Synthesis and biological activity of novel N-cycloalkyl-(cycloalkylaryl)-2- [(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-yl)thio]acetamides

Article abstract of DOI:10.1016/j.ejmech.2011.10.022

In this paper the novel N-cycloalkyl-(cycloalkylaryl)-2-[(3-R-2-oxo-2H-[1, 2,4]triazino[2,3-c]quinazoline-6-yl)thio]acetamides synthesis by aminolysis of activated by thionyl chloride or carbonyldiimidazole [(3-R-2-oxo-2H-[1,2,4] triazino[2,3-c]quinazolin

Full text of DOI:10.1016/j.ejmech.2011.10.022

European Journal of Medicinal Chemistry 46 (2011) 6066e6074
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activity of novel N-cycloalkyl-(cycloalkylaryl)-2-[(3-R-2-
oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-yl)thio]acetamides
,
a
a
a
Galyna G. Berest a , Olexii Yu. Voskoboynik , Sergiy I. Kovalenko , Olexii M. Antypenko ,
*
Inna S. Nosulenko a, Andrii M. Katsev b, Olena S. Shandrovskaya b
a Pharmaceutical Chemistry Department, Zaporozhye State Medical University, 26, Mayakovsky ave., 69035 Zaporozhye, Ukraine
b Department of Pharmacy, Crimean State Medical University, 95006 Simferopol, Ukraine
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper the novel N-cycloalkyl-(cycloalkylaryl)-2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-
6-yl)thio]acetamides synthesis by aminolysis of activated by thionyl chloride or carbonyldiimidazole [(3-
R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazolin-6-yl)-thio]acetic acids and alkylation of the 3-R-6-thio-6,7-
dihydro-2H-[1,2,4]triazino[2,3-c]quinazoline-2-ones potassium salts with N-cycloalkyl-(cycloalkylaryl)-
2-chloroacetamides are proposed. The structures of compounds are determined by 1H, 13C NMR, LC-MS
and EI-MS analysis. The in vitro anticancer, antibacterial activity and Photobacterium leiognathi Sh1
bioluminescence inhibition of synthesized compounds were revealed. SAR results were discussed.
Compound 4.10 was found to be the most anticancer active one, selectively influenced on the non-
small cell lung and CNS cancer cell lines, especially on the HOP-92 (log GI50 ¼ ꢀ6.01) and U251 (log
GI50 ¼ ꢀ6.00).
Received 7 June 2011
Received in revised form
7 October 2011
Accepted 11 October 2011
Available online 20 October 2011
Keywords:
N-cycloalkyl-(cycloalkylaryl)-2-[(3-R-2-oxo-
2H-[1,2,4]triazino[2,3-c]quina-zoline-6-yl)
thio]acetamides
Anticancer
Antibacterial
Ó 2011 Elsevier Masson SAS. All rights reserved.
Bioluminescence inhibition
1. Introduction
prostate (DU145) and renal (ACHN) cancer cell lines in concentra-
tion 1e8 mN in vitro [16] Fig. 1.
Nowadays heterocyclic compounds are intensively studied to
enhance the range of anticancer agents [1]. During the last decade
lots of quinazoline anticancer drugs were discovered [2e13].
Among them, “Iressa” (1), “Erlotinib”, its hydrochloride (2) and
“Vandetanib” (3) are already being used. In spite of the fact, that the
mentioned compounds are derivatives of 4-aminoquinazoline, the
other substituted quinazolines also have antiproliferative activity,
which is not limited only by the indicated pharmacophore [2e13].
One of the perspective antitumor heterocycles are azino- and
azolo-annelated quinazolines [14e16]. Recently, group of Slovak’s
scientists have found that [1,2,4]triazolo[4,3-c]quinazoline deriva-
tives (4) possessed considerable in vitro antitumor activity against
cell line HeLa and B16 [14]. Also anticancer activity is characteristic
for 6-cyanobenzimidazole[1,2-c]quinazolines (5), indolo[20,30:3,4]
pyrido[2,1-b]quinazolin-5(7H)-ones (6) [15,16]. The latter
substances inhibited breast (MCF7/ADR), CNS (U251), colon
(SW620), lung (H522), melanoma (UACC62), ovarian (SKOV3),
Furthermore, several biological activities have been found for
[1,2,4]triazino[c]quinazolines [17e32]. 3-R-6-thio-6,7-dihydro-2H-
[1,2,4]triazino[2,3-c]quinazolin-2-ones and their potassium salts,
that were synthesized by us, have already shown antibacterial
properties against Escherichia coli, Aspergillus niger, Mycobacterium
luteum and antifungal activity against Candida albicans and Candida
tenuis [31,32]. Additionally, combination of such pharmacological
fragments in our compounds as 1-adamantylamine, [4-(1-
adamantyl)phenyl]amine, (3-ethylcyclo[2.2.1]hept-2-yl)amine and
triazinoquinazoline skeleton, might cause the appearance of other
biological activities. Therefore, “structureeactivity” relationship for
substances 7e10 has already demonstrated that the adamantyl
group is essential in maintaining anticancer activity as an oxygen-
sensitive hypoxia-inducible factor-1, which is found in many
human tumours resistant to radiation treatment and in animal
models associated with increased tumour growth, vascularization
and metastasis inhibitor [33] Fig. 2.
So, the aim of this work was the development of synthesis
methods for novel N-cycloalkyl-(cycloalkylaryl)-2-[(3-R-2-oxo-2H-
[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]acetamides, investigation
of their physicoechemical properties, cytotoxicity, antitumor and
* Corresponding author. Tel.: þ38 097 687 68 89; fax: þ38 0612 34 15 71.
E-mail address: kovalenkosergiy@gmail.com (G.G. Berest).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.022

G.G. Berest et al. / European Journal of Medicinal Chemistry 46 (2011) 6066e6074
6067
Fig. 1. Structures of quinazoline-based compounds.
antibacterial activities. The obtained results could serve as
a powerful tool for the further rational anticancer drug design.
developed an alternative way of amides synthesis 4.1e4.10, namely,
alkylation of potassium salts 2.1e2.4 by N-cyclo-
alkyl(cycloalkylaryl)-2-chloroacetamides (Method C) in aqueous-
propanol-2 mixture, propanol-2 or dioxane (Scheme 2). The
results of the experiment showed that the last mentioned synthetic
method was a preparative one and had advantages such as short
duration of synthesis (60e90 min), high yielding and purity of the
obtained substances.
2. Results and discussion
Synthesis of the 3-R-6-thio-6,7-dihydro-2O-[1,2,4]triazino[2,3-c]
quinazoline-2-ones potassium salts (2.1e2.4) was carried out by two
approaches. First, by treatment of 6-R-3-(2-aminophenyl)-1,2,4-
triazin-5-ones (1.1e1.4) with carbon disulfide in ethanol in pres-
ence of potassium hydroxide (Method A). The second one, by inter-
action of compounds 2 with potassium ethylxanthogenate in the
propanol-2 (Method B, Scheme 1) [31,32]. Subsequent alkylation of
the potassium salts 2.1e2.4 with chloroacetic acid and their deriva-
tives in presence of basic reagents in aqueous-propanol-2 solution
lead to the corresponding acids 3.1e3.4 (Scheme 1) [33].
The direct aminolysis of carboxylic acids and their esters is well-
known method of amides synthesis, but, unfortunately, [(3-R-2-
oxo-6,7-dihydro-2H-[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]ace-
tic acids 3.1e3.4 and their esters appeared to be inert to ammonium
and primary amines. It was necessary to activate carboxylic group
of acids 3.1e3.4 for further successful amidation. The thionyl
chloride or N,N0-carbonyldiimidazole (CDI) were selected as acti-
vating agents. The experiments showed that synthesis with
appropriate acid chlorides (Method A) or acid imidazolides
(Method B) underwent quite easily with good yields. It is important
to mention that reactive capacity of the N-nucleophilic reagents
was sufficient for reaction and only two conditions were necessary
for the whole transformation to appropriate amides 4.1e4.10:
anhydrous dioxane and refluxing during 2e3 h. Besides, we have
Elemental analysis, IR, 1H and 13C NMR, LS-MS and EI-MS data
evaluated the structure and purity of synthesized substances. In the
LS-MS spectra compounds 4.1e4.10 were characterized by positive
ions [M þ 1] and [M þ 3], which simultaneously proved the
structure and characterized isotopic type of Sulfur.
