Synthesis, evaluation of anticancer activity and COMPARE analysis of N-bis(trifluoromethyl)alkyl-N'-substituted ureas with pharmacophoric moieties

Article abstract of DOI:10.1016/j.ejmech.2012.03.026

A series of new synthesized N-bis(trifluoromethyl)alkyl-N'-substituted ureas have been tested in the National Cancer Institute (NCI, Bethesda, USA) by Program NCI-60 DTP Human Tumor Cell Line Screen at a single high dose (10 ^-5 M). COMPARE analysis has been carried out for all tested compounds. The tested compounds showed antitumor activity against individual cell lines. The most sensitive cell lines relative to the tested compounds are: 5g Leukemia RPMI-8226 (GI% 52.7), Non-Small Cell Lung cancer HOP-92 (GI % 88.53), NCI-H522 (GI % 64.41), Melanoma UACC-62 (GI% 53.08), SK-MEL-5 (GI % 74.63), Breast cancer MDA-MB-468 (GI% 51.29), T-47D (GI % 65.1), 5b Leukemia K-562 (GI % 55.55), 7m Leukemia HL-60(TB) (GI % 51.76).

Full text of DOI:10.1016/j.ejmech.2012.03.026

European Journal of Medicinal Chemistry 53 (2012) 364e373
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, evaluation of anticancer activity and COMPARE analysis
of N-bis(trifluoromethyl)alkyl-N’-substituted ureas with pharmacophoric
moieties
,
b
Elena L. Luzina a , Anatoliy V. Popov
*
a Institute of Physiologically Active Compounds, Severnyi pr. 1, Chernogolovka, Moscow region, 142432, Russia
b University of Pennsylvania, Department of Radiology, 231 South 34th Street, Philadelphia, PA 19104, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new synthesized N-bis(trifluoromethyl)alkyl-N’-substituted ureas have been tested in the
National Cancer Institute (NCI, Bethesda, USA) by Program NCI-60 DTP Human Tumor Cell Line Screen at
a single high dose (10ꢀ5 M). COMPARE analysis has been carried out for all tested compounds. The tested
compounds showed antitumor activity against individual cell lines. The most sensitive cell lines relative
to the tested compounds are: 5g Leukemia RPMI-8226 (GI% 52.7), Non-Small Cell Lung cancer HOP-92
(GI % 88.53), NCI-H522 (GI % 64.41), Melanoma UACC-62 (GI% 53.08), SK-MEL-5 (GI % 74.63), Breast
cancer MDA-MB-468 (GI% 51.29), T-47D (GI % 65.1), 5b Leukemia K-562 (GI % 55.55), 7m Leukemia HL-
60(TB) (GI % 51.76).
Received 6 December 2011
Received in revised form
7 March 2012
Accepted 12 March 2012
Available online 23 March 2012
Keywords:
Fluorinated ureas
Anticancer activity
Synthesis
Ó 2012 Elsevier Masson SAS. All rights reserved.
COMPARE analysis
1. Introduction
[25e28] and other ones, are present in many drugs and biologically
active compounds or represent drugs themselves (Fig. 1).
We have previously reported the potent anticancer activity of
unsymmetrical N-bis(trifluoromethyl)alkyl-N’-thiazolyl, -N’-ben-
zothiazolyl, -N’-(1,2,4-triazolyl), and -N’-polychlorophenyl ureas
[1,2]. Some of these ureas showed an interesting antiproliferative
profile against different human tumor-derived cell lines.
The urea backbone has been chosen because ureas possess
better inhibition properties as compared with amides due to
(enzymatic) hydrolysis resistance [3e6]. This adds considerable
support in favor of urea as a core moiety of new potential anti-
cancer compounds. The best known cyclic fluorinated urea is 5-
fluorouracil (also known as 5-FU, Adrucil, Efudex, Fluoroplex, NCS
19893), an NCI standard, which has been used as an anticancer drug
for more than 40 years [7e10].
Based on these findings, we have synthesized N-bis(tri-
fluoromethyl)alkyl-N’-substituted ureas with various substituents.
Many of these substituents are fragments found in drugs or bio-
logically active compounds, such as 3,4-dimethoxyphenethyl
(fragment of Verapamil) [11e13], isoxazolyl [14,15], thiodiazolyl
[16], benzothiazolyl [17e19], sulfonamides [20e24], pyrazolone
It should be noted that these fragments are used as pharmaco-
phores in many QSAR programs [26,27,29], therefore we regard
pharmacophores as real substituents. In other words, the SAR
purpose includes the study of antiproliferative anticancer activity
for a number of polyfluoroalkylureido derivatives of the real drugs
or their (substituted) fragments.
Part of this work included modification of known drugs such as
sulfanilamide, sulfomethoxazole and ampyrone in order to inves-
tigate their biological activity. These ureas were synthesized
keeping in mind that the fragments responsible for antitumor,
antimicrobial, antioxidant, anti-inflammatory, analgesic, antipy-
retic and other activities may provide multitarget action. The fluo-
rinated portion of a molecule may be responsible for anticancer
activity [1,2]. Not by chance, 30% of known pharmaceuticals contain
fluorine [30]. We designed and synthesized a significant number of
fluorinated ureas with various substituents and, as a result, this set
of ureas with potential multitarget activity, presented in this paper,
was selected by the National Cancer Institute (NCI) (Bethesda, MD,
USA) under Development Therapeutic Program (NCI-60 DTP Human
Tumor Cell Line Screen) for screening in human cancer cell lines.
This screen is unique in that the 60 cell line dose response
produced by each compound results in a biological response pattern
which can be analyzed with pattern recognition algorithms
* Corresponding author. Tel./fax: þ7 496 524 9508.
E-mail addresses: luzina@ipac.ac.ru (E.L. Luzina), avpopov@mail.med.upenn.edu
(A.V. Popov).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.026

E.L. Luzina, A.V. Popov / European Journal of Medicinal Chemistry 53 (2012) 364e373
365
compounds most likely to interact with one (or several) specific
molecular target(s).
The single-dose data is graphed as the mean of the percent
growth of the treated cells, relative to the no-drug control, and
relative to the time zero number of cells. This allows for detection of
both growth inhibition (values between 0 and 100) and cytotoxicity
(values less than 0). The screening begins with the evaluation of all
compounds against the 60 cell lines at a single dose of 10ꢀ5 M (the
methodology is presented in SI1). The output from the single dose
screen is available for COMPARE analysis [31e33].
Compounds 5aeg, 6a, d, hel, 7meo have been evaluated by the
NCI, on all the 60 human cancer cell lines organized into subpanels
derived from nine different human cancer types: leukemia, mela-
noma, lung, colon, renal, ovarian, breast, prostate and CNS, at one
dose (10ꢀ5 M concentration) primary anticancer assay (Table 2).
Table 2 presents the data reported as a mean graph of the
percent growth of the treated cells. For comparison, we used the
NCI standard compound 5-FU [67] (see SI2) and three N-bis(tri-
fluoromethyl)alkyl-N0-benzothiazolyl ureas with various substitu-
ents in the side of fluorine (H-BZT for compounds 5 with R ¼ H, Me-
BZT for compounds 6 with R ¼ Me and Et-BZT for compounds 7
with R ¼ Et) that were previously tested [1] as the closest structural
analogs of the present ureas (Fig 1S, SI1).
Fig. 1. Examples of drugs with some fragments presented in this paper.
(COMPARE program [31e33]). Using these algorithms, it is possible
to assign a putative mechanism of action to each test compound,
and to determine whether the response pattern is similar to that of
any of the standard prototype compounds included in the NCI
database. The output from the single dose screen is reported as
a mean graph and is available for analysis by the COMPARE program.
The current communication is an extension of our publications
covering the general idea of developing new physiologically active
fluorinated compounds from perfluoroisobutene [1,2,34e40], which
is an excellent precursor of trifluoromethyl-, bis(trifluoromethyl)
alkyl-, and nonafluoro-tert-butyl-containing substances [41].
