-(4-chloro-2-methylphe-
nylamino)-6-(trifluoromethyl)nicotinate (21). Yield 73%.
1
Mp 184–185 ꢁC (2-propanol). H NMR (DMSO-d6): d
2.20 (s, 3H, CH3), 7.25–7.57 (m, 6H, aryl), 8.01 (d,
1H, J = 6.4 Hz, pyridyl), 8.76 (d, 1H, J = 6.4 Hz, pyri-
dyl), 9.70 (s, 1H, NH). IR (Nujol) 3348, 3310, 1720,
1620, 1591 cmꢀ1. Anal. Calcd for C20H12Cl3F3N2O2:
C, 50.50; H, 2.54; N, 5.89. Found: C, 50.43; H, 2.55;
N, 5.94.
3.1.3.1. 2-Chlorophenyl 2-(4-chloro-2-methylphenyla-
mino)-6-(trifluoromethyl)nicotinate (15). Yield 86%. Mp
1
194–195 ꢁC (2-propanol). H NMR (DMSO-d6): d 2.20
(s, 3H, CH3), 7.24–7.65 (m, 7H, aryl), 8.03 (d, 1H,
J = 8.8 Hz, pyridyl), 8.77 (d, 1H, J = 8.8 Hz, pyridyl),
9.74 (s, 1H, NH). IR (Nujol) 3344, 3311, 1712, 1605,
1590 cmꢀ1. Anal. Calcd for C20H13Cl2F3N2O2: C,
54.44; H, 2.97; N, 6.35. Found: C, 54.37; H, 2.98; N, 6.37.
3.1.3.8. 2,4-Dichlorophenyl 2-(5-chloro-2-methylphe-
nylamino)-6-(trifluoromethyl)nicotinate (22). Yield 82%.
Mp 164–165 ꢁC (cyclohexane). 1H NMR (CDCl3): d
2.31 (s, 3H, CH3), 7.04–7.54 (m, 6H, aryl), 8.25 (d, 1H,

V. Onnis et al. / Bioorg. Med. Chem. 16 (2008) 2367–2378
2375
J = 6.3 Hz, pyridyl), 8.68 (d, 1H, J = 6.3 Hz, pyridyl),
9.87 (s, 1H, NH). IR (Nujol) 3301, 1720, 1666, 1620,
1592 cmꢀ1. Anal. Calcd for C20H12Cl3F3N2O2: C, 50.50;
H, 2.54; N, 5.89. Found: C, 50.54; H, 2.55; N, 5.86.
3.1.3.15. 3,5-Dimethoxyphenyl 2-(4-chloro-2-methyl-
phenylamino)-6-(trifluoromethyl)nicotinate (29). Yield
87%. Mp 184–185 ꢁC (2-propanol). H NMR (DMSO-
d6): d 2.19 (s, 3H, CH3), 3.72 (s, 6H, OCH3), 6.44 (s,
1H, aryl), 6.53 (s, 2H, aryl), 7.24–7.36 (m, 3H, aryl),
8.07 (d, 1H, J = 8.1 Hz, pyridyl), 8.68 (d, 1H,
J = 8.1 Hz, pyridyl), 9.86 (s, 1H, NH). IR (Nujol)
3336, 3295, 3223, 3148, 3114, 1703, 1617 cmꢀ1. Anal.
Calcd for C22H18ClF3N2O4: C, 56.60; H, 3.89; N, 6.00.
Found: C, 56.67; H, 3.90; N, 5.96.
1
3.1.3.9. 2,4,6-Trichlorophenyl 2-(4-chloro-2-methyl-
phenylamino)-6-(trifluoromethyl)nicotinate (23). Yield
78%. Mp 179–180 ꢁC (cyclohexane). 1H NMR (CDCl3):
d 2.30 (s, 3H, CH3), 7.04–7.46 (m, 5H, aryl), 8.26 (d, 1H,
J = 6.7 Hz, pyridyl), 8.70 (d, 1H, J = 6.7 Hz, pyridyl),
9.78 (s, 1H, NH). IR (Nujol) 3324, 1725, 1621,
1591 cmꢀ1. Anal. Calcd for C20H11Cl4F3N2O2: C,
47.09; H, 2.17; N, 5.49. Found: C, 47.03; H, 2.19; N,
5.52.
