Synthesis and evaluation of anticancer activity of 2-arylamino-6-trifluoromethyl-3-(hydrazonocarbonyl)pyridines

Article abstract of DOI:10.1016/j.bmc.2009.07.066

The synthesis and anticancer activity of 2-arylamino-6-trifluoromethyl-3-(hydrazonocarbonyl)pyridines is described. The new trifluoromethylpyridine derivatives were evaluated for their anticancer activity toward human cancer cell lines by the National Can

Full text of DOI:10.1016/j.bmc.2009.07.066

Bioorganic & Medicinal Chemistry 17 (2009) 6158–6165
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of anticancer activity of 2-arylamino-6-trifluoro-
methyl-3-(hydrazonocarbonyl)pyridines
*
Valentina Onnis , Maria T. Cocco, Roberta Fadda, Cenzo Congiu
Dipartimento di Tossicologia, Università degli Studi di Cagliari, via Ospedale 72, Cagliari I-09124, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and anticancer activity of 2-arylamino-6-trifluoromethyl-3-(hydrazonocarbonyl)pyridines
is described. The new trifluoromethylpyridine derivatives were evaluated for their anticancer activity
toward human cancer cell lines by the National Cancer Institute (NCI). Most of them had excellent growth
inhibition activity, having GI50 values in the low micromolar to nanomolar concentration range. The most
potent 2,6-dichlorobenzaldehydehydrazone 29 inhibited the growth of all tested cancer cell lines with
nanomolar potency, and did not show animal toxicity. Hydrazone 29 has been selected by the Biological
Evaluation Committee of NCI for testing in vivo Hollow Fiber Assay.
Received 17 June 2009
Revised 24 July 2009
Accepted 26 July 2009
Available online 30 July 2009
Keywords:
Hydrazones
Anticancer activity
SAR
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
lines.6 At the same time as thieno[2,3-d]pyrimidin-4-yl hydrazones
have been identified as cyclin-dependent kinase 4 (CDK4) inhibitors.
In previous studies, our research group has reported the
interesting anticancer activity of a new class of 2-arylamino-6-tri-
fluoromethylnicotinamides. Most compounds of the series were
found to have GI50 values in the low micromolar or submicromolar
concentration range against human cancer cell lines and reaching,
in the case of most active derivative AM-44 (Fig. 1), averaged activ-
ity value on all tested cell lines of 2.88 ꢀ 10ꢁ6 M.1
Inaddition, thesecompoundshaveantiproliferativeactivitiesandact
as cytotoxic agents with the ability to prevent cell progression.7 Fur-
thermore phenylhydrazonopyrazolone sulfonate is as a potent and
cell permeable inhibitor of the protein tyrosine phosphatase Shp2 a
positive regulator of growth factor signaling. Activating mutations
of SHP2 have been identified in about 30% of the most common pedi-
atric leukemia, juvenile myelomonocytic leukemia (JMML), and in
myelodysplastic syndrome, acute myeloid leukemia, and some solid
tumors.8 Sulfonylhydrazone substituted imidazo[1,2-a]pyridines
On the other hand several studies have been devoted to the anti-
proliferative activity of aroylhydrazone derivatives. Thus ben-
zoxazolylhydrazones derived from alpha-(N)-acylheteroaromatics
are endowed with anticancer activity in vitro against colon, ovarian,
and renal cancer cells.2 While salicylaldehyde hydrazones showed
inhibitory effects on the growth of A549 lung cancer cells.3 A number
of hydrazone have been reported to induce apoptosis. For example
aroyl hydrazones of 2-phenylindole-3-carbaldehydes were capable
to cause cell cycle arrest accompanied by apoptosis in MDA-MB
231and MCF-7 breastcancer cells.4 Furthermore thearoylhydrazone
PAC-1 directly activates procaspase-3 to caspase-3 in vitro and in-
duces apoptosis in cancerous cells isolated from primary colon
tumorsin a mannerdirectlyproportional totheconcentrationof pro-
caspase-3 inside these cells.5 A variety of hydrazones demonstrated
their antiproliferative activity through inhibition of kinases. As a re-
sult 4-amino-6-arylamino-pyrimidine-5-carbaldehyde hydrazones
were identified as potent dual ErbB-2/EGFR kinase inhibitors, result-
ingingrowthinhibitionof ErbB-2over-expressing humancancercell
are potent inhibitors of PI3K p110a enzymatic activity and of tumor
cell growth in vitro and in vivo.9 Hydrazone derivatives, such us
EPH136, have been shown to exhibit anticancer activity in vitro
and in vivo, through generation of radicals and dissipation of the
mitochondrial membrane potential.10 Related molecular mecha-
nisms responsible of anticancer activity has been reported for guan-
ylhydrazone derivatives. These were found to be inhibitors of
OMe
OMe
O
N
H
OMe
CF3
N
NH
Cl
* Corresponding author. Tel.: +39 0706758632; fax: +39 0706758612.
E-mail address: vonnis@unica.it (V. Onnis).
Figure 1. 2-(5-Chloro-2-methylphenylamino)-6-(trifluoromethyl)-N-(3,4,5-(trimeth-
oxy)phenyl)nicotinamide (AM-44).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.066

V. Onnis et al. / Bioorg. Med. Chem. 17 (2009) 6158–6165
6159
NH2
Complex III of the mitochondrial respiratory chain and were able to
induce apoptosis in the cell lines HT29 and HL60,11 in addition they
wereabletocauseoxidativestressandtointerferewithcellularener-
getics.12 Further guanylhydrazones have been reported to be coacti-
vator binding inhibitors (CBIs), which block estrogen receptor
transcription through a different mechanism than traditional antag-
onists tamoxifen and related drugs. These molecules have been pro-
posed as potential alternative therapy in breast cancer resistant to
tamoxifen.13
COOEt
COOEt
NHAr
R'
+
(i)
H2N
OEt
H2N
R
1
6 : Ar = 2-Me-4-Cl-Ph
7 : Ar = 2-Me-5-Cl-Ph
8 : Ar = 2-OMe-5-Cl-Ph
9 : Ar = 3-CF3-Ph
2 : R = 2-Me; R' = 4-Cl
3 : R = 2-Me; R' = 5-Cl
4 : R = 2-OMe; R' = 5-Cl
5 : R = 3-CF3, R' = H
(ii)
In this context we decided to investigate if the presence of an
hydrazono moiety on the 2-arylamino-6-trifluoromethylpyridine
skeleton can also give rise to classes of biologically active
compounds. Therefore, in the present work we proposed the
synthesis and evaluation for in vitro anticancer efficacy against
human cancer cell lines of a series of novel 6-trifluomethylpyri-
dines bearing the hydrazone moiety at C-3, expecting that the
incorporation of these substituents may improve the anticancer
activities of 2-arylamino-6-trifluoromethylpyridines.
COOEt
CONHNH2
(iii)
CF3
N
NHAr
CF3
N
NHAr
10 : Ar = 2-Me-4-Cl-Ph
11 : Ar = 2-Me-5-Cl-Ph
12 : Ar = 2-OMe-5-Cl-Ph
13 : Ar = 3-CF3-Ph
14 : Ar = 2-Me-4-Cl-Ph
15 : Ar = 2-Me-5-Cl-Ph
16 : Ar = 2-OMe-5-Cl-Ph
17 : Ar = 3-CF3-Ph
(iv)
O
N
N
Ar'
H
2. Results and discussion
2.1. Chemistry
CF3
N
NHAr
18-65
Scheme 1. Reagents and conditions: (i) MeCN, rt; (ii) 1,1,1-trifluoro-4-iso-butoxy-
3-buten-2-one, reflux; (iii) NH2NH2ꢂH2O, EtOH, reflux; (iv) Ar0CHO, EtOH, reflux.