Comparison of the anticancer activity of ureas 5aeg (R ¼ H), 6a,
d, hel (R ¼ Me), and 7oem (R ¼ Et) showed that these compounds
only outperformed H-BZT, Me-BZT and Et-BZT against individual
cell lines.
In the present series, compound 5g was the most active. Urea 5g
demonstrated antitumor activity against all nine subpanels of the
standard type of NCI cancer panel. Mean Percent Growth equals
75.16 (GI ¼ 24.84%) (see SI1 Table 1S).
Despite the average activity of derivative 5g being less than that
of 5-FU,urea 5g outperformed 5-FU against several cell lines,
namely HOP-92, NCI-H522 (Non-Small Cell Lung Cancer), SF-295
(CNS), MALME-3, SK-MEL-2, SK-MEL-5 (Melanoma), T-47D (Breast
cancer) (see Table 2).
2. Results and discussion
2.1. Chemistry
Compound 5g outperformed H-BZT against all cancer cell line
subpanels, including Leukemia, Lung, Melanoma and Breast cancer
(See SI1 Table 1S). The most sensitive cell lines for urea 5g are
Leukemia RPMI-8226 (GI% 52.7 or GP% 47.30), Non-Small Cell Lung
cancer HOP-92 (GI % 88.53 or GP% 11.47) and NCI-H522 (GI % 64.41
or GP% 35.59), Melanoma UACC-62 (GI% 53.08 or GP% 46.92) and
SK-MEL-5 (GI % 74.63 or GP% 25.37), Breast cancer MDA-MB-468 (GI
% 51.29 or GP% 48.71) and T-47D (GI % 65.1 or GP% 34.90) (See
Tables 2 and 3).
Compounds 5a, 5b, 5e outperformed H-BZT (Mean GP, Tables 1,
2S, SI1) only against the Leukemia subpanel. Rather selective
anticancer activity against the individual Leukemia cell lines was
demonstrated by the following compounds: 5b (K-562 (GP% 44.45),
RPMI-8226 (GP% 57.62), 5g (CCFR-CEM (GP% 58.89) and RPMI-8226
(GP% 47.30) and 7m (HL-60(TB) (GP% 48.24) in comparison with H-
BZT and Et-BZT, respectively.
The preparation of N-bis(trifluoromethyl)alkyl-N0-substituted
ureas 5aeg, 6a, d, hel, and 7meo has been carried out by the
addition of amines 4aeo to hexafluoroalkyl isocyanates 1e3
(Scheme 1). The synthesis of isocyanates 1e3 was described in
our previous publications [1,39].
All 17 synthesized polyfluorinated ureas 5aeg, 6a, d, hel and
7meo are in agreement with the Lipinski’s rules of five [42,43] for
new potential drug candidates (MW ꢁ 500; CLogP ꢁ 5; number of
H-bond donors ꢁ 5; number of H-bond acceptors ꢁ 10; Table 1).
Additionally, all these ureas meet the requirements of the proper-
ties for prospective small molecular drugs (“the Gelovani’s rules”
[44]): molecular polar surface area (PSA) [45e56] <140 Å2; molar
refractivity (MR) [57e62] within the range of 40e130 cm3/mol; the
number of atoms in the molecule 20e70; Table 1). The parameters
in Table 1 have been calculated with the ChemBioDraw Ultra,
version 12 [63].
In particular the most sensitive cell lines for compound 5g are
Non-Small Lung cancer (HOP-92 (GP% 11.47) and NCI-H522 (GP%
35.59) (see Table 2).
2.2. Biological activity
Ureas 5a, 5d, 5f, 6d, 6i, 6h, and 7o outperformed their unsub-
stituted benzothiazolyl counterparts H-BZT, Me-BZT and Et-BZT
against individual cell lines of some subpanels of human cancer cell
lines (Tables 1S, 2S, 3S, 4S SI1). Namely, a selective antitumor
activity was shown by compounds 5a (Leukemia, Renal), 5d (Lung),
5f (Lung, CNS), 6d (Leukemia, Renal), 6i (Leukemia), 6h (Leukemia,
Melanoma, Renal, Breast cancer), and 7o (Leukemia).
Compounds 5e, 6j, 6k and 6l showed lower activity in
comparison with the other tested compounds (see Tables 2 and 3).
Ureas 5c, 6a and 7n were inactive.
2.2.1. Anticancer activity at one dose (10ꢀ5 M concentration)
All compounds were tested at a single high dose (10ꢀ5 M) in the
full NCI 60 cell panel [64e66]. Using this data assay, it is possible to
assign a putative mechanism of action to each test compound, or to
determine that the response pattern is unique and not similar to
that of any of the standard prototype compounds included in the
NCI database. In addition, following characterization of various
molecular targets in the 60 cell lines, it may be possible to select the

366
E.L. Luzina, A.V. Popov / European Journal of Medicinal Chemistry 53 (2012) 364e373
Scheme 1. Synthesis of polyfluorinated ureas 5aeg, 6a, d, hel and 7meo.
Table 1
Properties of the ureas calculated with ChemBioDraw Ultra, v. 12. The Lipinski’s and Gelovani’s parameters.
Compound
MW
CLogP
H-bond
donor
H-bond
acceptor
Lipinski
(score out of 4)
MR (cm3/mol)
Molecular Formula
(number of atoms)
PSA (Å2)
5a
5b
5c
5d
5e
5f
5g
6a
6d
6h
6i
6j
6k
6l
7m
7n
7o
374.28
288.15
295.21
357.27
371.30
362.18
354.30
388.30
371.30
391.72
360.25
379.28
460.35
410.31
402.33
404.30
319.20
2.673
2
2
2
2
2
2
2
2
2
2
2
3
3
2
2
2
2
9
9
8
8
8
12
9
9
8
8
9
9
11
9
9
4
4
4
4
4
3
4
4
4
4
4
4
3
4
4
4
4
77.58
50.52
50.79
71.21
77.11
55.87
67.44
82.12
75.75
74.46
72.1
C14H16F6N2O3 (41)
C8H6F6N4O (25)
59.59
66.85
53.49
53.49
53.49
65.85
65.85
59.59
53.49
53.49
59.59
101.29
108.89
64.68
59.59
68.82
62.72
1.4252
2.6893
4.0516
4.5506
2.2007
2.6685
2.982
4.3606
4.4857
3.0129
1.417
2.5525
0.7367
4.5999
2.8053
2.598
C7H7F6N3OS (25)
C12H9F6N3OS (32)
C13H11F6N3OS (35)
C7H3F9N4OS (25)
C8H8F6N4OS2 (29)
C15H18F6N2O3 (44)
C13H11F6N3OS (35)
C12H8ClF6N3OS (32)
C13H14F6N2O3 (38)
C11H11F6N3O3S (35)
C15H14F6N4O4S (44)
C16H16F6N4O2 (44)
71.2
92.34
87.96
86.3
83.95
59.88
C16H20F6N2O3 (47)
C15H18F6N2O4 (45)
C10H11F6N3O2 (32)
10
9

Table 2
Anticancer screening data (growth percentage) on the 60 human cancer cell line in single dose assay (10ꢀ5 M concentration) in vitro for tested compounds 5aeg, 6a, d, hel, 7meo in comparison with the data of NCI’s compounds
5FU, H-BZT, Me-BZT, Et-BZT.a-d
Panel/Cell line
Compound/Growth percent
5FUa
H-
Me-
Et-
5a
5b
5c
5d
5e
5f
5g
6a
6d
6h
6i
6j
6k
6l
7m
7n
7o
BZTb
BZTc
BZTd
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
57.1
47.9
42.3
43.1
41.4
24.8
91
115
79
89
90
92
80
113
62
64
110
4
78
nt
101
113
nt
nt
nt
91.56
nt
109.65
83.33
99.00
89.94
nt
130.53
nt
97.96
91.38
nt
nt
83.45
nt
98.32
93.19
86.22
107.44
58.89
nt
61.68
67.34
47.30
97.75
nt
nt
nt
nt
nt
132.44
nt
114.45