3.1.3.16. 3,5-Dimethoxyphenyl 2-(5-chloro-2-methyl-
phenylamino)-6-(trifluoromethyl)nicotinate (30). Yield
1
77%. Mp 185–186 ꢁC (2-propanol). H NMR (DMSO-
d6): d 2.22 (s, 3H, CH3), 3.71 (s, 6H, OCH3), 6.44–7.36
(m, 6H, aryl), 8.06 (d, 1H, J = 8.1 Hz, pyridyl), 8.68
(d, 1H, J = 8.1 Hz, pyridyl), 9.85 (s, 1H, NH). IR (Nu-
jol) 3334, 3295, 1704, 1619 cmꢀ1. Anal. Calcd for
C22H18ClF3N2O4: C, 56.60; H, 3.89; N, 6.00. Found:
C, 56.54; H, 3.88; N, 6.04.
3.1.3.10. 2,4,6-Trichlorophenyl 2-(5-chloro-2-methyl-
phenylamino)-6-(trifluoromethyl)nicotinate (24). Yield
84%. Mp 180–182 ꢁC (cyclohexane). H NMR (CDCl3)
d 2.32 (s, 3H, CH3), 7.16–8.73 (m, 7H, aryl and pyridyl),
9.80 (s, 1H, NH). IR (Nujol) 3322, 3301, 1725, 1621,
1592 cmꢀ1. Anal. Calcd for C20H11Cl4F3N2O2: C,
47.09; H, 2.17; N, 5.49. Found: C, 47.15; H, 2.18; N,
5.46.
1
3.1.3.17. 3-Pyridyl 2-(4-chloro-2-methylphenylamino)-
6-(trifluoromethyl)nicotinate (31). Yield 88%. Mp 194–
1
195 ꢁC (2-propanol). H NMR (DMSO-d6): d 2.22 (s,
3.1.3.11. 3-Methoxyphenyl 2-(4-chloro-2-methylphe-
nylamino)-6-(trifluoromethyl)nicotinate (25). Yield 87%.
3H, CH3), 7.24–8.75 (m, 9H, aryl and pyridyl), 9.77 (s,
1H, NH). IR (Nujol) 3348, 3305, 1720, 1624,
1
Mp 184–185 ꢁC (2-propanol). H NMR (DMSO-d6): d
1592 cmꢀ1
. Anal. Calcd for C19H13ClF3N3O2: C,
2.23 (s, 3H, CH3), 3.55 (s, 3H, OCH3), 7.20–7.66 (m,
7H, aryl), 8.16 (d, 1H, J = 6.9 Hz, pyridyl), 8.41 (d,
1H, J = 6.9 Hz, pyridyl), 10.57 (s, 1H, NH). IR (Nujol)
3344, 1748, 1705, 1621, 1591 cmꢀ1. Anal. Calcd for
C21H16ClF3N2O3: C, 57.74; H, 3.69; N, 6.41. Found:
C, 57.79; H, 3.70; N, 6.38.
55.96; H, 3.21; N, 10.30. Found: C, 55.91; H, 3.22; N,
10.34.
3.1.3.18. 3-Pyridyl 2-(5-chloro-2-methylphenylamino)-
6-(trifluoromethyl)nicotinate (32). Yield 86%. Mp 184–
1
185 ꢁC (2-propanol). H NMR (DMSO-d6): d 2.21 (s,
3H, CH3), 7.25–8.76 (m, 9H, aryl and pyridyl), 9.80
(s, 1H, NH). IR (Nujol) 3345, 3304, 1720, 1668,
1624, 1592 cmꢀ1. Anal. Calcd for C19H13ClF3N3O2:
C, 55.96; H, 3.21; N, 10.30. Found: C, 56.01; H,
3.20; N, 10.27.
3.1.3.12. 3-Methoxyphenyl 2-(5-chloro-2-methylphe-
nylamino)-6-(trifluoromethyl)nicotinate (26). Yield 88%.
Mp 164–165 ꢁC (cyclohexane). 1H NMR (CDCl3): d
2.32 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 6.76–7.40 (m,
7H, aryl), 8.25 (d, 1H, J = 6.3 Hz, pyridyl), 8.65 (d,
1H, J = 6.3 Hz, pyridyl), 10.03 (s, 1H, NH). IR (Nujol)
3337, 3309, 1705, 1619, 1590 cmꢀ1. Anal. Calcd for
C21H16ClF3N2O3: C, 57.74; H, 3.69; N, 6.41. Found:
C, 57.80; H, 3.70; N, 6.38.