The two stretching bands of associated NH group were detected
in the range of 3516e3058 cmꢀ1 in the IR spectra. Besides,
compounds 4.1e4.10 showed stretching vibrations of nco-group
(band “Amide I”) at 1678e1633 cmꢀ1 and mixed stretch-
ingedeformation vibration of the band NeH and CeN (“Amide II”) at
1597e1510 cmꢀ1. Substances 4.1e4.10 had low intensity stretching
vibrations of C¼C bond of aromatic ring at 1589e1468 cmꢀ1, out-of-
plane deformation stretching at 850e666 cmꢀ1 and strong peak at
2960e2850 cmꢀ1 of the symmetric and antisymmetric stretchings
of CH3e and CH2e moieties.
In 1H NMR spectra of amides 4.1e4.10 S-triazinoqunazoline
carcass was characterized by sub-spectra that had appropriate
signals and chemical shifts [29,31,32]. Two one-proton triplets of H-
10 and H-9 resonated at 7.69e7.66 ppm and 8.01e7.95 ppm, two
one-proton doublets of H-8 and H-11 e at 7.76e7.70 ppm and
8.49e8.45 ppm. The last one was considerably shifted in the low
Fig. 2. Structures of adamantyl-based compounds.

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G.G. Berest et al. / European Journal of Medicinal Chemistry 46 (2011) 6066e6074
Scheme 1. Synthesis of the 3-R-6-thio-6,7-dihydro-2H-[1,2,4]triazino[2,3-s]guinazoline-2-ones Potassium salts (2.1e2.4) and [(3-R-2-oxo-2H- [1,2,4]triazino[2,3-s]quinazoline-6-
yl)thio]acetic acids (3.1e3.4).
field due to the electron-acceptive influence of Nitrogen. Charac-
teristic signals of compounds 4.1e4.10 were two-proton singlet of
SCH2e group at 4.25e3.94 ppm and one-proton singlet of C(O)NH-
group at 10.40e7.74 ppm. It is important to notice that singlet of
amide’s 4.4e4.7 C(O)NH-group, which contained 4-(1-adamantyl)
phenyl substituent, resonated in strong field and for amides 4.1e4.3,
4.8e4.10 with donor substituents it was observed in “aromatic
region” of spectra. The protons of cycloalkyl substituents of
compounds 4.1e4.10 were observed in the strong field. The ada-
mantyl fragment exhibited six two-proton singlets at
1.73e1.62 ppm (H-40, H-60, H-100) and at 1.98e1.84 ppm (H-20, H-80,
H-90), that was characteristic for the near bridge Carbon type
protons [34,35]. The three proton singlet at 2.05e2.01 ppm (H-30, H-
50, H-70) characterized protons near nodal atom of Carbon. More
complicated situation was observed in 1H NMR spectra of
compounds 4.8e4.10, whose axial and equatorial protons have
formed multiples at 1.51e0.92 ppm, 2.16e2.09 ppm, 2.67e2.60 ppm
and 3.55e3.39 ppm.
pathways of [Mþ]ꢁ fragmentation were caused by the cleavage of
the amide bond (m/z 347 and m/z 285) and C(2)eC(3) and N(3)e
N(4) bonds of triazine system (m/z 244), that had the strongest
intensity in spectrum. Fragmentary ion with m/z 244 further
eliminated fragments of CO, SCH2 and CNO. Additionally for the
substance 3.5 fragmentation of the adamantane substituent was
revealed.
3. Pharmacology
3.1. Bioluminescence inhibition test
The results of the bioluminescence inhibition of luminescent
bacteria Photobacterium leiognathi strain Sh1 showed that synthe-
sized acids 3.1e3.4 and amides 4.1e4.10 possessed inhibition
properties in the acute and chronic action tests in the majority of
experiments (Table 1) [36]. Compound 1.1 showed cytotoxicity in
concentration of 0.1 and 0.25 mg/mL in acute action test.
Replacement of methyl substituent of substance 3.1 by aryl for
substances 3.2e3.4 lead to the reduction of their cytotoxicity.
Compound 3.2 revealed the 50% of inhibition at 0.25 mg/mL.
Similar results were observed for substances 3.1e3.4 in chronic
action test. Inhibition of bacteria’s bioluminescence by compounds
3.1, 3.2 and 3.3 was demonstrated only in concentration of 0.25 mg/
mL in the mentioned test. While compound 3.4 inhibited bacteria
bioluminescence in concentration of 0.25 mg/mL at 45% versus
control. Amide 4.1 demonstrated inhibition properties with the
increasing of concentration in the both acute and chronic action
tests. Replacement of the 3-methyl group of 4.1 by phenyl for 4.2
leads to a considerable reduction of inhibition properties as well as
introduction of the methoxyphenyl fragment in the structure of
compound 4.3 in chronic action test. Moderate inhibition activity in
In 13C NMR spectra signals of Carbon C-6, C-2 and C(O)NH (4.1,
4.3, 4.8) were the most shifted ones and appeared at
155.20e155.16 ppm, 161.02e160.13 ppm and 166.39e166.17 ppm
appropriately. The additional confirmation of structure and S-
regioselectity of alkylation was the position of the Carbon of SCH2e
group that resonated at 36.79e35.78 ppm. The signals of nodal
atoms of adamantan’s Carbon of amides 4.1, 4.3 appeared at
36.49 ppm (C-40, C-60, C-100) and at 41.43 ppm (C-20, C-80, C-90),
while near bridge type Carbon was located at 29.30e29.23 ppm
[34]. sp3-hybridizated Carbon atoms of the 3-ethylbicyclo[2.2.1]
hepthyl substituent (4.8) were observed in the strong field
according to the suggested structure.
EI-MS spectra of substances 4.5 and 4.8 were characterized by
absence of the molecular ion causes by its low stability. Main
O
S
N
N
X
N
H
O
N
O
S
N
N
N
OH
Method A, B
4.1-4.7
Method C
N
N
SK
N
N
O
R
N
3.1-3.4
N
O
R
O
2.1-2.4
S
N
R
N
H
N
N
4.8-4.10
N
R
O
Method A: SOCl2, dioxane, NH2R1; Method B: CDI, dioxane or DMF, NH2R1; Method C: propanol-2 or dioxane, ClCH2C(O)NHR1;
R=Me, Ph, 4-MePh, 4-MeOPh; X=H, C6H4; R1=1-adamantyl; 4-(1-adamantyl)phenyl; (3-ethylbicyclo[2.2.1]hept-2-yl
Scheme 2. Synthesis of the N-cycloalkyl-(cycloalkylaryl)-2-[(3-R-2-oxo-2H-[1,2,4]-triazino[2,3-s]quinazolin-6-yl)thio]acetamides (4.1e4.10).

G.G. Berest et al. / European Journal of Medicinal Chemistry 46 (2011) 6066e6074
6069
Table 1
Table 2
Bioluminescence intensity, %.
Antimicrobial activity of synthesized compounds.
Compd.
