Novel N-aryl(alkaryl)-2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6- yl)thio]acetamides: Synthesis, cytotoxicity, anticancer activity, COMPARE analysis and docking

MEDICINAL

CHEMISTRY

RESEARCH

Med Chem Res (2013) 22:2610–2632

DOI 10.1007/s00044-012-0257-x

ORIGINAL RESEARCH

Novel N-aryl(alkaryl)-2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-

c]quinazoline-6-yl)thio]acetamides: synthesis, cytotoxicity,

anticancer activity, COMPARE analysis and docking

•

Sergey I. Kovalenko Inna S. Nosulenko Alexey Yu. Voskoboynik

•

Galina G. Berest Lyudmila N. Antipenko Alexey N. Antipenko

Andrey M. Katsev

Received: 21 May 2012 / Accepted: 21 September 2012 / Published online: 7 October 2012

ꢀ Springer Science+Business Media New York 2012

Abstract The series of novel N-aryl(alkaryl)-2-[(3-R-2-

oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-yl)thio]acetami

des were obtained by aminolysis of activated acids 1a–1d

and by alkylation of potassium salts 2a–2d by N-aryl-2-

chloroacetamies. The structures of compounds were

determined by IR, ¹H, ¹³C NMR, LC- and EI-MS analyses.

The results of cytotoxicity evaluation by bioluminescence

inhibition of bacterium Photobacterium leiognathi Sh1

showed that compounds reveal moderate cytotoxicity.

Screening of anticancer activity in vitro yielded the most

active compounds 3t, 4b, 4f, 4l and 4n in micromolar

concentrations with the GI₅₀level (logGI₅₀is from -7.57

to -4.05 for different cell lines, and logGI₅₀mean graph

midpoint varied from -5.30 to -4.50). Moreover, com-

pound 4b had a distinctive selectivity against renal cancer,

4f—colon cancer and melanoma and 4l—renal cancer. The

highest sensitivity to compound 4b showed renal cancer

cell lines A498 (logGI₅₀= -7.57). SAR-analysis was

discussed, COMPARE analysis and docking was studied.

Introduction

Quinazoline derivatives is a classical group of natural and

synthetic biologically active compounds in modern medical

chemistry, which are characterized by a wide and diversiﬁed

spectra of biological activities (Abou-Seri et al., 2011;

Alafeefy et al., 2010; Aniszewski, 2007; Armarego, 2008;

Bertelli et al., 2000; Michael, 2005; Nosova et al., 2009,

Rather et al., 2010; Redondo et al., 2012; Rohini et al., 2009).

Recently, the search of antitumour agents based on quinaz-

oline backbone has become one of the priority areas of sus-

tainable design ‘drug-like molecules’ (Al-Obaid et al., 2009;

Al-Omary et al., 2012; Cao et al., 2009; Chandrika et al.,

2008; Denny, 2001a, b; Kidwai et al., 2002; Li et al., 2012;

¨

Lu¨th and Lowe, 2008; Pawar et al., 2010; Richter and Gi-

offreda, 2011; Sharma et al., 2004; Yadav et al., 2012; Zhang

et al., 2009). Synthetic and screening studies of condensed

quinazolines, which were conducted by our research school

in cooperation with the National Cancer Institute (NCI),

revealed the signiﬁcant antitumour potential among

S-substituted 6-thio-3-R-6,7-dihydro-2H-[1,2,4]triazin[2,3-

c]quinazolin-2-ones (Berest et al., 2011, 2012). These

derivatives exhibited a broad spectrum of antitumour activ-

ity in micromole concentrations with the GI₅₀level (logGI₅₀

is from -6.29 to -5.01 for different cell lines, and logGI₅₀

mean graph midpoint varied from -5.57 up to -5.30).

Conducted SAR-analysis discovered a number of critical

‘pharmacophore’ fragments in the structure of the synthe-

sized compounds and determined the best areas for the fur-

ther modiﬁcation of the molecule, namely functionalizing

substituents in position 3 by introduction of the aryl radical

and substituents in position 6 by alkylating reagents. Thus,

synthesis of unknown S-substituted 6-thio-3-R-2-oxo-2H-

[1,2,4]triazino[2,3-c]quinazolines, detailed SAR-analysis

database and investigation of their possible molecular

Keywords

N-Aryl(alkaryl)-2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-

c]guinazoline-6-yl)thio]acetamides ꢀ Bioluminescence ꢀ

Cytotoxicity ꢀ Anticancer ꢀ Docking

S. I. Kovalenko (&) ꢀ I. S. Nosulenko ꢀ

A. Yu. Voskoboynik ꢀ G. G. Berest ꢀ L. N. Antipenko ꢀ

A. N. Antipenko

Organic and Bioorganic Chemistry Department, Zaporozhye

State Medical University, 26, Mayakovsky ave., Zaporozhye

69035, Ukraine

e-mail: kovalenkosergiy@gmail.com

A. M. Katsev

Pharmacy Department, Crimean State Medical University,

Simferopol 95006, Ukraine

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Med Chem Res (2013) 22:2610–2632

2611

mechanisms of action may be considered as a promising

approach for creating of anticancer products.

method of amides synthesis (4a–4r method B) was more

effective and led to quantitative yields of the products

(Scheme 2). Experimental data proved that benzyl- and

phenethylamines were more evident nucleophiles.

Results and discussion

The identiﬁcation of compounds by elemental analysis,

IR, ¹H and ¹³C NMR, LS-MS and EI-MS data proved

purity and structure of synthesized substances. The LC-MS

using positive-ion atmospheric pressure chemical ioniza-

tion (APCI) showed the appropriate molecular ions [M^1?],

which conﬁrmed the expected molecular weights of com-

pounds 3a–3z and 4a–4r. Besides, positive ions [M^2?] and

[M^3?] were present in spectra—the last one characterizes

the presence of Sulphur isotope.

Synthesis of amides 3a–3z was conducted by known meth-

ods: by aminolysis of activated acids 1a–1d, with thionyl

chloride or N,N⁰-carbonyldiimidazole (CDI). Experiments

showed that appropriate acid chloride anhydrides (method

A) or imidazolides (method B) of acids 1a–1d were obtained

quite easily with good yields. It is important to mention that

they were highly reactive with substituted anilines. Two

necessary conditions for the transformation to appropriate

amides 3a–3j were anhydrous dioxane, and heating for

3–6 h. Amides 3a–3z were also obtained by the alternative

approach—alkylation of potassium salts 2a–2d by N-aryl-2-

chloroacetamies (method C) in aqueous–propanol-2 mix-

ture, propanol-2 or dioxane (Scheme 1). The last method had

a range of advantages, notably insigniﬁcant duration of time

(60–90 min), quantitative yields and highly pure products.

We obtained a series of the novel N-aryl-2-[(3-R-2-oxo-

2H-[1,2,4]triazino[2,3-c]quinazoline-6-yl)thio]acetamides

(4a–4r) as a logical conclusion to the systematic search of

‘leader-compound’ with anticancer activity among 2-[(3-R-

2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-yl)thio]aceta

mides and the research of correlation peculiarities of

‘structure-anticancer activity’. Synthesis of these deriva-

tives from pharmacological aspect was determined by the

established fact of decisive inﬂuence of substituent’s

character in 6 position at anticancer activity.

IRspectrashowedwidestretchingsofassociatedNHgroup

at 3,626–3,004 cm^-1in spectra of amides 3a–3z and 4a–4z,

stretchings of m_CO-group (‘Amid I’) at 1,665–1,587 cm^-1and

mixed stretching–deformation vibrations of bonds N–H and

C–N (‘Amid II’) at 1,596–1,510 cm^-1. In addition, amides

3a–3z and 4a–4r could be characterized by triazinoquinazo-

line cycle vibrations of m_CO-group at 1,748–1,649 cm^-1

,

character outline of low-intensive vibrations of m-_C=C–bond of

aromatic ring at 1,589–1,468 cm^-1, out-of-plane vibrations

c

_(=C–H)at 850–666 cm^-1, and intensive vibrations at 2,992–

,829 cm^-1, which were related to symmetric and asymmetric

stretchings of m_CH3– and m_CH2–group.

Protons signals of triazino[2,3-c]quinazoline cycle of

ABCD-system in ¹H NMR spectra of compounds 3a–3z and

4a–4r were observed as two doublet signals of protons H-11

(8.51–7.70 ppm) and H-8 (7.82–7.44 ppm) and two triplets of

protons H-9 (8.01–7.66 ppm) and H-10 (7.71–7.37 ppm)

(Berest etal., 2010, 2011, 2012). Protons of–CONH– and –S–

CH₂–group were character for compounds 3a–3z and 4a–4r.

Thus, proton of –CONH–group according to the surrounding

resonated as singlet (compounds 3a–3z) at 11.77–9.61 ppm

and as triplet (4a–4r) at 8.97–8.36 ppm. Two-proton singlets

of –SCH₂–group of mentioned compounds were registered at

The most convenient method of synthesis of compounds

(4a–4r) was alkylation of potassium salts of 3-R-6-thio-6,7

-dihydro-2H-[1,2,4]triazino[2,3-c]quinazolin-2-ones (2a, 2b)

by N-aryl-2-chloroacetamies (method A) in a mixture of

propanol-2–water (2:1). However, carbonyldiimidazol’s

O

R₁

N

SK

N

S

N

S

N

OH

iii

i, ii

H

N

O

N

O

N

R

O

3a-3z

1a-1d

2a-2d

(i): SOCl₂, dioxane, NH₂C₆H₄R₁; (ii): CDI, dioxane or DMF, NH₂C₆H₄R₁; (iii): propanol-2, or dioxane or propanol-2 - water (2:1), ClCH₂C(O)NHC₆H₄R₁;

1a, 2a R=Me; 1b, 2b R=Ph; 1c, 2c R=4-MeC₆H₄; 1d, 2d R=4-MeOC₆H₄; 3a R=Me, R₁=H; 3b R=Ph, R₁=H; 3c R=4-MeC₆H₄, R₁=2-Me; 3d R=4-MeO₆H₄, R₁=2-Me; 3e R=Me, R₁=3-