3.1.4. General procedure for the synthesis of amides (33–
58). A mixture of acids 10 or 11 (0.33 g, 1 mmol),
EDCI (1.92 g, 1.1 mmol), and HOBt (0.13 g, 1 mmol)
in dry MeCN (10 mL) was stirred at room tempera-
ture for 30 min and then treated with the appropriate
amine (1 mmol). The mixture was stirred at room tem-
perature for an additional 24 h. Then the solution was
evaporated to dryness in vacuo. The residue was dis-
solved in ethyl acetate (20 mL) and washed sequen-
tially with brine (2· 5 mL), 10% aqueous sodium
carbonate (2· 5 mL), 10% aqueous citric acid (2·
5 mL), and water (2· 5 mL). The organic layer was
dried over anhydrous magnesium sulfate. Concentra-
tion of the dried extracts yielded a solid residue which
was washed with ethyl ether, filtered off, and dried to
give the amide derivatives in analytically pure form
without additional purification by crystallization if
not elsewhere indicated.
3.1.3.13. 4-Methoxyphenyl 2-(4-chloro-2-methylphe-
nylamino)-6-(trifluoromethyl)nicotinate (27). Yield 80%.
1
Mp 169–170 ꢁC (2-propanol). H NMR (DMSO-d6): d
2.22 (s, 3H, CH3), 3.74 (s, 3H, OCH3), 6.97–7.36 (m,
7H, aryl), 8.10 (d, 1H, J = 8.1 Hz, pyridyl), 8.71 (d,
1H, J = 8.1 Hz, pyridyl), 9.91 (s, 1H, NH). IR (Nujol)
3336, 3299, 1713, 1619, 1590 cmꢀ1. Anal. Calcd for
C21H16ClF3N2O3: C, 57.74; H, 3.69; N, 6.41. Found:
C, 57.69; H, 3.68; N, 6.42.
3.1.3.14. 4-Methoxyphenyl 2-(5-chloro-2-methylphe-
nylamino)-6-(trifluoromethyl)nicotinate (28). Yield 92%.
1
Mp 171–173 ꢁC (2-propanol). H NMR (DMSO-d6): d
2.21 (s, 3H, CH3), 3.74 (s, 3H, OCH3), 6.97–7.35 (m,
7H, aryl), 8.09 (d, 1H, J = 8.1 Hz, pyridyl), 8.70 (d,
1H, J = 8.1 Hz, pyridyl), 9.90 (s, 1H, NH). IR (Nujol)
3336, 3298, 1713, 1619, 1590 cmꢀ1. Anal. Calcd for
C21H16ClF3N2O3: C, 57.74; H, 3.69; N, 6.41. Found:
C, 57.79; H, 3.70; N, 6.37.
3.1.4.1. 2-(4-Chloro-2-methylphenylamino)-N-(3-meth-
ylphenyl)-6-(trifluoromethyl)nicotinamide (33). Yield
95%. Mp 179–180 ꢁC. H NMR (DMSO-d6): d 2.25 (s,
3H, CH3), 2.28 (s, 3H, CH3), 6.94–7.48 (m, 7H, aryl),
8.11 (d, 1H, J = 8.1 Hz, pyridyl), 8.43 (d, 1H,
1

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V. Onnis et al. / Bioorg. Med. Chem. 16 (2008) 2367–2378
J = 8.1 Hz, pyridyl), 10.29 (s, 1H, NH), 10.55 (s, 1H,
NH). IR (Nujol) 3293, 1639, 1609 cmꢀ1. Anal. Calcd
for C21H17ClF3N3O: C, 60.08; H, 4.08; N, 10.01. Found:
C, 60.14; H, 4.06; N, 10.04.
3.1.4.8. 2-(5-Chloro-2-methylphenylamino)-6-(trifluo-
romethyl)-N-(3,4-(dimethoxy)phenyl)nicotinamide (40).
Yield 79%. Mp 156–157 ꢁC. 1H NMR (DMSO-d6):
d 2.24 (s, 3H, CH3), 3.71 (s, 6H, OCH3), 6.94–7.35
(m, 6H, aryl), 8.12 (d, 1H, J = 8.4 Hz, pyridyl), 8.41
(d, 1H, J = 8.4 Hz, pyridyl), 10.37 (s, 1H, NH), 10.49
(s, 1H, NH). IR (Nujol) 3371, 3284, 1637, 1611,
3.1.4.2. 2-(5-Chloro-2-methylphenylamino)-N-(3-methyl-
phenyl)-6-(trifluoromethyl)nicotinamide (34). Yield 85%.