Control Acute action test, mg/mL
Chronic action test,
mg/mL
Compd.a
Conc, mg/ml The inhibitory zones of the investigated
compounds, mm
E. coli S. aureus M. luteum C. tenuis A. niger
0.025
0.1
0.25
0.025
0.1
0.25
DMSO
3.1
3.2
3.3
3.4
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
66.7
100.0
91.7
105.0
36.00
86.96
85.6
90.00
102.94
84.62
76.60
100.0
0
75.0
20.0
80.0
0
80.43
87.5
65.71
80.88
96.15
59.57
100.0
0
50.0
0
0
0
56.52
83.3
32.86
54.41 118.75 111.25
50.00 170.00 140.00
59.57
62.50
100.0
36.4
34.0
93.3
88.9
25.00
97.14
127.3
86.96
100.0
61.5
40.9
255.3
191.5
1.88
85.71
107.7
94.57
100.0
0
3.6
0
45.0
0
78.57
116.7
50.00
66.25
16.00
76.92
86.11
44.00
80.0
0
4.3
0.5
0.1
0.5
0.1
0.5
0.1
0.5
0.1
0
0
0
0
0
0
0
0
16
0
9
0
0
0
7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
18b
0
4.4
21b
0
4.7
0
0
4.10
18b
0
Vancomicin 0.01
Nystatin
Oxacillin
18
11
21
58
15
0
0
25
0
0.01
0.01
24
0
0
48.72
90.28
34.62
93.06
a
Compounds 3.1, 3.2, 3.3, 3.4, 4.1, 4.2, 4.5, 4.6, 4.8 and 4.9 at concentration of 1.0
91.67 104.17
110.00
38.5
and 5.0 mg/ml did not inhibit investigated bacteria.
96.00 120.00 100.00 120.00
84.0
9.1
b
Zone of late spore formation of Aspergillus niger (without inhibition of fungus
90.9
0
85.3
0
144.0
0
mycelium).
Tetracycline 100.0
80.7
Therapeutic Program (www.dtp.nci.nih.gov). Compounds 3.1e3.4,
4.3, 4.10 were submitted and evaluated according to the US NCI
protocol, which was described elsewhere [38e42]. The compounds
were first evaluated at one dose primary anticancer assay towards
or approximately 60 cell lines (concentration 10ꢀ5 M). The human
tumor cell lines were derived from nine different cancer types:
leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate and
breast cancers. In the screening protocol, each cell line was inocu-
lated and preincubated for 24e48 h on a microtiter plate. Test
agents were then added at a single concentration and the culture
was incubated for further 48 h. The end point determinations were
made with a protein binding dye, sulforhodamine B (SRB). Results
for each test agent were reported as the percent growth of the
treated cells when compared to the untreated control cells. The
preliminary screening results are shown in Table 3.
Compounds 3.1e3.4 showed cytotoxicity against cancer cell
lines of leukemia, CNS cancer, renal cancer and prostate cancer.
More evident cytotoxic activity against tumor cell lines of CCRF-
CEM, HL-60(TB), K-562, MOLT-4 and SR was characteristic for
compound 3.4 with p-methoxyphenyl substituent. While
compounds 3.1e3.3 demonstrated higher antitumor activity
against CNS cancer cell lines SNB-75, U251, renal cancer UO-31 and
prostate cancer PC-3. It is important that compound 4.3, opposite to
substances 3.1e3.4, inhibited practically all tumor cell lines in the
range of 54e90% versus control (Table 3). Compound 4.3 inhibited
growth of leukemia SR cell line, non-small cell lung cancer EKVX,
SNB-75 of the CNS cancer, A498, CAKI-1, UO-31 of the renal cancer,
PC-3 of the prostate cancer and MDA-MB-231/ATCC, T-47D of the
breast cancer. While compound 4.10 appeared to be the more active
cytotoxic agent comparing to compound 4.3. So, substance 4.10 was
effective against tumor cell lines of leukemia (K-562, MOLT-4,
RPMI-8226, SR), non-small cell lung cancer (A549/ATCC, EKVX,
HOP-92, NCI-H23, NCI-H322M, NCI-H460), colon cancer (COLO 205,
HCT-116, HCT-15, HT29, KM12, SW-620), CNS cancer (SF-268, SF-
295, SNB-19, SNB-75), melanoma (LOX IMVI, M14, MDA-MB-435,
SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257), ovarian cancer
(IGROV1, OVCAR-8, NCI/ADR-RES), renal cancer (786-0, CAKI-1, RXF
393, RXF-393, SN12C, TK-10), prostate cancer (PC-3), and breast
cancer (MCF7, MDA-MB-231/ATCC, BT-549). It is interesting to
mention that substance 4.10 exhibited noticeable antiproliferative
effect against non-small cell lung cancer (HOP-62, NCI-H522),
ovarian cancer (OVCAR-3), (OVCAR-4, SK-OV-3), renal cancer
(ACHN, UO-31), and breast cancer (HS 578T, T-47D, MDA-MB-468).
Substance 4.10 was subsequenly investigated for dose-
dependent action in 5 concentrations (10ꢀ4e10ꢀ8 M). In Table 4
the acute and chronic action tests was characteristic for compounds
4.4e4.7, where adamantyl fragment was replaced by 4-(1-
adamantyl)phenyl substituent. It is interesting to mention that
compounds 4.4e4.7 showed effect of hormesis in chronic action
test. Amides 4.8e4.10 with 3-ethylcyclo[2.2.1]heptyl substituent
moderately inhibited bioluminescence of bacteria in concentration
of 0.25 mg/mL in chronic action test.
The SAR study elucidated that: (1) the most cytotoxic
compounds against luminescent bacteria P. leiognathi strain Sh1 in
acute and chronic test are acids 3.1e3.4; (2) cytotoxicity of the
compounds (3.1e3.4) increases in such order of derivatives
PhMeO > Ph > PhMe > Me; (3) functionalization of acids 3.1e3.4 to
amides 4.1e4.10 leads to the decrease of cytotoxicity and biolumi-
nescence inhibition; (4) it’s important that, the similar relationship
of the cytotoxicity from the type of substituent at 3 position is
observed for amides 4.1e4.10.
3.2. Antimicrobial and antifungal activities
The results of antimicrobial screening showed that substances
3.1e3.4 and 4.1e4.10 had moderate antimicrobial activity (Table 2).
The highest bactericidal data were estimated for substances 4.3 and
4.7 against S. aureus. While other cultures of bacteria revealed to be
insensitive to synthesized compounds in investigated concentra-
tions. It is interesting to mention, that compounds 4.3, 4.4 and 4.10
showed considerable fungicidal activity against A. niger, and in
concentration of 0.5 mg/mL caused the late spore formation against
mentioned fungus at 18e23 mm.
The SAR study revealed that: (1) substances 3.1e3.4 and
4.1e4.10 show moderate antimicrobial and antifungal activity, with
most active ones e amides 4.3, 4.4, 4.7 and 4.10; (2) amides 4.3 and
4.7 with adamantyl and aryl substituents at 3 position possess
antimicrobial activity against Staphylococcus aureus; (3) antifungal
activity of amides 4.3, 4.4 and 4.10 against A. niger is also deter-
mined by cycloalkyl-(cycloalkylaryl-) substituent in thioacetic
fragment and aryl moiety at 3 position.
3.3. Anticancer assay for preliminary in vitro testing
The moderate cytotoxicity of the synthesized substances against
luminescent bacteria P. leiognathi strain Sh1 and antimicrobial
activity against E. coli, S. aureus, M. luteum, C. tenuis and A. niger
appeared to be the reason for the in vitro cell line anticancer activity
screening in National Cancer Institute (NCI) under Developmental

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G.G. Berest et al. / European Journal of Medicinal Chemistry 46 (2011) 6066e6074
Table 3
Cytotoxic activity of the compounds in concentration 10ꢀ5 M against 60 cell cancer lines.