Me; 3f R=4-MeOC₆H₄, R₁=4-Me; 3g R=Ph, R₁=2-MeO; 3h R=4-MeC₆H₄, R₁=4-MeO; 3i R=Me, R₁=2-CF₃; 3j R=4-MeOC₆H₄, R₁=2-CF₃; 3k R=Ph, R₁=3-CF₃; 3l R=4-MeC₆H₄,

R₁=3-CF₃; 3m R=4-MeC₆H₄, R₁-4-CF₃; 3n R=4-MeOC₆H₄, R₁=2-F; 3o R=4-MeC₆H₄, 2-F; 3p R=Me, R₁=2-F; 3q R=Ph, R₁=3-F; 3r R=4-MeC₆H₄, R₁-3-F; 3s R=4-MeC6H4, R1=4-

F; 3t R=4-MeOC₆H₄, R₁=2-Cl; 3u R=Ph, R1=3-Cl; 3v R=Me, R₁=3-Cl; 3w R=Ph, R₁=4-Cl; 3x 4-MeC₆H₄, R=4-Cl; 3y R=4-MeC₆H₄, R₁=2-NO₂; 3z R=4-MeC₆H₄, R₁=2-COOH

Scheme 1 Synthesis of the N-aryl-2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-yl)thio]acetamides

123

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Med Chem Res (2013) 22:2610–2632

O

N

S

N

S

N

SK

N

n

N

OH

ii

i

H

R₁

N

O

N

O

R

O

2a, 2b

1a, 1b

4a-4r

(i): propanol-2, dioxane or propanol-2 - water (2:1), ClCH₂C(O)NH(CH₂)nC₆H₄R₁; (ii) CDI, dioxane, or DMF, NH₂(CH₂)nC₆H₄R₁;1a, 2a R=Me; 1b, 2b R=Ph; 4a R=Me, R1=H, n=1;

4b R=Ph, R1=H, n=1; 4c R=Me, R1=4-Me, n=1; 4d R=Ph, R1=4-Me, n=1; 4e R=Me, R1=2-MeO, n=1; 4f R=Ph, R1=2-MeO, n=1; 4g R=Me, R1=4-MeO, n=1; 4h R=Ph, R1=4-MeO,

n=1; 4i R=Me, R1=3-CF₃, n=1; 4j R=Ph, R1=3-CF₃, n=1; 4k R=Me, R1=2-F, n=1; 4l R=Ph, R1=2-F, n=1; 4m R=Me, R1=4-F, n=1; 4n R=Ph, R1=4-F, n=1; 4o R=Me, R1=2-Cl, n=1;

4p R=Ph, R1=2-Cl, n=1; 4q R=Me, R1=H, n=2; 4r R=Ph, R1=H, n=2

Scheme 2 Synthesis of the N-alkaryl-2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-yl)thio]acetamides

4.35–4.02 ppm. It is important to note that proton’s shift of –

CONH– and –S–CH₂– groups of benzyl- (compounds 4a–4p)

and phenacethylamides (4q, 4r) atstrongmagnetic ﬁeld could

be explained by more evident positive inductive effect of

benzyl and phenetyl substituents than that of amides 3a–3z. It

is also important to point that proton’s shift of mentioned

functional groups of amides 3a–3z was strongly inﬂuenced by

the substituent’s character and its position (‘o-effect’) at a-

rylamid substituent. Two-proton doublets of –CH2Ph-group

at 4.45–4.26 ppm in benzylamides (4a–4p) and two two-

proton triplets of –CH₂CH₂Ph-group at 2.75–2.74 and

3.36–3.35 ppm appropriate in phenetylamides demonstrated

anadditionalconformationofthestructure(4q, 4r).Protonsof

methyl group at position 3 in ¹H NMR spectra of compounds

3a, 3e, 3i, 3p, 3v, 4a, 4c, 4e, 4 g, 4i, 4 k, 4 m, 4o and 4q

resonated as three-proton singlets at 2.41–25 ppm, methyl

group of thiol substituent at position 3 of compounds 3c, 3h,

3l, 3m, 3o, 3r, 3s, 3x, 3y and 3z had multiplicity at

2.56–2.38 ppm. On the other hand, methyl group of

4-methoxyphenyl substituent at 3 position of compounds 3d,

3f, 3j, 3n and 3t was more deshielded and could be observed

as three-proton singlet at low-intensive ﬁeld at 3.89–3.57

ppm. Aliphatic protons of amide substituents of compounds

3a–3z and 4a–4r, notably methyl and methoxy group, were

registered as three-proton singlets and had classical chemical

shifts. Aromaticprotonsat3positionofcompounds3a, 3d, 3f,

3h, 3j, 3l–3o, 3r–3t and 3x–3z were observed as classical

A₂B₂-system, two two-proton doublets (H-3, H-5 and H-2,

H-6)—at 8.39–8.07 and 7.41–7.16 ppm. On the other hand,

phenyl substituent at 3 position of synthesized compounds

resonated as multiplet (H-3, H-4, H-5) at 7.69–7.57 ppm and

two-proton doublet (H-2 and H-6) at 8.31–8.29 ppm. Multi-

plicity and chemical shifts of aromatic protons of amide

substituents of compounds 3a–3z and 4a–4r were determined

to their substituents.

163.53–160.07 ppm and 167.59–166.16 ppm appropriately.

The presence of SCH₂-group Carbon at 36.83–35.35 ppm

was an additional conformation of structure and S-regiose-

lectivity of alkylation.

Low intensive peaks of molecular ions in EI-MS spectra of

arylamides 3b, 3g and 3v were present, charactering the break

of amid bond with the formation of intensive ions [heteryl-

SCH₂CO]^?(F₁) with m/z 347 (52.1 %), 347 (100.0 %), 285

(100.0 %) appropriate. Further fragmentation of F₁was the

break of bonds C(2)–C(3) and N(3)–N(4) of triazinoquinazo-

line system with the formation of F₂with m/z 244

(28.4–52.8 %). F₂eliminate fragments CO, SCH₂CO, CNO

forming ions with m/z 216 (20.2–49.2 %), 170 (27.2–37.3 %),

143 (24.0–100 %) and 129 (26.9–70.1 %). b-Break was more

character for benzylamides 4a and 4d in EI-MS spectra with the

formation of ions [Heteryl-S]^?with (F₁) m/z 243/245

(38.7–50.1 %), 305/307 (19.5–56.7 %), which besides proved

the ‘isotopic proﬁle of Sulphur’. The difference of compounds

4a and 4d fragmentation in EI-MS spectra from previous ones

was probably caused by moderate inductive effect of mole-

cule’s benzyl substituent. Further fragmentation of F₁was

caused by break of bonds C(2)–C(3) and N(3)–N(4) of triazi-

noquinazoline system with formation of F₂with m/z 202/204

(7.1–58.6 %). F₂eliminate fragments, CNS and CO forming

ions with m/z 170, 144 and 174 of appropriate intensity in

spectra. Tropilium ions were the most intensive with m/z 91

(100 %) for compound 4a and m/z 105 (100 %) for compound

4d. Speaking about EI-MS spectrum of phenethylamide 4q,

ﬁrst, phenethyl substituent stabilized molecular ion (M^•?-64.

5 %); secondary, M^•?under the inﬂuence of electronic impact

formed a range of fragmentary ions: F₁–[M–C₆H₅C₂H₄]^•?-m/

z 301 (26,4 %); F₂–[M–C₆H₅C₂H₄NH]^•?-m/z 285 (69.6%);

F₃–[M–C₆H₅C₂H₄NHC(O)CH₂]^•?-m/z 243/245 (52.1 %). It

is important to note that further fragmentation was due to the

breaking of the C(2)–C(3) and N(3)–N(4) bonds of triazino-

quinazoline system of the less stable ion F₂with the formation

of F₄p m/z 244 (68.1 %). F₄eliminated fragments CO,

SCH₂CO, forming high stable ions with m/z 216 (100), 170

In ¹³C NMR spectra signals of Carbon in 6, 2 positions and

C(O)NH group of compounds 3a–3z and 4a–4r were the

most shifted one’s and appeared at 160.11–154.83 ppm,

123

Med Chem Res (2013) 22:2610–2632

2613

(22.8), 143 (46.3) and 129 (39.2 %). Ion with m/z 129 in EI-MS

spectra of researched compounds was correspondent to calcu-

lated mass of quinazoline, for which typical mass-fragmenta-

tion was character. So, the fact that triazino[2,3-c]quinazoline

systems had ‘speciﬁc’ degradation of cycle by bond C(2)–C(3)

and N(4)–N(5) ensured the structure of compounds.

further 48 h. End point determinations were made with a

protein binding dye, sulforhodamine B (SRB). Results for

each tested agent were reported as the percent growth of

the treated cells comparing to the untreated control cells.

The preliminary screening results are shown in Table 2.

Screening researches of synthesized compounds in concen-

tration 10 lm showed that individual cell lines have different

sensitivities to synthesized compounds (Tables 2,3). Thus, N-(2-

methylphenyl)-2-[(2-oxo-3-(4-methylphenyl)-2H-[1,2,4]triazi-

no[2,3-c]quinazolin-6-yl)thio]acetamide (3.3) showed a high

cytotoxic activity against cell lines of CNS cancer (SNB-75,

U251) and antiproliferative effect against leukaemia (CCRF–

CEM). Replacement of methyl substituent at 2 position by car-

boxylic group did not lead to changing and extension of com-

pounds 3.26 cytotoxic activity. The introduction of Fluorine at

position 2 of aryl substituent (compound 3o) led to over expres

sion of cytotoxic activity against all cell lines of leukaemia

(inhibit growth up to 6–26 %). It is important to note that com-

pounds 3c, 3o and 3z at 3 position of triazinoquinazoline system

have 4-methylphenyl substituent. The introduction of Fluorine

(3n) or Chlorine (3t) at 2 position of aryl’s and replacement of

4-methylphenyl substituent at 3 position by 4-methoxyphenyl

led to substantial growth of cytotoxic activity. Thus, compound

3n inhibited cell lines of leukaemia (CCRF–CEM, HL-60,

K-562, MOLT-4, RPMI-8226, SR) by 23–52 %, and colon

cancer (HCT-116, HCT-15, HT29, KM12, SW-620) by

22–43 %, whereas compound 3t had wider range of anticancer

activity (48 cell lines) and possessed a considerable cytotoxic

activity against cell lines of leukaemia (HL-60(TB), K-562,

MOLT-4, SR), non-small cell lung (A549/ATCC, EKVX, HOP-

62, NCI-H460, NCI-H522), colon (HCT-116, HT29, KM12,

SW-620), CNS (SF-268, SF-295, SNB-75, U251), ovarian

(IGROV1, OVCAR-3, OVCAR-4, OVCAR-8), renal (786-0,

CAKI-1, RXF 393), prostate (DU-145), breast cancer (MCF7,

HS 578T) and melanoma (LOX IMVI, MALME-3M, MDA-

MB-435, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62).