76.67
nt
nt
88.74
nt
101.40
90.19
99.46
100.95
nt
nt
nt
nt
nt
nt
nt
nt
73.30
92.77
93.42
67.59
nt
99.56
44.45
97.05
57.62
nt
105.02
82.18
75.46
76.58
nt
108.15
101.95
102.28
103.54
nt
99.30
103.22
90.31
72.69
nt
98.55
97.63
76.40
80.24
nt
90.86
98.54
110.32
56.01
nt
105.74
106.24
100.95
107.38
nt
48.24
100.40
65.28
80.96
nt
91.88
105.36
106.40
64.22
nt
81
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
HOP-62
34.2
58.4
47.8
50.6
69.5
39.0
59.5
13.0
58.0
87
79
99
91
95
nt
77
90
66
63
95
88
93
84
nt
75
77
77
85
93
92
101
84
101.59
97.71
105.85
91.40
103.47
103.28
98.54
108.40
158.28
95.23
nt
89.51
98.61
124.85
95.81
120.49
94.35
106.22
117.56
63.17
85.24
86.89
121.27
86.90
119.26
98.61
95.72
106.03
85.60
nt
nt
90.02
96.07
110.92
94.41
101.01
104.68
103.37
114.10
104.03
88.76
94.11
120.54
92.38
111.09
102.73
105.68
106.09
122.31
82.71
90.82
122.27
99.17
111.31
89.49
104.69
102.53
129.66
93.49
101.61
97.93
85.26
100.52
94.57
96.05
110.13
119.42
93.46
97.55
105.36
91.05
98.18
101.39
89.50
116.58
86.89
101.60
95.42
112.40
94.40
93.02
112.13
97.52
nt
101.10
96.69
99.16
106.83
103.78
105.33
95.17
106.37
104.70
102.59
94.95
102.32
94.22
103.20
102.95
96.31
107.35
94.80
104.67
98.21
102.66
104.93
85.83
102.07
102.22
94.17
95.30
112.05
97.27
95.82
100.55
107.30
111.29
109.44
92.45
107.81
60.91
97.25
97.88
110.38
108.76
92.80
68.44
101.69
11.47
82.60
60.19
97.89
85.80
35.59
94.32
108.04
86.56
96.59
105.60
97.09
108.16
100.14
116.40
100.66
102.42
103.47
96.81
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
nt
151
114
93
120.42
102.27
109.17
78.43
107.90
81.06
40.2
96
81
85
84
94
96
84
102
88
110.92
nt
111.74
99.11
98.83
121.08
103.30
98.04
119.64
116.64
98.67
105.96
103.26
102.66
103.10
122.86
92.95
91.27
107.95
103.43
95.72
118.71
102.21
97.61
89.35
94.02
89.85
111.19
124.84
87.55
98.50
104.68
95.36
104.62
102.98
108.65
74.89
73.20
79.27
81.09
74.78
99.10
111.07
100.78
108.13
110.74
103.50
98.10
110.11
nt
112.75
94.69
105.55
102.18
111.69
98.77
116.44
92.95
116.87
94.21
98.28
114.81
102.76
100.01
105.01
112.72
98.35
102.24
113.19
104.69
95.53
116.83
99.72
99.38
109.80
104.20
106.72
102.05
112.78
94.89
101.60
104.76
112.90
93.56
105.99
99.81
99.06
105.83
109.50
108.09
113.65
114.14
91.03
97.19
114.08
97.26
92.31
105.07
ꢀ17.8 97
102
100
94
108
91
17.8
26.5
27.1
40.7
50.1
81
91
94
94
99
110.13
111.69
98.17
112.10
100.01
101.67
106.04
112.77
104.71
103.64
105.14
114.55
112.65
107.95
104.71
HT29
KM12
SW-620
107
117.43
102.80
107.55
110.87
105.03
95.76
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
59.0
69.1
97
84
98
95
95
77
47
86
94
89
98
118
34
95
107.79
100.01
92.19
103.20
nt
102.70
98.05
103.20
109.93
nt
104.29
90.32
108.16
96.55
94.83
97.47
96.59
93.22
119.39
103.33
nt
99.05
81.31
123.93
118.01
nt
100.85
76.03
101.98
88.59
96.41
79.92
82.75
65.81
88.57
86.25
113.03
68.89
103.80
95.58
112.30
111.85
nt
103.75
98.05
101.09
96.72
nt
92.32
98.74
108.97
116.57
nt
104.84
100.18
101.54
121.05
nt
95.74
104.14
103.31
101.61
nt
104.91
98.96
105.02
114.08
nt
108.59
94.20
118.88
99.25
99.22
98.29
102.03
105.04
103.43
95.23
nt
107.50
96.79
101.31
110.56
nt
101.11
101.34
102.12
107.80
nt
ꢀ19.6 104
65.9
65.9
50.3
100
74
96
SNB-75
U251
94.41
97.56
87.48
92.94
101.06
82.00
87.01
97.09
96.70
100.28
96.49
93.90
99.26
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer
786e0
30.4
58.2
nt
36.6
95.5
nt
33.7
19.5
39.7
100
90
88
95
108
94
83
88
86
93
67
87
84
84
91
90
88
88
99
84
104
101
87
99
99
109
84
103.41
98.41
109.08
98.39
nt
105.07
103.02
98.76
93.65
104.20
80.52
106.48
102.73
nt
107.96
105.11
97.30
93.32
100.46
110.22
109.07
96.22
111.77
108.44
102.26
104.65
101.03
93.72
114.69
105.92
92.10
nt
114.66
107.34
91.77
89.71
93.99
101.36
99.98
96.07
nt
112.79
96.61
94.15
93.47
99.43
92.40
81.10
53.34
96.32
67.64
81.10
83.35
25.37
69.24
46.92
98.03
106.00
115.01
101.69
nt
116.95
99.52
92.48
101.31
87.49
101.36
106.02
87.53
nt
111.36
87.16
100.33
86.58
62.86
95.88
105.45
90.46
nt
122.04
86.86
99.31
85.05
99.20
91.76
104.91
98.20
nt
114.15
100.98
96.25
89.05
95.78
85.92
104.79
102.29
nt
105.04
101.40
84.18
88.08
96.39
97.83
109.33
105.21
nt
109.55
101.13
90.13
95.45
101.35
100.87
110.14
95.46
117.21
107.95
98.30
101.06
99.01
101.97
107.66
nt
111.81
109.03
91.04
85.67
96.75
97.54
106.91
101.65
nt
106.92
104.68
90.63
100.11
95.10
91.20
100.26
101.19
nt
102.33
108.60
93.18
83.83
115.95
103.35
109.07
105.72
82.43
99.49
95.72
102.86
105.78
51.2
47.4
59.4
44.3
nt
97
68
77
82
92
98
90
77
84
69
107
78
94
87
98
110
101
96
96.11
99.85
102.99
118.09
101.84
100.72
104.56
100.57
109.87
110.14
107.35
96.89
120.00
110.95
103.59
113.86
102.00
98.34
95.84
105.01
107.88
103.00
114.45
104.28
125.10
94.84
96.39
104.40
95.59
83.95
67.67
66.18
98.01
87.67
87.04
96.03
115.08
111.10
104.95
107.95
110.40
103.54
110.81
103.08
125.20
101.33
109.31
97.50
94.85
98.99
100.24
108.45
102.41
96.76
88.84
99.98
106.00
105.95
97.20
96.39
99.26
102.88
118.37
106.56
100.44
104.35
108.20
104.52
91.77
110.11
115.76
108.78
105.48
109.27
98.70
100.45
105.51
110.24
96.84
118.64
101.92
109.56
129.41
107.61
89.90
102.64
96.11
110.42
110.46
94.11
102.79
99.01
120.02
102.91
120.10
109.20
95.92
118.89
115.45
92.23
107.13
101.29
103.66
114.72
109.27
97.52
104.88
104.77
107.48
47.6
77.5
101
98
95.82
106.40
103.04
107.67
91
112.81
101.81
105.75
107.34
106.66
104.19
48.7
ꢀ16.3 82
98
72
92
112
73
103.77
65.11
106.78
125.07
102.39
89.77
97.21
108.91
94.79
114.28
99.10
84.46
79.47
60.21
108.74
97.24
101.87
62.98
98.52
68.40
103.41
99.54
102.19
115.09
108.02
83.97
104.75
106.29
104.48
86.53
108.52
96.25
105.01
104.74
A498
(continued on next page)

368
E.L. Luzina, A.V. Popov / European Journal of Medicinal Chemistry 53 (2012) 364e373
Table 3 shows the overview of antitumor activity as a percentage of
growth inhibition (GI%) of the bis(trifluoromethyl)alkyl ureas
against all panels of the tested DTP cell lines and presents the most
sensitive cell lines in the subpanels.
