Anticancer activity of N-bis(trifluoromethyl)alkyl-N′- (polychlorophenyl) and N′-(1,2,4-triazolyl) ureas

Article abstract of DOI:10.1016/j.ejmech.2010.08.057

A number of N-bis(trifluoromethyl)alkyl-N′-substituted ureas have been synthesized and evaluated for their in vitro anticancer activity against the human cancer cell lines at the National Cancer Institute (NCI, USA). Marked activity was shown for compounds 4a and 5a. The most sensitive cell lines relative to the tested compounds were: 4a UO-31 (renal cancer, log GI ₅₀ -5.62), HS 578T (breast cancer, log GI₅₀ -5.50), 5a HCC-2998 (colon cancer, log GI₅₀ -5.94), NCI-H322M (lung cancer, log GI₅₀ -5.75) and PC-3 (prostate cancer, log GI₅₀ -5.66).

Full text of DOI:10.1016/j.ejmech.2010.08.057

European Journal of Medicinal Chemistry 45 (2010) 5507e5512
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Anticancer activity of N-bis(trifluoromethyl)alkyl-N0-(polychlorophenyl)
and N0-(1,2,4-triazolyl) ureas
,
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
A number of N-bis(trifluoromethyl)alkyl-N0-substituted ureas have been synthesized and evaluated for
their in vitro anticancer activity against the human cancer cell lines at the National Cancer Institute (NCI,
USA). Marked activity was shown for compounds 4a and 5a. The most sensitive cell lines relative to the
tested compounds were: 4a UO-31 (renal cancer, log GI50 ꢀ5.62), HS 578T (breast cancer, log GI50 ꢀ5.50),
5a HCC-2998 (colon cancer, log GI50 ꢀ5.94), NCI-H322M (lung cancer, log GI50 ꢀ5.75) and PC-3 (prostate
cancer, log GI50 ꢀ5.66).
Article history:
Received 29 June 2010
Received in revised form
20 August 2010
Accepted 25 August 2010
Available online 20 September 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Fluorinated ureas
Anticancer activity
Synthesis
1. Introduction
derivatives are found to be useful for the treatment of inflammatory
diseases [21,22]. One of unsymmetrical cyclic fluorinated urea is
In the first paper of this series [1] we described the potent anti-
cancer activity of unsymmetrical N-bis(trifluoromethyl)alkyl-
N0-thiazolyl and N0-benzothiazolyl ureas. In continuation of our
study herewedescribed the synthesis and invitroanticanceractivity
of new polyfluoroalkyl ureas where we will try to combine poly-
chlorophenyl or 1,2,4-triazolyl subsistent moieties in a molecule of
fluoroalkyl urea as a series of unsymmetrical N-bis(trifluoromethyl)
alkyl-N0-polychlorophenyl and N0-1,2,4-triazolyl ureas.
Organofluorine compounds are of exceptional interest in medicinal
applications [2e7]. In fact, approximately 20% of all currently approved
drugs contain at least one fluorine atom. An increasing number of
fluorinated antitumor agents have now become available for cancer
treatment [8e11]. At the same time, it is well known that urea is
a functional moiety commonly found in natural products and often
displays a wide range of biological activities [12e17]. An asymmetri-
cally substituted urea is a common structural unit of many biologically
active compounds, such as enzyme inhibitors and pseudopeptides [18].
Ureas asymmetrically substituted at amino groups have also been
shown to have a potent HIV-1 protease inhibitory activity [19] equi-
potent toward both wild and mutant types and exhibit strong
inhibition against growth of several cancer cell lines [20]. Some urea
5-fluorouracil (5-FU, an NCI standard) that has been used as an anti-
cancer drug for more than 40 years (Fig. 1) [23,24].
Polychloro (di- or tri-) phenyl substituents, which are present in
many drugs and potentially biologically active compounds, are the
important and responsible part of molecules for their action, for
example, as insecticide [25,26], herbicide (e.g. inhibitor of photosyn-
thesis DMCU (Fig. 1) [27]), and tyrosine kinase inhibitors [28]. On the
other hand, 1,2,4-triazole derivatives are known to exhibit antimi-
crobial [29e33], antitubercular [34], anticancer [35,36], anticonvul-
sant [37], antihypertensive [38], anti-inflammatory and analgesic
properties [19,39]. In many cases, this is the 1,2,4-triazole ring that is
responsible for antiviral activities in the compounds containing the
triazole ring [40], as well as for biological activity of interesting drug
candidates including H1/H2 histamine receptor blockers, cholines-
terase active agents, CNS stimulants, antianxiety agents and sedatives
[41], and antimycotic agents, such as Fluconazole and Voriconazole
(Fig. 2) [42e44]. At the same time, the combination of the 1,2,4-tri-
azole ring with the 2,4-dichloro-5-fluorophenyl moiety at position 3
of the condensed triazole ring showed very good antiproliferative
activity against most of cancer cell lines screened [45].
The current communication extends our publications combined
under the general idea of developing new physiologically active
fluorinated compounds from perfluoroisobutene [1,46e52], which
is an excellent precursor of trifluoromethyl-, bis(trifluoromethyl)
alkyl-, and nonafluoro-tert-butyl-containing substances [53].
* 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 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.057

5508
E.L. Luzina, A.V. Popov / European Journal of Medicinal Chemistry 45 (2010) 5507e5512
Fig. 1. 5-Fluorouracyl (5-FU) and dichlorophenyl urea (DCMU).