Interestingly, the relocation of triﬂuoromethyl group (3) from o-

at m-position (3l) of aryl substituent led to wider range of cyto-

toxic activity (from 19 to 30 cell lines) and increased expression

of inhibition of cell lines (Table 2).

Pharmacology

Bioluminescence inhibition test

The results of bioluminescence research showed that lumines-

cent bacteria Photobacterium leiognathi Sh1 was sensitive to

synthesized compounds in acute and chronic action tests

(Table 1). Thus, among N-aryl-2-[(3-R-2-oxo-2H-[1,2,4]triazi-

no[2,3-c]quinazoline-6-yl)thio]acetamides (3a–3z) the highest

inhibition properties (inhibition of the growth up to 50 %) in

acute test at concentration 0.25 mg/ml have compounds 3b, 3k,

3n,3o,3s and 3y. Exact inﬂuence of the arylamide substituent on

inhibition properties of synthesized compounds was not dis-

covered. However, the highest biocide action (inhibition of the

growth up to 24.5–97.84 %) in acute test in explored concen-

trations was characteristic for compound 3s that has Fluorine at 4

position. Changing of arylamide by benzyl- (4a–4p) or phen-

ethyl- (4q, 4r) fragment showed that the predominant number of

compounds in acute test initiate growth of luminescent bacteria

Photobacterium leiognathi strain Sh1. And only compound 4j

that has at 3 position benzyamid fragment triﬂuoromethyl sub-

stituent in given concentrations had inhibition properties (inhi-

bition of the growth 33.3–82.2 %). It is important to note that the

majority of compounds in acute test had inhibition activities, but

in chronic test, had initiative properties. The most active ones

were compounds 3g, 3r and 3t that at 3 position of triazino-

quinazoline system had aryl substituents.

Anticancer assay for preliminary in vitro testing

Newly synthesized compounds were selected by the

National Cancer Institute (NCI) Developmental Thera-

peutic Program (www.dtp.nci.nih.gov) for the in vitro cell

line screening to investigate their anticancer activity.

Anticancer assays were performed according to the US

NCI protocol, which was described elsewhere (Boyd, 1997;

Boyd and Paull, 1995; Monks et al., 1991). The compounds

were ﬁrst evaluated at one dose primary anticancer assay

relative to *60 cell lines (concentration 10 lM). The

human tumour cell lines were derived from nine different

cancer types: leukaemia, melanoma, lung, colon, CNS,

ovarian, renal, prostate and breast cancers. In the screening

protocol, each cell line was inoculated and preincubated for

24–48 h on a microtiter plate. Test agents were then added

at a single concentration and the culture was incubated for

The replacement of arylamide (compounds 3a–3z) frag-

ment by benzylamide (4a–4p) led to similar relationship

‘structure–cytotoxic activity’. First, phenyl substituent at 3

position in triazinoquinazoline system (compounds 4b, 4d,

4f, 4h, 4l, 4n, 4p) was important for revealing of anticancer

activity. Thus, compound 4b showed selective cytotoxic

activity against cell lines of leukaemia, non-small cell lung

cancer (A549/ATCC, HOP-92, NCI-H460), colon cancer

(HCT-116, KM-12 and SW-620), CNS cancer (SF-268, SF-

295, U251), melanoma (LOX IMVI, MDA-MB-435,

UACC-62), ovarian cancer (OVCAR-3), prostate cancer

(PC-3, DU-145) and antiproliferative action against cell

lines of CAKI-1 renal cancer (Table 2). Second, character

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Med Chem Res (2013) 22:2610–2632

Table 1 Bioluminescence inhibition assay data

Compd.

Control

Action test, mg/ml

0.025

Chronic action test, mg/ml

0.1

0.25

0.025

0.1

0.25

DMSO

3a

100.00

100.00^a

95.59

95.24

119.70

96.49

96.77

112.50

71.15

88.30

88.89

97.80

96.67

61.10

103.30

90.00

89.86

84.27

94.55

97.83

75.51

83.30

104.76

99.30

100.00

68.09

83.30

66.70

125.00

83.30

84.60

89.20

111.50

92.30

111.50

66.70

66.67

116.70

118.80

77.80

91.70

108.30

72.20

109.50

80.70

100.00

98.53

78.57

109.70

82.46

98.92

120.00

63.46

106.00

86.67

106.50

100.00

78.80

113.30

84.00

86.96

73.03

90.91

93.48

36.73

87.50

109.52

99.10

120.00

63.83

36.00

87.50

112.50

100.00

106.00

110.00

99.10

130.00

106.20

75.00

33.33

66.70

125.00

78.80

110.00

80.00

87.50

121.50

9.10

100.00

95.59

47.62

193.50

68.42

73.12

146.70

53.85

142.90

88.89

113.40

46.67

83.30

142.50

40.00

47.83

50.56

96.36

65.22

8.16

100.00

88.89

100.00

61.11

100.00

57.78

80.56

93.30

94.55

14.29

27.30

18.18

75.00

97.14

102.90

187.50

200.00

100.00

142.86

92.59

41.46

166.67

33.30

101.39

42.90

87.50

37.50

50.00

250.00

78.20

150.00

140.00

188.00

73.30

180.00

40.00

193.30

0.00

3b

3c

90.28

83.33

100.00

90.91

66.70

3d

3e

94.55

196.43

69.80

178.57

76.80

3f

3g

22.73

11.36

3h

3i

95.60

117.00

100.00

200.00

203.13

81.50

100.00

185.20

187.50

118.20

51.20

3j

3k

3l

3m

3n

3o

116.70

96.67

100.00

112.50

142.86

44.44

87.50

3p

3q

3r

196.43

38.89

69.51

32.93

3s

250.00

54.50

133.33

35.40

3t

75.00

59.52

104.50

142.90

31.91

62.50

91.70

100.00

128.60

145.50

139.40

122.60

151.50

138.70

100.00

17.78

170.00

100.00

91.70

142.90

155.00

100.00

119.40

0.00

3u

3v

101.39

185.20

88.90

100.00

250.00

212.80

93.75

3w

3x

75.00

3y

93.00

250.00

115.40

113.60

212.80

112.00

160.00

80.00

4a

72.70

4b

4c

84.90

122.20

80.00

4d

4e

93.30

4f

150.00

138.70

50.00

4g

176.00

44.40

4h

4i

123.60

13.33

92.30

4j

10.67

4k

4l

130.20

90.60

233.30

75.00

200.00

54.50

200.00

166.70

125.00

110.00

133.30

57.10

0.00

4m

4n

4o

118.20

90.90

123.10

76.90

148.90

93.00

180.90

133.30

100.00

400.00

0.00

4p

4q

4r

136.40

222.20

0.00

Tetracycline

a

Bioluminescence intensity, %

123

Med Chem Res (2013) 22:2610–2632

2615

Table 2 Percentage of in vitro tumour cell lines growth at 10 lM for N-aryl-(alkaryl-)-2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-

yl)thio]acetamides (3a–3z, 4a–4r)

Compd. Mean Range of

growth, % growth, %

Most sensitive cell line growth, %*

3c

92.37

-49.64 to

113.32

-49.64 (CCRF-CEM/L), 46.68 (SNB-75/CNSC), 63.52 (U251/CNSC)

3f

87.77

90.79

24.37–114.72 59.00 (SNB-19/CNSC), 55.98 (SNB-75/CNSC), 24.37 (U251/CNSC)

57.93–127.94 57.93 (NCI-H522/nscLC), 61.08 (U251/CNSC)

85.06–130.04 85.06 (RPMI-8226/L)

3h

3j

104.77

98.61

3l

66.45–120.52 66.45 (SR/L), 70.96 (HOP-92/nscLC), 80.81 (SF-539/CNSC)

66.02–135.04 66.02 (SR/L)

3m

3n

103.01

87.90

48.25–117.25 67.54 (CCRF-CEM/L), 67.91 (HL-60(TB)/L), 60.18 (K-562/L), 52.73 (MOLT-4/L), 67.61 (RPMI-8226/

L), 48.25 (SR/L), 56.68 (HCT-116/ColC), 92.23 (HCT-15/ColC), 67.78 (HT29/ColC), 51.31 (KM12/

ColC), 65.60 (SW-620/ColC)

3o

3t

97.74

47.16

73.66–124.13 76.80 (CCRF-CEM/L), 84.47 (HL-60(TB)/L), 77.45 (K-562/L), 82.28 (MOLT-4/L), 94.42 (RPMI-8226/

L), 73.66 (SR/L)

-29.58 to

105.89

38.56 (HL-60(TB)/L), 26.92 (K-562/L), 36.30 (MOLT-4/L), 36.99 (SR/L), -29.58 (A549/ATCC/

nscLC), 27.20 (EKVX/nscLC), 30.32 (HOP-62/nscLC), 24.15 (NCI-H460/nscLC), 36.45 (NCI-H522/

nscLC), 12.22 (HCT-116/ColC), 71.91 (HCT-15/ColC), 5.16 (HT29/ColC), 34.82 (KM12/ColC),

13.89 (SW-620/ColC), 53.93 (SF-268/CNSC), 23.86 (SF-295/CNSC), 55.64 (SNB-75/CNSC), -22.59

(U251/CNSC), 42.14 (LOX IMVI/M), 18.81 (MALME-3 M/M), 15.70 (MDA-MB-435/M), -5.98

(SK-MEL-28/M), 41.37 (SK-MEL-5/M), –17.13 (UACOLC-257/M), 18.10 (UACOLC-62/M), 38.44

(IGROV1/OV), 7.18 (OVCAR-3/OV), 45.00 (OVCAR-4/OV), 31.50 (OVCAR-8/OV), 32.90 (786-0/

RC), 54.67 (CAKI-1/RC), 37.41 (RXF 393/RC), -17.92 (DU-145/PC), 32.78 (MCF7/BC), 44.31

(MDA-MB-468/BC)