The target nicotinohydrazones 18–65 (Table 1) were synthe-
sized as shown in Scheme 1. We have previously reported an easy
and convenient method for the synthesis of alkyl esters of 2-aryla-
mino-6-trifluoromethyl-3-pyridinecarboxylic acids.1 As reported
in Scheme 1, ethyl 3-amino-3-ethoxypropenoate 1 was sequen-
tially treated with the appropriate substituted arylamine 2–5, then
with 1,1,1-trifluoro-4-isobutoxy-3-buten-2-one in MeCN solution
to give pyridine derivatives 10–13. High yields of 3-hydrazinocar-
bonylpyridines 14–17 were achieved upon refluxing for 3 h an eth-
anolic solution of esters 10–13 and hydrazine hydrate.
ate aldehydes in ethanol. All the newly synthesized compounds
gave corrected analytical data. The IR and NMR spectral data are
consistent with the assigned structure. According to literature,14
the presence of single downfield resonating (8.43–8.99 ppm)
CH@N signal accounts for formation of E-isomers exclusively.
The antiproliferative properties of this first series of hydrazones
18–65 were then evaluated (see Pharmacological results, Tables 2
and 3). According to our best compounds for a biological point of
view, we decided to realize novel modulations on their structure
through introduction of seven atom linker between 2,6-dichloro-
Hydrazones 18–65 were obtained in good to excellent yield by
coupling 3-hydrazinocarbonylpyridines 14–17 with the appropri-
Table 1
2-Arylamino-6-trifluomethylpyridin-hydrazone derivatives 18–65
O
N
Ar'
N
H
CF3
N
NHAr
Compd
Ar
Ar0
Compd
Ar
Ar0
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
2-Me-4-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-4-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-5-Cl-phenyl
2-Me-5-Cl-phenyl
4-NO2-phenyl
4-N(Me)2-phenyl
4-F-phenyl
3-Pyridyl
4-Pyridyl
4-OH-3-OMe-phenyl
3-OH-phenyl
3,4,5-(OMe)3-phenyl
2-Cl-phenyl
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
2-OMe-5-Cl-phenyl
2-OMe-5-Cl-phenyl
2-OMe-5-Cl-phenyl
2-OMe-5-Cl-phenyl
2-OMe-5-Cl-phenyl
2-OMe-5-Cl-phenyl
2-OMe-5-Cl-phenyl
2-OMe-5-Cl-phenyl
2-OMe-5-Cl-phenyl
2-OMe-5-Cl-phenyl
2-OMe-5-Cl-phenyl
2-OMe-5-Cl-phenyl
3-CF3-phenyl
3-CF3-phenyl
3-CF3-phenyl
3-CF3-phenyl
3-CF3-phenyl
3-CF3-phenyl
3-CF3-phenyl
3-CF3-phenyl
3-CF3-phenyl
4-NO2-phenyl
4-N(Me)2-phenyl
4-F-phenyl
3-Pyridyl
4-Pyridyl
4-OH-3-OMe-phenyl
3-OH-phenyl
3,4,5-(OMe)3-phenyl
2-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
2,4-Cl2-phenyl
2,6-Cl2-phenyl
4-NO2-phenyl
4-N(Me)2-phenyl
4-F-phenyl
3-Pyridyl
4-Pyridyl
4-OH-3-OMe-phenyl
3-OH-phenyl
3,4,5-(OMe)3-phenyl
2-Cl-phenyl
2,4-Cl2-phenyl
2,6-Cl2-phenyl
4-NO2-phenyl
4-N(Me)2-phenyl
4-F-phenyl
3-Pyridyl
4-Pyridyl
4-OH-3-OMe-phenyl
3-OH-phenyl
3,4,5-(OMe)3-phenyl
2-Cl-phenyl
4-Cl-phenyl
2,4-Cl2-phenyl
2,6-Cl2-phenyl
3-CF3-phenyl
3-CF3-phenyl
3-CF3-phenyl
4-Cl-phenyl
2,4-Cl2-phenyl
2,6-Cl2-phenyl

6160
V. Onnis et al. / Bioorg. Med. Chem. 17 (2009) 6158–6165
Table 2
Overview of the resultsa of the anticancer screening for compounds 18–20, 23, 24, 26, 27, 29 33, 39, 41, 42, 45, 49–51, 53, 56, 58, 63, 65, 75, Chlorambucil (NSC 3088) and AM-44b
b
Compd
No. of the cell lines investigated
Number of the cell lines giving positive log GI50, log TGI and log LC50
Log of GI50 (M) Log of TGI (M)
range
Log of LC50 (M)
range
No.
range
MG_MIDc
No.
MG_MIDc
No.
19
MG_MIDc
18
19
20
23
24
26
27
29
33
39
41
42
45
49
50
51
53
56
58
63
65
75
AM-44
NSC 3088
58
56
59
58
59
60
60
60
58
57
59
56
59
58
59
59
58
55
56
53
55
59
59
60
58
56
59
58
59
60
60
60
58
57
59
56
59
57
59
57
52
55
56
53
55
59
59
60
ꢁ5.37 to ꢁ7.37
ꢁ5.23 to ꢁ6.39
ꢁ5.46 to ꢁ6.31
ꢁ5.29 to ꢁ6.38
ꢁ5.20 to ꢁ7.77
ꢁ5.73 to ꢁ8.00
ꢁ5.44 to ꢁ6.42
ꢁ5.87 to ꢁ7.90
ꢁ5.27 to ꢁ7.01
ꢁ5.46 to ꢁ6.59
ꢁ7.25 to ꢁ5.62
ꢁ4.51 to ꢁ6.01
ꢁ5.11 to ꢁ6.28
ꢁ5.29 to ꢁ 6.19
ꢁ5.05 to ꢁ7.56