2. Results and discussion
2.1. Chemistry
The preparation of N-bis(trifluoromethyl)alkyl-N0-polychloroph
enyl ureas 4aec and 5aec, N-bis(trifluoromethyl)alkyl-N0-3-(4H-
1,2,4-triazol-3(4)-yl) ureas 4dee and 5dee has been carried out by
the addition of dicloroanilines 3a,b, 2,4,6-tricloroaniline 3c, 3- or 4-
amino-1,2,4-triazoles 3d,e to fluoroalkyl isocyanates 1,2 (Scheme
1). The synthesis of isocyanates 1 and 2 was described in our
previous papers [1,51].
2.2. Biological activity
2.2.1. Pharmacology
2.2.1.1. Primary anticancer assay. The synthesized ureas were
evaluated in the anticancer screen for the human disease-oriented
tumor cell lines developed at the NCI [54e56]. This in vitro screen is
subdivided into a pre-test and a main test. Within the one dose the
pre-test consisting of three tumor cell lines: MCF7 (breast), NCI-
H460 (lung), and SF-268 (CNS) test agents were added at only one
concentration (10ꢀ4 M) to each cell line inoculated and pre-
incubated on a microtiter plate, and the culture was then incubated
for 48 h. End point determinations are made with a protein binding
Scheme 1. Synthesis of polyfluorinated ureas 4aee and 5aee.
moiety, a part of “active” compounds 4a and 5a, to the best of our
knowledge, is not an element of any existing drug. A possible
explanation for bioactivity of 2,5-dichlorophenyl containing ureas
4a and 5a can be a unique combination of Mþ and Iꢀ effects of
chlorine atoms [57] situated in 2 and 5-positions of the phenyl ring
that can stabilize such an active tautomer as isourea [1]. The isoureas
contain OH group that can be chemically (or biochemically) modi-
fied (alkylated, acylated, phosphorylated) inside cancer cells thus
interfering with physiological processes [1]. On the other hand the
substitution of two chlorine atoms at 2 and 6 positions of phenyl ring
makes 2,6-dichlorophenyl and 2,4,6-trichlorophenyl groups bulky
dye Sulphorhodamine
B (SRB). The results for each tested
compound are reported as the percentage of growth of the treated
cells in comparison with that of the untreated control cells. The
percentage growth was evaluated spectrophotometrically versus
controls untreated with test agents (Table 1). Negative numbers
indicate cell death. Compounds reducing the growth of any of the
three cell lines by 32% or more are called “active”, while others are
called “inactive”.
Primary screening demonstrated that polyfluoroalkyl ureas with
1,2,4-triazolyl residues 4d,e and 5d,e did not display sufficient
anticancer activity at concentration 10ꢀ4 M. Compounds with
2,6-dichlorophenyl residue 4b, 5b and 2,4,6-trichlorophenyl residue
4c, 5c also were “inactive”. Polyfluoroalkyl ureas with 2,5-dichlor-
ophenyl residue 4a and 5a were “active” in primary test and passed
on for evaluation in the main test, in this case consisting of
approximately 60 cell lines over a 5-log dose range (10ꢀ4e10ꢀ8 M).
At the same time at primary screening compound 5a showed
negative value against all three cell lines, so it would be suggested
that this compound revealed cytotoxic activity. 2,5-Dichlorophenyl
Table 1
In vitro anticancer activity e NCI’s 3 cell line preliminary test.a
Compound
Growth of cells (%)
NCI-H460 (lung)
MCF7 (breast)
SF-268 (CNS)
Resultb
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
17
85
34
82
50
84
Active
Inactive
Inactive
Inactive
Inactive
Active
Inactive
Inactive
Inactive
Inactive
103
111
94
89
98
81
96
111
105
ꢀ99
101
98
ꢀ100
ꢀ89
79
75
81
88
86
94
102
84
102
105
a
Data were obtained from the NCI’s in vitro anticancer 3 cell line pre-test.
Negative numbers indicate cell kill. Each cell line was inoculated and preincubated
on a microtiter plate. Test agents (10ꢀ4 M) were added and the culture incubated for
48 h. Results for each test agent were reported as the percentage of growth of the
treated cells when compared to untreated control cells.
b
Compounds which reduce the growth of any one of the cell lines to 32% or less
Fig. 2. Fluorine-containing 1,2,4-triazoles.
are called “active” and passed on for evaluation in the main test.

E.L. Luzina, A.V. Popov / European Journal of Medicinal Chemistry 45 (2010) 5507e5512
5509
(in “inactive” compounds 4b, 5b, 4c and 5c) and may have dramatic
effects on the conformation of such tautomeras isourea compared to
2,5-dichlorophenyl group.
Table 3 demonstrates the antiproliferative data for N-bis(tri-
floromethyl)alkyl-N0-substituted ureas 4a and 5a against 60 human
cancer cell lines. For visibility in Tables 3 and 4 we added N0-2-ben-
zothiazolyl analogs of 4a (Me-BZT e NCS 713412) and 5a (Et-BZT e
NCS 713404), ureas from our previous article (Fig. 3) [1], as well as the
NCI standard anticancer agent 5-fluorouracil (5-FU) [58] to thor-
oughly compare anticancer activity of the fluoroalkyl ureas. A
comparison of the anticancer activity of compounds 4a and 5a with
the standard values for (5-FU) revealed the following: moderate
growth inhibition of all cell lines of 5-FU (MG_MID ꢀ6.05) is higher
than that of urea derivatives 4a and 5a. Nevertheless, compounds 4a
and 5a demonstrate higher activity than standard 5-fluorouracil 5-FU
against NCI-H226, HOP-92, and NCI-H522 (Non-Small Cell Lung
cancer), as well as SNB-75 (CNS cancer) and SK-MEL-2 (melanoma)
and HS 578T (breast cancer). Derivative 4a demonstrated higher
activity than cyclic urea 5-FU against cell line T-47D (breast cancer).