3v

3w

3z

103.36

102.03

97.88

57.19–129.11 57.19 (K-562/L)

77.46–145.28 77.46 (MOLT-4/L)

78.15–124.86 78.15 (NCI-H522/nscLC)

78.50–128.26 78.50 (MOLT-4/L)

4a

4b

101.78

69.35

-7.56 to

110.77

37.85 (CCRF-CEM/L), 51.60 (HL-60(TB)/L), 29.68 (K-562/L), 42.96 (MOLT-4/L), 44.50 (RPMI-8226/

L), 41.93 (SR/L), 12.80 (A549/ATCC/nscLC), 24.65 (HOP-92/nscLC), 1.57 (NCI-H460/nscLC), 48.62

(HCT-116/ColC), 11.96 (KM12/ColC), 19.38 (SW-620/ColC), 53.11 (SF-268/CNSC), 55.74 (SF-295/

CNSC), 30.11 (U251/CNSC), 29.49 (LOX IMVI/M), 21.70 (MDA-MB-435/M), 13.69 (OVCAR-3/

OV), -7.56 (CAKI-1/RC), 18.11 (PC-3/PC), 21.81 (DU-145/PC)

4c

104.10

79.47

81.81–134.17 81.81 (SR/L)

4d

26.93–110.25 71.36 (SR/L), 63.96 (A549/ATCC/nscLC), 85.57 (HOP-92/nscLC), 72.09 (NCI-H460/nscLC), 32.54

(HCT-116/ColC), 78.77 (HT29/ColC), 80.05 (SW-620/ColC), 73.27 (SF-268/CNSC), 48.00 (SF-539/

CNSC), 62.54 (SNB-75/CNSC), 62.97 (U251/CNSC), 78.62 (LOX IMVI/M), 77.23 (MALME-3 M/

M), 67.65 (MDA-MB-435/M), 81.85 (SK-MEL-5/M), 71.08 (UACC-257/M), 74.91 (OVCAR-4/OV),

60.36 (OVCAR-8/OV), 61.42 (NCI/ADR-RES/OV), 80.24 (786-0/RC), 75.50 (A498/RC), 85.84

(ACHN/RC), 77.19 (RXF 393/RC), 78.62 (SN12C/RC), 61.89 (UO-31/RC), 82.96 (PC-3/PC), 79.13

(MCF7/BC), 70.52 (MDA-MB-231/ATCC/BC), 62.30 (HS 578T/BC), 26.93 (BT-549/BC)

4e

4f

107.51

63.71

75.58–131.55 75.58 (SR/L)

-23.09 to

112.61

41.93 (HL-60(TB)/L), 26.38 (K-562/L), 52.32 (MOLT-4/L), 42.15 (SR/L), 39.66 (A549/ATCC/nscLC),

31.44 (EKVX/nscLC), 26.22 (HOP-92/nscLC), 8.38 (NCI-H460/nscLC), 33.10 (HCT-116/ColC),

43.31 (KM12/ColC), 30.18 (SW-620/ColC), 43.50 (SF-268/CNSC), 18.75 (SF-295/CNSC), 27.70

(LOX IMVI/M), 27.67 (MDA-MB-435/M), 18.77 (IGROV1/OV), 21.28 (OVCAR-3/OV), 35.27 (786-

0/RC), -23.09 (A498/RC), -13.55 (CAKI-1/RC), 43.79 (UO-31/RC), 35.58 (PC-3/PC), 0.48 (DU-

145/PC)

4g

4h

4i

103.67

103.82

100.00

103.75

71.48–131.78 71.48 (SR/L)

78.69–127.18 78.69 (MOLT-4/L)

54.41–123.36 54.41 (UO-31/RC)

77.77–129.15 77.77 (K-562/L)

4k

123

2616

Med Chem Res (2013) 22:2610–2632

Table 2 continued

Compd. Mean Range of

growth, % growth, %

Most sensitive cell line growth, %*

4l

53.65

-50.46 to

108.44

26.01 (A549/ATCC/nscLC), 5.50 (NCI-H460/nscLC), 34.52 (NCI-H522/nscLC), (HCT-116/ColC),

33.10 (HT29/ColC), 31.04 (SW-620/ColC), 20.88 (SF-539/CNSC), 15.79 (SNB-75/CNSC), 2.29

(U251/CNSC), -50.46 (LOX IMVI/M), 49.58 (MDA-MB-435/M), 50.30 (SK-MEL-2/M), 47.32 (SK-

MEL-5/M), 41.25 (UACOLC-62/M), 9.50 (OVCAR-3/OV), -39.48 (OVCAR-4/OV), 36.25

(OVCAR-8/OV), 38.81 (NCI/ADR-RES/OV), 16.54 (786-0/RC), 42.30 (RXF 393/RC), 46.27 (TK-10/

RC), 35.89 (DU-145/PC), 38.25 (MCF7/BC), 48.89 (MDA-MB-231/ATCOLC/BC)

4m

4n

100.25

50.29

61.57–134.52 61.57 (UO-31/RC)

-25.39 to

120.40

-25.39 (NCI-H460/nscLC), 30.81 (COLO 205/ColC), 38.88 (HCT-116/ColC), 30.91 (HT29/ColC), 9.97

(KM12/ColC), 45.68 (SW-620/ColC), 51.25 (SF-268/CNSC), 42.88 (SF-539/CNSC), 44.31 (SNB-75/

CNSC), 19.91 (U251/CNSC), 30.80 (MALME-3 M/M), 17.90 (SK-MEL-5/M), 24.76 (A498/RC),

43.74 (ACHN/RC), -5.36 (CAKI-1/RC), 12.69 (UO-31/RC), 13.67 (MDA-MB-231/ATCC/BC),

43.87 (BT-549/BC)

4o

4p

100.22

76.14

79.81–123.73 79.88 (NCI-H522/nscLC), 79.81 (MDA-MB-435/M)

7.01–116.51

47.44 (SR/L), 44.79 (HOP-92/nscLC), 43.24 (NCI-H522/nscLC), 7.01 (HCT-116/ColC), 9.93 (SF-539/

CNSC), 27.86 (LOX IMVI/M), 47.85 (OVCAR-3/OV)

4q

4r

105.56

93.73

77.27–126.98 77.27 (UO-31/RC)

61.06–121.84 61.06 (LOX IMVI/M)

L leukaemia, nscLC non-small cell lung cancer, ColC colon cancer, CNSC CNS cancer, M melanoma, OV ovarian cancer, RC renal cancer, PC

prostate cancer, BC breast cancer

Table 3 Summary of anticancer screening data at dose-dependent assay

Compd.

N^a

log GI₅₀

log TGI

log LC₅₀

N1^b

Range

MG_MID

N2^b

Range

MG_MID

N3^b

Range

MG_MID

3t

4b

4f

4l

4n

a

59

58

48

32

40

8

-5.86 to -4.66

-7.57 to -4.20

-5.93 to -4.79

-5.89 to -5.07

-5.95 to -4.05

-5.30

-4.69

-5.26

-4.50

-4.71

33

16

13

0

-5.48 to -4.07

-5.15 to -4.20

-5.39 to -4.17

[4.00

-4.43

-4.17

-4.23

-4.00

-4.07

18

10

4

-4.73 to -4.01

-4.52 to -4.04

-5.13 to -4.12

[4.00

-4.09

-4.03

-4.04

-4.00

0

36

7

-5.16 to -4.32

1

-4.13 to [4.00

N number of human tumour cell lines tested at the 2nd stage assay

b

N1, N2, N3 number of sensitive cell lines, against which the compound possessed considerable growth inhibition according to mentioned

parameters log GI₅₀, log TGI and log LC₅₀B 4.00

and position of substituent at benzylamide fragment played

an important role in anticancer activity. Thus, introduction of

methyl group at 4 position of benzylamide substituent (4.4)

led to wider range of anticancer activity. Compound 4d was

effective against cell lines of breast cancer (MCF7, MDA-

MB-231/ATCC, HS 578T and BT-549). It is important to

note that replacement of methyl group (4d) by methoxy (4h)

led to the loss of anticancer activity (Table 2). On the other

hand, movement of methoxy group at 2 position of ben-

zylamide substituent (4f) led to the expansion of activity

spectrum (40 cell lines) and to increased expression of

anticancer activity. Halogen derivatives of benzylamides (4l,

4n, 4p) had substantial anticancer activity. Among them

compound 4n, that has ﬂuorine at 4 position, had the highest

activity. So, compound 4n more evidently inhibited cell lines

of colon cancer (COLO 205, HCT-116, HT29, KM12, SW-

620), CNS cancer (SF-268, SF-539, SNB-75, U251), renal

cancer (A498, ACHN, CAKI-1, UO-31) and breast cancer

(MDA-MB-231/ATCC, BT-549) in comparison to 4b and

4f. Prolongation of benzylamide substituent (4a–4p) by

methylene group (4q, 4r) led to the loss of anticancer

activity. Thus, valuation of anticancer activity of pheneth-

ylamids (4q, 4r) in researched concentrations 10 lM

showed that they in most of cases initiation growth of cell

lines (Table 2).

Through dose-dependent study in vitro on 60 cancer

cell lines

For compounds 3t, 4b, 4f, 4l and 4n dose-dependent action in

5 concentrations according to standard procedure of NCI at

57–59 cell lines of 9 types of cancer (100–0.0 lm) was

123

Med Chem Res (2013) 22:2610–2632

2617

Table 4 The inﬂuence of

compounds 3t, 4b, 4f, 4l and 4n

Compd.