ꢁ4.13 to ꢁ6.97
ꢁ4.41 to ꢁ5.98
ꢁ5.58 to ꢁ6.49
ꢁ5.36 to ꢁ6.57
ꢁ5.30 to ꢁ7.36
ꢁ5.48 to ꢁ7.35
ꢁ6.49 to ꢁ4.95
ꢁ4.97 to ꢁ8.00
ꢁ4.50 to ꢁ 6.10
ꢁ5.99
ꢁ5.77
ꢁ5.80
ꢁ5.67
ꢁ5.70
ꢁ6.13
ꢁ5.74
ꢁ6.51
ꢁ5.63
ꢁ5.83
ꢁ6.18
ꢁ5.70
ꢁ5.53
ꢁ5.70
ꢁ5.73
ꢁ5.40
ꢁ5.09
ꢁ5.98
ꢁ5.69
ꢁ5.83
ꢁ6.07
ꢁ5.56
ꢁ5.54
ꢁ5.05
54
8
ꢁ4.42 to ꢁ7.11
ꢁ4.35 to ꢁ5.33
ꢁ4.72 to ꢁ5.65
ꢁ4.67 to ꢁ5.51
ꢁ4.74 to ꢁ5.81
ꢁ6.11 to ꢁ4.75
ꢁ4.55 to ꢁ5.47
ꢁ4.82 to ꢁ6.53
ꢁ4.54 to ꢁ5.82
ꢁ4.92 to ꢁ5.97
ꢁ6.31 to ꢁ4.32
ꢁ4.20 to ꢁ5.49
ꢁ4.12 to ꢁ5.33
ꢁ4.39 to ꢁ5.64
ꢁ4.16 to ꢁ6.26
ꢁ4.16 to ꢁ6.03
ꢁ4.14 to ꢁ 5.36
ꢁ4.92 to ꢁ5.82
ꢁ4.77 to ꢁ5.76
ꢁ4.79 to ꢁ6.23
ꢁ4.03 to ꢁ6.38
ꢁ5.31 to ꢁ4.14
ꢁ4.48 to ꢁ7.23
ꢁ4.10 to ꢁ5.10
ꢁ5.33
ꢁ4.18
ꢁ5.15
ꢁ4.98
ꢁ4.96
ꢁ5.29
ꢁ5.12
ꢁ4.93
ꢁ4.90
ꢁ5.28
ꢁ5.21
ꢁ5.06
ꢁ4.10
ꢁ4.98
ꢁ4.86
ꢁ4.30
ꢁ4.13
ꢁ5.35
ꢁ5.06
ꢁ5.18
ꢁ4.99
ꢁ4.40
ꢁ4.78
ꢁ4.45
ꢁ4.00 to ꢁ5.52
ꢁ4.32
59
58
59
56
59
32
56
57
50
53
10
51
47
27
12
55
56
52
44
41
59
59
44
51
58
25
45
5
52
52
15
8
ꢁ4.03 to ꢁ5.21
ꢁ4.05 to ꢁ5.12
ꢁ4.31 to ꢁ4.98
ꢁ5.35 to ꢁ4.03
ꢁ4.18 to ꢁ5.22
ꢁ4.86 to ꢁ5.17
ꢁ4.02 to ꢁ5.14
ꢁ4.02 to ꢁ5.31
ꢁ5.52 to ꢁ4.18
ꢁ4.06 to ꢁ5.11
ꢁ4.34
ꢁ4.37
ꢁ4.46
ꢁ4.34
ꢁ4.38
ꢁ4.09
ꢁ4.37
ꢁ4.18
ꢁ4.08
28
9
9
ꢁ4.07 to ꢁ5.17
ꢁ4.14 to ꢁ5.33
ꢁ4.02 to ꢁ5.17
ꢁ4.30
ꢁ4.10
54
55
49
9
3
52
27
ꢁ4.19 to ꢁ5.29
ꢁ4.25 to ꢁ5.12
ꢁ4.11 to ꢁ5.21
ꢁ4.11 to ꢁ5.14
ꢁ4.30 to ꢁ4.07
ꢁ4.04 to ꢁ5.14
ꢁ4.01 to ꢁ 4.04
ꢁ4.70
ꢁ4.49
ꢁ4.51
ꢁ4.10
ꢁ4.01
ꢁ4.27
ꢁ4.02
a
Data obtained from the NCI’s in vitro disease-oriented human tumor cells screen (see Refs. 15–17 for details).
The response parameters: log GI50, log TGI and log LC50 are interpolated values representing the molar concentrations at which percentage growth is +50, 0 and ꢁ50,
b
respectively.
c
MG_MID = mean graph midpoint = arithmetical mean value for all tested cancer cell lines. If the indicated effect was not attainable within the used concentration interval,
the highest concentration was used for the calculation.
Table 3
The in vitro activitya and selectivity toward most sensitive tumor cell lines for compounds 18, 24, 26, 29, 33, 41, 50, 51, 63, and 65
Compd
Most sensitive tumor cell lines
Log GI50
(M)
Log TGI
(M)
Log LC50
(M)
Selectivity toward tumor cell lines
(d) for log GI50/TGI/LC50 (M).
The value is shown if d >1b,c
GI50
MG_MID
TGI
MG_MID
LC50
MG_MID
18
24
Leukemia: CCRF-CEM
Renal: CAKI-1
Renal: RXF 393
Breast: T-47D
Ovarian: OVCAR-3
Renal: A498
Non small cell lung: EKVX
Non small cell lung: HOP-92
Non small cell lung: NCI-H226
CNS: SF-295
Renal: CAKI-1
Renal: TK-10
Prostate: PC-3
Breast: MCF7
Breast: MDA-MB-231/ATCC
Breast: BT-549
Breast: T-47D
Breast: MDA-MB-468
Leukemia: SR
Leukemia: SR
Non small cell lung: HOP-62
Colon: HCT-116
Renal: 786-0
ꢁ7.37
ꢁ7.77
ꢁ6.71
ꢁ7.45
ꢁ6.72
<8.00
ꢁ6.71
ꢁ6.72
ꢁ6.87
ꢁ7.62
ꢁ7.90
ꢁ6.65
ꢁ6.64
ꢁ6.63
ꢁ6.81
ꢁ6.71
ꢁ6.65
ꢁ6.56
ꢁ7.01
ꢁ7.25
ꢁ6.60
ꢁ6.67
ꢁ6.60
ꢁ7.56
ꢁ6.97
ꢁ7.36
ꢁ7.35
ꢁ6.72
ꢁ6.72
ꢁ7.11
ꢁ5.48
ꢁ4.95
ꢁ5.67
ꢁ6.29
ꢁ5.57
ꢁ6.15
ꢁ6.27
ꢁ6.28
ꢁ6.53
ꢁ6.49
ꢁ6.10
ꢁ6.06
ꢁ6.13
ꢁ6.07
ꢁ6.17
ꢁ6.11
ꢁ6.07
GI50 1.38/ TGI 1.12
GI50 2.07
GI50 1.01
GI50 1.75
TGI 1.0
GI50 1.87
TGI 1.22
TGI 1.34
TGI 1.35
GI50 1.11/ TGI 1.60
GI50 1.39/ TGI 1.56
TGI 1.17
TGI 1.13
TGI 1.20
TGI 1.14
TGI 1.24
TGI 1.18
TGI 1.14
GI50 1.38
GI50 1.07
LC50 1.06
TGI 1.10 / LC50 1.30
TGI 1.01 / LC50 1.34
GI50 1.83
GI50 1.57/ TGI 1.73
GI50 1.53/ TGI 1.05
GI50 1.28/ TGI 1.39
TGI 1.20
ꢁ5.99
ꢁ5.70
ꢁ5.33
ꢁ4.62
ꢁ4.67
ꢁ5.08
ꢁ4.75
26
29
ꢁ6.13
ꢁ6.51
ꢁ5.29
ꢁ4.93
ꢁ4.36
33
41
ꢁ5.63
ꢁ6.18
ꢁ5.57
ꢁ6.11
ꢁ6.31
ꢁ6.22
ꢁ6.26
ꢁ6.03
ꢁ6.23
ꢁ6.38
ꢁ6.19
ꢁ6.19
ꢁ4.38
ꢁ5.24
ꢁ5.48
ꢁ5.52
ꢁ5.33
ꢁ5.21
ꢁ4.99
ꢁ4.18
50
51
63
65
Leukemia: SR
Breast: BT-549
Non small cell lung: HOP-92
Non small cell lung: HOP-92
Ovarian: OVCAR-3
Renal: A498
ꢁ5.73
ꢁ5.40
ꢁ5.83
ꢁ6.07
ꢁ4.68
ꢁ4.11
ꢁ5.03
TGI 1.20
a
Data obtained from the NCI’s in vitro disease-oriented human tumor cells screen (see Refs. 15–17 for details).