Compound 5a is more active than cyclic urea 5-FU against CCRF-CEM
(leukemia), EKVX (Non-Small Cell Lung cancer) OVCAR-5 and SK-OV-
3 (ovarian cancer), PC-3 (prostate cancer), MDA-MB-231/ATCC and
BT-549 (breast cancer).
2.2.1.2. Determination of GI50, TGI, and LC50 values. Within the
main test the antitumor activity of a test compound is expressed
by three different doseeresponse parameters for each of the 60
cell lines derived from nine different types of cancer, namely,
leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate,
and breast: GI50 (molar concentration required for half-growth
inhibition), TGI (molar concentration leading to total growth
inhibition), and LC50 (molar concentration required for 50% cell
death). Furthermore, a mean graph midpoint (MG_MID) is
calculated for each of the above-mentioned parameters, which
displays an averaged activity parameter over all cell lines, as well
as the Delta parameter that is the difference between the highest
and the average values [54].
Table 2 represents a general overview of the main parameters
that characterize anticancer activity of the tested compounds 4a
and 5a (GI50, TGI, LC50) and most sensitive tumor cell lines for these
compounds. From the data reported in Table 2 it is evident that the
highest activity is demonstrated by urea derivative 5a (MG_MID:
log GI50 ꢀ5.37, log TGI ꢀ4.87, log LC50 ꢀ4.38). Polyfluoroalkyl ureas
with 2,5-dichlorophenyl residues 4a and 5a showed no selectivity
Compound 4a is more active than its analog Me-BZT: the former
was active against 25 cancer cell lines (log GI50 < ꢀ5.00), whereas
the latter demonstrated such anticancer activity against only 3 cell
lines. Urea derivative 4a showed the selectivity for several cell lines
on all 60 cell lines: the values of
0.56 (5a) ( < 1 is considered low). Nevertheless, the selectivity can
be observed for separate cancers and cell lines. Compound 4a
demonstrated moderate selectivity for TGI ( log TGI: 1.18). Selec-
tivity can be seen for several cells within the subpanel cell line.
Ethyl (R ¼ Et) compound 5a demonstrated the cytotoxic activity for
all three indicators (GI50, TGI, LC50): from 58 tested cell lines
D
log GI50 are equal to 0.82 (4a) and
only by log TGI (Dlog TGI: 1.18): K-562, MOLT-4, SR (leukemia),
D
SNB-75 (CNS cancer), UO-31 (log TGI ꢀ5.24, renal cancer), HS 578T
(breast cancer). Overall activity of compound 5a on all cell lines in
the test was higher than that of its analog Et-BZT but without
sufficient selectivity. As for structural fragment comparison, ethyl
(R ¼ Et) ureas are more active than methyl (R ¼ Me) ureas, i.e. the
activity of ethyl derivatives 5a and Et-BZT is higher than that of
methyl derivatives 4a and Me-BZT. All the latter four ureas (4a, 5a,
Me-BZT and Et-BZT) demonstrate good results on cell lines SR
leukemia, SNB-75 CNS cancer, and UO-31 renal cancer
(log GI50 < ꢀ5.00).
D
(57 log GI50
, 58 log TGI and 53 log LC50, respectively). Methyl
(R ¼ Me) compound 4a showed the cytostatic activity for all three
indicators as well (GI50, TGI, LC50): from 57 tested cell lines
(41 log GI50, 6 log TGI and 1 log LC50, respectively).
The most potent compounds relative to the cell lines are: 4a
against lung cancer NCI-H522 (log GI50 ꢀ5.51), renal cancer UO-31
(log GI50 ꢀ5.62, log TGI ꢀ5.24), breast cancer HS 578T
(log GI50 ꢀ5.50, log TGI ꢀ4.31); 5a against lung cancer NCI-H322 M
(log GI50 ꢀ5.75, log TGI ꢀ5.27, log LC50 ꢀ4.66), colon cancer HCC-
2998 (log GI50 ꢀ5.94, log TGI ꢀ5.43, log LC50 ꢀ4.87), melanoma
SK-MEL-2 (log GI50 ꢀ5.66, log TGI ꢀ5.26, log LC50 ꢀ4.74), prostate
cancer PC-3 (log GI50 ꢀ5.66, log TGI ꢀ5.31, log LC50 ꢀ4.91), and
renal cancer TK-10 (log GI50 ꢀ5.65, log TGI ꢀ5.06, log LC50 ꢀ4.52).
When these compounds are passed on for extensive evaluation
in the full panel of 60 cell lines over a five log dose range, they
showed variable antitumor properties against most of the tested
subpanel tumor cell lines at the GI50 level. The subpanel tumor cell
lines median growth inhibitory concentration (the average sensi-
tivity of each subpanel toward each test compound) and the full
panel mean graph midpoint (MG_MID: the average of all cell lines
toward each test compounds) are reported in Table 4. The active
compound 5a in the present study is proven to be nonselective
against any of nine tumor subpanels tested.