Test compounds Mean growth, % Range of growth, %
Most sensitive cell line growth, %*
3.1
3.2
109.80
105.33
83.45e142.51
82.53e127.73
93.33 (CCRF-CEM/L), 83.45 (SNB-75/CNS), 96.05 (CAKI-1/RC), 87.14 (UO-31/RC), 84.45 (PC-3/PC)
92.52 (CCRF-CEM/L), 91.97 (K-562/L), 91.87 (SR/L), 82.53 (A549/ATCC/nscLC), 86.57 (SNB-75/CNSC),
91.36 (U251/CNSC), 96.84 (M14/M), 93.24 (UACC-257/M), 96.97 (SK-OV-3/OC), 89.01 (UO-31/RC),
90.76 (PC-3/PC)
3.3
3.4
103.01
105.05
74.22e123.43
63.47e145.27
95.57 (CCRF-CEM/L), 94.48 (K-562/L), 84.86 (MOLT-4/L), 68.04 (SR/L), 96.51 (A549/ATCC/nscLC),
94.95 (EKVX/nscLC), 84.35 (SF-295/CNSC), 74.22 (SNB-75/CNSC), 96.04 (SK-MEL-5/M), 95.09
(UACC-257/M), 89.89 (SK-OV-3/OV), 82.80 (CAKI-1/RC), 85.32 (UO-31/RC), 86.48 (PC-3/PC)
90.66 (CCRF-CEM/L), 63.47 (HL-60(TB)/L), 80.90 (K-562/L), 84.78 (MOLT-4/L), 65.78 (SR/L), 95.86
(A549/ATCC/nscLC), 94.90 (NCI-H522/nscLC), 94.68 (HCC-2998/CC), 93.93 (SNB-75/CNSC), 86.92
(LOX IMVI/M), 95.40 (SK-MEL-5/M), 91.41 (CAKI-1/RC), 91.64 (UO-31/RC), 91.74 (PC-3/PC), 89.86
(MDA-MB-231/ATCC/BC)
4.3
85.70
31.39
49.65e121.05
85.05 (RPMI-8226/L), 69.62 (SR/L), 62.96 (EKVX/nscLC), 74.21 (HOP-92/nscLC), 79.88 (NCI-H322M/nscLC),
82.14 (HCT-116/ColC), 85.84 (SF-268/CNSC), 81.45 (SF-295/CNSC), 85.79 (SNB-19/CNSC), 63.03
(SNB-75/CNSC), 82.93 (LOX IMVI/M), 74.60 (OVCAR-3/OV), 83.19 (OVCAR-4/OV), 81.61 (SK-OV-3/OV),
49.65 (A498/RC), 56.92 (CAKI-1/RC), 83.27 (SN12C/RC), 51.06 (UO-31/RC), 54.14 (PC-3/PC), 76.88 (MCF7/BC),
64.80 (MDA-MB-231/ATCC/BC), 82.53 (BT-549/BC), 70.68 (T-47D/BC)
4.10
ꢀ98.94e110.28 39.23 (K-562/L), 11.38 (MOLT-4/L), 47.38 (RPMI-8226/L), 7.67 (SR/L), 50.38 (A549/ATCC/nscLC), 25.61
(EKVX/nscLC), ꢀ19.46 (HOP-62/nscLC), 56.97 (HOP-92/nscLC), 62.84 (NCI-H23/nscLC), 65.58
(NCI-H322M/nscLC), 56.05 (NCI-H460/nscLC), ꢀ49.92 (NCI-H522/nscLC), 28.64 (COLO 205/ColC),
64.33 (HCT-116/ColC), 67.94 (HCT-15/ColC), 42.76 (HT29/ColC), 68.27 (KM12/ColC), 45.93 (SW-620/ColC),
66.33 (SF-268/CNSC), 69.86 (SF-295/CNSC), 39.82 (SNB-19/CNSC), 4.94 (SNB-75/CNSC), ꢀ98.94 (U251/CNSC),
70.53 (LOX IMVI/M), ꢀ29.17 (MALME-3M/M), 44.16 (M14/M), 58.58 (MDA-MB-435/M), 56.52
(SK-MEL-2/M), 68.65 (SK-MEL-28/M), 59.80 (SK-MEL-5/M), 55.64 (UACC-257/M), 16.96 (IGROV1/OV),
ꢀ69.71 (OVCAR-3/OV), ꢀ58.54 (OVCAR-4/OV), 53.06 (OVCAR-8/OV), 73.65 (NCI/ADR-RES/OV), ꢀ5.12
(SK-OV-3/OV), 77.65 (786-0/RC), ꢀ17.15 (ACHN/RC), 36.10 (CAKI-1/RC), 9.46 (RXF 393/RC),
9.46 (RXF-393/RC), 57.78 (SN12C/RC), 13.78 (TK-10/RC), ꢀ12.73 (UO-31/RC), 55.08 (PC-3/PC), ꢀ31.96
(DU-145/PC), 48.17 (MCF7/BC), 22.47 (MDA-MB-231/ATCC/BC), ꢀ9.67 (HS 578T/BC), 90.86 (BT-549/BC),
ꢀ11.26 (T-47D/BC), ꢀ59.54 (MDA-MB-468/BC)
L e leukemia, nscLC e non-small cell lung cancer, ColC e colon cancer, CNSC e CNS cancer, M e melanoma, OVe ovarian cancer, RC e renal cancer, PC e prostate cancer, BC e
breast cancer.
results of log GI50, log TGI, log LC50 are shown. (GI50 e molar
concentration of the compound that inhibits 50% net cell growth;
TGI e molar concentration of the compound leading to total inhi-
bition of cell growth; LC50 e molar concentration of the compound
leading to 50% net cell death). It is important to mention that
substance 4.10 had strong anticancer effect only in the concentra-
tion of 10ꢀ4 and 10ꢀ5 M (Table 4).
The SAR study revealed that: (1) newly synthesized 6-
substituted 3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazolines are
previous unknown anticancer agents; (2) anticancer activity among
compounds 3.1e3.4 is determined by substituent at 3 position and
increases in such order Me > Ph > 4-MePh > 4-MeOPh; (3) func-
tionalization of the carboxylic group of thioacetic moiety at 6
position by introduction of 1-aminoadamantyl (4.3) or (3-
ethylbicyclo[2.2.1]hept-2-yl)amine (4.10) leads to the increasing
of anticancer activity; (4) further modification of carboxylic group
by aryl-(aralkyl-, heteryl-)amides is a perspective way for modifi-
cation of the compounds for further elucidation of their mechanism
of action.
Table 4
The influence of compounds 4.10 on the growth of individual tumor cell lines (log
GI50 ꢂꢀ5.40).
Disease
Cell line
log GI50 log TGI Log LC50
Leukemia
CCRF-CEM
MOLT-4
RPMI-8226
SR
ꢀ5.70
ꢀ5.79
ꢀ5.53
ꢀ5.87
ꢀ5.71
ꢀ5.61
ꢀ6.01
ꢀ5.87
ꢀ5.46
ꢀ5.49
ꢀ5.46
ꢀ5.46
ꢀ5.51
ꢀ6.00
ꢀ5.45
ꢀ5.41
ꢀ5.45
ꢀ5.57
ꢀ5.83
ꢀ5.78
ꢀ5.52
ꢀ5.72
ꢀ5.43
ꢀ5.83
ꢀ5.55
ꢀ5.50
>ꢀ4.30 >ꢀ4.30
>ꢀ4.30 >ꢀ4.30
ꢀ4.42 >ꢀ4.30
ꢀ5.13 >ꢀ4.30
>ꢀ4.30 >ꢀ4.30
Non-small cell lung cancer EKVX
HOP-62
ꢀ4.94
ꢀ5.40
ꢀ5.10
ꢀ4.44
ꢀ4.59
ꢀ4.41
HOP-92
NCI-H522
COLO 205
HT29
Colon cancer
CNS cancer
>ꢀ4.30 >ꢀ4.30
>ꢀ4.30 >ꢀ4.30
ꢀ4.66 >ꢀ4.30
ꢀ4.65 >ꢀ4.30
4. Conclusion
SF-295
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
UACC-62
IGROV1
OVCAR-3
OVCAR-4
786e0
ACHN
CAKI-1
RXF 393
PC-3
DU-145
In the present paper, synthesis of novel [(3-R-2-oxo-2H-[1,2,4]
triazino[2,3-s]quinazoline-6-yl)thio]acetic acids and N-cycloalkyl-
(cycloalkylaryl)-2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazo-
line-6-yl)thio]acetamides were described. The antibacterial, anti-
fungal and bioluminiscence inhibition activities of the synthesized
substances against E. coli, S. aureus, M. luteum, C. tenuis, A. niger and
P. leiognathi and were investigated and determinated as moderate.
Six compounds (3.1-3.4, 4.3, 4.10) were tested for anticancer
activity against leukemia, melanoma, lung, colon, CNS, ovarian,
renal, prostate and breast cancers cell lines. The most potent anti-
cancer compound appeared to be 4.10 selectively influencing the
non-small cell lung and CNS cancer cell lines, especially the HOP-92
(log GI50 ¼ ꢀ6.01) and U251 (log GI50 ¼ ꢀ6.00). The obtained
results have proved the necessity of the further investigations of
the antitumor potential of [1,2,4]triazino[2,3-c]quinazoline
derivatives.