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
b
low if <1, moderate >1 and <3, high if >3.
c
The value is shown if d >1.

V. Onnis et al. / Bioorg. Med. Chem. 17 (2009) 6158–6165
6161
phenyl and trifluoromethylpyridine rings (Scheme 2), by removal
of the 6-trifluoromethyl group or replacement of pyridine ring
with a phenyl (Scheme 3), or by replacement of 2-arylamino group
with a primary amino group (Scheme 4).
Hydrazone 69 was prepared as reported in Scheme 2. Treatment
of acid 66 with ethyl glycinate hydrochloride in the presence of
1-(3-dimethylaminopropyl)-3-ethylcardodiimide
hydrochloride
(EDCI) and hydroxybenzotriazole (HOBt) in MeCN solution gave
ester 67. Following the reaction sequence described for hydrazones
18–65, high yields of 69 were obtained.
As shown in Scheme 3, the preparation of hydrazones 74 and 75
was accomplished starting from methyl esters of flufenamic and
niflumic acids, using a procedure identical to that described for
the preceding hydrazones.
The synthesis of hydrazone 78 was accomplished by refluxing
compound 1 with 1,1,1-trifluoro-4-isobutoxy-3-buten-2-one in
presence of ammonium acetate, in ethanol, followed by reaction of
ester 76with hydrazinehydrate, in ethanol, under reflux (Scheme4).
Reaction of 77 with 2,6-dichlorobenzaldehyde produces the tar-
get hydrazone 78.
O
COOH
N
H
COOEt
(i)
CF3
N
NH
CF3
N
NH
67
66
Cl
Cl
(ii)
O
O
Cl
2.2. Pharmacology
H
N
N
H
CONHNH2
N
N
H
The synthesized hydrazones were submitted to the US National
Cancer Institute (NCI; Bethesda, MD), for in vitro testing against a
panel of approximately 60 tumor cell lines, derived from nine differ-
ent cancer types: leukemia, lung, colon, CNS, melanoma, ovarian,
renal, prostate and breast. First the compounds have been evaluated
in primary anticancer assay at 10ꢁ5 M concentration. The com-
poundsshowing antiproliferativeactivity intheprimaryassay(Table
S1, Supplementary data), were then 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 encoded
by the activity pattern over all cell lines, have been published.15–17
The anticancer activity of tested compounds is given by three param-
eters for each cell line: log GI50 value (GI50 = molar concentration of
the compound that inhibits 50% net cell growth), log TGI value
(TGI = molar concentration of the compound leading to total inhibi-
tion)andlogLC50 value(LC50 = molarconcentration ofthecompound
leading to 50% net cell death). Furthermore, a mean graph midpoint
(MG_MID) is calculated for eachof the mentioned 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 compound with respect to
one or more cell lines of the screen is characterized by a high devia-
O
Cl
CF3
N
NH
CF3
N
NH
68
69
(iii)
Cl
Cl
Scheme 2. Reagents and conditions: (i) EtOOCCH2NH2ꢂHCl, EDCI, HOBt, TEA, MeCN,
rt; (ii) NH2NH2ꢂH2O, EtOH, reflux; (iii) 2,6-dichlorobenzaldehyde, EtOH, reflux.
COOMe
NH
CONHNH2
NH
i
X
X
CF3
CF3
72 : X = CH
70 : X = CH
71 : X = N
73 : X = N
(ii)
Cl
N
O
N
H
Cl
X
NH
tion (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 chlorine atoms on particular positions of benzylidene
moiety appears to favorably modulate antiproliferative activity.
Thus 2,6-dichlorobenzaldehydehydrazones 29, 41, and 65 inhib-
ited the growth of all tested cell lines with micro and submicrom-
olar GI50 values.
CF3
74 : X = CH
75 : X = N
Scheme 3. Reagents and conditions: (i) NH2NH2ꢂH2O, EtOH, reflux; (ii) 2,6-
dichlorobenzaldehyde, EtOH, reflux.
Hydrazone 29 showed the best activity, as a matter of fact it
inhibited the growth of all tested cell lines at nanomolar to micro-
molar concentrations with MG_MID value ꢁ6.51. Compound 29
displayed cytostatic activity at nanomolar concentrations against
SF-295 and CAKI-1 as well as high selectivity on the same cell lines
(log GI50 ꢁ7.62 and ꢁ7.90). In addition micromolar concentration
of 29 totally inhibited the growth of the above cell lines as well
COOEt
COOEt
(i)
NH4OAc
+
CF3
N
NH2
H2N
OEt
76
1
(ii)
Cl
as of further 7 cell lines of renal, prostate and breast panels (
D
O
CONHNH2
NH2
>1). Although less potent when compared to 29, hydrazones 41
and 65 (GI50 MG_MID ꢁ6.18 and ꢁ6.07) displayed selectivity on
particular cell lines. Compound 41 showed a log GI50 value ꢁ7.25
against SR leukemia cell line. The same compound exhibited good
selectivity at TGI and LC50 levels against HCT-116 and 786-0 cell
lines. Hydrazone 65 showed selectivity against HOP-92 at GI50
N
(iii)
N
H
Cl
N
CF3
CF3
N
NH2
77
78
Scheme 4. Reagents and conditions: (i) 1,1,1-trifluoro-4-iso-butoxy-3-buten-2-
one, EtOH, reflux; (ii) NH2NH2ꢂH2O, EtOH, reflux; (iii) 2,6-dichlorobenzaldehyde,
EtOH, reflux.
and TGI levels (log GI50 ꢁ7.35, TGI ꢁ6.19;
D 1.28 and 1.39, respec-
tively) and against OVCAR-3 and A498 at TGI level (log TGI ꢁ6.19;

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V. Onnis et al. / Bioorg. Med. Chem. 17 (2009) 6158–6165
Table 4
D
1.20). The shift of one chlorine atom from the 6-position to the 4-
COMPARE correlation coefficients (PCC) using inhibitory values of compound 29 as
seeds, tested in the US NCI 60 Cell lines in vitro screen
position on phenyl ring leads to the inactive compounds 28, 40, 52,
and 64. Substitution of the 2,6-dichlorobenzylidene moiety by a 2-
chlorobenzylidene resulted in reduction (compound 26, GI50
MG_MID value ꢁ6.13) or loss of activity (compound 62). However
a 2-chlorobenzylidene (compound 50, GI50 MG_MID ꢁ5.73) caused
an increase in activity respect to the 2,6-dichloro analog 53 (GI50
MG_MID ꢁ5.09). Compound 50 showed high selectivity against
Standard agent
Endpoint level
PCC
No. of common cell lines
Spiromustine
GI50
GI50
TGI
0.415
0.402
0.431
0.373
0,507
0.498
0.484
53
43
48
51
53
51
51
Cyanomorpholino-ADR
Piperazinedione
Cyclopentenylcytosine
Penclomedine
TGI
LC50
LC50
LC50
leukemia SR cell line (log GI50 ꢁ7.56,
D 1.83).