Table 2
Overview of the results of the in vitro anticancer activity screening for compounds 4a and 5a.a
b log TGI,c log LC50
MG_MIDe and Df for:
Most sensitive cell lines
d
Cd
N
Number of cell lines giving positive log GI50
,
log TGIc
log LC50
b
d
log GI50
N1
41
Rangeg
N2
6
Rangeg
N3
1
Rangeg
log GI50
log TGI
log LC50
4a
5a
57
58
ꢀ5.62 to ꢀ4.12
ꢀ5.94 to ꢀ4.92
ꢀ5.24 to ꢀ4.31
ꢀ5.43 to ꢀ4.56
ꢀ4.09 to ꢀ4.00
ꢀ4.81, 0.82
ꢀ4.06, 1.18
ꢀ4.00, 0.09
Renal UO-31, Breast HS 578T,
Lung NCI-H522
Lung NCI-H322M, Colon HCC-2998,
Melanoma SK-MEL-2, Renal TK-10,
Prostate PC-3
57
58
53
ꢀ4.87 to ꢀ4.04
ꢀ5.37, 0.56
ꢀ4.87, 0.57
ꢀ4.38, 0.91
N e number of tested human tumor cell lines; N1, N2, N3 e number of cell lines giving positive log GI50, log TGI, log LC50
.
a
Data obtained from the NCI’s in vitro disease-oriented human tumor cells.
The log of the molar concentration that inhibits 50% net cell growth.
The log of the molar concentration leading to total growth inhibition.
The log of the molar concentration leading to 50% net cell death.
b
c
d
e
MG_MID ¼ mean graph midpoint ¼ arithmetical mean value for all tested cell lines. If the indicated effect was not attainable within the used concentration interval, the
highest tested concentration was used for the calculation.
f
The reported data represent the logarithmic difference between the parameter value referred to the most sensible cell line and the same mean parameter. Delta is
considered low if <1, moderate if >1 and <3, high if >3.
g
The values > ꢀ4.00 were excluded.

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E.L. Luzina, A.V. Popov / European Journal of Medicinal Chemistry 45 (2010) 5507e5512
Table 3 (continued)
Table 3
In vitro anticancer activity of compounds 4a and 5a against 60 human cancer cell
Panel/cell line
Compound/growth inhibitory activity (log GI50)
lines in comparison with data of NCI’s compounds.a,b,c
4a
5a
Me-BZTa
Et-BZTb
5-FUc
Panel/cell line
Compound/growth inhibitory activity (log GI50)
BT-549
T-47D
>ꢀ4.00
ꢀ5.46
ꢀ4.81
ꢀ4.98
e
>ꢀ4.00
ꢀ4.42
ꢀ4.45
ꢀ4.74
ꢀ4.81
ꢀ4.77
ꢀ4.97
ꢀ5.09
ꢀ6.05
4a
5a
Me-BZTa
Et-BZTb
5-FUc
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
MG_MIDd
ꢀ5.37
e
ꢀ5.39
ꢀ5.38
ꢀ5.45
ꢀ5.43
e
ꢀ4.56
ꢀ4.01
ꢀ4.77
ꢀ4.74
ꢀ4.41
ꢀ5.44
ꢀ4.63
e
ꢀ5.01
ꢀ5.64
ꢀ5.45
ꢀ6.45
ꢀ7.35
ꢀ7.62
If the indicated effect was not attainable within the used concentration interval, the
highest tested concentration was used for the calculation.
ꢀ4.76
ꢀ5.23
ꢀ5.02
e
ꢀ4.63
ꢀ4.53
e
a
NCI’s data for N0-2-benzothiazolyl analog of 4a: NCS 713412: 1-Benzothiazol-2-
yl-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethyl-ethyl)-urea (Fig. 3) [1].
b
NCI’s data for N0-2-benzothiazolyl analog of 5a: NCS 713404:: 1-Benzothiazol-2-
ꢀ5.13
ꢀ5.47
ꢀ4.77
yl-3-(1,1-bis-trifluoromethyl-propyl)-urea (Fig. 3) [1].
c
NCI’s data for 5-fluorouracil NCS 19893 (NCI standard compound).
MG_MID ¼ mean graph midpoint ¼ arithmetical mean value for all tested cell
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H322M
NCI-H460
NCI-H522
NCI-H23
d
ꢀ5.25
ꢀ5.46
ꢀ5.50
ꢀ4.97
ꢀ5.33
ꢀ5.21
ꢀ5.75
ꢀ5.66
ꢀ5.55
e
ꢀ4.79
ꢀ4.13
ꢀ4.50
ꢀ4.12
ꢀ4.41
ꢀ4.57
ꢀ4.61
ꢀ4.61
e
ꢀ4.80
ꢀ4.77
ꢀ4.75
ꢀ4.72
ꢀ4.80
ꢀ4.59
ꢀ4.67
ꢀ4.77
e
ꢀ6.72
ꢀ4.21
ꢀ6.40
ꢀ4.11
ꢀ4.26
ꢀ6.75
ꢀ7.25
ꢀ5.14
ꢀ6.48
lines.
e
ꢀ4.20
ꢀ5.16
ꢀ4.71
e
Compound 4a exhibited values of MG_MID log GI50 < ꢀ5.00
against subpanels leukemia (ꢀ5.04), lung cancer (ꢀ5.01), colon
cancer (ꢀ5.15), and prostate cancer (ꢀ5.24) whereas for full panel
MG_MID log GI50 ꢀ4.81. On other subpanels some selectivity for
several cell lines can be observed: CNS cancer SF-295 log GI50 ꢀ5.46
(MG_MID log GI50 ꢀ4.70), melanoma LOX IMVI log GI50 ꢀ5.11
(MG_MID log GI50 ꢀ4.44), ovarian cancer OVCAR-3 log GI50 ꢀ5.38
(MG_MID log GI50 ꢀ4.62), renal cancer UO-31 log GI50 ꢀ5.62
(MG_MID log GI50 ꢀ4.82), and breast cancer HS 578T log GI50 ꢀ5.50
(MG_MID log GI50 ꢀ4.21).