Disease

Cell line

log GI^a₅₀

log TGI^b

Log LC^c₅₀

on the growth of individual

tumour cell lines (log GI₅₀

-5.50)

3t

NSC lung cancer

HOP-62

HOP-92

SF-268

-5.54

-5.74

-5.53

-5.70

-5.75

-5.73

-5.58

-5.56

-5.86

-5.58

-5.66

-5.86

-5.63

-5.71

-5.56

-5.66

-7.57

-5.59

-5.52

-5.63

-5.57

-5.61

-5.74

-5.65

-5.68

-5.57

-5.52

-5.65

-5.93

-5.73

-5.72

-5.75

-5.73

-5.51

-5.67

-5.55

-5.74

-5.57

-5.89

-5.57

-5.76

-5.59

-5.60

-5.52

-5.95

-5.01

-5.04

-4.45

-4.37

B

CNS cancer

Melanoma

-4.86

-4.14

SNB-75

U251

-5.22

-4.60

-5.33

-4.73

MALME-3M

M14

-5.10

[-4.00

-4.40

[-4.00

-4.17

UACC-62

OVCAR-3

RXF 393

TK-10

Ovarian cancer

Renal cancer

-5.48

-5.11

-5.14

-4.47

Prostate cancer

Breast cancer

DU-145

MCF7

[-4.00

-5.17

[-4.00

-4.52

HS 578T

HOP-62

SNB-75

A498

4b

4f

NSC lung cancer

CNS cancer

-5.15

-4.87

-4.36

Renal cancer

-4.43

[-4.00

-4.71

UO-31

[-4.00

-5.11

Breast cancer

BT-549

NSC lung cancer

A549/ATCC

HOP-62

HCT-116

SF-295

[-4.00

-5.26

Colon cancer

CNS cancer

[-4.00

-5.07

[-4.00

-4.36

SF-539

[-4.00

-5.18

SNB-75

LOX IMVI

SK-MEL-5

IGROV1

786-0

Melanoma

[-4.00

-5.28

[-4.00

-5.13

Ovarian cancer

Renal cancer

A498

-5.39

ACHN

[-4.00

-5.04

CAKI-1

RXF 393

UO-31

Breast cancer

HS 578T

BT-549

-5.40

a

GI50—concentration of

4l

NSC lung cancer

CNS cancer

HOP-62

SNB-75

U251

[-4.00

-5.16

[-4.00

substance resulting in a 50 %

lower net protein increase in the

treated cells

b

Renal cancer

A498

TGI—concentration of

substance resulting in a total

growth inhibition

Breast cancer

NSC lung cancer

MDA-MB-231/ATCC

HOP-62

NCI-H460

NCI-H522

SK-MEL-5

A498

4n

c

LC50 concentration of

[-4.00

-4.50

substance resulting in a 50 %

reduction in the measured

protein at the end of the drug

treatment as compared to that at

the beginning

Melanoma

[-4.00

-4.46

Renal cancer

123

2618

Med Chem Res (2013) 22:2610–2632

researched. Three dose-dependent parameters were calcu-

lated in the result of research: (1) GI₅₀—molar concentration

of the compound that inhibits 50 % net cell growth; (2)

TGI—molar concentration of the compound leading to total

inhibition of cell growth; (3) LC₅₀—molar concentration of

the compound leading to 50 % net cell death. If logarithmic

data of researched parameters (log GI₅₀, log TGI and log

LC₅₀) were\4.00, compounds were marked as active.

For each of the parameters, average value from the

experimental data were calculated (mean graph midpoints,

MG_MID) (Table 3). From the data, it can be seen that the

most wide spectral range and the expressed anticancer

activities were characteristic for compounds 3t and 4f with

MG_MID log GI₅₀-5.3 and -5.26 as appropriate.

improvement and expansion of antitumour activity spectrum

among S-substituted 3-R-6-thio-6,7-dihydro-2H-[1,2,4]tria-

zin[2,3-c]quinazolin-2-ones is a functionalization of sub-

stituent in 6 position by extending the combinatorial bases of

aminestoformappropriate amides, replacementofthio-group

by oxo-, alkyl-(aryl-, heteryl) amino group or functionaliza-

tion of ‘leader-compounds’ by introducing substituents at

positions 8, 9, 10, 11 of triazin[2,3-c]quinazoline cycle, such

as alkyl-, -alkoxy, aminoalkyloxy-, heterocyclil-, alkyloxy-,

halogen- and other functional groups. The latter aspect was

used by New Zealand scientiﬁc school in the creation of

anticancer agents among 4-anilinoquinazolin inhibitors of

kinases, in order to enhance performance and expand its range

(Denny, 2001a, b; Rewcastle et al., 2000).

Parameters of compounds’ activities against the most

sensitive cell lines are shown in Table 4 (log GI₅₀B-

5.65). It is necessary to mention the selective sensitivity

against cell lines of NSC lung cancer (HOP-62), CNS

cancer (SNB-75), Renal cancer (A498) to compounds 3t,

4b, 4f, 4l and 4n. Hence, substance 4b revealed the high

level of inhibition (log GI₅₀= -7.57) against cell line

A498 of renal cancer, compound 3t (log GI₅₀= -5.86)—

against cell line DU-145 of prostate Cancer (MG_MID log

GI₅₀= -5.3 against 48 cell lines), and compound 4f (log

GI₅₀= -5.93)—against cell line A498 of renal cancer

(MG_MID log GI₅₀= -5.26 against 40 cell lines).

COMPARE analysis and molecular mechanism

assumptions

We have also performed COMPARE analyses for all the

active compounds in order to investigate the similarity of their

cytotoxicity (mean graph ﬁngerprints) with those of known

anticancer standard agents, NCI active synthetic compounds

and natural extracts, which are present in public available

databases (Paull et al., 1989; Shoemaker et al., 2002; Wein-

stein et al., 1997; Zaharevitz et al., 2002). Such analysis was

based on comparing the patterns of differential growth inhi-

bition for cultured cell lines and could potentially gain insight

into the mechanism of the cytotoxic action. It determines

Pearson correlation coefﬁcient (PCC) for the degree of simi-

larity of mean graph ﬁngerprints obtained from in vitro anti-

cancer screen with patterns of activity of standard agents. We

performed COMPARE computations for synthesized com-

poundsagainsttheNCI‘StandardAgents’ databaseattheGI₅₀

level (correlations PCC C0.4 Table 6).

It is important, that among researched substances com-

pound 4f had a high anticancer activity against cell lines of

cancer (average data logGI₅₀= -5.69), melanoma (aver-

age data logGI₅₀= -5.62) and CNS cancer (average data

logGI₅₀= -5.59), compound 4l—against cell lines of

renal cancer (average data logGI₅₀= -5.89) (Table 5).

Analysis of the ‘structure–activity’ relationship (SAR-

analysis) showed that the antitumour activity of synthesized

compounds was deﬁned as by the basic structure of the mol-

ecule ([1,2,4]triazin[2,3-c]quinazoline cycle) so by substitu-

ents in 3 and 6 positions. An important direction of further

Results of COMPARE analysis showed that the investi-

gated compounds poorly correlated with the speciﬁcity of the

Table 5 Anticancer selectivity pattern (average value of LogGI₅₀) of the most active compounds 3t, 4b, 4f, 4l and 4n

Disease

Compound

3t

4b

-4.69

4f

4l

-4.50

4n

-4.71

MGMID

L

-5.30

-5.12

-5.39

-5.35

-5.39

-5.32

-5.26

-5.29

-5.52

-5.46

-5.26

-5.33

-5.55

-5.69

-5.59

-5.62

-5.56

-5.58

-5.54

-5.43

[-4.00

-5.04

-5.34

-5.24

-5.11

-4.56

-5.61

-4.49

-5.22

[-4.00

-5.55

[-4.0

-5.46

[-4.0

-5.30

-5.89

[-4.0

-5.43

[-4.00

-5.05

-5.42

-5.12

-5.31

-4.46

-5.03

-5.33

-5.20

NSCLC

ColC

CNSC

M

OV

RC

PC

BC

123

Med Chem Res (2013) 22:2610–2632

2619

cancer activity of the tested compounds in the NCI drugs and

nature biological active compounds. Only compound 3t had

a reasonable correlation on the level GI₅₀with the inhibitor

or promoter of polymerization microtubules—alkaloid

Vinca Rosea Linn L. vinblastine (Table 6), On the other

hand, quinazoline and their condensed analogues according

to the literature data are inhibitors of kinases (CDK2, p. 38),

in particular vascular endothelial growth factor-1 (Denny,

2001a, b; Rewcastle et al., 2000), and others.

the mechanism of action of 4-R-anilinoquinazolines—known

anticancer products (Denny, 2001a, b; Rewcastle et al., 2000).

The methodology of research consisted of the following

steps: generation of R-, S- and cis-, trans-isomers of ligands

(program Flipper), for production of isomer’s range of studied

compounds; generation of 3D-structure of the obtained iso-

meric forms—molecular modelling [program Hyper Chem

7.5 using the method of molecular mechanics (MM^?) and

semiempirical quantum mechanical method with Polak–

Ribiere algorithm until the root-mean-square gradient of

0.01 kcal mol^-1(PM3)]; and generation of ligands’ confor-

mations (Omega2.4.3). The number of conformations

obtained was not signiﬁcant due to the further selection by

program Fred2.2.5 most optimal conformer; carrying out

molecular docking with the resulting geometrics (Fred2.2.5).

A number of scoring functions that use smooth Gaussian

functions represent the shape and chemistry of molecules

(Shapegauss, PLP, Chemgauss2, Chemgauss3, Chemscore,

OEChemscore, Screenscore, CGO, CGTand Zapbind) and

Consensus Score (Table 7) was obtained as a result of

studies, values of which assess speciﬁc characteristics of the

ligand–protein complex, indicating the possibility of their

matching. All the mentioned compounds in this study and

drugs used for comparison (Geﬁtinib, Lapatinib) were

docked to the active site of EGFR.

Docking

In silico methodology was used for establishing the likely

mechanism for the compound’s antitumour activity, namely

molecular docking, and evaluating the binding of ligands to

the cancer targets (Avendano and Menendez, 2008; Baxter

et al., 2000, Cummings et al., 2005, Prendergast, 2003).

Research was conducted by ﬂexible molecular docking, as an

approach of ﬁnding molecules with afﬁnity to a speciﬁc bio-

logical target (Schulz-Gasch and Stahl, 2003, 2004) using the

software package OpenEye (2005) that include related utili-

ties: Fred Receptor2.2.5, Vida4.1.1, Flipper, Babel3,

Omega2.4.3 and Fred2.2.5. The crystal structure of the

enzyme, namely epidermal growth factor receptor (EGFR)

(2ITY) with Geﬁtinib was obtained from the protein data bank

and was used as the biological target (Degtever et al., 2001;

Lee et al., 2010). This choice was due to the literature data on

Molecular docking study indicated that compound 4b was

nicely bonded to the EGFR. Based on the obtained data of

Consensus score, it was revealed that compound 4b has the

Table 6 COMPARE analysis results for tested compounds

Compd no.