Timetrexate
O6-Methylguanine
A comparison of substituent effects revealed that the introduc-
tion of 4-chlorobenzylidene led to compounds 27, 39 and 63
endowed with reduced antiproliferative effect (GI50 MG_MD
ꢁ5.74, ꢁ5.83 and ꢁ5.83, respectively) as compared with 2,6-dichlo-
robenzylidene analogs 29, 41 and 65. Hydrazone 63 displayed good
selectivity against HOP-92 non small cell lung line (log GI50 value
determine the degree of similarity of mean graph fingerprints
obtained from the in vitro anticancer screen with patterns of activ-
ity 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 standard agent database, the compound of inter-
est may have the same mechanism of action as those agents with
known mechanism. A correlation coefficient of 0.55–0.6 is consid-
ered the lowest correlation that suggests a relationship with
another compound.18 Using GI50 values of hydrazone 29
(NSC744613) as seed, COMPARE analysis shown that compounds
in the database (Table 4) had a Pearson’s correlation coefficient
(PCC) <0.55. The weakly correlated compounds, showed in Table
4, are cytotoxic through diverse mechanisms of action, including
DNA alkylation, inhibition of cytidine triphoshate synthase (cyclo-
pentenylcytosine) and dihydrofolate reductase (trimetrexate). All
in all the COMPARE analysis for the representative compound 29
against the standard agent database showed poor or no correlation
indicating that mechanism of action for the novel hydrazones may
differ from that of the standard antitumor drugs. Therefore, anti-
cancer activity of the novel hydrazones may be caused by a new
and unknown mechanism. Compound 29 that showed reproduc-
ible activity in the in vitro anticancer screening as well as no
in vivo toxicity, and because it lies outside the category of ade-
quately studied classes of antitumor agents was one of the small
percentage which has been selected by the Biological Evaluation
Committee of NCI for testing in vivo Hollow Fiber Assay.19,20
In conclusion, we have synthesized a series of N0-(arylidene)-2-
(arylamino)-6-(trifluoromethyl) nicotinohydrazides. Several of
these had excellent growth inhibition activity against the cancer
cell lines tested. Furthermore the displacement of 3-amide moiety
of 2-arylamino-6-trifluoromethylnicotinamides with an hydrazono
group led to new derivatives 18–65, endowed with about 10-fold
enhanced antiproliferative activity with respect to the reference
nicotinamides. The most potent compound, 29, inhibited the
growth of all tested cancer cell lines with nanomolar potency,
and did not show animal toxicity. The potent anticancer activity
presented for the synthesized compounds, especially 26, 29, 41,
and 65, together with their easiness of synthesis makes these com-
pounds useful as a template for the design of promising anticancer
hydrazone agents.
ꢁ7.36, log TGI value ꢁ6.23,
D1.53 and 1.05, respectively). As general
trend, hydrazone derived from 4-nitro-, 4-dimethylamino- 3-hydro-
xy-, 3,4,5-trimethoxy- and 4-hydroxy-3-methoxybenzaldehydes
showed reduced antiproliferative activity respect to that derived
from 2,6-dichlorobenzaldehyde. As example, hydrazones of the 2-
(4-chloro-2-methylphenylamino) series show antiproliferative
activity decreasing approximately in the order: 29 (2,6-
dichloro) ꢃ 18 (4-nitro) > 19 (4-dimethylamino) > 24 (3-hydro-
xy) > 23
Although hydrazone 24 showed ꢁ5.70 GI50 MG_MID value it was
high selective against renal CAKI-1 cell line (log GI50 ꢁ7.77;
(4-hydroxy-3-methoxy) ꢃ 25
(3,4,5-trimethoxy).
D
2.07). Whereas the presence of 4-fluorobenzylidene versus 2,6-
dichlorobenzylidene produces variable effects: in compound 20
led to clear reduction in inhibitory activity (GI50 MG_MID ꢁ5.80
against ꢁ6.51 of 29), while in compound 56 resulted in marginal
reduction of activity (GI50 MG_MID ꢁ5.98 against ꢁ6.07 of 65).
Taking in account the 2-(5-chloro-2-methoxyphenylamino) series,
influence of benzylidene substituents on antiproliferative activity
is extremely different with respect to hydrazones of the other three
series. As matter of fact the antiproliferative activity decreased
approximately in the order: 50 (2-chloro, GI50 MG_MID
ꢁ5.73) > 42 and 41 (4-nitro and 3,4,5-trimethoxy, GI50 MG_MID
ꢁ5.70) > 51 (4-chloro, GI50 MG_MID ꢁ5.40) > 53 (2,6-dichloro, GI50
MG_MID ꢁ5.09).
Next, the influence on antiproliferative activity of connecting
patterns between 2,6-dichlorophenyl and trifluoromethylpyridine
rings was evaluated. Introduction of an extra two atom linker
between these rings led to drop in activity, as illustrated by the
homologous pair 29/69. As matter of fact compound 69 did not
show antiproliferative activity in the primary anticancer assay.
The importance of the 6-trifluoromethyl group on pyridine ring
was explored by comparing the antiproliferative activity of hydra-
zones 65 and 75. The presence of the 6-trifluoromethyl substituent
served to enhance activity by a factor of about 3 (compound 75
GI50 MG_MID ꢁ5.56 vs ꢁ6.07 of 65).
The replacement of pyridine ring of 75 with a phenyl to afford
hydrazone 74 caused a further reduction of activity (compound
74, G% 90.62). The importance of the 2-arylamino group on the tri-
fluoromethylpyridine ring is underlined by the dramatic decrease
in activity upon replacement of this group by a primary amino
group (compound 78, G% 101.6).
3. Experimental
3.1. Chemistry
Animal toxicity experiments were performed on the most active
hydrazone 29. To determine maximum tolerated dose (MTD) this
compound was intraperitoneal (ip) administrated to athymic nude
mice at the doses of 100, 200 and 400 mg/kg. The highest dose
resulted in no body weight loss or lethality, indicating that 29
was well tolerated.
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 part per million (d, ppm) downfield from tetra-
methylsilane (TMS), which was used as internal standard. Infrared
spectra were obtained with a Bruker Vector 22 spectrophotometer.
Elemental analyses were carried out with a Carlo Erba model 1106
Elemental Analyzer and the values found were within 0.4% of
A COMPARE18 analysis was performed with the most active
compound 29 to investigate whether it resembles anticancer drugs
of the NCI standard agent database and to probably predict its
mechanism of action. The COMPARE algorithm was developed to

V. Onnis et al. / Bioorg. Med. Chem. 17 (2009) 6158–6165
6163
theoretical values. The compounds 1,21 10–11,1 66,22 70,23 71,24
and 1,1,1-trifluoro-4-iso-butoxy-3-buten-2-one25 were obtained
with previously described procedures.
Ar), 7.02 (d, J = 8.8, 1H, Ar), 7.3 (d, J = 7.7, 1H, Py), 8.21 (d, J = 7.7, 1H, Py),
8.65 (d, J = 2.3, 1H, Ar), 10.21 (s, 1H, NH), 11.21 (s, 1H, NH). IR (Nujol)
3287, 1630 cmꢁ1. Anal. Calcd for C15H15ClF3N4O2: C, 52.94; H, 4.44;
N, 16.46. Found: C, 53.00; H, 4.45; N, 16.41.