ꢀ5.24
ꢀ5.51
e
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
ꢀ5.38
ꢀ5.42
ꢀ5.31
e
ꢀ5.74
ꢀ5.94
ꢀ5.49
ꢀ5.39
ꢀ5.48
ꢀ5.39
ꢀ5.28
ꢀ4.42
ꢀ4.41
ꢀ4.38
>ꢀ4.00
ꢀ4.71
ꢀ4.33
ꢀ4.34
ꢀ4.79
ꢀ4.72
ꢀ4.74
ꢀ4.75
ꢀ4.71
ꢀ4.69
ꢀ4.68
ꢀ6.80
ꢀ7.28
ꢀ6.64
ꢀ6.96
ꢀ6.75
ꢀ6.67
ꢀ6.03
ꢀ5.43
ꢀ5.36
>ꢀ4.00
Compound 5a showed the most activity (MG_MID log GI50
ꢀ5.37) on 9 subpanels of cells with no sufficient selectivity and
following cell lines were most sensitive in the subpanels: lung
cancer NCI-H322M log GI50 ꢀ5.75 (MG_MID log GI50 ꢀ5.43), colon
cancer HCC-2998 log GI50 ꢀ5.94 (MG_MID log GI50 ꢀ5.33), mela-
noma SK-MEL-2 log GI50 ꢀ5.66 (MG_MID log GI50 ꢀ5.28), prostate
cancer PC-3 log GI50 ꢀ5.66 (MG_MID log GI50 ꢀ5.50), and renal
cancer TK-10 log GI50 ꢀ5.65 (MG_MID log GI50 ꢀ5.41).
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
ꢀ4.14
ꢀ5.46
ꢀ4.52
>ꢀ4.00
ꢀ5.39
e
ꢀ5.32
ꢀ5.46
ꢀ5.02
ꢀ5.33
ꢀ5.44
ꢀ5.53
ꢀ4.53
ꢀ4.22
ꢀ4.61
ꢀ4.61
ꢀ5.12
ꢀ4.47
ꢀ4.77
ꢀ4.77
ꢀ4.74
ꢀ4.68
ꢀ5.84
ꢀ4.76
ꢀ5.80
ꢀ6.64
ꢀ7.20
ꢀ5.42
ꢀ4.10
ꢀ6.04
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
We used the COMPARE algorithm to look at the overall patterns
of responsiveness of the cell lines [59e61]. Strong COMPARE
correlations (empirically defined as correlations greater than 0.6)
among the cell line responses suggest that the compounds may be
ꢀ5.11
ꢀ4.75
ꢀ5.39
ꢀ5.09
ꢀ5.29
ꢀ5.66
ꢀ4.92
ꢀ5.18
ꢀ5.32
ꢀ5.38
ꢀ4.44
ꢀ4.82
ꢀ4.43
ꢀ4.52
ꢀ4.14
ꢀ4.31
ꢀ4.29
>ꢀ4.00
ꢀ4.74
ꢀ4.81
ꢀ4.73
ꢀ4.80
ꢀ4.75
ꢀ4.75
ꢀ4.70
ꢀ4.81
ꢀ6.61
ꢀ7.29
ꢀ6.01
ꢀ4.25
ꢀ5.99
ꢀ6.33
ꢀ5.45
ꢀ6.28
>ꢀ4.00
ꢀ4.91
>ꢀ4.00
ꢀ4.60
ꢀ4.12
Table 4
>ꢀ4.00
Median growth inhibitory concentration (log GI50) for compounds 4a and 5a of in
vitro subpanel tumor cell lines.
Ovarian cancer
IGROV1
ꢀ4.75
ꢀ5.38
ꢀ4.97
>ꢀ4.00
e
ꢀ5.25
ꢀ5.51
ꢀ5.25
ꢀ5.32
ꢀ5.28
ꢀ4.97
ꢀ4.38
ꢀ4.68
ꢀ4.66
ꢀ4.09
ꢀ4.28
ꢀ4.28
ꢀ4.77
ꢀ4.78
ꢀ4.73
ꢀ4.71
ꢀ4.74
ꢀ4.75
ꢀ5.91
ꢀ7.80
ꢀ5.35
ꢀ4.96
ꢀ5.76
ꢀ4.66
Panel/cell line
Compound/growth inhibitory activity (log GI50)
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
4a
5a
Me-BZTe
Et-BZTf
5-FUd
Leukemia
ꢀ5.04
ꢀ5.01
ꢀ5.15
ꢀ4.70
ꢀ4.44
ꢀ4.62
ꢀ4.82
ꢀ5.24
ꢀ4.21
ꢀ4.81
0.82
ꢀ5.42
ꢀ5.43
ꢀ5.33
ꢀ5.35
ꢀ5.28
ꢀ5.26
ꢀ5.41
ꢀ5.50
ꢀ5.26
ꢀ5.37
0.56
ꢀ4.66
ꢀ4.47
ꢀ4.37
ꢀ4.59
ꢀ4.37
ꢀ4.40
ꢀ4.55
ꢀ4.25
ꢀ4.30
ꢀ4.45
1.21
ꢀ4.64
ꢀ4.73
ꢀ4.73
ꢀ4.93
ꢀ4.76
ꢀ4.75
ꢀ4.82
ꢀ4.74
ꢀ4.74
ꢀ4.77
1.08
ꢀ6.25
ꢀ5.70
ꢀ6.73
ꢀ5.87
ꢀ6.03
ꢀ5.74
ꢀ6.24
ꢀ6.04
ꢀ5.86
ꢀ6.05
1.75
Non-small cell lung cancer
Colon cancer
CNS cancer
>ꢀ4.00
Renal cancer
786-0
A498
Melanoma
ꢀ5.01
>ꢀ4.00
ꢀ5.29
ꢀ4.72
ꢀ4.11
ꢀ5.06
ꢀ4.71
ꢀ5.62
ꢀ5.36
ꢀ5.38
ꢀ5.41
ꢀ5.34
ꢀ5.00
ꢀ5.55
ꢀ5.65
ꢀ5.58
ꢀ4.69
>ꢀ4.00
ꢀ4.54
ꢀ4.41
ꢀ4.19
ꢀ4.59
ꢀ4.31
ꢀ5.66
ꢀ4.70
ꢀ4.87
ꢀ4.74
ꢀ4.72
ꢀ4.76
ꢀ4.79
ꢀ4.66
ꢀ5.33
ꢀ6.14
ꢀ6.40
ꢀ6.53
ꢀ7.14
ꢀ5.58