Table 4 represents the overview of antitumor activity (GI) of the
bis(trifluoromethyl)alkyl ureas against all panels of the tested DTP
cell lines without concrete values of activity (for comparison with
our compounds reported previously [1]). The spectrum of anti-
tumor activity of polyfluorinated ureas revealed the next results.
On the Leukemia subpanel, thirteen polyfluorinated ureas were
tested and showed low to moderate activity. The most active
compounds were ureas 5b GI% individual cell lines ꢂ 40e50, 5g GI
% ꢂ 30e50, 6i GI% ꢂ 40, 7m GI% ꢂ 30e50. Furthermore, for Non-
Small Cell Lung cancer GI values were within the range of
10e80% (for 13 compounds) with urea 5g being the most active (GI
% ꢂ 30e80). Thus, there was the same pattern of activity, but with
smaller values than those seen for Leukemia. The Melanoma cell
lines were inhibited by ten compounds in the 10e70% range (urea
5g was again the most active with GI values of 10e70%). For Renal
cancer cell lines, only ten compounds showed GI of 10e30% (the
most active compounds were ureas 5a, 5g, 6d and 6h). In Breast
cancer cell lines, there were eight active compounds; for CNS
cancer, six active compounds; for Colon cancer, three active
compounds (5eeg, GI ¼ 10e20%); and for Ovarian cancer, three (5a,
5g, 6j; GI ¼ 10e30%). A more detailed comparative analysis of the
all tested compounds with the H-BZT, Me-BZT and Et-BZT is pre-
sented in SI1 (Tables 1Se4S).
The comparison with our previous data [1] shows, that bis(tri-
fluoromethyl)alkyl ureas are active against some subpanels of
human cancer cell lines of leukemia, renal, lung, melanoma and less
so for CNS and breast cancers.
The tested compounds show different strength of anticancer
activity: some have moderate overall values of anticancer activity,
but possess a significant specific influence on several cancer cell
lines. This activity pattern is likely due to distinctive molecular
mechanisms of action for the mentioned substances (vide infra).
2.2.2. Structureeactivity relationships
2.2.2.1. N’-benzothiazolyl ureas. Four tested ureas bear substituted
benzothiazoles at the N’-position. Comparison of N-bis(tri-
fluoromethyl)alkyl-N’-substituted benzothiazolyl ureas 5d,e and
6d,h and their unsubstituted analogs H-BZT, Me-BZT, H-BZT
(Fig. 1S, SI1) from our previous paper [1], revealed a number of
observations (Table 4S, SI1). Additional Me-substituents in the
benzothiazolyl moiety improves activity in several cell lines, for
example (in Percent Growth): Leukemia MOLT-4 (75.46%), RPMI-
8226 (76.58%), Renal cancer CAKI-1 (84%), MCF7 (89.63%), MDA-
MB-231/ATCC (85.96%) for urea 5e compared to H-BZT (89%, 90%,
91%, 94% and 109%, respectively). Likewise, for substituted ureas 6d
and 6h in comparison to Me-BZT we found: Leukemia RPMI-8226:
6d e 72.69%, 6h e 80.24% and Me-BZT e 110%; Melanoma LOX
IMVI: 6de87,49%, 6he62.86% and Me-BZT e 93%; Renal cancer
A498 6de62,98%, 6he68.40%, while Me-BZT scored 94%. At the
same time, urea 6d (R ¼ Me) was more active than its analog 5d
(R ¼ H) against cell lines RPMI-8226 Leukemia, U251 CNS cancer,
LOX IMVI, MDA-MB-435, SK-MEL-5, UACC-62 Melanona, A498,
SN12C Renal cancer, MDA-MB-231/ATCC, HS 578T Breast cancer,
but against NCI-H522 Non-Small Cell Lung cancer 5d (63.17%) was
more active than 6d (122.31%). Insertion of a chlorine atom at
position C-4 of the benzothiazole ring (6h) leads to increased
activity against Leukemia MOLT-4 (76.4%), Non-Small Cell Lung
cancer NCI-H23 (89.49%), Melanoma LOX IMVI (62.86%), Renal
cancer A498 (68.4%), SN12C (84%), Breast cancer MDA-MB-231/
ATCC (69.61%) cell lines, compared to benzothiazoles 5d,e, 6d,
which have methyl substituents in the benzothiazole part.

E.L. Luzina, A.V. Popov / European Journal of Medicinal Chemistry 53 (2012) 364e373
369
Table 3
Percent of growth inhibition (GI%) for the tested compounds against some selected tumor cell lines at the single dose assay (10ꢀ5 M concentration) in vitro.a
Compound
NSC No.
Panel
GI% of tumor cell lines
5a
D-758741
Leukemia
HL-60(TB) (26.7), RPMI-8226 (32.41)
Ovarian cancer
Renal cancer
OVCAR-5 (10.1)
A498 (34.89), UO-31 (13.88)
Breast cancer
Leukemia
HS 578T (13.35)
K-562 (55.55), RPMI-8226 (42.38)
5b
D-758746
Non-Small Cell Lung cancer
Melanoma
HOP-92 (14.17)
MALME-3M (19.48)
Renal cancer
SN12C (11.28), TK-10 (10.58), UO-31 (12.68)
Breast cancer
Leukemia
Renal cancer
Non-Small Cell Lung cancer
CNS cancer
MDA-MB-231/ATCC (11.09)
MOLT-4 (16.67), SR (10.06)
A498 (10.23)
A549/ATCC (10.49), NCI-H522 (36.83)
5c
D-758751
D-758731
5d
U251 (12.52)
Melanoma
UACC-62 (10.29)
Renal cancer
UO-31 (12.45)
5e
5f
D-758732
D-758750
Leukemia
Non-Small Cell Lung cancer
Colon cancer
CNS cancer
Renal cancer
K-562 (17.82), MOLT-4 (24.54), RPMI-8226 (23.42)
A549/ATCC (14.76), EKVX (13.11), HOP-92 (13.1), NCI-H522 (14.4)
HCT-15 (10.65), KM12 (10.15)
SF-295 (18.69)
CAKI-1 (16.0)
Breast cancer
Leukemia
MCF7 (10.37), MDA-MB-231/ATCC (14.04)
CCRF-CEM (16.55), RPMI-8226 (13.78)
Non-Small Cell Lung cancer
Colon cancer
HOP-92 (39.09)
HCC-2998 (12.45)
CNS cancer
Melanoma
CS-295 (23.97), SNB-19 (11.41), U251 (20.08)
SK-MEL-5 (17.57)
Renal cancer
A498 (15.54)
Breast cancer
Leukemia
Non-Small Cell Lung cancer
MDA-MB-231/ATCC (10.57)
5g
D-758749
CCRF-CEM (41.11), K-562 (38.32), MOLT-4 (32.66), RPMI-8226 (52.7)
EKVX (31.56), HOP-92 (88.53), NCI-H226 (17.4), NCI-H23 (39.81), NCI-H460 (14.2),
NCI-H522 (64.41)
Colon cancer
CNS cancer
Melanoma
HCC-2998 (25.11), HCT-116 (26.8), HCT-15 (20.73), HT29 (18.91), KM12 (25.22)