1
Mp 174–175 ꢁC (cyclohexane). H NMR (DMSO-d6): d
1598 cmꢀ1
. Anal. Calcd for C22H19ClF3N3O3: C,
2.24 (s, 3H, CH3), 2.28 (s, 3H, CH3), 6.93–7.48 (m, 7H,
aryl), 8.11 (d, 1H, J = 7.7 Hz, pyridyl), 8.42 (d, 1H,
J = 7.7 Hz, pyridyl), 10.29 (s, 1H, NH), 10.54 (s, 1H,
NH). IR (Nujol) 3294, 1639, 1610 cmꢀ1. Anal. Calcd for
C21H16ClF3N2O2: C, 59.94; H, 3.83; N, 6.66. Found: C,
60.00; H, 3.82; N, 6.70.
56.72; H, 4.11; N, 9.02. Found: C, 56.78; H, 4.12; N,
8.97.
3.1.4.9. 2-(4-Chloro-2-methylphenylamino)-6-(trifluo-
romethyl)-N-(3,5-(dimethoxy)phenyl)nicotinamide (41).
1
Yield 90%. Mp 148–150 ꢁC. H NMR (DMSO-d6): d
2.25 (s, 3H, CH3), 3.70 (s, 6H, OCH3), 6.29 (s, 1H,
aryl), 6.93 (s, 2H, aryl), 7.20–7.35 (m, 3H, aryl), 8.08
(d, 1H, J = 8.1 Hz, pyridyl), 8.40 (d, 1H, J = 8.1 Hz,
pyridyl), 10.20 (s, 1H, NH), 10.53 (s, 1H, NH). IR (Nu-
jol) 3429, 3402, 3248, 1661, 1613 cmꢀ1. Anal. Calcd for
C22H19ClF3N3O3: C, 56.72; H, 4.11; N, 9.02. Found: C,
56.66; H, 4.10; N, 9.05.
3.1.4.3. 2-(4-Chloro-2-methylphenylamino)-6-(trifluo-
romethyl)-N-(3-(trifluoromethyl)phenyl)nicotinamide (35).
1
Yield 77%. Mp 130–132 ꢁC. H NMR (DMSO-d6): d
2.24 (s, 3H, CH3), 7.06–8.46 (m, 9H, aryl and pyridyl),
10.12 (s, 1H, NH), 10.89 (s, 1H, NH). IR (Nujol) 3452,
3184, 1671, 1613, 1536 cmꢀ1
.
Anal. Calcd for
C21H14ClF6N3O: C, 53.24; H, 2.98; N, 8.87. Found: C,
53.29; H, 2.97; N, 8.83.
3.1.4.10. 2-(5-Chloro-2-methylphenylamino)-6-(trifluo-
romethyl)-N-(3,5-(dimethoxy)phenyl)nicotinamide (42).
Yield 96%. Mp 123–125 ꢁC. H NMR (DMSO-d6): d
1
3.1.4.4. 2-(5-Chloro-2-methylphenylamino)-6-(trifluo-
romethyl)-N-(3-(trifluoromethyl)phenyl)nicotinamide (36).
Yield 73%. Mp 149–150 ꢁC (cyclohexane). 1H NMR
(DMSO-d6): d 2.23 (s, 3H, CH3), 7.18–7.74 (m, 5H,
aryl), 7.95–8.07 (m, 3H, aryl and pyridyl), 8.44 (m,
1H, pyridyl), 10.12 (s, 1H, NH), 10.86 (s, 1H, NH). IR
2.25 (s, 3H, CH3), 3.70 (s, 6H, OCH3), 6.29 (s, 1H, aryl),
6.93 (s, 2H, aryl), 7.20–7.35 (m, 3H, aryl), 8.08 (d, 1H,
J = 8.1 Hz, pyridyl), 8.40 (d, 1H, J = 8.1 Hz, pyridyl),
10.20 (s, 1H, NH), 10.53 (s, 1H, NH). IR (Nujol)
3401, 3246, 1661, 1610 cmꢀ1
. Anal. Calcd for
(Nujol) 3453, 3233, 3182, 3133, 1670, 1613, 1537 cmꢀ1
.
C22H19ClF3N3O3: C, 56.72; H, 4.11; N, 9.02. Found:
C, 56.68; H, 4.10; N, 8.99.