ꢀ6.30
ꢀ5.95
ꢀ5.85
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
MG_MIDa
ACHN
CAKI-1
RXF 393
SN12C
TK-10
Deltab
Rangec
1.62
1.01
1.66
1.32
3.70
UO-31
a
MG_MID ¼ mean graph midpoint ¼ arithmetical mean value for all tested cell
lines. If the indicated effect was not attainable within the used concentration
interval, the highest tested concentration was used for the calculation.
Prostate cancer
PC-3
DU-145
ꢀ5.46
ꢀ5.02
ꢀ5.66
ꢀ5.33
ꢀ4.46
ꢀ4.04
ꢀ4.74
ꢀ4.73
ꢀ5.63
ꢀ6.44
b
Delta e difference in log GI50 value of the most sensitive cell lines and the
Breast cancer
MCF7
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MG_MID value. Delta is considered low if <1, moderate if >1 and <3, high if >3.
c
ꢀ4.93
ꢀ5.23
ꢀ5.14
ꢀ5.38
ꢀ5.48
ꢀ5.28
ꢀ5.29
ꢀ5.38
ꢀ4.42
>ꢀ4.00
ꢀ4.46
ꢀ4.73
ꢀ4.38
ꢀ4.01
ꢀ4.80
ꢀ4.71
ꢀ4.79
ꢀ4.67
ꢀ4.70
ꢀ4.73
ꢀ7.10
ꢀ6.50
ꢀ5.18
ꢀ5.01
ꢀ7.14
e
Range e difference in log GI50 value between the most and least sensitive cell
lines. The values > ꢀ4.00 were excluded.
d
ꢀ4.52
5-fluorouracil (NCI standard compound).
NCI’s data for N0-2-benzothiazolyl analog of 4a: NCS 713412: 1-Benzothiazol-2-
e
ꢀ5.50
MDA-MB-435
MDA-N
>ꢀ4.00
>ꢀ4.00
yl-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethyl-ethyl)-urea (Fig. 3) [1].
f
NCI’s data for N0-2-benzothiazolyl analog of 5a: NCS 713404: 1-Benzothiazol-2-
yl-3-(1,1-bis-trifluoromethyl-propyl)-urea (Fig. 3) [1].

E.L. Luzina, A.V. Popov / European Journal of Medicinal Chemistry 45 (2010) 5507e5512
5511
NMR (DMSO-d6): 1.93 (s, 3H), 6.86 (dd, J ¼ 8.7 Hz, J ¼ 2.5 Hz, 1H),
7.22 (d, J ¼ 8.5 Hz, 1H), 7.85 (s, 1H), 8.22 (d, J ¼ 2.5 Hz, 1H), 8.48 (s,
1H). 19F NMR (DMSO-d6)
calcd. for C11H8Cl2F6N2O: C, 35.80%; H, 2.18%; F, 30.88%; N, 7.59%.
Found: C, 35.53%; H, 2.21%; F, 30.57%; N, 7.40%.
d
1.56 s. EI-MS (m/z): 369 (Mþ þ 1). Anal.
Fig. 3. 1-Benzothiazol-2-yl-3-(2,2,2-trifluoro-1-methyl-1-trifluoromethylethyl)urea
(Me-BZT) and 1-Benzothiazol-2-yl-3-(1,1-bis(trifluoromethyl)propyl)urea (Et-BZT).
4.1.1.2. 1-(2,6-Dichlorophenyl)-3-(1,1,1,3,3,3-hexafluoro-2-methyl-
propan-2-yl)urea (4b). Yield 88%, white solid, m.p. 213e214 ꢁC. 1H
acting through similar mechanisms whose nature is still to be
determined. At the GI50 endpoint (Tables S1 and S2 in
Supplementary data), the pairwise correlations among two of the
compounds (4a and 5a) has not identified any single strong
candidates for mechanism of action or molecular targets that may
be targeted by the fluoroalkylureas. It may be that the fluo-
roalkylureas are affecting several different cellular processes at the
same time, producing cell response patterns that do not correlate
well with public compounds previously submitted to COMPARE.