SF-268 (17.25), SF-295 (34.19), SF-539 (11.43), SNB-19 (13.75), U251 (31.11)
LOX IMVI (18.9), MALME-3M (46.66), MDA-MB-435 (32.36), SK-MEL-2 (18.9),
SK-MEL-28 (16.65), SK-MEL-5 (74.63), UACC-257 (30.76), UACC-62 (53.08)
IGROV1 (16.05), OVCAR-3 (32.33), OVCAR-4 (33.82), OVCAR-8 (12.33), NCI/ADR-RES (12.96)
786-0 (20.53), A498 (39.79), ACHN (13.49), CAKI-1 (37.75), SN12C (18.020, UO-31 (38.38)
PC-3 (17.1)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
MCF7 (34.5), MDA-MB-231/ATCC (13.95), HS 578T (13.69), T-47D (65.1), MDA-MB-468 (51.29)
6a
6d
D-758740
D-758733
Non-Small Cell Lung cancer
Leukemia
A549/ATCC (9.98)
RPMI-8226 (27.31)
Non-Small Cell Lung cancer
CNS cancer
A549/ATCC (11.24)
U251 (18.0)
Melanoma
Renal cancer
Breast cancer
Leukemia
Non-Small Cell Lung cancer
CNS cancer
LOX IMVI (12.51), MDA-MB-435 (12.47), SK-MEL-5 (12.84), UACC-62 (13.42)
A498 (37.02), SN12C (10.34), UO-31 (19.47)
MDA-MB-231/ATCC (17.87), HS 578T (11.83)
MOLT-4 (23.6), RPMI-8226 (19.76)
A549/ATCC (17.29), NCI-H23 (10.51)
U251 (12.99)
6h
D-758734
Melanoma
Renal cancer
Breast cancer
Leukemia
LOX IMVI (37.14), SK-MEL-5 (13.14), UACC-62 (14.95)
A498 (31.6), SN12C (16.0), UO-31 (18.33)
MDA-MB-231/ATCC (30.39)
RPMI-8226 (43.99)
6i
6j
D-758742
D-758747
Non-Small Cell Lung cancer
Melanoma
Renal cancer
HOP-92 (14.74)
UACC-62 (10.95)
SN12C (11.8)
RPMI-8226 (23.33)
Leukemia
Non-Small Cell Lung cancer
Melanoma
Ovarian cancer
Renal cancer
NCI-H322M (10.5), NCI-H522 (13.11)
MALME-3M (14.08), UACC-257 (15.82), UACC-62 (11.92)
OVCAR-8 (11.16)
SN12C (15.36)
6k
6l
D-758748
D-758752
D-758743
Non-Small Cell Lung cancer
Renal cancer
Leukemia
Non-Small Cell Lung cancer
Leukemia
Melanoma
NCI-H522 (21.57)
A498 (16.03)
CCRF-CEM (11.26)
HOP-92 (13.44)
HL-60(TB) (51.76), MOLT-4 (34.72), RPMI-8226 (19.04)
UACC-62 (14.33)
7m
Renal cancer
Breast cancer
A498 (13.47), CAKI-1 (12.96), UO-31 (13.57)
BT-549 (10.37)
7n
7o
D-758744
D-758745
Leukemia
RPMI-8226 (35.78)
Non-Small Cell Lung cancer
Melanoma
NCI-H522 (18.94)
UACC-62 (16.17)
GI% >10%; GI% ¼ 100 e Growth Percent in Table 2.
The inhibition percentages are calculated by subtracting the growth percentages from 100.
a
The data obtained from NCI in vitro disease-oriented human tumor cell screen at 10ꢀ5 M concentration.

370
E.L. Luzina, A.V. Popov / European Journal of Medicinal Chemistry 53 (2012) 364e373
Table 4
The highest percent growth inhibition (GIa) of compounds 5aeg, 6a, d, hel, 7meo and NCI’s H-BZTb, Me-BZTb and Et-BZTb in vitro on
several subpanels of human cancer cell lines at 10ꢀ5 M concentration.
b
H, Me, Et ¼ R of the isocyanate part of ureas, BZT e 2-benzothiazolyl ¼ R’ of the amine part of ureas [1].
2.2.2.2. Electron withdrawing/donating groups. For all 17 tested
compounds an additional structure-activity analysis could be done
by comparison of contribution of the electron-donating (EDG) and
electron-withdrawing (EWG) groups. While all three bis(tri-
fluoromethyl)alkyl groups represent EWG, the pharmacophore
moieties can be conditionally divided into two groups: (hetero)aryl
EWG (5b, c, d, e, f, g, 6d, h, i, j, k, l, 7m,n,o) and alkyl EDG (5a and
6a). No sharp correlation was found. For instance, EDG-bearing urea
5a revealed activity, whereas its homolog 6a did not (Tables 2and
4). The three most active compounds 5b, g and 7m bear EWG
(hetero)aryl pharmacophores, whilst twelve other EWG ureas were
less active.
On another hand, there is a pair of 1-(1,3,4-thiadiazol-2-yl)-3-
[2,2,2-trifluoro-1-(trifluoromethyl)ethyl]ureas 5f and 5g, which
differ from each other only by the substituents at position-5 of the
1,3,4-thiadiazole ring. Thiadiazolyl urea 5f bears a strong I-/M-
trifluoromethyl EWG - CF3, whereas its structural analog 5g has
a strong Mþ ethylsulfanyl EDG e SCH2CH3 (Scheme 1). In spite of
the structural similarity, the physiological activity of the two
compounds is drastically different (Table 3 and 2S, 3S in SI1).
Compound 5g revealed activity against all cancers: 54 of the 57
tested cell lines, what is more, the GI(%) values for 45 cell lines fall
in the interval of 10e89%. Ethylsulfanyl compound 5g is most active
against Leukemia, Non-Small Cell Lung cancer, Melanoma and
Breast cancer. In contrast, the trifluoromethyl derivative 5f was
active against 35 of 57 cell lines, and the GI(%) values for only 10 cell
lines were in the range of 10e39%; no activity against ovarian and
prostate cancer was detected.
The uncertainties in SAR data led us to run a COMPARE analysis,
which studies the link between the inhibition results for the novel
ureas with standard agents for which the mechanism of action has
previously been reported (vide infra).
Table 5
COMPARE analysis results (interval of PCC values) for tested compounds 5aeg, 6a, d,
heland 7meo.
Compound
NSC
Maximum value of PCC
Interval of PCC value
5a
5b
5c
5d
5e
5f
5g
6a
6d
6h
6i
6j
6k
6l
7m
7n
7o
758741
758746
758751
758731
758732
758750
758749
758740
758733
758734
758742
758747
758748
758752
758743
758744
758745
0.354
0.572
0.494
0.344
0.400
0.405
0.806
0.312
0.400
0.351
0.347
0.335
0.407
0.403
0.699
0.350
0.237
0.201e0.354
0.202e0.572
0.259e0.494
0.211e0.344
0.313e0.400
0.208e0.405
0.584e0.806
0.202e0.312
0.206e0.400
0.229e0.351
0.201e0347
0.211e0.335
0.21e0.407
0.239e0.403
0.319e0.699
0.200e0.350
0.201e0.237

E.L. Luzina, A.V. Popov / European Journal of Medicinal Chemistry 53 (2012) 364e373
371
Table 6
COMPARE analysis results for tested compounds 5b, 5g and 7m.