Anal. Calcd for C21H14ClF6N3O: C, 53.24; H, 2.98; N,
8.87. Found: C, 53.20; H, 2.99; N, 8.90.
3.1.4.11. 2-(4-Chloro-2-methylphenylamino)-6-(trifluo-
romethyl)-N-(3,4,5-(trimethoxy)phenyl)nicotinamide (43).
Yield 84%. Mp 208–210 ꢁC. 1H NMR (DMSO-d6): d
2.24 (s, 3H, CH3), 3.61 (s, 3H, OCH3), 3.73 (s, 6H,
OCH3), 7.06 (m, 2H, aryl), 7.20–7.35 (m, 3H, aryl), 8.05
(d, 1H, J = 7.7 Hz, pyridyl), 8.40 (d, 1H, J = 7.7 Hz, pyri-
dyl), 10.22 (s, 1H, NH), 10.53 (s, 1H, NH). IR (Nujol)
3411, 3118, 1666, 1610, 1540 cmꢀ1. Anal. Calcd for
C23H21ClF3N3O4: C, 55.71; H, 4.27; N, 8.47. Found: C,
55.80; H, 4.28; N, 8.43.
3.1.4.5. 2-(4-Chloro-2-methylphenylamino)-6-(trifluo-
romethyl)-N-(3-(methoxy)phenyl)nicotinamide (37). Yield
73%. Mp 155–156 ꢁC (cyclohexane). 1H NMR (CDCl3):
d 2.34 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 6.74–8.29 (m,
9H, aryl and pyridyl), 10.21 (s, 1H, NH), 10.53 (s, 1H,
NH). IR (Nujol) 3362, 3273, 1642, 1613, 1595 cmꢀ1
.
Anal. Calcd for C21H17ClF3N3O2: C, 57.87; H, 3.93;
N, 9.64. Found: C, 57.82; H, 3.94; N, 9.67.
3.1.4.6. 2-(5-Chloro-2-methylphenylamino)-6-(trifluoro-
methyl)-N-(3-(methoxy)phenyl)nicotinamide (38). Yield
3.1.4.12. 2-(5-Chloro-2-methylphenylamino)-6-(trifluo-
romethyl)-N-(3,4,5-(trimethoxy)phenyl)nicotinamide (44).
Yield 91%. Mp 210–211 ꢁC. H NMR (DMSO-d6): d
2.24 (s, 3H, CH3), 3.61 (s, 3H, OCH3), 3.73 (s, 6H,
OCH3), 7.06 (m, 2H, aryl), 7.19–7.35 (m, 3H, aryl),
8.05 (d, 1H, J = 7.7 Hz, pyridyl), 8.39 (d, 1H,
J = 7.68 Hz, pyridyl), 10.24 (s, 1H, NH), 10.52 (s, 1H,
NH). IR (Nujol) 3407, 3117, 1666, 1613 cmꢀ1. Anal.
Calcd for C23H21ClF3N3O4: C, 55.71; H, 4.27; N, 8.47.
Found: C, 55.66; H, 4.26; N, 8.51.
1
1
83%. Mp 154–155 ꢁC. H NMR (DMSO-d6): d 2.25 (s,
3H, CH3), 3.72 (s, 3H, OCH3), 6.80–7.36 (m, 7H, aryl),
8.10 (d, 1H, J = 7.7 Hz, pyridyl), 8.42 (d, 1H,
J = 7.7 Hz, pyridyl), 10.25 (s, 1H, NH), 10.59 (s, 1H,
NH). IR (Nujol) 3361, 3273, 1642, 1613, 1595 cmꢀ1
.
Anal. Calcd for C21H17ClF3N3O2: C, 57.87; H, 3.93; N,
9.64. Found: C, 57.93; H, 3.92; N, 9.60.
3.1.4.7. 2-(4-Chloro-2-methylphenylamino)-6-(trifluo-
romethyl)-N-(3,4-(dimethoxy)phenyl)nicotinamide (39).
Yield 86%. Mp 174–175 ꢁC. H NMR (DMSO-d6): d
3.1.4.13. 2-(4-Chloro-2-methylphenylamino)-6-(trifluo-
romethyl)-N-(3-(chloro)phenyl)nicotinamide (45). Yield
93%. Mp 165–166 ꢁC. H NMR (DMSO-d6): d 2.24 (s,
3H, CH3), 7.17–7.82 (m, 7H, aryl), 8.07 (d, 1H,
J = 7.7 Hz, pyridyl), 8.41 (d, 1H, J = 7.7 Hz, pyridyl),
10.16 (s, 1H, NH), 10.72 (s, 1H, NH). IR (Nujol)
3451, 3345, 3289, 3190, 1639, 1613, 1588 cmꢀ1. Anal.