NMR (DMSO-d6):
d
1.97 (s, 3H), 7.21 (m, 2H, p-C þ NH), 7.40 (d, m-
CH, 2H, J ¼ 8.4 Hz,), 8.16 (s, 1H, NH). 19F NMR (DMSO-d6)
d 1.19 s. EI-
MS (m/z): 369 (Mþ þ 1). Anal. calcd. for C11H8Cl2F6N2O: C, 35.80%;
H, 2.18%; F, 30.88%; N, 7.59%; Found: C, 35.72%; H, 2.41%; F, 30.65%;
N, 7.70%.
4.1.1.3. 1-(2,5-Dichlorophenyl)-3-[1,1,1-trifluoro-2-(trifluoromethyl)
butan-2-yl]urea (5a). Yield 83%, white solid, m.p. 165e166 ꢁC. 1H
NMR (DMSO-d6):
d
1.08 (t, J ¼ 7.3 Hz, 3H), 2.51 (q, J ¼ 8.5 Hz, 2H),
6.90 (dd, J ¼ 8.5 Hz, J ¼ 2.1 Hz, 1H), 7.27 (d, J ¼ 8.5 Hz, 1H), 7.86 (s,
1H), 8.30 (d, J ¼ 2.1 Hz, 1H), 8.53 (s, 1H). 19F NMR (DMSO-d6)
d 5.58
3. Conclusion
s. EI-MS (m/z): 382 (Mþ). Anal. calcd. for C12H10Cl2F6N2O: C, 37.62%;
H, 2.63%; F, 29.75%; N, 7.31%; Found: C, 37.53%. H, 2.71%; F, 29.57%;
N, 7.40%.
Herein we demonstrated in vitro anticancer activity of some
new e N,N0-asymmetrically substituted ureas. N-bis(trifluorome-
thyl)alkyl ureas with N0-2,6-dichlorophenyl and 2,4,6-tri-
chlorophenyl substituents, as well as N0-1,2,4-triazolyl ureas
showed a low anticancer activity in primary test, whereas N0-2,5-
dichlorophenyl ureas 4a and 5a showed an interesting anti-
proliferative profile against different human tumor-derived cell
lines. Urea derivative 4a demonstrated low selectivity on subpanel
leukemia, lung cancer, colon cancer, and prostate cancer and was
active against cell lines UO-31 (renal cancer), HS 578T (breast
cancer), and NCI-H522 (lung cancer) cell lines. Compound 5a was
nonselective but was active against NCI-H322M (lung cancer), HCC-
2998 (colon cancer), SK-MEL-2 (melanoma), PC-3 (prostate cancer).
Thus N-bis(trifluoromethyl)alkyl-N0-substituted ureas represent
a new potential class of antitumor compounds.
4.1.1.4. 1-(2,6-Dichlorophenyl)-3-[1,1,1-trifluoro-2-(trifluoromethyl)
butan-2-yl]urea (5b). Yield 78%, white solid, m.p. 173e174 ꢁC. 1H
NMR (DMSO-d6): 1H NMR (DMSO-d6):
d
1.11 (t, J ¼ 6.7 Hz, 3H), 2.49
(q, J ¼ 8.5 Hz, 2H), 7.22 (m, p-CH þ NH, 2H), 7.40 (d, m-CH,
J ¼ 7.4 Hz, 2H), 8.14 (s, 1H, NH). 19F NMR (DMSO-d6)
d 5.09 s EI-MS
(m/z): 383 (Mþ þ 1). Anal. calcd. for C12H10Cl2F6N2O: C, 37.62%; H,
2.63%; F, 29.75%; N, 7.31%. Found: C, 37.94%; H, 2.41%; F, 29.81%; N,
7.37%.
4.1.1.5. 1-(4H-1,2,4-Triazol-3-yl)-3-[1,1,1-trifluoro-2-(tri-
fluoromethyl)butan-2-yl]urea (5d). Yield 77%, colorless crystals,
m.p. 310e312 ꢁC. 1H NMR (DMSO-d6):
d
1.12 (t, J ¼ 7 Hz, 3H), 2.44 (q,
J ¼ 7 Hz, 2H), 5.50 (br s, 1H, NH), 7.27 (s, 1H, CH), 9.90 (br s, 1H, NH).
19F NMR (DMSO-d6) 5.35 s. EI-MS (m/z): 305 (Mþ). Anal. calcd. for
4. Experimental
d
C8H9F6N5O: C, 31.48%; H, 2.97%; F, 37.35%; N, 22.95%; Found: C,
31.42%; H, 2.85%; F, 37.39%; N, 23.07%.
4.1. General methods
The 1H and 19F NMR spectra were recorded on a Bruker DXP
spectrometer at 200 and 188 MHz, respectively, in CDCl3, DMSO-d6
using tetramethylsilane (TMS) as an internal standard and
CF3COOH as an external standard. Chemical shifts are reported in
4.1.1.6. 1-(4H-1,2,4-Triazol-4-yl)-3-[1,1,1-trifluoro-2-(tri-
fluoromethyl)butan-2-yl]urea (5e). Yield 92%, yellow solid, m.p.
121e123 ꢁC. 1H NMR (DMSO-d6):
d
1.09 (t, J ¼ 7 Hz, 3H), 2.44 (q,
J ¼ 7 Hz, 2H), 7.91 (s,1H, NH), 8.35 (s, 2H, CH), 9.63 (br s,1H, NH). 19F
ppm units with the use of the
d
scale. Mass-spectra were recorded
NMR (DMSO-d6)
d
5.76 s. EI-MS (m/z): 306 (Mþ þ 1). Anal. calcd. for
on a Finnigan 4021 spectrometer. The elemental analyses (C, H, F, N)
were performed at the laboratory of analytical Chemistry of the
IPAC RAS. Melting points were measured in open capillary tubes
and were uncorrected. The starting materials isocyanates 1 and 2
were prepared by the Curtius reaction [1,51]. 2,5-Dichloroaniline
3a, 2,6-dichloroaniline 3b, 2,4,6-trichloroaniline 3c, 3-amino-
1,2,4-triazole 3d, and 4-amino-1,2,4-triazole 3e (Aldrich) were
sublimed in vacuum (10ꢀ2 Torr) before the reaction.