PCC

Target

Target vector NSC

Target mechanism of action^a

3t

4b

4f

4l

0.547

0.524

0.441

0.512

0.507

Vinblastine sulfate

O₆-methylquanine

Bleomycin

S49842

S37364

S125066

Inhibits or promotes polymerization of microtubules

Inhibitor of DNA methyltransferase

Inhibits incorporation of thymidine into DNA

Bleomycin

4n

Bleomycin

Only correlations with PCC C 0.4 were selected, as signiﬁcant

a

Putative mechanisms of action were identiﬁed with the use of literature sources

Table 7 Scoring functions of docked compounds comparing to Geﬁtinib and Lapatinib

Compd

no.

Consensus

score

Shapegauss PLP

Chemgauss2 Chemgauss3 Chem

score

OEChem

score

Screen

score

CGO

CGT

Zapbind

Geﬁtinib 159.000

Lapatinib 173.000

-507.265 -56.548 -59.782

-599.984 -61.497 -64.464

-477.858 -46.307 -49.016

-571.319 -55.156 -56.153

-535.246 -50.691 -53.942

-471.314 -45.614 -46.913

-435.202 -39.707 -42.895

-69.129

-71.097

-50.519

-64.322

-60.789

-42.431

-40.609

-20.412 -35.313

-16.219 -42.817

-15.637 -29.165

-21.925 -38.515

-15.342 -34.813

-14.085 -29.225

-15.494 -27.869

-126.058 -367.448 -0.611 -25.604

-131.696 -329.616 -0.433 -29.617

-117.703 -384.398 -0.554 -28.513

-132.347 -336.983 -0.505 -26.960

-118.736 -394.064 -0.593 -23.292

3t

4b

4f

4l

432.000

143.000

286.000

634.000

658.000

-105.957

299.978 -0.420 -25.725

4n

-101.414 -306.010 -0.415 -29.669

123

2620

Med Chem Res (2013) 22:2610–2632

Fig. 1 a Interaction of Geﬁtinib with the binding site of EGFR; b Interaction of compound 4b with the binding site of EGFR.; c overlapping of

Geﬁtinib and compound 4b. H-bond is shown as black line (Color ﬁgure online)

highest afﬁnity to EGFR. Therefore, ‘Geﬁtinib’ (treatment of

locally widespread or metastatic lung cancer nonsmallcell

cancer of lungs) and ‘Lapatinib’ (treatment of metastatic

breast cancer) have similar afﬁnities to compound 4b.

To visualize obtained interaction, we have been using

Vida4.4.1. The complex of active sites of protein and

Geﬁtinib was characterized by one hydrogen bond between

nitrogen atom position 1 of quinazoline and the rest of

Screening of anticancer activity in vitro yielded the most

active compounds 3t, 4b, 4f, 4l and 4n in micromolar con-

centrations with the GI₅₀level (logGI₅₀is from -7.57 to

-4.05 for different cell lines, logGI₅₀mean graph midpoint

varies from -5.30 to -4.50); moreover, compound 4b had a

distinctive selectivity against renal cancer; 4f—colon can-

cer and melanoma; and 4l—renal cancer. The highest sen-

sitivity to compound 4b was demonstrated against renal

cancer cell lines A498 (logGI₅₀= -7.57). Molecular

docking showed that compound 4b revealed afﬁnity to

EGFR similar to other known drugs.

˚

MET 793a (1.72 A) (Fig. 1).

The compound 4b did not form hydrogen bond; never-

theless, it has the highest afﬁnity among all compounds

accordingtotheConsensusscore,evenwhilelookingtoothers

that have hydrogen bonds. Comparing the docking mode, it

could be postulated that the designed compounds might act on

the same enzyme target where Geﬁtinib acted. Thus, the

ﬁndings are an argument for the study of metabolic pathways

involving EGFR and related kinases to establish molecular

mechanisms of action of new N-substituted 2-[(3-R-2-oxo-

2H-[1,2,4]triazino[2,3-c]quinazoline-6-yl)thio]acetamides.

Antitumour potential of new N-aryl(alkaryl)-2-[(3-R-2-

oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-yl)thio]acetami

des was veriﬁed and some regularities of ‘structure–activity’

were discussed, which formed the basis for further modiﬁ-

cation of ‘leader-compounds’ and research of antitumour

mechanism of action within this heterocyclic system.

Experimental protocols

Chemistry

Melting points were determined in open capillary tubes and

were uncorrected. The elemental analyses (C, H, N and S) were

performed using the ELEMENTAR vario EL cube analyzer

(USA). Analyses were indicated by the symbols of the elements

or functions within 0.3 % of the theoretical values. IR spectra

(4,000–600 cm^-1) were recorded on a Bruker ALPHA FT-IR

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)

spectrometers with TMS as internal standard in DMSO-d₆

solution. LC-MS were recorded using chromatography/mass

spectrometric system which consists of high-performance

liquid chromatography ‘Agilent 1100 Series’ (Agilent, Palo

Alto, CA, USA) equipped with diode-matrix and mass-selec-

tive detector ‘Agilent LC/MSD SL’ (APCI). Electron impact-

mass spectra (EI-MS) were recorded on a Varian 1,200 L

Conclusion

In the present article, 42 new N-aryl(alkaryl)-2-[(3-R-2-oxo-

2H-[1,2,4]triazino[2,3-c]quinazoline-6-yl)thio]acetamides

were described. Twenty nine of the synthesized com

pounds were tested, and most of them displayed antitumour

activities against leukaemia, melanoma, lung, colon, CNS,

ovarian, renal, prostate and breast cancers cell lines.

123

Med Chem Res (2013) 22:2610–2632

2621

instrument at 70 eV (Varian, USA). The purities of all the

obtained compounds were checked by ¹H-NMR and LC-MS.

Substances 1a–1d and 2a–2d were synthesized according

to the reported procedures (Berest et al., 2010). Other start-

ing materials and solvents were obtained from commercially

available sources and used without additional puriﬁcation.

151.92 (3), 154.64 (7a), 155.22 (6), 160.96 (2), 166.28

(CONH); LC-MS, m/z = 378 [M?1], 380 [M?3]; Anal.

calcd. for C₁₉H₁₅N₅O₂S: C, 60.7; H, 4.1; N, 18.6; S, 8.50;

Found: C, 60.47; H, 4.01; N, 18.56; S, 8.54.

N-Phenyl-2-[(2-oxo-3-phenyl-2H-[1,2,4]triazino[2,3-c]qui

nazolin-6-yl)thio]acetamide (3b) Yield: Method A,

73.1 %; Method B, 80.9 %; Method C, 85.3 %; M.p.

263–265 ꢁC; IR (cm^-1): 3575, 3259, 3193, 3116, 3063,

3046, 2982, 2919, 1682, 1647, 1585, 1563, 1549, 1535, 1495,

1481, 1468, 1443, 1377, 1338, 1322, 1310, 1285, 1262, 1242,

1193, 1181, 1160, 1137, 1102, 1079, 1030, 1018, 1001, 990,

941, 904, 873, 848, 815, 784, 773, 758, 716, 688, 653, 623;

¹H-NMR (400 MHz) d: 4.27 (s, 2H, –S–CH₂), 7.07 (t, 1H,

J = 6.7, H-5⁰Ph), 7.34 (t, 2H, J = 6.7, H-3⁰, 4⁰Ph),

7.69–7.57 (m, 6H, H-3⁰, 4⁰, 5⁰3-Ph; H-2⁰, 6⁰Ph; H-10), 7.71

(d, 1H, J = 7.9, H-8), 7.96 (t, 1H, J = 7.7, H-9), 8.30 (d, 2H,

J = 7.7, H-2⁰, 6⁰3-Ph), 8.48 (d, 1H, J = 7.9, H-11), 10.48 (s,

1H, –C(O)NH); ¹³C-NMR (100 MHz) d: 36.82 (–SCH₂),

118.35 (11a), 119.73 (2⁰, 6⁰NHPh), 123.98 (4⁰NHPh),

126.13 (8), 126.79 (10), 128.08 (11), 128.94 (3⁰, 5⁰NHPh),

129.29 (3⁰, 5⁰Ph), 129.83 (2⁰, 6⁰Ph), 132.05 (4⁰Ph), 132.12

(1⁰Ph), 136.08 (9), 139.47 (1⁰NHPh), 144.20 (11b), 149.70

(3), 151.05 (7a), 154.94 (6), 160.11 (2), 166.25 (CONH); EI-

MS, m/z (I_rel, %) = 439 (2.2), 349 (3.9), 348 (13.8), 347

(52.1), 244 (28.4), 243 (5.3), 218 (9.5), 217 (31.1), 216

(21.5), 204 (11.8), 203 (6.0), 189 (10.9), 188 (24.4), 187

(5.5), 174 (13.0), 173 (5.8), 172 (16.6), 171 (45.6), 170

(37.3), 167 (6.0), 161 (18.6), 160 (15.8), 156 (5.3), 149 (8.1),

148 (49.7), 145 (26.1), 144 (26.0), 143 (99.9), 142 (41.0), 134

(16.0), 133 (10.3), 132 (21.4), 131 (7.6), 130 (16.8), 129

(70.1), 119 (9.4), 118 (6.1), 117 (18.6), 116 (21.9), 115 (7.2),

106 (26.9), 105 (8.9), 104 (36.2), 103 (96.0), 102 (65.9), 94

(9.1), 93 (54.5), 92 (16.4), 91 (6.6), 90 (7.4), 89 (5.7), 88

(9.6), 86 (6.0), 79 (11.7), 78 (11.4), 77 (75.6), 76 (34.6), 75

(13.5), 74 (5.1), 66 (12.7), 65 (92.3), 64 (24.0), 63 (24.7), 56

(16.3), 52 (15.3), 51 (34.3), 50 (17.6), 47 (6.3), 46 (6.2), 45

(9.7), 43 (8.1), 42 (5.5); LC-MS, m/z = 440 [M?1], 442

[M?3]; Anal. calcd. for C₂₄H₁₇N₅O₂S: C, 65.59; H, 3.90; N,

15.93; S, 7.30; Found: C, 65.58; H, 3.91; N, 15.94; S, 7.31.