Com-pound
NSC
number
PCC
Target
Target vector NSC
No. of common
cell lines
Putative mechanism
of actionaeb
5b
5g
758,746
0.572
0.533
0.806
BCNUa
S409962
S95441
S15886
54
45
57
Alkylating Agenta
Alkylating Agenta
Inhibition of HIV reverse
transcriptasea
Methyl-CCNUa
758,749
1,8-Dichloro-3,6-dinitro-9H-carbazole
0.754
0.753
2,5,7-Trimethyl-3,4,6-trinitro-1H-indole
Fenticlor
S627390
S55636
41
55
Not determined
Inhibition of androgene or
estrogen receptorsb
0.704
0.703
0.686
1,3-Dinitrophenoxazine
S139948
S402779
S618214
56
51
45
Inhibition of factor Xa,
HIV reverse transcriptaseb
Not determined
5-(1,3-Benzodioxol-5-yl)-3-(3,4,5-trimethoxybenzyl)
dihydrofuran-2(3H)-one
(2,5-Dihydroxyphenyl)[2-(trichloromethyl)
phenyl]methanone
Estrogen receptor agonistb
7m
758,743
0.699
0.519
0.505
Spirohydantoin Mustarda
DTICa
S172112
S45388
S409962
52
51
55
Alkylating Agenta
Not determined
Alkylating Agenta
BCNUa
a
Standard agents from DTP NCI http://dtp.nci.nih.gov/docs/cancer/searches/standard_agent_table.html.
Putative mechanisms of action were identified with the use of literature sources: PUBLIC COMPARE database NCI, ChemSpider, and PubChem.
b
2.3. COMPARE analysis
anticancer activity on leukemia, renal, lung and melanoma
subpanels. Some ureas only showed activity against individual
cancer lines. Putative mechanisms of action were suggested based
upon the COMPARE analysis.
The most sensitive cell lines for the three most active tested
compounds were: for urea 5g - Leukemia RPMI-8226 (GI% 52.7),
Non-Small Cell Lung cancer HOP-92 (GI % 88.53), NCI-H522 (GI %
64.41), Melanoma UACC-62 (GI% 53.08), SK-MEL-5 (GI % 74.63),
Breast cancer MDA-MB-468 (GI% 51.29), T-47D (GI % 65.1); for urea
5b - Leukemia K-562 (GI % 55.55); for urea 7m: Leukemia HL-
60(TB) (GI % 51.76).
The work presented represents the first step in the investigation
of a new series of compounds. The data has revealed promising
clusters or even individual compounds fit for further analysis. Future
structure tailoring may lead to the discovery of more effective
anticancer agents.
Hence advanced target identification studies were beyond the
scope of this article, for the first assumption of molecular mecha-
nism we used accessible online tool - NCI COMPARE analysis
[31e33]. This analysis has been performed for all tested compounds
(see SI1 and SI2) to investigate whether there is resemblance to the
anticancer drugs from either the NCI standard agent database
[31e33], or the NCI active synthetic and natural compounds
(present in publicly-available databases) and to predict probable
mechanisms of action. The COMPARE analysis hypothesis is that
compounds may have the same mechanism of action as known
agents if the data pattern correlates well with the pattern of
compounds belonging to the standard agent database. Three ureas
showed a significant Pearson’s coefficient correlation (PCC): 0.572
for urea 5b, 0.806 for urea 5g and 0.699 for urea 7m (Tables 5and 6).
The comparison with NCI Standard agents for the other ureas is also
taken into account despite low correlations - the PCC ꢁ 0.4 (see SI1
and SI2). The analysis revealed that possible mechanisms of these
ureas include: alkylating agent (5b, 7m), DNA antimetabolites (5a,
5c, 5e), and inhibition of topoisomerase II (6h, 6l). Other possible
mechanisms for the tested urea 5g could be inhibition of top-
oisomerases, reverse transcriptases, aldose reductase, andro- and
estrogen receptors (Tables 6 and 5S (SI1)). The most active
compound 5g may act by a novel mechanism as no standard DTP
NCI compound was found with a similar pattern.
4. Experimental
4.1. General methods
The 1H and 19F NMR spectra were recorded on Bruker DXP at
200 and 188 MHz, respectively, in CDCl3, DMSO-d6 and acetone-d6
using tetramethylsilane (TMS) as an internal standard and
CF3COOH as an external standard. Chemical shifts are reported in
ppm units with the use of
d scale. Mass-spectra were recorded on
It is necessary to note that the physiological activity of poly-
fluoroalkyl- substituted ureas can be derived from urea <e> iso-
urea tautomerism as we reported previously [1,2]. The OH group in
the isourea molecule may be modified in vivo (alkylation, acylation,
phosphorylation, etc.).
a Finnigan 4021 spectrometer. The elemental (combustion) analysis
(C, H, F, N) was performed in the analytic chemistry laboratory of
IPAC RAS. Melting points were measured in open capillary tubes
and are uncorrected. HPLC analysis was performed on Waters 600
system as it was described previously [68].
These results illustrate that the mechanism of action of the
synthesized ureas may differ from that of standard antitumor
drugs. Therefore, their antitumor activity may be caused by new
and unknown mechanisms. The complete information on
COMPARE analysis for all reported ureas can be found in SI1
(Table 5S) and SI2.
4.1.1. General procedure for the synthesis of compounds 5a, ceg,
6a, d, h, l, 7meo
To a solution of 1 mmol of amine in 10 ml of dry diethyl ether or
dry benzene 1 mmol of isocyanate 1e3 was added. The mixture was
stirred during 4 h at rt and left overnight. The resulting precipitate
was filtered and recrystallized from benzene. HPLC analysis
revealed single peaks for all tested compounds.
3. Conclusion
In this paper, we report the synthesis and anticancer activity of
a series of new N-bis(trifluoromethyl)alkyl-N’-substituted ureas
bearing pharmacophoric fragments. These ureas have demonstrated
4.1.2. General procedure for the synthesis of compounds 5b, 6j, k
A solution of 1 mmol of isocyanate 1e2 in 5 ml of benzene was
added to 1 mmol of amine in 10 ml of dry benzene and 0.2 ml of

372
E.L. Luzina, A.V. Popov / European Journal of Medicinal Chemistry 53 (2012) 364e373
saturated ether solution of catalyst trimethylamine in a 50 ml glass
ampoule. This ampoule was sealed and heated at 100 ꢃC for 1.5e2 h.
The ampoule was opened, the solvent was evaporated, and the
residue was recrystallized from benzene.
d
0.85 s. EIeMS (m/z): 388 (Mþ). Anal. Calcd. for C15H18F6N2O3: C,
46.40%; H, 4.67%; F, 29.36%; N, 7.21%; Found: C, 46.53%; H, 4.51%;
F, 29.57%; N, 7.20%.
4.1.2.8. 1-(4-Methyl-1,3-benzothiazol-2-yl)-3-[2,2,2-trifluoro-1-methyl-
1-(trifluoromethyl)ethyl]urea (6d). Yield 91%, m.p. 186e188 ꢃC 1H NMR
4.1.2.1. 1-[2-(3,4-dimethoxyphenyl)ethyl]-3-[2,2,2-trifluoro-1-(trifluo-
romethyl)ethyl]urea (5a). Yield 89%, m.p. 144e145 ꢃC 1H NMR
(DMSO-d6):
d
2.00 (s, 3H, CH3C), 2.24 (s, 3H, CH3C), 7.12 (s þ d, 2H,
(DMSO-d6):
d
2.64 (t, 2H, CH2, J ¼ 7 Hz), 3.32 (q, 2H, J ¼ 7 Hz), 3.82 (s,
CHAr, J ¼ 7 Hz), 7.48 (s,1H, NH), 7.04 (s þ d, 2H, NHþCHAr, J ¼ 7 Hz). 19
F
3H, CH3O), 3.84 (s, 3H, CH3O), 5.84 (d sept, 1H, CH, JHF ¼ 8 Hz,
JHH ¼ 7 Hz), 5.92 (t, 1H, NH, J ¼ 7 Hz), 6.70 (s þ d, 2H, CHAr,
JHH ¼ 7 Hz), 6.78 (d,1H, CHAr, JHH ¼ 7 Hz), 7.26 (d,1H, NH, JHH ¼ 7 Hz).