C8H9F6N5O: C, 31.48%; H, 2.97%; F, 37.35%; N, 22.95%. Found: C,
31.58%; H, 2.79%; F, 37.23%; N, 22.87%.
4.1.2. General procedure for the synthesis of compounds 4cee, 5c
A
solution of isocyanate 1 or 2 (1 mmol) and 2,4,6-tri-
chloroaniline 3c or 3-amino- or 4-amino-1,2,4-triazole 3d or 3e
(1 mmol) in 10 ml of dry benzene was refluxed for 2 h and left
overnight. The resulting precipitate was filtered and recrystallized
from benzene.
4.1.1. General procedure for the synthesis of compounds 4a,b and
5a,b,d,e
4.1.2.1. 1-(2,4,6-Trichlorophenyl)-3-(1,1,1,3,3,3-hexafluoro-2-methyl-
propan-2-yl)urea (4c). Yield 87%, white solid, m.p. 272e274 ꢁC. 1H
Dichloroaniline 3a or 3b, 3-amino- or 4-amino-1,2,4-triazole 3d
or 3e in 10 ml of dry diethyl ether, and saturated ether solution
(0.2 ml) of catalyst trimethylamine were added to a solution of
isocyanate 1 or 2 (1 mmol). The mixture was stirred for 4 h at room
temperature and left overnight. The resulting precipitate was
filtered and recrystallized from benzene.
NMR (DMSO-d6):
d 1.93 (s, 3H), 7.19 (s, 1H, NH), 7.42 (s, 2H), 8.16 (s,
1H). 19F NMR (DMSO-d6)
d
1.15 s EI-MS (m/z): 403 (Mþ þ 1). Anal.
calcd. for C11H7Cl3F6N2O: C, 32.74%; H, 1.75%; F, 28.25%; N, 6.94%.
Found: C, 34.63%; H, 1.64%; F, 28.37%; N, 6.83%.
4.1.2.2. 1-(1,1,1,3,3,3-Hexafluoro-2-methylpropan-2-yl)-3-(4H-1,2,4-
triazol-3-yl)urea (4d). 88%, colorless crystals, m.p. 332e334 ꢁC. 1H
NMR (DMSO-d6): d 2.02 (s, 3H), 5.50 (br s, 1H, NH), 7.27 (s, 1H, CH),
4.1.1.1. 1-(2,5-Dichlorophenyl)-3-(1,1,1,3,3,3-hexafluoro-2-methylpro-
pan-2-yl)urea (4a). Yield 87%, white solid, m.p. 204e205 ꢁC. 1H

5512
E.L. Luzina, A.V. Popov / European Journal of Medicinal Chemistry 45 (2010) 5507e5512
9.88 (s, 1H, NH). 19F NMR (DMSO-d6) 1.21 s. EI-MS (m/z): 291 (Mþ).
d
Anal. calcd. for C7H7F6N5O: C, 28.88%; H, 2.42%; F, 39.15%; N, 24.05%.
Found: C, 29.04%; H, 2.55%; F, 39.31%; N, 24.27%.
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4.1.2.3. 1-(1,1,1,3,3,3-Hexafluoro-2-methylpropan-2-yl)-3-(4H-
1,2,4-triazol-4-yl)urea (4e). Yield 78%, colorless crystals, m.p.
242e243 ꢁC. 1H NMR (DMSO-d6):
d
1.95 (s, 3H), 7.88 (s, 1H, NH),
1.61 s. EI-MS
8.34 (s, 2H, CH), 9.59 (s, 1H, NH). 19F NMR (DMSO-d6)
d
(m/z): 292 (Mþ þ 1). Anal. calcd. for C7H7F6N5O: C, 28.88%; H, 2.42%;
F, 39.15%; N, 24.05%. Found: C, 28.96%; H, 2.61%; F, 39.28%; N,
24.17%.
4.1.2.4. 1-(2,4,6-Trichlorophenyl)-3-[1,1,1-trifluoro-2-(tri-
fluoromethyl)butan-2-yl]urea (5c). Yield 83%, colorless crystals,
m.p. 240e242 ꢁC. 1H NMR (DMSO-d6):
d
1.02 (t, J ¼ 7 Hz, 3H), 2.59
(q, J ¼ 7 Hz, 2H), 7.14 (s, 1H, NH), 7.37 (s, 2H), 8.10 (s, 1H). 19F NMR
(DMSO-d6)
d
5.05 s. EI-MS (m/z): 416 (Mþ). Anal. calcd. for
C12H9Cl3F6N2O: C, 34.52%; H, 2.17%; F, 27.30%; N, 6.71%. Found: C,
34.53%; H, 2.22%; F, 27.37%; N, 6.64%.
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Acknowledgments
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759e767.
We thank the National Cancer Institute (NCI) (Bethesda, MD,
USA) for screening our compounds in human cancer cell lines. The
synthetic part of this work was financially supported by the Russian
Foundation for Basic Research (Grant no 98-03-33007).
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Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2010.08.057.
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