Calcd for C20H14Cl2F3N3O: C, 54.56; H, 3.21; N, 9.54.
Found: C, 54.63; H, 3.22; N, 9.50.
1
1
2.24 (s, 3H, CH3), 3.71 (s, 6H, OCH3), 6.91–7.35
(m, 6H, aryl), 8.11 (d, 1H, J = 8.8 Hz, pyridyl), 8.41
(d, 1H, J = 8.8 Hz, pyridyl), 10.38 (s, 1H, NH), 10.50
(s, 1H, NH). IR (Nujol) 3370, 3286, 1637, 1611,
1598 cmꢀ1
. Anal. Calcd for C22H19ClF3N3O3: C,
56.72; H, 4.11; N, 9.02. Found: C, 56.67; H, 4.10; N,
9.06.

V. Onnis et al. / Bioorg. Med. Chem. 16 (2008) 2367–2378
2377
3.1.4.14. 2-(5-Chloro-2-methylphenylamino)-6-(trifluo-
romethyl)-N-(3-(chloro)phenyl)nicotinamide (46). Yield
Calcd for C21H22ClF3N4O3: C, 53.57; H, 4.71; N,
11.90. Found: C, 53.60; H, 4.72; N, 11.93.
1
86%. Mp 179–180 ꢁC. H NMR (DMSO-d6): d 2.24 (s,
3H, CH3), 7.16–8.46 (m, 9H, aryl and pyridyl), 10.17
(s, 1H, NH), 10.71 (s, 1H, NH). IR (Nujol) 3344,
3.1.4.21. (2-(4-Chloro-2-methylphenylamino)-6-(tri-
fluoromethyl)pyridin-3-yl)(4-(3-chlorophenyl) piperazin-1-
yl)methanone (53). Yield 91%. Mp 129–130 ꢁC (cyclo-
hexane). 1H NMR (DMSO-d6): d 2.10 (s, 3H, CH3),
3.24 (m, 4H, piperazinyl), 3.59 (m, 4H, piperazinyl),
6.75–7.82 (m, 9H, aryl and pyridyl) 8.50 (s, 1H, NH).
IR (Nujol) 3356, 1614, 1593 cmꢀ1. Anal. Calcd for
C24H21Cl2F3N4O: C, 56.59; H, 4.16; N, 11.19. Found:
C, 56.63; H, 4.27; N, 11.36.
3288, 1639, 1612, 1588 cmꢀ1
.
Anal. Calcd for
C20H14Cl2F3N3O: C, 54.56; H, 3.21; N, 9.54. Found:
C, 54.51; H, 3.20; N, 9.58.
3.1.4.15. 2-(4-Chloro-2-methylphenylamino)-N-(3-chloro-
4-methoxyphenyl)-6-(trifluoromethyl)nicotinamide (47).
Yield 80%. Mp 173–175 ꢁC. 1H NMR (DMSO-d6): d
2.24 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 7.12–8.78 (m,
8H, aryl and pyridyl), 10.28 (s, 1H, NH), 10.58 (s, 1H,
NH). IR (Nujol) 3454, 3294, 3254, 1641, 1613,
3.1.4.22. 2-(5-Chloro-2-methylphenylamino)-6-(trifluo-
romethyl)pyridin-3-yl)(4-(3-chlorophenyl) piperazin-1-yl)
1594 cmꢀ1
.
Anal. Calcd for C21H16Cl2F3N3O2: C,
53.63; H, 3.43; N, 8.94. Found: C, 53.67; H, 3.44; N, 8.90.
methanone (54). Yield 83%. Mp 178–180 ꢁC. H NMR
1
(DMSO-d6): d 2.11 (s, 3H, CH3), 3.23 (m, 4H, piperazi-
nyl), 3.59 (m, 4H, piperazinyl), 6.75–7.79 (m, 9H, Ar
and pyridyl) 8.49 (s, 1H, NH). IR (Nujol) 2770, 2721,
3.1.4.16. 2-(5-Chloro-2-methylphenylamino)-N-(3-chloro-
4-methoxyphenyl)-6-(trifluoromethyl)nicotinamide (48).