3.1.1. Ethyl 2-((5-chloro-2-methoxyphenyl)amino)-6-
trifluoromethylpyridine-3-carboxylate (12)
3.1.3.4. 6-(Trifluoromethyl)-2-(3-(trifluoromethyl) phenylami-
no)nicotinohydrazide (17). Yield 81%. Mp 113–115 °C. 1H NMR
(DMSO-d6): d 4.69 (s, 2H, NH2), 7.28–7.53 (m, 3H, Ar) 7.66 (d,
J = 8.1, 1H, Py), 8.2 (d, J = 7.7, 1H, Py), 8.40 (s, 1H, Ar), 10.28 (s,
1H, NH), 10.95 (s, 1H, NH). IR (Nujol) 3321, 1643, 1598 cmꢁ1. Anal.
Calcd for C14H10F6N4O: C, 46.16; H, 2.77; N, 15.38. Found: C, 46.10;
H, 2.78; N, 15.41.
5-Chloro-2-methoxyaniline (1.58 g, 10 mmol) was added to a
solution of ethyl 3-amino-3-ethoxypropenoate 1 (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-but-
oxy-3-buten-2-one (1.96 g, 10 mmol) was added. The resulting
mixture was stirred at room temperature for 0.5 h and then re-
fluxed for 3 h. Then solvent was removed to dryness and the result-
ing residue was treated with isopropyl ether, separated by
filtration and crystallized from n-hexane. Yield 80%. Mp 149–
150 °C. 1H NMR (DMSO-d6): d 1.47 (t, J = 6.9 Hz, 3H, CH3), 4.04 (s,
3H, OCH3), 4.52 (q, J = 6.9 Hz, 2H, CH2), 7.20 (m, 2H, aryl), 7.50
(d, 1H, J = 8.1 Hz, pyridyl), 8.64 (d, 1H, J = 8.1 Hz, pyridyl), 8.84 (s,
1H, aryl), 10.92 (s, 1H, NH). IR (Nujol) 3262, 1699, 1612 cmꢁ1. Anal.
Calcd for C16H14ClF3N2O3: C, 51.28; H, 3.77; N, 7.48. Found: C,
51.32; H, 3.76; N, 7.50.
3.1.4. General procedure for the synthesis of hydrazones (18–
65, 69, 74, 75, 78)
A mixture of hydrazides 14–17, 68, 72, 73, 77 (1 mmol) and the
appropriate aldehyde (1 mmol) in EtOH (10 mL) was refluxed for
3 h. After cooling the formed precipitate was filtered off and puri-
fied by crystallization from the adequate solvent to give the hydra-
zone derivatives. The hydrazones 29, 41, 50 and 65 are described as
examples. Physical, spectral and analytical data of hydrazones 18–
40, 42–49, and 51–64 are available in the Supplementary data.
3.1.2. Ethyl 2-(-3-(trifluoromethyl)phenylamino)-6-
trifluoromethylpyridine-3-carboxylate (13)
3.1.4.1. (E)-N0-(2,6-Dichlorobenzylidene)-2-(4-chloro-2-methyl-
phenylamino)-6-(trifluoromethyl) nicotinohydrazide (29). Yield
74%. Mp 201–203 °C (EtOH). 1H NMR (DMSO-d6): d 2.20 (s, 3H,
CH3), 7.05 (m, 2H, Ar), 7.25 (m, 2H, Ar), 7.49 (m, 1H, Ar), 7.71 (m,
1H, Ar), 8.02 (d, J = 8.0, 1H, Py), 8.37 (d, J = 8.0, 1H, Py), 8.73 (s
1H, CH), 10.28 (s, 1H, NH), 12.47 (s, 1H, NH). IR (Nujol) 3206,
3266, 1644, 1614 cmꢁ1. Anal. Calcd for C21H14Cl3F3N4O: C, 50.27;
H, 2.81; N, 11.17. Found: C, 50.34; H, 2.82; N, 11.13.
3-Trifluoromethylaniline (1.61 g, 10 mmol) was added to a
solution of ethyl 3-amino-3-ethoxypropenoate 1 (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-but-
oxy-3-buten-2-one (1.96 g, 10 mmol) was added. The resulting
mixture was stirred at room temperature for 0.5 h and then re-
fluxed for 3 h. Then solvent was removed to dryness and the result-
ing residue was treated with isopropyl ether, separated by
filtration and crystallized from 2-PrOH. Yield 90%. Mp 196–
197 °C. 1H NMR (CDCl3): d 0.71 (t, J = 7.1 Hz, 3H, CH3), 3.64 (q,
J = 7.1 Hz, 2H, CH2), 6.66 (m, 1H, aryl), 6.85 (m, 3H, Aryl and pyri-
dyl), 7.08 (d, 1H, J = 8.4 Hz, pyridyl), 7.10 (m, 1H, aryl), 8.98 (s, 1H,
NH). IR (Nujol) 3349, 1734, 1720, 1646 cmꢁ1. Anal. Calcd for
C16H12F6N2O2: C, 50.80; H, 3.20; N, 7.41. Found: C, 50.86; H,
3.19; N, 7.38.
3.1.4.2. (E)-N0-(2,6-Dichlorobenzylidene)-2-(5-chloro-2-methyl-
phenylamino)-6-(trifluoromethyl) nicotinohydrazide (41). Yield
62%. Mp 191–192 °C (EtOH). 1H NMR (DMSO-d6): d 2.21 (s, 3H,
CH3), 7.24 (m, 3H, Ar), 7.42 (m, 1H, Ar), 7.49 (m, 2H, Ar), 8.03 (d,
J = 7.8 1H, Py), 8.35 (d, J = 7.8 1H, Py), 8.56 (s, 1H, CH), 10.27 (s,
1H, NH), 12.47 (s, 1H, NH). IR (Nujol) 3221, 3056, 1644, 1614,
1597 cmꢁ1. Anal. Calcd for C21H14Cl3F3N4O: C, 50.27; H, 2.81; N,
11.17. Found: C, 50.22; H, 2.80; N, 11.22.
3.1.3. General procedure for the synthesis of hydrazides (14–17,
68, 72, 73, 77)
3.1.4.3. (E)-N0-(2-Chlorobenzylidene)-2-(5-chloro-2-methoxyphen-
ylamino)-6-(trifluoromethyl) nicotinohydrazide (50). Yield 89%.
Mp 250 °C (dec). 1H NMR (DMSO-d6): d 4.05 (s, 3H, CH3), 7.18 (m,
4H, Ar), 7.59 (d, J = 6.9, 2H, Ar), 8.20 (m, 1H, Py), 8.61 (d, J = 7.7, 1H,
Py), 8.83 (s, 1H, Ar), 8.98 (s, 1H, CH), 11.13 (s, 1H, NH), 12.55 (s, 1H,
A mixture of esters 10–13, 67, 70, 71, 76 (2 mmol), and hydra-
zine monohydrate (1 mL, 20.5 mmol) in EtOH (10 mL) was refluxed
for 1.5 h. Then solvent was removed to dryness and the resulting
residue tritured with diethyl ether. The resulting precipitate was
filtered off an used without further purification.
NH). IR (Nujol) 3236, 1645, 1601 cmꢁ1
.
Anal. Calcd for
C21H15Cl2F3N4O2: C, 52.19; H, 3.13; N, 11.59. Found: C, 52.15; H,
3.12; N, 11.63.