ꢀ4.94
ꢀ5.64
ꢀ4.51
ꢀ5.24
Melanoma
ꢀ4.74 >ꢀ4.30
ꢀ4.61 >ꢀ4.30
ꢀ4.58 >ꢀ4.30
ꢀ4.44 >ꢀ4.30
Ovarian cancer
Renal cancer
ꢀ5.25
ꢀ5.20
ꢀ4.55
ꢀ4.36
ꢀ4.80 >ꢀ4.30
>ꢀ4.30 >ꢀ4.30
ꢀ4.41 >ꢀ4.30
ꢀ5.38
ꢀ4.76
Prostate cancer
Breast cancer
ꢀ4.61 >ꢀ4.30
ꢀ4.43 >ꢀ4.30
ꢀ4.68 >ꢀ4.30
ꢀ4.70 >ꢀ4.30
>ꢀ4.30 >ꢀ4.30
MDA-MB-231/ATCC ꢀ5.51
HS 578T
ꢀ5.41
ꢀ5.43
ꢀ5.87
T-47D
MDA-MB-468
ꢀ5.40
ꢀ4.52

G.G. Berest et al. / European Journal of Medicinal Chemistry 46 (2011) 6066e6074
6071
5. Experimental protocols
90 Ad), 51.72 (10 Ad),118.55 (11a),126.05 (8), 126.65 (10),127.88 (11),
136.03 (9), 144.24 (11b), 151.94 (3), 154.94 (7a), 155.16 (6), 161.02
(2), 166.17 (CONH); LC-MS, m/z ¼ 436 [M þ 1], 438 [M þ 3]; Anal.
calcd. for C23H25N5O2S: C, 63.43; H, 5.79; N, 16.08; S, 7.36; Found: C,
63.42; H, 5.79; N, 16.08; S, 7.34.
5.1. Chemistry
Melting points were determined in open capillary tubes and
were uncorrected. Elemental analyses (C, H, N) performed at the
ELEMENTAR vario EL Cube analyzer (USA) and were within ꢃ0.3%
from the theoretical values. IR spectra (4000e600 cmꢀ1) were
recordedona Bruker ALPHAFTeIR spectrometer (Bruker Bioscience,
Germany) using a module for measuring attenuated total reflection
(ATR). 1H NMR spectra (400 MHz) and 13C NMR spectra (100 MHz):
were recorded on a Varian-Mercury 400 (Varian Inc., Palo Alto, CA,
USA) spectrometer with TMS as internal standard in DMSO-d6
solution. LC-MS were recorded using chromatography/mass spec-
trometric system which consists of high performance liquid chro-
matograph “Agilent 1100 Series” (Agilent, Palo Alto, CA, USA)
equipped with diode-matrix and mass-selective detector “Agilent
LC/MSD SL” (atmospheric pressure chemical ionization e APCI).
Electron impact mass spectra (EI-MS) were recorded on a Varian
1200 L instrument at 70 eV (Varian Inc., Palo Alto, CA, USA).
Substances 2.1e2.4 and 3.1e3.4 were synthesized according to
the reported procedures [31,32]. Other starting materials and
solvents were obtained from commercially available sources and
used without additional purification.
5.1.3. N-1-Adamantyl-2-[(3-phenyl-2-oxo-2H-[1,2,4]triazino[2,3-c]
quinazolin-6-yl)thio]acetamides (4.2)
Yield: Method A, 56.3%; Method B, 77.4%; Method C, 91.3%; M.p.
214e216 ꢄC; IR (cmꢀ1): 3287, 3067, 2904, 2848, 1674, 1644, 1590,
1555, 1509, 1487, 1469, 1455, 1359, 1337, 1310, 1286, 1269, 1242,
1183, 1161, 1136, 1102, 1020, 988, 939, 878, 844, 811, 782, 773, 753,
688, 668, 653, 615; 1H NMR (400 MHz) : 1.63 (s, 6H, H-40, 60, 100
d
Ad), 1.97 (s, 6H, H-20, 80, 90 Ad), 2.01 (s, 3H, H-30, 50, 70 Ad), 3.99 (s,
2H, eSeCH2e), 7.64e7.57 (m, 3H, H-30, 40, 50 3-Ph), 7.69 (t, 1H,
J ¼ 7.7 Hz, H-10), 7.76 (d, 1H, J ¼ 7.8 Hz, H-8), 7.88 (s, 1H, eNHC(O)),
8.01 (t,1H, J ¼ 7.7 Hz, H-9), 8.28 (d, 2H, J ¼ 8.1 Hz, H-20, 60 3-Ph), 8.49
(d, 1H, J ¼ 7.9 Hz, H-11); LC-MS, m/z ¼ 498 [M þ 1], 500 [M þ 3];
Anal. calcd. for C28H27N5O2S: C, 67.58; H, 5.47; N, 14.07; S, 6,44;
Found: C, 67.58; H, 5.47; N, 14.09; S, 6.46.
5.1.4. N-1-Adamantyl-2-[(3-(4-methylphenyl)-2-oxo-2H-[1,2,4]
triazino[2,3-c]quinazolin-6-yl)thio]acetamides(4.3)
Yield: Method A, 72.6%; Method C, 94.2%; M.p. 218e220 ꢄC; IR
(cmꢀ1): 3318, 3065, 3037, 2972, 2905, 2849, 1682, 1658, 1588, 1562,
1538, 1496, 1470, 1453, 1399, 1377, 1359, 1339, 1321, 1304, 1285,
1269, 1240, 1182, 1164, 1154, 1139, 1119, 1089, 1045, 989, 939, 876,
831, 810, 784, 770, 710, 701, 685, 653, 641, 625; 1H NMR (400 MHz)
5.1.1. General procedure for synthesis of N-cycloalkyl-(cycloalkaryl)-
2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]
acetamides (4.1e4.10)
Method A. The thionyl chloride (1.2 g, 0.011 mol) was added to
the solution of proper acid (3.1e3.4) (0.01 mol) in 10 mL of anhy-
drous dioxane with subsequent adding of the 2e3 drops of DMF.
Mixture was heated at the water bath at 60e80ꢄS till full elimi-
nation of hydrochloric acid. Then proper amine (0.01 mol) was
added to the resulting mixture with stirring and refluxing for
2e3 h. The mixture was poured in the water, neutralized to pH 6e7
by acetic acid. The precipitate was filtered, dried and recrystallized
from dioxaneewater (1:1).
d
: 1.63 (s, 6H, H-40, 60, 100 Ad), 1.98 (s, 6H, H-20, 80, 90 Ad), 2.01 (s, 3H,
H-30, 50, 70 Ad), 2.42 (s, 3H, 3-(4-CH3Ph)), 3.99 (s, 2H, eSeCH2e),
7.39 (d, 2H, H-30, 50 3-(4-CH3Ph)), 7.68 (t, 1H, J ¼ 7.7 Hz, H-10),
7.82e7.74 (m, 2H, H-8, eNHC(O)), 7.98 (t, 1H, J ¼ 7.7 Hz, H-9), 8.24
(d, 2H, H-20, 60 3-(4-CH3Ph)), 8.48 (d, 1H, J ¼ 7.9 Hz, H-11); 13C NMR
(100 MHz): d
: 21.66 (CH3), 29.30 (30, 50, 70 Ad), 36.49 (40, 60, 100 Ad),
36.79 (eSCH2), 41.43 (20, 80, 90 Ad), 51.74 (10 Ad), 118.25 (11a), 126.09
(8), 126.73 (10), 127.93 (11), 129.32 (10 Ph), 129.53 (20, 60 Ph), 129.78
(30, 50 Ph), 135.99 (9), 142.20 (40 Ph), 144.20 (3), 149.29 (11b), 150.80
(7a), 155.20 (6), 160.13 (2), 166.17 (CONH); Anal. calcd. for
C29H29N5O2S: C, 68.08; H, 5.71; N, 13.69; S, 6.27; Found: C, 69.00; H,
5.71; N, 13.70; S, 6.27.