2486,
1595,
1571 cmꢀ1
.
Anal.
Calcd
for
1
Yield 90%. Mp 168–170 ꢁC. H NMR (DMSO-d6): d
C24H21Cl2F3N4O: C, 56.59; H, 4.16; N, 11.19. Found:
C, 56.63; H, 4.17; N, 11.16.
2.24 (s, 3H, CH3), 3.81 (s, 3H, OCH3), 7.12–8.42 (m,
8H, aryl and pyridyl), 10.28 (s, 1H, NH), 10.59 (s, 1H,
NH). IR (Nujol) 3288, 3251, 1640, 1612, 1594 cmꢀ1
.
3.1.4.23. (2-(4-Chloro-2-methylphenylamino)-6-(tri-
fluoromethyl)pyridin-3-yl)(4-(4-fluorophenyl) piperazin-1-
yl)methanone (55). Yield 87%. Mp 169–170 ꢁC. 1H
NMR (DMSO-d6): d 2.10 (s, 3H, CH3), 3.10 (m, 4H,
piperazinyl), 3.56 (m, 4H, piperazinyl), 6.93–7.77 (m,
9H, aryl and pyridyl) 8.49 (s, 1H, NH). IR (Nujol)
Anal. Calcd for C21H16Cl2F3N3O2: C, 53.63; H, 3.43;
N, 8.94. Found: C, 53.58; H, 3.44; N, 8.97.
3.1.4.17. 2-(4-Chloro-2-methyl-phenylamino)-N-pyri-
din-3-yl-6-trifluoromethylnicotinamide (49). Yield 89%.
1
Mp 175–176 ꢁC. H NMR (DMSO-d6): d 2.25 (s, 3H,
3374, 1630, 1615, 1599 cmꢀ1
.
Anal. Calcd for
CH3) 7.08–8.83 (m, 9H, aryl and pyridyl) 10.24 (s, 1H,
NH), 10.85 (s, 1H, NH). IR (Nujol) 3370, 3299, 3251,
3191, 3134, 3082, 1672, 1623, 1592 cmꢀ1. Anal. Calcd
for C19H14ClF3N4O: C, 56.10; H, 3.47; N, 13.77. Found:
C, 56.15; H, 3.45; N, 13.81.
C24H21ClF4N4O: C, 58.48; H, 4.29; N, 11.37. Found:
C, 58.42; H, 4.27; N, 11.40.
3.1.4.24. (2-(5-Chloro-2-methyl-phenylamino)-6-(tri-
fluoromethyl)pyridin-3-yl)(4-(4-fluorophenyl) piperazin-1-
yl)methanone (56). Yield 87%. Mp 139–140 ꢁC. 1H
NMR (DMSO-d6): d 2.11 (s, 3H, CH3), 3.10 (m, 4H,
piperazinyl), 3.57 (m, 4H, piperazinyl), 6.93–7.77 (m,
9H, aryl and pyridyl) 8.48 (s, 1H, NH). IR (Nujol)
3.1.4.18. 2-(5-Chloro-2-methyl-phenylamino)-N-pyri-
din-3-yl-6-trifluoromethylnicotinamide (50). Yield 91%.
1
Mp 194–195 ꢁC. H NMR (DMSO-d6): d 2.25 (s, 3H,
CH3) 7.06–8.84 (m, 9H, aryl and pyridyl) 10.23 (s, 1H,
NH), 10.81 (s, 1H, NH). IR (Nujol) 3419, 3311, 3248,
. Anal. Calcd for
3374, 1679, 1614, 1599 cmꢀ1
.
Anal. Calcd for
C24H21ClF4N4O: C, 58.48; H, 4.29; N, 11.37. Found:
C, 58.53; H, 4.30; N, 11.33.
3188, 1672, 1623, 1593 cmꢀ1
C19H14ClF3N4O: C, 56.10; H, 3.47; N, 13.77. Found:
C, 56.16; H, 3.46; N, 13.80.
3.1.4.25. (2-(4-Chloro-2-methylphenylamino)-6-(tri-
fluoromethyl)pyridin-3-yl)(4-(4-methoxyphenyl) pipera-
zin-1-yl)methanone (57). Yield 83%. Mp 179–180 ꢁC.