NMR (DMSO-d6): d
5.79 s. EIeMS (m/z): 371 (Mþ). Anal. Calcd. for
C13H11F6N3OS: C, 42.05%; H, 2.99%; F, 30.70%; N, 11.32%; Found: C,
42.27%; H, 3.11%; F, 30.89%; N, 11.23%.
19F NMR (DMSO-d6):
d
5.85 (d, JHF ¼ 8 Hz). EIeMS (m/z): 374 (Mþ).
Anal. Calcd. for C14H16F6N2O3: C, 44.93%; H, 4.31%; F, 30.46%; N,
7.48%; Found: C, 45.03%; H, 4.51%; F, 30.57%; N, 7.29%.
4.1.2.9. 1-(4-Chloro-1,3-benzothiazol-2-yl)-3-[2,2,2-trifluoro-1-methyl-
1-(trifluoromethyl)ethyl]urea (6h). Yield 92%, m.p. 230e231 ꢃC 1H
NMR (DMSO-d6):
d
1.94 (s, 3H, CH3C), 6.12 (t, 1H, NH, J ¼ 7 Hz), 6.28
4.1.2.2. 1-(4,5-Dihydrothiazol-2-yl)-3-[2,2,2-trifluoro-1-(trifluorom-
ethyl)ethyl]urea (5c). Yield 78%, m.p. 201e202 ꢃC 1H NMR (DMSO-
(dd, 1H, CHAr, JHH ¼ 7 Hz, JHH ¼ 1.5 Hz), 7.38 (d,1H, CHAr, JHH ¼ 1.5 Hz),
7.84 (s,1H, NH), 8.18 (d,1H, CHAr, JHH ¼ 7 Hz), 8.54 (s,1H, NH).19F NMR
d6):
d
3.22 (t, 2H, CH2N, J ¼ 7 Hz), 3.62 (t, 2H, CH2N, J ¼ 7 Hz), 5.42
(DMSO-d6): d
5.94 s. EIeMS (m/z): 391 (Mþ). Anal. Calcd. for
(d sept, 1H, CH, JHF ¼ 8 Hz, JHH ¼ 7 Hz), 9.24 (s, 1H, NH), 11.42 (d, 1H,
C12H8ClF6N3OS: C, 36.79%; H, 2.06%; F, 29.10%; N, 10.73%; Found: C,
36.58%; H, 2.22%; F, 29.04%; N, 10.64%.
CH, JHF ¼ 8 Hz). 19F NMR (DMSO-d6):
d
5.85 (d, JHF ¼ 8 Hz). EIeMS
(m/z): 295 (Mþ). Anal. Calcd. for C7H7F6N3OS: C, 28.48%; H, 2.39%;
F, 38.61%; N, 14.23%; Found: C, 28.43%; H, 2.50%; F, 38.57%; N,
14.29%.
4.1.2.10. 1-(2,5-dimethoxyphenyl)-3-[2,2,2-trifluoro-1-methyl-1-(tri-
fluoromethyl)ethyl]urea (6i). Yield 99%, m.p. 186e188 ꢃC 1H NMR
(acetone-d6):
d 2.10 (s, 3H, CH3C), 3.80 (s, 3H, CH3O), 3.90 (s, 3H,
4.1.2.3. 1-(4-Methyl-1,3-benzothiazol-2-yl)-3-[2,2,2-trifluoro-1-(tri-
fluoromethyl)ethyl]urea (5d). Yield 91%, m.p. 189e190 ꢃC 1H NMR
CH3O), 6.50 (dd,1H, JHH ¼ 7 Hz, JHH ¼ 3 Hz), 6.92 ( d,1H, JHH ¼ 7 Hz),
(7,20 s, 1H, NH), 7.90 (d,1H, JHH ¼ 3 Hz), 8.20 (br. s, 1H, NH). 19F NMR
(DMSO-d6):
d
2.35 (s, 3H, CH3C), 5.32 (d sept, 1H, CH, JHF ¼ 8 Hz,
(acetone-d6): d
1.85 s. EIeMS (m/z): 360 (Mþ). Anal. Calcd. for
C13H14F6N2O3 : C, 43.34%; H, 3.92%; F, 31.64%; N, 7.78%; Found: C,
43.53%; H, 3.71%; F, 31.37%; N, 7.60%.
JHH ¼ 7 Hz), 7.12 (m, 2H, NHþCHAr), 7.94 (s þ d, 2H, NHþCHAr,
J ¼ 7 Hz), 8.14 (d, 1H, CHAr, J ¼ 7 Hz). 19F NMR (DMSO-d6):
d 5.95 (d,
JHF ¼ 8 Hz). EIeMS (m/z): 357 (Mþ). Anal. Calcd. for C12H9F6N3OS: C,
40.34%; H, 2.54%; F, 31.91%; N, 11.76%; Found: C, 40.54%; H, 2.51%; F,
31.77%; N, 11.70%.
4.1.2.11. 1-(1,5-Dimethyl-3-oxo-2-phenyl-pyrazol-4-yl)-3-[2,2,2-tri-
fluoro-1-methyl-1-(trifluoromethyl)ethyl]urea (6l). Yield 97%, m.p.
206e208 ꢃC 1H NMR (DMSO-d6):
d
1.96 (s, 3H, CH3), 2.22 (s, 3H,
4.1.2.4. 1-(4,6-Dimethyl-1,3-benzothiazol-2-yl)-3-[2,2,2-trifluoro-1-
(trifluoromethyl)ethyl]urea (5e). Yield 98%, m.p. 302e304 ꢃC 1H
CH3), 3.04 (s, 3H, CH3), 7.16 (s, 1H, NH), 7.24 (m, 1H, CHAr), 7.46 (m,
4H, CHAr), 7.88 (s, 1H, NH). 19F NMR (DMSO-d6):
d 5.90 s. EIeMS
NMR (DMSO-d6):
d
2.45 (s, 3H, CH3C), 2.54 (s, 3H, CH3C), 5.52 (d
(m/z): 411 (Mþþ1). Anal. Calcd. for C16H16F6N4O2: C, 46.84%; H,
3.93%; F, 27.78%; N, 13.65%; Found: C, 46.72%; H, 3.71%; F, 27.57%;
N, 13.86%.
sept, 1H, CH, JHF ¼ 8 Hz, JHH ¼ 7 Hz), 6.84 (s, 1H, HAr), 7.35 (s, 1H,
CHAr), 8.54 (br. s, 1H, NH), 10.75 (s, 1H, NH). 19F NMR (DMSO-d6):
d
5.95 (d, JHF ¼ 8 Hz). EIeMS (m/z): 371 (Mþ). Anal. Calcd. for
C13H11F6N3OS: C, 42.05%; H, 2.99%; F, 30.70%; N, 11.32%; Found: C,
42.23%; H, 2.81%; F, 30.57%; N, 11.20%.
4.1.2.12. 1-[1,1-bis(trifluoromethyl)propyl]-3-(2,5-diethoxyphenyl)urea -
(7m). Yield 98%, m.p. 160e161 ꢃC 1H NMR (acetone-d6):
d 1.20 (t, 3H,
CH3CC, JHH ¼ 7 Hz), 1.28 (t, 6H, CH3CO), 2.68 (q, 2H, CH2C, JHH ¼ 7 Hz),
4.2 (q, 2H, CH2O, JHH ¼ 7 Hz), 4.08 (q, 2H, CH2O, JHH ¼ 7 Hz), 6.64 (dd
1H, JHH ¼ 7 Hz, JHH ¼ 2 Hz), 6.92 ( d,1H, JHH ¼ 7 Hz), 7,05 s,1H, NH), 7.94
4.1.2.5. 1-[5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl]-3-[2,2,2-trifluoro-
1-(trifluoromethyl)ethyl]urea (5f). Yield 87%, m.p. 132e134 ꢃC 1H
NMR (acetone-d6):
d
5.54 (d sept,1H, CH, JHF ¼ 8 Hz, JHH ¼ 7 Hz), 8.20
(d 1H, JHH ¼ 2 Hz), 8.15 (br. s, 1H, NH). 19F NMR (acetone-d6):
d 5.34 s.