Method B. N,N0-carbonyldiimidazole (1.95 g, 0.011 mol) was
added to a solution of proper acid (3.1e3.4) (0.01 mol) in 10 mL of
anhydrous dioxane or DMF and heated at the water bath at 60-80ꢄC
for 1 h with calcium chloride tube. The proper amine (0.01 mol) was
added with stirring to the resulting mixture and refluxed for 2e3 h.
The mixture was poured in the water, neutralized to pH 6e7 by
acetic acid. The precipitate was filtered, dried and recrystallized
from the dioxaneewater (1:1).
5.1.5. N-[4-(1-Adamantyl)phenyl]-2-[3-methyl-2-oxo-2H-[1,2,4]
triazino[2,3-c]quinazolin-6-yl thio]acetamides (4.4)
Yield: Method A, 72.0%; Method B, 81.2%; Method C, 97.3%; M.p.
225e227 ꢄC; IR (cmꢀ1): 3251, 3184, 2897, 2845, 1655, 1583, 1558,
1503, 1466, 1449, 1433, 1405, 1393, 1361, 1332, 1315, 1286, 1259,
1220, 1206, 1190, 1163, 1131, 1101, 1046, 1016, 969, 955, 832, 807,
Method
C.
N-cycloalkyl-(cycloalkaryl)-2-chloroacetamides
(0.011 mol) were added to a suspension of Potassium salt of 3-R-
6-thio-6,7-dihydro-2H-[1,2,4]triazino[2,3-c]quinazolin-2-ones
(2.1e2.6) (0.01 mol) in 20 ml of propanol-2, water-propanol-2 (1:2),
or dioxane and refluxed for 60e90 min. The mixture was cooled
and poured in the water. The precipitate was filtered, dried and
recrystallized from the dioxaneewater (1:1).
772, 715, 699, 685, 630, 607; 1H NMR (400 MHz) : 1.73 (s, 6H, H-40,
d
60,100 Ad),1.84 (s, 6H, H-20, 80, 90 Ad), 2.05 (s, 3H, H-30, 50, 70 Ad), 2.39
(s, 3H, 3-CH3e), 4.21 (s, 2H, eSeCH2e), 7.30 (d, 2H, J ¼ 8.5 Hz, H-30,
50 eNHC6H4-Ad), 7.55 (d, 2H, J ¼ 8.5 Hz, H-20, 60 eNHC6H4-Ad), 7.66
(t, 1H, J ¼ 7.7 Hz, H-10), 7.70 (d, 1H, J ¼ 7.9 Hz, H-8), 7.95 (t, 1H,
J ¼ 7.7 Hz, H-9), 8.45 (d, 1H, J ¼ 7.9 Hz, H-11), 10.36 s, 1H, eNHC(O);
LC-MS, m/z ¼ 512 [M þ 1], 514 [M þ 3]; Anal. calcd. for
C29H29N5O2S: C, 68.08; H, 5.71; N, 13.69; S, 6.27; Found: C, 68.10; H,
5.71; N, 13.70; S, 6.29.
5.1.2. N-1-Adamantyl-2-[(3-methyl-2-oxo-2H-[1,2,4]triazino[2,3-c]
quinazolin-6-yl)thio]acetamides (4.1)
Yield: Method A, 48.4%; Method B, 64.3%; Method C, 89.2%; M.p.
160e162 ꢄC; IR (cmꢀ1): 3516, 3358, 3259, 3077, 2907, 2849, 1676,
1663, 1585, 1558, 1505, 1470, 1425, 1388, 1360, 1344, 1306, 1286,
1265, 1210, 1165, 1139, 1103, 1045, 998, 956, 885, 862, 813, 773, 687,
5.1.6. N-[4-(1-Adamantyl)phenyl]-2-[3-phenyl-2-oxo-2H-[1,2,4]
triazino[2,3-c]quinazolin-6-yl thio]acetamides (4.5)
633, 608; 1H NMR (400 MHz)
d
: 1.62 (s, 6H, H-40, 60, 100 Ad), 1.96 (s,
Yield: Method A, 78.4%; Method C, 94.5%; M.p. 314e318 ꢄC; IR
(cmꢀ1): 3349, 3047, 2982, 2899, 2849, 1678, 1657, 1596, 1587, 1564,
1546, 1519, 1498, 1486, 1470, 1444, 1407, 1380, 1341, 1331, 1311, 1287,
1263, 1246, 1237, 1187, 1171, 1138, 1100, 1076, 1033, 1017, 1002, 988,
963, 941, 895, 877, 848, 836, 810, 779, 768, 751, 706, 687, 665, 654,
6H, H-20, 80, 90 Ad), 2.01 (s, 3H, H-30, 50, 70 Ad), 2.39 (s, 3H, 3-CH3),
3.94 (s, 2H, -S-CH2-), 7.67 (t, 1H, J ¼ 7.7 Hz, H-10), 7.74 (d, 1H,
J ¼ 7.9 Hz, H-8), 7.84 (s, 1H, eNHC(O)), 7.98 (t, 1H, J ¼ 7.7 Hz, H-9),
8.46 (d, 1H, J ¼ 7.9 Hz, H-11); 13C NMR (100 MHz)
d: 18.18 (CH3),
29.23 (30, 50, 70 Ad), 36.49 (40, 60, 100 Ad), 36.68 (eSCH2), 41.43 (20, 80,
636; 1H NMR (400 MHz) : 1.73 (s, 6H, H-40, 60, 100 Ad), 1.84 (s, 6H,
d

6072
G.G. Berest et al. / European Journal of Medicinal Chemistry 46 (2011) 6066e6074
H-20, 80, 90 Ad), 2.05 (s, 3H, H-30, 50, 70 Ad), 4.25 (s, 2H, eSeCH2e),
7.31 (d, 2H, J ¼ 8.5 Hz, H-30, 50 eNHC6H4-Ad), 7.56 (d, 2H, J ¼ 8.5 Hz,
H-20, 60 eNHC6H4-Ad), 7.77e7.59 (m, 5H, H-30, 40, 50 3-Ph, H-8, 10),
7.98 (t,1H, J ¼ 7.7 Hz, H-9), 8.30 (d, 2H, J ¼ 7.3, H-20, 60 3-Ph), 8.49 (d,
1173, 1133, 1102, 1045, 955, 880, 856, 768, 698, 685, 630, 607; 1H
NMR (400 MHz)
: 1.48e0.94 (m, 11H, 3-CH3CH2-, 3-CH3CH2-, 5, 50,
d
6, 60, 7 bicyclo[2.2.1]heptyl), 2.16e2.09 (m, 2H, H-30, 40 bicyclo[2.2.1]
heptyl), 2.38 (s, 3H, 3-CH3), 2.66 (m, 1H, H-10 bicyclo[2.2.1]heptyl),
3.50e3.39 (m, 1H, H-20 bicyclo[2.2.1]heptyl), 3.96 (s., 2H,
eSeCH2e), 7.66 (t, 1H, J ¼ 7.7 Hz, H-10), 7.73 (d, 1H, J ¼ 7.9 Hz, H-8),
7.97 (t, 1H, J ¼ 7.7 Hz, H-9), 8.05 (d, 1H, J ¼ 8.5 Hz, eNHC(O)e), 8.46
1H, J ¼ 7.9 Hz, H-11), 10.40 (s, 1H, eNHC(O); EI-MS, m/z (Irel
,
%) ¼ 396 (6.3), 395 (5.7), 348 (16.9), 347 (73.5), 346 (49.1), 345
(16.1), 339 (7.2), 316 (5.4), 307 (8.7), 302 (15.9), 301 (64.8), 269
(16.0), 268 (5.2), 258 (5.1), 254 (13.8), 253 (57.9), 252 (10.8), 246
(7.0), 245 (16.1), 244 (99.9), 243 (26.4), 240 (5.4), 228 (5.8), 227
(49.5), 226 (8.0), 218 (25.8), 217 (29.8), 216 (58.8), 213 (8.9), 212
(7.1), 211 (7.0), 210 (16.6), 197 (9.5), 196 (55.8), 195 (5.6), 189 (5.4),
188 (9.0), 186 (7.2), 185 (20.9), 184 (11.2), 183 (7.0), 182 (8.6), 180
(6.1), 179 (8.3), 178 (43.2), 170 (42.5), 161 (5.0), 160 (5.6), 159 (17.8),
158 (9.6), 157 (9.0), 156 (20.0),155 (8.7), 154 (9.4), 153 (12.0), 152
(9.6), 148 (21.9), 145 (8.0), 144 (7.8), 143 (16.9), 142 (7.5), 141 (6.0),
135 (22.4), 134 (8.4), 133 (24.1), 132 (32.9),131 (12.6),130 (16.0),129
(22.9), 128 (18.2), 127 (12.5), 119 (10.4), 118 (21.9), 117 (22.4), 116
(18.3), 115 (12.5), 106 (5.3), 105 (7.2), 104 (14.4), 103 (80.9), 101
(12.8), 95 (7.0), 94 (27.6), 93 (34.4), 92 (12.9), 90 (12.7), 89 (10.5), 86
(13.8), 79 (26.3), 77 (10.2), 75 (5.4), 67 (16.5), 66 (7.4), 65 (21.6), 64
(14.3), 63 (15.4), 56 (14.9), 55 (19.1), 53 (6.1), 47 (9.2), 43 (15.2), 41
(21.1)LC-MS, m/z ¼ 574 [M þ 1], 575 [M þ 2], 576 [M þ 3]; Anal.