General procedure for synthesis of N-aryl-2-

(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-

yl)thio]acetamides (3a–3z)

Method A The thionyl chloride (1.2 g, 0.011 mol) was added

to the solution of proper acids (1a–1a) (0.01 mol) in 10 ml of

anhydrous dioxane with subsequent adding of the 2–3 drops of

DMFA. Mixture was heated at the water bath at 60–80 ꢁC till

full elimination of hydrochloric acid. Then proper aniline

(0.01 mol) was added to the resulting mixture with stirring and

reﬂuxed for 3–6 h. The mixture was poured into water, neu-

tralized to pH 6–7 by acetic acid. The precipitate was ﬁltered,

dried and recrystallized from DMF–water (1:1).

Method B N,N⁰-CDI (1.95 g, 0.011 mol) was added to a

solution of proper acids (1a–1d) (0.01 mol) in 10 ml of

anhydrous dioxane or DMFA and heated at the water bath at

60–80 ꢁC for 1 h with calcium chloride tube. The proper ani-

line (0.01 mol) was added with stirring to the resulting mixture

and reﬂuxed for 3–6 h. The mixture was poured into water, and

neutralized to pH 6–7 by acetic acid. The precipitate was ﬁl-

tered, dried and recrystallized from the DMF–water (1:1).

Method C N-Aryl-2-chloroacetamides (0.011 mol) were

added to a suspension of potassium salts of 3-R-6-thio-6,7-

dihydro-2H-[1,2,4]triazino[2,3-c]quinazolin-2-ones (2a–

2d) (0.01 mol) in 20 ml of propanol-2, water-propanol-2

(1:2), or dioxane and reﬂuxed for 60–90 min. The mixture

was cooled and poured into water. The precipitate was ﬁl-

tered, dried and recrystallized from the DMF–water (1:1).

N-Phenyl-2-[(2-oxo-3-methyl-2H-[1,2,4]triazino[2,3-c]qui

nazolin-6-yl)thio]acetamide (3a) Yield: Method A,

66.2 %; Method B, 79.3 %; Method C, 86.2 %; M.p.

262–265 ꢁC; IR (cm^-1): 3295, 3197, 3122, 3042, 2973,

2894, 1658, 1598, 1579, 1555, 1496, 1463, 1434, 1386,

1360, 1330, 1307, 1284, 1259, 1243, 1223, 1201, 1164,

1130, 1100, 1044, 1020, 951, 901, 860, 773, 752, 715, 687,

629, 605; ¹H-NMR (400 MHz) d: 2.39 (s, 3H, 3-CH₃), 4.23

(s, 2H, –S–CH₂), 7.06 (t, 1H, J = 6.7, H-5⁰Ph), 7.32 (t,

2H, J = 6.7, H-3⁰, 4⁰Ph), 7.69–7.61 (m, 4H, H-8, 10, 2⁰, 6⁰

Ph), 7.92 (t, 1H, J = 7.7, H-9), 8.43 (d, 1H, J = 7.9,

H-11), 10.46 (s, 1H, –C(O)NH); ¹³C-NMR (100 MHz) d:

18.19 (–CH₃), 36.80 (–SCH₂), 118.56 (11a), 119.72 (2⁰, 6⁰

Ph), 123.95 (4⁰Ph), 125.99 (8), 126.67 (10), 127.93 (11),

129.25 (3⁰, 5⁰Ph), 135.98 (9), 139.47 (1⁰Ph), 144.17 (11b),

N-(2-Methylphenyl)-2-[(2-oxo-3-(4-methylphenyl)-2H-

[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]acetamide

(3c)

Yield: Method A, 73.8 %; Method C, 80.2 %; M.p.

264–267 ꢁC; IR (cm^-1): 3248, 3118, 3099, 3062, 3033,

2992, 1669, 1649, 1589, 1552, 1534, 1502, 1468, 1388, 1364,

1335, 1321, 1308, 1284, 1270, 1244, 1207, 1186, 1161, 1135,

1105, 1071, 1040, 1020, 991, 970, 940, 901, 885, 855, 835,

785, 774, 753, 710, 698, 686, 641, 626; ¹H-NMR (400 MHz)

d: 2.22 (s, 3H, 2-CH₃Ph), 2.38 (s, 3H, 4-CH₃Ph), 4.30 (s, 2H,

–S–CH₂), 6.91 (m, 2H, H-4⁰, 5⁰2-CH₃Ph), 7.35 (d, 2H, H-3⁰,

5⁰4-CH₃Ph); 7.47–7.39 (m, 2H, H-3⁰2-CH₃Ph, H-10), 7.52

(d, 2H, J = 7.9, H-8), 8.01–7.94 (m, 2H, H-6⁰2-CH₃Ph,

123

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Med Chem Res (2013) 22:2610–2632

H-9), 8.11 (d, 2H, H-2⁰, 6⁰4-CH₃Ph), 8.26 (d, 1H, H-11), 9.77

(s, 1H, –C(O)NH); LC-MS, m/z = 468 [M?1], 469 [M?2];

Anal. calcd. for C₂₆H₂₁N₅O₂S: C, 66.79; H, 4.53; N, 14.98;

S, 6.86; Found: C, 66.80; H, 4.53; N, 15.00; S, 6.88.

3H, 4-CH₃Ph), 3.89 (s, 3H, 4-CH₃OPh), 4.26 (s, 2H, –S–

CH₂), 7.17–7.10 (m, 4H, H-3⁰, 5⁰4-CH₃OPh); H-3⁰, 5⁰

4-CH₃Ph), 7.50 (d, 2H, J = 8.1, H-2⁰, 6⁰4-CH₃Ph), 7.68 (t,

2H, J = 7.7, H-10), 7.75 (d, 2H, J = 7.9, H-8), 7.96 (t, 1H,

J = 7.7, H-9), 8.39 (d, 2H, J = 8.9, H-2⁰, 6⁰4-CH₃OPh),

8.50 (d, 1H, J = 7.9, H-11), 10.13 (s, 1H, –C(O)NH); LC-

MS, m/z = 484 [M?1], 486 [M?3]; Anal. calcd. for

C₂₆H₂₁N₅O₃S: C, 64.58; H, 4.38; N, 14.48; S, 6.63; Found:

C, 64.58; H, 4.38; N, 14.48; S, 6.63.

N-(2-Methylphenyl)-2-[(2-oxo-3-(4-methoxyphenyl)-2H-

[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]-acetamide (3d)

Yield: Method C, 77.6 %; M.p. 238–240 ꢁC; IR (cm^-1):

3251, 3062, 3010, 2930, 2838, 1669, 1651, 1630, 1591, 1562,

1545, 1500, 1470, 1423, 1386, 1369, 1341, 1320, 1263, 1244,

1178, 1163, 1139, 1108, 1072, 1024, 990, 940, 908, 873, 853,

N-(2-Methoxyphenyl)-2-[(2-oxo-3-phenyl-2H-[1,2,4]triazi-

no[2,3-c]quinazolin-6-yl)thio]acetamide

1

838, 824, 810, 767, 750, 700, 684, 642, 622; H-NMR

(3g) Yield:

(400 MHz) d: 2.24 (s, 3H, 2-CH₃Ph), 3.88 (s, 3H,

4-CH₃OPh), 4.31 (s, 2H, –S–CH₂), 7.11 (t, 1H, H-4⁰

2-CH₃Ph), 7.19–7.14 (m, 3H, H-3⁰, 5⁰4-CH₃OPh; H-5⁰

2-CH₃Ph), 7.22 (d, 1H, J = 7.5, H-3⁰2-CH₃Ph), 7.41 (d, 1H,

J = 7.5, H-6⁰2-CH₃Ph), 7.70 (t, 2H, J = 7.7, H-10), 7.79 (d,

2H, J = 7.9, H-8), 7.99 (t, 1H, J = 7.7, H-9), 8.39 (d, 2H,

J = 8.9, H-2⁰, 6⁰4-CH₃OPh), 8.51 (d, 1H, J = 7.9, H-11),

9.72 (s, 1H, –C(O)NH); LC-MS, m/z = 484 [M?1], 486

[M?3]; Anal. calcd. for C₂₆H₂₁N₅O₃S: C, 64.58; H, 4.38; N,

14.48; S, 6.63; Found: C, 64.58; H, 4.38; N, 14.48; S, 6.63.

Method A, 73.2 %; Method B, 76.3 %; Method C, 87.3 %;

M.p. 226–227 ꢁC; IR (cm^-1): 3368, 3069, 3004, 2951, 2881,

2832, 1670, 1587, 1556, 1531, 1507, 1485, 1459, 1442, 1407,

1371, 1340, 1311, 1286, 1269, 1250, 1179, 1154, 1134, 1116,

1101, 1077, 1045, 1020, 1002, 988, 962, 939, 925, 877, 847,

1

811, 783, 763, 753, 728, 706, 686, 653, 628; H-NMR

(400 MHz) d: 3.79 (s, 3H, 2-CH₃OPh), 4.32 (s, 2H, –S–

CH₂), 6.90 (t, 1H, J = 7.3, H-5⁰2-CH₃OPh), 7.10–7.03 (m,

2H, H-3⁰, 4⁰, 2-CH₃OPh), 7.66–7.57 (m, 3H, H-3⁰, 4⁰, 5⁰Ph),

7.71 (t, 1H, J = 7.7, H-10), 7.82 (d, 1H, J = 7.9, H-8),

8.04–7.97 (m, 2H, H-6⁰2-CH₃OPh; H-9), 8.30 (d, 2H,

J = 7.7, H-2⁰, 6⁰Ph), 8.50 (d, 1H, J = 7.9, H-11), 9.61 (s,

1H, –C(O)NH); EI-MS, m/z (I_rel, %) = 469 (0.8), 349 (5.9),

348 (20.5), 347 (99.9), 346 (65.8), 345 (30.7), 261 (7.4), 245

(10.6), 244 (52.8), 242 (8.3), 218 (7.9), 217 (25.6), 216

(49.2), 203 (9.0), 197 (5.0), 189 (5.9), 188 (9.6), 185 (5.1),

172 (5.0), 171 (21.7), 170 (28.5), 165 (5.6), 163 (5.2), 162

(9.0), 161 (8.2), 160 (6.8), 149 (13.7), 148 (21.8), 145 (9.9),

144 (6.5), 143 (24.0), 142 (13.4), 136 (9.1), 135 (12.2), 134

(13.0), 132 (5.7), 130 (6.4), 129 (26.9), 123 (38.3), 122 (6.8),

121 (7.5), 120 (17.8), 117 (7.9), 116 (8.3), 108 (52.1), 107

(18.4), 106 (13.7), 104 (11.5), 103 (84.0), 102 (29.2), 95

(5.0), 94 (17.3), 93 (5.9), 92 (16.5), 90 (14.5), 89 (6.3), 80

(26.6), 79 (9.6), 78 (14.0), 77 (24.4), 76 (40.5), 75 (14.5), 74

(6.1), 66 (6.1), 65 (45.4), 64 (13.1), 63 (17.1), 56 (11.4), 53

(8.3), 52 (22.2), 51 (21.7), 50 (18.6), 45 (6.4), 44 (9.0), 43

(14.3), 42 (6.2), 41 (8.1); LC-MS, m/z = 470 [M?1], 472

[M?3]; Anal. calcd. for C₂₅H₁₉N₅O₃S: C, 63.95; H, 4.08; N,

14.92; S, 6.83; Found: C, 63.95; H, 4.08; N, 14.91; S, 6.82.