(d, 1H, NH, JHH ¼ 7 Hz), 11.30 (br s, 1H, NH). 19F NMR (acetone-d6):
EIeMS (m/z): 402 (Mþ). Anal. Calcd. for C16H20F6N2O3: C, 47.77%; H,
5.01%; F, 28.33%; N, 6.96%; Found: C, 47.51%; H, 5.17%; F, 28.49%; N,
7.19%.
d
5.94 (d, 6F JHF ¼ 8 Hz), 18.40 (s, 3F). EIeMS (m/z): 362 (Mþ). Anal.
Calcd. for C7H3F9N4OS: C, 23.21%; H, 0.83%; F, 47.21%; N, 15.47%;
Found: C, 23.23%; H, 0.81%; F, 47.57%; N, 15.20%.
4.1.2.13. 1-[1,1-bis(trifluoromethyl)propyl]-3-(3,4,5-trimethoxyphenyl)-
urea (7n). Yield 96%, m.p. 145e147 ꢃC 1H NMR (acetone-d6):
4.1.2.6. 1-(5-Ethylsulfanyl-1,3,4-thiadiazol-2-yl)-3-[2,2,2-trifluoro-1-
(trifluoromethyl)ethyl]urea (5g). Yield 98%, m.p. 152e154 ꢃC 1H
d
1.10 (t, 3H, CH3C, JHH ¼ 7 Hz), 2.58 (d, 2H, CH2C, JHH ¼ 7 Hz),
NMR (acetone-d6):
d
1.40 (t, 3H, CH3, J ¼ 7 Hz), 3.25 (q, 2H, J ¼ 7 Hz),
3.70 (s, 3H, CH3O), 3.90 (s, 6H, CH3O), 6.92 (s,1H, NH), 6.85 (s, 2H,
5.84 (d sept, 1H, CH, JHF ¼ 8 Hz, JHH ¼ 7 Hz), 7.86 (d, 1H, NH,
CHAr), 8.20 (br. s, 1H, NH). 19F NMR (acetone-d6):
d 5.52 s. EIeMS
JHH ¼ 7 Hz), 10.60 (s, 1H, NH). 19F NMR (acetone-d6):
d
5.9 (d,
(m/z): 404 (Mþ). Anal. Calcd. for C15H18F6N2O4: C, 44.56%; H,
4.49%; F, 28.19%; N, 6.93%; Found: C, 44.73%; H, 4.54%; F, 28.41%;
N, 7.21%.
JHF ¼ 8 Hz). EIeMS (m/z): 355 (Mþ þ 1). Anal. Calcd. for
C8H8F6N4OS2: C, 27.12%; H, 2.28%; F, 32.17%; N, 15.81%; Found: C,
27.03%; H, 2.51%; F, 32.37%; N, 15.60%.
4.1.2.14. 1-[1,1-bis(trifluoromethyl)propyl]-3-(5-methylisoxazol-3-yl)
4.1.2.7. 1-[2-(3,4-dimethoxyphenyl)ethyl]-3-[2,2,2-trifluoro-1-methyl-
1-(trifluoromethyl)ethyl]urea (6a). Yield 92%, m.p. 126e127 ꢃC 1H
urea (7o). Yield 98%, m.p. 110e112 ꢃC 1H NMR (acetone-d6):
d 1.14
(t, 3H, CH3CC, JHH ¼ 7 Hz), 2.42 (s, 3H, CH3), 2.66 (d, 2H, CH2C,
NMR (DMSO-d6):
d
1.92 (s, 3H, CH3C), 2.62 (t, 2H, CH2, J ¼ 7 Hz),
JHH ¼ 7 Hz), 6.34 (s, 1H, CH), 7.55 (s, 1H, NH), 8.95 (br. s, 1H, NH). 19
F
3.24 (q, 2H, J ¼ 7 Hz), 3.78 (s, 3H, CH3O), 3.84 (s, 3H, CH3O), 6.12 (t,
NMR (acetone-d6): d
5.56 s. EIeMS (m/z): 319 (Mþ). Anal. Calcd. for
1H, NH, J ¼ 7 Hz), 6.68 (s, 1H, NH), 6.72 (s þ d, 2H, CHAr
,
C10H11F6N3O2: C, 37.63%; H, 3.47%; F, 35.71%; N, 13.16%; Found: C,
37.55%; H, 3.56%; F, 35.78%; N, 13.46%.
JHH ¼ 7 Hz), 6.74 (d, 1H, CHAr, JHH ¼ 7 Hz). 19F NMR (DMSO-d6):

E.L. Luzina, A.V. Popov / European Journal of Medicinal Chemistry 53 (2012) 364e373
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4.1.2.15. 1-Pyrimidin-2-yl-3-[2,2,2-trifluoro-1-(trifluoromethyl)ethyl]
urea (5b). Yield 89%, m.p. 148e149 ꢃC 1H NMR (acetone-d6):
5.74
d
(d sept, 1H, CH, JHF ¼ 8 Hz, JHH ¼ 7 Hz), 7.14 (t, 1H, JHH ¼ 7 Hz), 8.74
(d, 2H, JHH ¼ 7 Hz), 9.80 (br s, 1H, NH), 10.50 (d, 1H, NH, JHH ¼ 7 Hz).
19F NMR (acetone-d6):
d
5.90 (d, JHF ¼ 8 Hz). EIeMS (m/z): 289
(Mþþ1). Anal. Calcd. for C8H6F6N4O: C, 33.35%; H, 2.10%; F, 39.56%;
N, 19.44%; Found: C, 33.23%; H, 2.21%; F, 39.57%; N, 19.29%.
4.1.2.16. 1-(4-sulfamoylphenyl)-3-[2,2,2-trifluoro-1-methyl-1-(trifluo-
romethyl)ethyl]urea (6j). Yield 91%, m.p. 189e190 ꢃC 1H NMR
(DMSO-d6): d 1.40 (s, 3H, CH3), 7.16 (s, 2H, NH2), 7.26 (s,1H, NH), 7.62
(d, 2H, CHAr, JHH ¼ 7 Hz), 7.74 (d, 2H, CHAr, JHH ¼ 7 Hz), 9.06 (s, 1H,
NH). 19F NMR (DMSO-d6):
Calcd. for C11H11F6N3O3S: C, 34.83%; H, 2.92%; F, 30.05%; N, 11.08%;
Found: C, 34.63%; H, 2.78%; F, 30.12%; N, 11.20%.
d
0.36 s. EIeMS (m/z): 380 (Mþþ1). Anal.
4.1.2.17. 1-[4-[(5-methylisoxazol-3-yl)sulfamoyl]phenyl]-3-[2,2,2-trif-
luoro-1-methyl-1-(trifluoromethyl)ethyl]urea (6k). Yield 92%, m.p.
190e191 ꢃC 1H NMR (DMSO-d6):
d 1.94 (s, 3H, CH3), 2.32 (s, 3H,
CH3), 5.94 (s, 1H, CH), 6.06 (s, 1H, NH), 7.04 (s, 1H, NH), 7.52 (d, 2H,
CHAr, JHH ¼ 7 Hz), 7.70 (d, 2H, CHAr, JHH ¼ 7 Hz), 8.96 (s, 1H, NH). 19
F
NMR (DMSO-d6):
d
0.46 s. EIeMS (m/z): 460 (Mþ). Anal. Calcd. for
C15H14F6N4O4S: C, 39.14%; H, 3.07%; F, 24.76%; N, 12.17%; Found: C,
39.03%; H, 3.21%; F, 24.57%; N, 12.20%.
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Acknowledgments
We thank the National Cancer Institute (NCI) (Bethesda, MD,
USA) for screening our compounds in human cancer cell lines. Our
special thanks are addressed to Dr. J. G. Gelovani (The University of
Texas M.D. Anderson Cancer Center, Houston, TX, USA) for
permission to publish for first time “the Gelovani’s Rules” for
potential small molecule drug candidates [44].
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