calcd. for C34H31N5O2S: C, 71.18; H, 5.45; N, 12.21; S, 5.59; Found: C,
71.19; H, 5.45; N, 12.23; S, 5.61.
(d, 1H, J ¼ 7.9 Hz, O-11); 13C NMR (100 MHz):
d: 18.17 (CH3), 18.79
(CH3CH2), 20.70 (60), 28.87 (CH3CH2), 30.33 (50), 35.78 (eSCH2),
36.33 (70), 36.85 (10), 38.95 (40), 48.51 (30), 49.37 (20), 118.50 (11a),
125.99 (8), 126.70 (10), 127.90 (11), 135.91 (9), 144.21 (3), 151.92
(11b), 154.74 (7a), 155.16 (6), 160.98 (2), 166.39 (CONH); EI-MS, m/z
(Irel, %) ¼ 303 (7.2), 302 (45.1), 285 (31.8), 246 (7.9), 245 (24.1), 244
(100.0), 143 (16.3), 218 (5.6), 217 (17.7), 216 (51.5), 188 (4.9), 179
(6.3), 170 (7.9), 148 (12.6), 143 (7.7), 129 (7.0), 123 (5.9), 122 (9.0), 95
(24.4), 93 (14.0), 91 (5.3), 90 (8.6), 81 (11.8), 67 (20.2), 57 (8.7), 56
(9.4), 55 (12.9); LC-MS, m/z ¼ 424 [Mþ1], 426 [Mþ3]; Anal. calcd.
for C22H25N5O2S: C, 62.39; H, 5.95; N, 16.54; S, 7.57; Found: C,
62.40; H, 5.95; N, 16.54; S, 7.58.
5.1.10. N-(3-ethylbicyclo[2.2.1]hept-2-yl)-2-[3-(4-methylphenyl)-
2-oxo-2H-[1,2,4]triazino[2,3-c]quinazolin-6-yl thio]acetamides (4.9)
Yield: Method A, 67.3%; Method C, 87.3%; M.p. 248e250 ꢄC; IR
(cmꢀ1): 3285, 3078, 2950, 2868, 1668, 1633, 1589, 1562, 1549, 1501,
1468,1454,1391,1372,1335,1308,1270,1239,1183,1135,1104,1019,
991, 939, 830, 782, 770, 712, 699, 684, 641, 626; 1H NMR (400 MHz)
5.1.7. N-[4-(1-Adamantyl)phenyl]-2-[3-(4-methylphenyl)-2-oxo-
2H-[1,2,4]triazino[2,3-c]quinazolin-6-yl thio]acetamides (4.6)
Yield: Method B, 84.3%; Method C, 99.9%; M.p. 266e268 ꢄC; IR
(cmꢀ1): 3344, 2982, 2915, 2898, 2882, 2849, 1675, 1655, 1584, 1562,
1542, 1517, 1490, 1469, 1451, 1406, 1368, 1340, 1329, 1308, 1285,
1264, 1237, 1189, 1139, 1125, 1106, 1071, 1036, 1017, 989, 961, 942,
893, 875, 834, 807, 779, 768, 708, 686, 665, 643, 624; 1H NMR
d
: 1.51e0.94 (m, 11H, 3-CH3CH2e, 3-CH3CH2e, 5, 50, 6, 60, 7 bicyclo
[2.2.1]heptyl, 2.16e2.09 (m, 2H, H-30, 40 bicyclo[2.2.1]heptyl), 2.42
(s, 3H, 3-(4-CH3Ph), 2.67 (m, 1H, H-10 bicyclo[2.2.1]heptyl),
3.60e3.41 (m, 1H, H-20 bicyclo[2.2.1]heptyl), 4.00 (s., 2H,
eSeCH2e), 7.41 (d, 2H, J ¼ 7.5 Hz, H-3, H-5 4eCH3Ph), 7.68 (t, 1H,
J ¼ 7.7 Hz, H-10), 7.76 (d, 1H, J ¼ 7.9 Hz, H-8), 7.98 (t, 1H, J ¼ 7.7 Hz,
H-9), 8.07 (d, 1H, J ¼ 8.5 Hz, eNHC(O)e), 8.24 (d, 2H, J ¼ 7.5 Hz, H-2,
H-6 4eCH3Ph), 8.48 (d, 1H, J ¼ 7.9 Hz, H-11); LC-MS, m/z ¼ 500
[M þ 1], 502 [M þ 3]; Anal. calcd. for C28H29N5O2S: C, 67.31; H, 5.85;
N, 14.02; S, 6.42; Found: C, 67.31; H, 5.85; N, 14.02; S, 6.42.
(400 MHz) d
: 1.73 (s, 6H, H-40, 60, 100 Ad), 1.84 (s, 6H, H-20, 80, 90 Ad),
2.05 (s, 3H, H-30, 50, 70 Ad), 2.42 (s, 3H, 3-(4-CH3Ph)), 4.25 (s, 2H,
eSeCH2e), 7.31 (d, 2H, J ¼ 8.5, H-30, 50 eNHC6H4-Ad), 7.41 (d, 2H,
J ¼ 7.9 Hz, H-30, 50 3-(4eCH3Ph)), 7.56 (d, 2H, J ¼ 8.5 Hz, H-20, 60
eNHC6H4-Ad), 7.68 (t,1H, J ¼ 7.7 Hz, H-10), 7.74 (d, 1H, J ¼ 7.8 Hz, H-
8), 7.99 (t, 1H, J ¼ 7.7 Hz, H-9), 8.25 (d, 2H, J ¼ 7.9 Hz, H-20, 60 3-(4-
CH3Ph)), 8.49 (d, 1H, J ¼ 7.9 Hz, H-11), 10.39 (s, 1H, eNHC(O); LC-
5.1.11. N-(3-ethylbicyclo[2.2.1]hept-2-yl)-2-{[3-(4-
methoxyphenyl)-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazolin-6-yl
thio]acetamides (4.10)
MS, m/z
¼
588 [M
þ
1], 590 [M
þ
3]; Anal. calcd. for
C35H33N5O2S: C, 71.53; H, 5.66; N, 11.92; S, 5.46; Found: C, 71.53; H,
5.66; N, 11.92; S, 5.44.