3.1.3.1. 2-(4-Chloro-2-methylphenylamino)-6-(trifluoromethyl)-
nicotinohydrazide (14). Yield 88%. Mp 144–145 °C. 1H NMR
(DMSO-d6): d 2.26 (s, 3H, CH3), 7.18–7.26 (m, 6H, Ar, Py and NH2),
8.18 (d, J = 9.2 Hz, 1H Py), 8.21 (s, 1H, NH), 10.80 (s, 1H, NH). IR (Nu-
. Anal. Calcd for
C14H12ClF3N4O: C, 48.78; H, 3.51; N, 16.25. Found: C, 48.84; H,
3.50; N, 16.23.
3.1.4.4. (E)-N0-(2,6-Dichlorobenzylidene)-6-(trifluoromethyl)-2-
(3-(trifluoromethyl)phenylamino) nicotinohydrazide (65). Yield
68%. Mp 250 °C dec. (EtOH). 1H NMR (DMSO-d6): d 7.04 (m, 1H,
Ar), 7.31 (m, 2H, Ar), 7.65 (m, 4H, Ar), 7.96 (d, J = 8.0, 1H, Py),
8.36 (d, J = 8.0, 1H, Py), 8.57 (s, 1H, CH), 10.46 (s, 1H, NH), 12.09
(s, 1H, NH). IR (Nujol) 3359, 3192, 1645, 1606 cmꢁ1. Anal. Calcd
for C21H12Cl2F6N4O: C, 48.39; H, 2.32; N, 10.75. Found: C, 48.33;
H, 2.30; N, 10.72.
jol) 3332, 3280, 3127, 1657, 1612 cmꢁ1
3.1.3.2. 2-(5-Chloro-2-methylphenylamino)-6-(trifluoromethyl)
nicotinohydrazide (15). Yield 86%. Mp 149–150 °C. 1H NMR
(DMSO-d6): d 2.26 (s, 3H, CH3), 7.19–8.21 (m, 8H, Ar, Py, NH), 10.80
(s, 1H, NH). IR (Nujol) 3311, 1616 cmꢁ1
.
Anal. Calcd for
3.1.5. Ethyl 2-(2-(4-chloro-2-methylphenylamino)-6-(trifluoro-
methyl)nicotinamido)acetate (67)
C14H12ClF3N4O: C, 48.78; H, 3.51; N, 16.25. Found: C, 48.74; H,
3.49; N, 16.28.
A mixture of acid 66 (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 ethyl gly-
cinate hydrochloride (0.28 g, 2 mmol) and TEA (0.2 mL, 2 mmol).
The mixture was stirred at room temperature for an additional
3.1.3.3. 2-(5-Chloro-2-methoxyphenylamino)-6-(trifluoromethyl)
nicotinohydrazide (16). Yield 92%. Mp 250 °C (dec). 1H NMR (DMSO-
d6): d 3.85 (s, 3H, OCH3), 4.65 (s, 2H, NH2), 6.96 (dd, J = 8.8, 2.3 Hz, 1H,

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V. Onnis et al. / Bioorg. Med. Chem. 17 (2009) 6158–6165
2 h. Then the solution evaporated to dryness in vacuo. The residue
was dissolved in ethyl acetate (20 mL) and washed with brine
(2 ꢀ 5 mL), 10% aqueous citric acid (2 ꢀ 5 mL), satd aqueous so-
dium hydrogencarbonate (2 ꢀ 5 mL) and water (2 ꢀ 5 mL). The or-
ganic layer was dried over anhydrous magnesium sulfate.
Concentration of the dried extract yielded a residue which was tri-
tured with isopropyl ether. The formed precipitate was filtered off
and utilized in the step without further purification. Yield 87%. Mp
128–130 °C. 1H NMR (DMSO-d6): d 1.34 (t, J = 6.9, 3H, CH3), 2.38 (s,
3H, CH3), 4.12 (m, 2H, CH2), 4.18 (q, J = 6.9, 2H, CH2), 7.37 (d, J = 8.7
1H, Ar), 7.44 (s, 1H, Ar), 7.48 (d, J = 8.1 1H, Py), 8.29 (d, J = 8.7 1H,
Ar), 8.47 (d, J = 8.1 1H, Py), 9.62 (m, 1H, NH), 10.76 (s, 1H, NH).
IR (Nujol) 3431, 1723, 1656 cmꢁ1. Anal. Calcd for C18H17ClF3N3O3:
C, 52.00; H, 4.12; N, 10.11. Found: C, 52.06; H, 4.14; N, 10.07.
1604 cmꢁ1. Anal. Calcd for C21H14Cl2F3N3O: C, 55.77; H, 3.12; N,
9.29. Found: C, 55.71; H, 3.13; N, 9.25.
3.1.11. (E)-N0-(2,6-Dichlorobenzylidene)-2-(3-(trifluoromethyl)
phenylamino)nicotinohydrazide (75)
Following the general procedure for the synthesis of hydrazones
the title compound was obtained in quantitative yield. Mp 250 °C
dec. (1-PrOH). 1H NMR (DMSO-d6): d 7.07 (m, 1H, Ar), 7.43 (m,
2H, Ar), 7.64 (m, 4H, Ar), 7.98 (d, J = 8.4 Hz, 1H, Py), 8.39 (m, 1H,
Py), 8.54 (s, 1H, CH), 8.79 (m, 1H, Py), 10.75 (s, 1H, NH), 12.44 (s,
1H, NH). IR (Nujol) 3322, 3197, 3042, 1641, 1607 cmꢁ1. Anal. Calcd
for C20H13Cl2F3N4O: C, 53.00; H, 2.89; N, 12.63. Found: C, 52.96; H,
2.90; N, 12.61.
3.1.12. Ethyl 2-amino-6-(trifluoromethyl)nicotinate (76)
Ammonium acetate (2 g, 26 mmol) was added to a solution of
ethyl 3-amino-3-ethoxypropenoate 1 (0.80 g, 5 mmol) and 1,1,1-
trifluoro-4-iso-butoxy-3-buten-2-one (0.98 g, 5 mmol) in anhy-
drous EtOH (10 mL). The resulting mixture was refluxed for 3 h.
Then solvent was removed to dryness. The residue was dissolved
in diethyl ether (20 mL) and washed with water (2 ꢀ 5 mL). The or-
ganic layer was dried over anhydrous magnesium sulfate. Concen-
tration of the dried extract yielded a residue which was tritured
with isopropyl ether, separated by filtration to give quantitative
yield of the title compound. Mp 66–68 °C (n-hexane). 1H NMR
(DMSO-d6): d 1.47 (t, J = 7.0 Hz, 3H, CH3), 3.80 (q, J = 7.0 Hz, 2H,
CH2), 7.15 (d, J = 8.1, 1H, Py), 7.70 (s, 2H, NH2), 8.41 (d, J = 8.1 1H,
Py). IR (Nujol) 3440, 3299, 3233, 3185, 1707 cmꢁ1. Anal. Calcd for
C9H9F3N2O2: C, 46.16; H, 3.87; N, 11.96. Found: C, 46.20; H, 3.88;
N, 11.92.
3.1.6. 2-(2-(4-Chloro-2-methylphenylamino)-6-(trifluoromethyl)-
nicotinamido)acetohydrazide (68)
Following the general procedure for the synthesis of hydrazides
the title compound was obtained in 76% yield. Mp 218–220 °C dec.