1H NMR (DMSO-d6): d 2.25 (s, 3H, CH3), 3.24 (m,
4H, piperazinyl), 3.59 (s, 4H, piperazinyl), 3.63 (s, 3H,
OCH3), 6.76–8.48 (m, 9H, aryl and pyridyl) 11.17 (s,
1H, NH). IR (Nujol) 3464, 3357, 3189, 1681, 1614,
. Anal. Calcd for C25H24ClF3N4O2: C,
59.47; H, 4.79; N, 11.10. Found: C, 59.50; H, 4.80; N,
11.14.
3.1.4.19. Ethyl 4-(2-(4-chloro-2-methylphenylamino)-
6-(trifluoromethyl)nicotinoyl)piperazine-1-carboxylate (51).
1
Yield 84%. Mp 188–190 ꢁC. H NMR (DMSO-d6): d
1.13 (t, 3H, J = 6.9 Hz, CH3), 2.10 (s, 3H, CH3), 3.42
(m, 8H, piperazinyl), 4.02 (q, 2H, J = 6.9 Hz, CH2),
7.17–8.48 (m, 5H, aryl and pyridyl) 11.17 (s, 1H, NH).
IR (Nujol) 3463, 3300, 3190, 1703, 1680, 1613,
1598 cmꢀ1
1597 cmꢀ1
. Anal. Calcd for C21H22ClF3N4O3: C,
53.57; H, 4.71; N, 11.90. Found: C, 53.53; H, 4.72; N,
11.87.
3.1.4.26. 2-(5-Chloro-2-methyl-phenylamino)-6-(tri-
fluoromethyl)pyridin-3-yl)(4-(4-methoxyphenyl) pipera-
zin-1-yl)methanone (58). Yield 95%. Mp 198–200 ꢁC. 1H
NMR (DMSO-d6): d 2.24 (s, 3H, CH3), 3.24 (m, 4H, pip-
erazinyl), 3.59 (m, 4H, piperazinyl), 3.65 (s, 3H, OCH3),
6.75–7.82 (m, 9H, aryl and pyridyl) 11.18 (s, 1H, NH). IR
(Nujol) 3310, 3183, 1680, 1609, 1532 cmꢀ1. Anal. Calcd
for C25H24ClF3N4O2: C, 59.47; H, 4.79; N, 11.10. Found:
C, 59.45; H, 4.78; N, 11.16.
3.1.4.20. Ethyl 4-(2-(5-chloro-2-methylphenylamino)-
6-(trifluoromethyl)nicotinoyl)piperazine-1-carboxylate (52).
1
Yield 88%. Mp 123–125 ꢁC. H NMR (DMSO-d6): d
1.13 (t, 3H, J = 6.8 Hz, CH3), 2.10 (s, 3H, CH3), 3.40
(m, 8H, piperazinyl), 4.01 (q, 2H, J = 6.8 Hz, CH2),
7.19–7.76 (m, 5H, aryl and pyridyl) 8.45 (s, 1H, NH).
IR (Nujol) 3319, 1703, 1634, 1613, 1597 cmꢀ1. Anal.

2378
V. Onnis et al. / Bioorg. Med. Chem. 16 (2008) 2367–2378
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3.2. Determination of GI50, TGI, and LC50 values
A total of 60 human tumor cell lines, derived from nine
cancer types (leukemia, lung, colon, brain, melanoma,
ovarian, renal, prostate, and breast) formed the basis
of this test. The tumor cells were cultured in
RPMI1640 medium supplemented with 5% fetal calf
serum and 2 mM L-glutamine. The tumor cells are
inoculated over a series of standard 96-well microtiter
plates in 100 mL of medium.32,33 Density of inoculum
depends on the type of tumor cell and its growth char-
acteristics.27 These cells are then preincubated on the
microtiter plate for 24 h before adding the compounds.
These were tested in DMSO solution at five different
concentrations (10ꢀ4, 10ꢀ5, 10ꢀ6, 10ꢀ7, and 10ꢀ8 M).
After an incubation of the chemical agent for 48 h with
the tumor cell lines a sulforhodamine B (SRB) protein
assay was used to estimate cell viability or growth. The
cytotoxic effects are evaluated and the assay results and
dose–response parameters were calculated as previously
described.34
Acknowledgment
We thank the Antitumor Evaluation Branch of the Na-
tional Cancer Institute for performing biological
evaluations.
22. Girgis, A. S.; Hosni, H. M.; Barsoum, F. F. Bioorg. Med.
Chem. 2006, 14, 4466–4476.
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