Yield: Method B, 73.8%; Method C, 89.6%; M.p. 230e233 ꢄC; IR
(cmꢀ1): 3274, 3067, 2945, 2866, 1668, 1643, 1590, 1563, 1545, 1500,
1469, 1454, 1421, 1372, 1340, 1317, 1306, 1288, 1272, 1259, 1238,
1174, 1137, 1105, 1049, 1016, 989, 965, 940, 878, 840, 810, 766, 723,
5.1.8. N-[4-(1-Adamantyl)phenyl]-2-[3-(4-methoxyphenyl)-2-oxo-
2H-[1,2,4]triazino[2,3-c]quinazolin-6-yl thio]acetamides (4.7)
Yield: Method B, 64.7%; Method C, 93.2%; M.p. 259e262 ꢄC; IR
(cmꢀ1): 3346, 2982, 2898, 2849, 1678, 1654, 1597, 1563, 1538, 1518,
1504, 1490, 1469, 1452, 1406, 1370, 1341, 1315, 1304, 1255, 1244,
1177, 1140, 1117, 1050, 1036, 1016, 989, 942, 879, 843, 808, 780, 769,
700, 684, 641, 622; 1H NMR (400 MHz)
d: 1.47e0.92 (m, 11H, 3-
CH3CH2-, 3-CH3CH2e, 5, 50, 6, 60, 7 bicyclo[2.2.1]heptyl), 2.16e2.11
(m, 2H, H-30, 40 bicyclo[2.2.1]heptyl), 2.60 (m,1H, H-10 bicyclo[2.2.1]
heptyl), 3.55e3.43 (m, 1H, H-20 bicyclo[2.2.1]heptyl), 3.87 (s, 3H, 3-
(4-CH3OPh), 4.00 (s, 2H, eSeCH2e), 7.16 (d, 2H, J ¼ 7.9 Hz, H-3, H-5
4eCH3OPh), 7.68 (t,1H, J ¼ 7.7 Hz, H-10), 7.76 (d,1H, J ¼ 7.9 Hz, H-8),
7.98 (t, 1H, J ¼ 7.7 Hz, H-9), 8.08 (d, 1H, J ¼ 8.5 Hz, eNHC(O)e), 8.37
(d, 2H, J ¼ 7.9 Hz, H-2, H-6 4eCH3OPh), 8.48 (d,1H, J ¼ 7.9 Hz, H-11);
LC-MS, m/z ¼ 516 [M þ 1], 518 [M þ 3]; Anal. calcd. for
C28H29N5O3S: C, 65.22; H, 5.67; N, 13.58; S, 6.22; Found: C, 65.24; H,
5.67; N, 13.56; S, 6.20.
722, 701, 687, 665, 643, 621; 1H NMR (400 MHz) : 1.73 (s, 6H, H-40,
d
60,100 Ad),1.84 (s, 6H, H-20, 80, 90 Ad), 2.05 (s, 3H, H-30, 50, 70 Ad), 3.88
(s, 3H, 3-(4-CH3OPh)), 4.25 (s, 2H, eSeCH2e), 7.17 (d, 2H, J ¼ 8.3, H-
30, 50 3-(4eCH3OPh)), 7.31 (d, 2H, J ¼ 8.5 Hz, H-30, 50eNHC6H4-Ad),
7.56 (d, 2H, J ¼ 8.5 Hz, H-20, 60 eNHC6H4-Ad), 7.68 (t, 1H, J ¼ 7.7 Hz,
H-10), 7.74 (d, 1H, J ¼ 7.8 Hz, H-8), 7.97 (t, 1H, J ¼ 7.7 Hz, H-9), 8.39
(d, 2H, J ¼ 8.3, H-20, 60 3-(4-CH3OPh)), 8.49 (d, 1H, J ¼ 7.9 Hz, H-11),
10.40 (s, 1H, eNHC(O); LC-MS, m/z ¼ 604 [M þ 1], 607 [M þ 3];
Anal. calcd. for C35H33N5O3S: C, 69.63; H, 5.51; N, 11.60; S, 5,31;
Found: C, 69.64; H, 5.51; N, 11.61; S, 5.32.
5.2. Biological assay
5.2.1. Bioluminescence inhibition test
The marine luminescent bacteria P. leiognathi strain Sh1, isolated
from the Azov Sea Shrimp, were used for the bioluminescence
analysis [36]. Bacteria were cultivated on a nutrient environment
containing (g/L): pepton e 5, yeast extract e 1.5, meat extract e 1.5,
sodium chloride e 30, pH ¼ 7.4. In the acute action test (inhibiting
luminescence of bacteria), bacteria were diluted in the 3% sodium
5.1.9. N-(3-ethylbicyclo[2.2.1]hept-2-yl)-2-[3-methyl-2-oxo-2H-[1,2,4]
triazino[2,3-c]quinazolin-6-yl thio]acetamides (4.8)
Yield: Method A, 36.9%; Method B, 81.3%; Method C, 84.6%; M.p.
217e219 ꢄC; IR (cmꢀ1): 3287, 2944, 2866, 1667, 1636, 1584, 1558,
1538, 1509, 1469, 1454, 1418, 1386, 1360, 1337, 1287, 1261, 1208,

G.G. Berest et al. / European Journal of Medicinal Chemistry 46 (2011) 6066e6074
6073
chloride solution up to a concentration of 105 cells/mL. 5e50 lg/mL
of the studied substances suspended in DMSO was mixed with 1 mL
of the diluted bacterial suspension. Vials were incubating for
10 min at 25 ꢄC, then the intensity of bioluminescence was
measured in percent (%) relative to the control, which was prepared
without the studied compounds. In the chronic action test (inhib-
iting growth and luminescence of bacteria), growth medium was
added for potential breeding in a ratio of 1:50 and the mix was
incubated for 16e18 h at 30 ꢄC. Whereupon the intensity of
bioluminescence was measured the same way as in acute action
testing. Tetracycline was used as a reference. The bacterial lumi-
calculated from [(Ti ꢀ Tz)/Tz] ꢅ 100 ¼ ꢀ50. Values were calculated
for each of these three parameters if the level of activity was
reached; however, if the effect was not reached or was exceeded,
the value for that parameter was expressed as greater or less than
the maximum or minimum concentration tested. The log GI50, log
TGI, log LC50 were then determined, defined as the mean of the
log’s of the individual GI50, TGI, LC50 values. The lowest values were
obtained with the most sensitive cell lines.
Acknowledgements
nescence was measured with
(“Science”, Krasnoyarsk, Russia).
a Bioluminometer BLM-8801
The authors gratefully acknowledge “Enamine Ltd.” (Kiev,
Ukraine) for financial support of this work, and team of the Drug
Synthesis and Chemistry Branch, National Cancer Institute,
Bethesda, MD, USA, for in vitro evaluation of anticancer activity. We
are grateful to Lviv Polytechnic National University for in vitro
evaluation of antibaterial activity. The authors have declared no
conflict of interest.
5.2.2. Antimicrobial and antifungal test
The investigation of antimicrobial and antifungal activity of
compounds 1.1e1.4 and .3.1e3.10 was carried out using the stiff
plate agar diffusion method against E. coli, S. aureus, M. luteum,
C. tenuis and A. niger [37]. The amount of microbial cells was
109 c.f.u./mL. Incubation period of bacteria was 24 h at 35 ꢄC, yeast
e 48e72 h at 28e30 ꢄC. Antibiotics vancomicin, oxacillin, nystatin
were used as standards. The bacterial cultures, standards and the
obtained substances were streaked across grooves in 5 mg/mL
concentration, and then allowed to diffuse in the agar nutrient
plate. The antimicrobial effect and degree of activity of the tested
compounds were evaluated by measuring of the inhibition zone
diameters (not sensitive: 11e15 mm; sensitive: 16e25 mm; highly
sensitive >25 mm). The results were compared with well-known
drug standards vancomicin, nystatin, and oxacillin. All experi-
ments were repeated three times.
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