(EtOH). 1H NMR (DMSO-d6): d 2.40 (s, 3H, CH3), 4.01 (d, J = 5.7, 2H,
CH2), 6.92 (s, 2H, NH2), 7.37 (d, J = 8.7 1H, Ar), 7.44 (s, 1H, Ar), 7.46
(d, J = 7.8 1H, Py), 8.30 (d, J = 8.7 1H, Ar), 8.48 (d, J = 7.8 1H, Py),
9.37 (m, 1H, NH), 10.85 (s, 1H, NH), 11.28 (s, 1H, NH). IR (Nujol)
3331, 1673, 1616 cmꢁ1. Anal. Calcd for C16H15ClF3N5O2: C, 47.83;
H, 3.76; N, 17.43. Found: C, 47.89; H, 3.75; N, 17.40.
3.1.7. (E)-N0-(2,6-Dichlorobenzylidene)-2-(2-(4-chloro-2-methyl-
phenylamino)-6-(trifluoromethyl)nicotinamido)acetohydrazide
(69)
Following the general procedure for the synthesis of hydrazones
the title compound was obtained in 77% yield. Mp 230–232 °C
(EtOH). 1H NMR (DMSO-d6): d 2.39 (s, 3H, CH3), 4.53 (d, J = 5.4,
2H, CH2), 7.45 (m, 5H, Ar), 7.70 (d, J = 8.1, 1H, Py), 8.31 (d, J = 8.1,
1H, Py), 8.39 (s, 1H, CH), 8.51 (d, J = 7.3, 1H, Ar), 9.43 (m, 1H,
NH), 10.84 (s, 1H, NH), 11.99 (s, 1H, NH). IR (Nujol) 3194, 3100,
3.1.13. 2-Amino-6-(trifluoromethyl)nicotinohydrazide (77)
Following the general procedure for the synthesis of hydrazides
the title compound was obtained in 87% yield. Mp 159–160 °C. (2-
PrOH). 1H NMR (DMSO-d6): d 7.14 (d, J = 7.7 Hz, 1H, Py), 7.40 (s, 4H,
NH2), 8.15 (d, J = 7.7 Hz, 1H, Py), 10.70 (s, 1H, NH). IR (Nujol) 3439,
3338, 3220, 3036, 1658, 1623 cmꢁ1. Anal. Calcd for C7H7F3N4O: C,
38.19; H, 3.20; N, 25.45. Found: C, 38.21; H, 3.22; N, 25.40.
1689, 1632, 1612 cmꢁ1
. Anal. Calcd for C23H17Cl3F3N5O2: C,
49.44; H, 3.07; N, 12.53. Found: C, 49.50; H, 3.06; N, 12.50.
3.1.8. 2-(3-(Trifluoromethyl)phenylamino) benzohydrazide (72)
Following the general procedure for the synthesis of hydrazides
the title compound was obtained in 96% yield. Mp 134–136 °C dec.
(2-PrOH). Lit.26 135–137 °C (EtOH). 1H NMR (DMSO-d6): d 7.36 (m,
1H, Ar), 7.33 (d, J = 7.3 Hz, 1H, Ar), 7.49 (m, 6H, Ar and NH2), 7.58
(d, J = 7.7 Hz, 1H, Ar), 7.70 (d, J = 7.7 Hz, 1H, Ar), 9.97 (s, 1H, NH),
12.16 (s, 1H, NH). IR (Nujol) 3324, 1636, 1584 cmꢁ1. Anal. Calcd
for C14H12F3N3O: C, 56.95; H, 4.10; N, 14.23. Found: C, 56.89; H,
4.12; N, 14.27.
3.1.14. (E)-N0-(2,6-Dichlorobenzylidene)-2-amino-6-(trifluoro-
methyl)nicotinohydrazide (78)
Following the general procedure for the synthesis of hydrazones
the title compound was obtained in 60% yield. Mp 250 °C dec. (1-
PrOH). 1H NMR (DMSO-d6): d 6.87 (br s, 2H, NH2), 7.21 (m, 1H,
Ar), 7.58 (m, 2H, Ar and Py), 7.69 (m, 1H, Ar), 8.34 (m, 1H, Py),
8.72 (s, 1H, CH), 12.34 (s, 1H, NH). IR (Nujol) 3479, 3356, 3180,
3038, 1660, 1618 cmꢁ1. Anal. Calcd for C14H9Cl2F3N4O: C, 44.58;
H, 2.41; N, 14.86. Found: C, 52.96; H, 2.90; N, 12.61.
3.1.9. 2-(3-(Trifluoromethyl)phenylamino) nicotinohydrazide (73)
Following the general procedure for the synthesis of hydrazides
the title compound was obtained in 73% yield. Mp 139–140 °C dec.
(2-PrOH). 1H NMR (DMSO-d6): d 7.07 (m, 1H, Ar), 6.24 (s, 2H, NH2),
7.39 (d, J = 8.1 Hz, 1H, Py), 7.63 (m, 1H, Ar), 7.93 (d, J = 8.1 Hz, 1H,
Py), 8.23 (m, 1H, Ar), 8.39 (d, J = 8.1 Hz, 1H, Py), 8.49 (m, 1H, Ar),
10.60 (s, 1H, NH), 10.86 (s, 1H, NH). IR (Nujol) 3316, 3204, 3029,
1630, 1602 cmꢁ1. Anal. Calcd for C13H11F3N4O: C, 52.71; H, 3.74;
N, 18.91. Found: C, 52.76; H, 3.73; N, 18.96.
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, pros-
tate and breast) formed the basis of this test. The tumor cells were
cultured in RPMI1640 medium supplemented with 5% foetal calf
serum and 2 mM L-glutamine. The tumor cells are inoculated over
a series of standard 96-well microtrite plates in 100 mL of med-
ium.27,28 Density of inoculum depends on the type of tumor cell
and from its growth characteristics.17 These cells are then preincu-
bated on the microtrite plate for 24 h before adding the com-
pounds. These were tested in DMSO solution at five different
concentrations (10ꢁ4, 10ꢁ5, 10ꢁ6, 10ꢁ7 and 10ꢁ8 M). After an incu-
bation 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
3.1.10. (E)-N0-(2,6-Dichlorobenzylidene)-2-(3-(trifluoromethyl)
phenylamino)benzohydrazide (74)
Following the general procedure for the synthesis of hydrazones
the title compound was obtained in quantitative yield. Mp 237–
239 °C. (EtOH). 1H NMR (DMSO-d6): d 7.00 (m, 2H, Ar), 7.14 (m,
1H, Ar), 7.33 (m, 7H, Ar), 7.68 (m, 1H, Ar), 8.52 (s, 1H, CH), 9.10
(s, 1H, NH), 12.10 (s, 1H, NH). IR (Nujol) 3292, 3174, 3042, 1643,

V. Onnis et al. / Bioorg. Med. Chem. 17 (2009) 6158–6165
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The determination of maximum tolerated dose (MTD) is per-
formed in a way that conserves compound and minimizes the num-
ber of animals sacrificed. Thus, a single mouse is given a single
injection (IP) of 400 mg/kg; a second mouse receives a dose of
200 mg/kg and a third mouse receives a single dose of 100 mg/kg.
The mice are observed for a period of 2 weeks. They are sacrificed
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material administered to mice during antitumor testing. The mice
are allowed ad libitum feed and water. Injections are administered
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5
l
L, 2.5
lL and 1.25 lL/g of body weight.
Acknowledgement
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We thank the Antitumor Evaluation Branch of the National Can-
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.07.066.
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