N-(3-Methylphenyl)-2-[(2-oxo-3-methyl-2H-[1,2,4]triazi-

no[2,3-c]quinazolin-6-yl)thio]acetamide (3e) Yield: Method

A, 67.3 %; Method B, 80.4 %; Method C, 79.2 %; M.p.

248–249 ꢁC; IR (cm^-1): 3514, 3254, 3200, 3140, 3077, 3004,

2912, 2831, 1683, 1648, 1612, 1580, 1556, 1495, 1465, 1428,

1383, 1364, 1328, 1305, 1283, 1259, 1208, 1185, 1171, 1145,

1135, 1044, 1005, 951, 904, 886, 860, 772, 753, 692, 682, 632,

608; ¹H-NMR (400 MHz) d: 2.27 (s, 3H, 3-CH₃Ph), 2.39 (s, 3H,

CH₃), 4.21 (s, 2H, –S–CH₂), 6.88 (d, 1H, J = 7.5, H-4⁰

3-CH₃Ph), 7.19 (t, 1H, J = 7.6, H-5⁰, 3-CH₃Ph), 7.42 (d, 1H,

J = 7.9, H-6⁰3-CH₃Ph), 7.47 (s, 1H, H-2⁰3-CH₃Ph), 7.68–7.60

(m, 2H, H-8, 10), 7.91 (t, 1H, J = 7.7, H-9), 8.41(d, 1H, J = 7.9,

H-11), 10.37 (s, 1H, –C(O)NH); ¹³C-NMR (100 MHz) d: 17.00

(–CH₃), 21.25 (3-CH₃Ph), 36.83 (–SCH₂), 116.89 (2⁰,3-CH₃Ph),

118.49 (11a), 120.20 (8), 124.64 (4⁰3-CH₃Ph), 125.96 (10),

126.64 (5⁰3-CH₃Ph), 127.90 (6⁰3-CH₃Ph), 129.09 (11), 135.97

(9), 138.45 (3⁰3-CH₃Ph), 139.39 (1⁰3-CH₃Ph), 144.13 (11b),

151.85 (3), 154.61 (7a), 155.21 (6), 160.95 (2), 166.16 (CONH);

LC-MS, m/z = 392 [M?1], 394 [M?3]; Anal. calcd. for

C₂₀H₁₇N₅O₂S: C, 61.37; H, 4.38; N, 17.89; S, 8.19; Found: C,

61.34; H, 4.35; N, 17.89; S, 8.17.

N-(4-Methoxyphenyl)-2-[(2-oxo-3-(4-methylphenyl)-2H-

[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]acetamide

(3h)

Yield: Method A, 69.8 %; Method B, 75.4 %; Method C,

79.6 %; M.p. 279–282 ꢁC; IR (cm^-1): 3242, 3122, 3064,

3038, 3007, 2976, 2901, 2837, 1666, 1652, 1625, 1590,

1562, 1548, 1498, 1469, 1455, 1402, 1365, 1337, 1309,

1295, 1283, 1270, 1248, 1185, 1163, 1155, 1135, 1104,

1072, 1029, 995, 977, 966, 940, 903, 866, 834, 823, 770,

757, 714, 701, 683, 638, 626, 612; ¹H-NMR (400 MHz) d:

2.42 (s, 3H, 4-CH₃Ph), 3.72 (s, 3H, 4-CH₃OPh), 4.23 (s,

2H, –S–CH₂), 6.92–6.84 (m, 4H, H-3⁰, 5⁰4-CH₃OPh; H-3⁰,

N-(4-Methylphenyl)-2-[(2-oxo-3-(4-methoxyphenyl)-2H-

[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]acetamide (3f)

Yield: Method C, 69.3 %; M.p. 272–274 ꢁC; IR (cm^-1):

3266, 3130, 3058, 3012, 2976, 2953, 2915, 2839, 1658,

1591, 1562, 1546, 1498, 1469, 1421, 1374, 1339, 1317,

1304, 1288, 1274, 1258, 1240, 1175, 1163, 1139, 1107,

1072, 1018, 992, 973, 940, 906, 874, 838, 813, 779, 768,

1

722, 702, 685, 642, 622; H-NMR (400 MHz) d: 2.27 (s,

123

Med Chem Res (2013) 22:2610–2632

2623

5⁰4-CH₃Ph), 7.53 (d, 2H, J = 8.9, H-2⁰, 6⁰4-CH₃OPh),

7.68 (t, 2H, J = 7.7, H-10), 7.73 (d, 2H, J = 7.9, H-8),

7.96 (t, 1H, J = 7.7, H-9), 8.25 (d, 2H, J = 8.1, H-2⁰, 6⁰

4-CH₃Ph), 8.48 (d, 1H, J = 7.9, H-11), 10.32 (s, 1H, –

C(O)NH); LC-MS, m/z = 484 [M?1], 485 [M?2]; Anal.

calcd. for C₂₆H₂₁N₅O₃S: C, 64.58; H, 4.38; N, 14.48; S,

6.63; Found: C, 64.57; H, 4.39; N, 14.47; S, 6.61.

1000, 988, 941, 918, 894, 875, 849, 812, 799, 778, 763,

1

752, 698, 684, 650, 636; H-NMR (400 MHz) d: 4.33 (s,

2H, –S–CH₂), 7.43 (d, 1H, J = 7.6, H-4⁰3-CF₃Ph),

7.69–7.57 (m, 4H, H-5⁰3-CF₃Ph; H-3⁰, 4⁰, 5⁰Ph), 7.68 (t,

1H, J = 7.7, H-10), 7.71 (d, 1H, J = 7.9, H-8), 7.89 (d,

1H, J = 8.1, H-6⁰, 3-CF₃Ph), 7.95 (t, 1H, J = 7.7, H-9),

8.17 (s., 1H, H-2⁰3-CF₃Ph), 8.31 (d, 2H, J = 7.1, H-2⁰, 6⁰

Ph), 8.49 (d, 1H, J = 7.9, H-11), 11.05 (s, 1H, –C(O)NH);

¹³C-NMR (100 MHz) d: 36.71 (–SCH₂), 115.72 (CF₃),

118.36 (2⁰3-CF₃Ph), 120.25 (6⁰3-CF₃Ph), 120.28 (11a),

123.24 (4⁰3-CF₃Ph), 125.12 (8), 126.80 (10), 128.11 (11),

130.11 (4⁰Ph), 130.57 (3⁰3-CF₃Ph), 128.96 (3⁰, 5⁰Ph),

129.83 (2⁰, 6⁰Ph), 132.07 (5⁰3-CF₃Ph), 132.11 (1⁰Ph),

136.08 (9), 140.32 (1⁰3-CF₃Ph), 144.18 (3), 149.70 (11b),

151.08 (7a), 154.83 (6), 160.14 (2), 167.05 (CONH); LC-

MS, m/z = 508 [M?1]; Anal. calcd. for C₂₅H₁₆F₃N₅O₂S:

C, 59.17; H, 3.18; N, 13.80; S, 6.32; Found: C, 59.19; H,

3.21; N, 13.80; S, 6.34.

N-(2-Triﬂuorophenyl)-2-[(2-oxo-3-methyl-2H-[1,2,4]triaz-

ino[2,3-c]quinazolin-6-yl)thio]-acetamide

(3i) Yield:

Method A, 54.2 %; Method B, 78.1 %; Method C, 56.1 %;

M.p. 231–233 ꢁC; IR (cm^-1): 3298, 3077, 3042, 3014,

2960, 2925, 2855, 1680, 1662, 1623, 1596, 1566, 1537,

1508, 1472, 1454, 1438, 1384, 1359, 1335, 1312, 1292,

1276, 1266, 1242, 1202, 1174, 1138, 1122, 1111, 1055,

1037, 967, 951, 875, 853, 776, 766, 700, 685, 667, 650,

630, 615; ¹H-NMR (400 MHz) d: 2.39 (s, 3H, 3-CH₃), 4.27

(s, 2H, –S–CH₂), 7.51–7.44 (m, 2H, H-3⁰, 6⁰2-CF₃Ph),

7.68 (t, 2H, J = 7.7, H-4⁰, 5⁰2-CF₃Ph), 7.78–7.72 (m, 2H,

H-8, 10), 7.99 (t, 1H, J3 = 7.7, J4 = 1.5, H-9), 8.47 (d,

1H, J3 = 7.9, J4 = 1.2, H-11), 10.03 (s, 1H, –C(O)NH);

LC-MS, m/z = 446 [M?1], 447 [M?2]; Anal. calcd. for

C₂₀H₁₄F₃N₅O₂S: C, 53.93; H, 3.17; N, 15.72; S, 7.20;

Found: C, 53.93; H, 3.17; N, 15.72; S, 7.20.

N-(3-Triﬂuorophenyl)-2-[(2-oxo-3-(4-methylphenyl)-2H-

[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]acetamide

(3l)

Yield: Method C, 84.4 %; M.p. 268–270 ꢁC; IR (cm^-1):

3336, 3248, 2978, 2922, 2896, 2854, 1685, 1649, 1625,

1591, 1564, 1538, 1528, 1487, 1469, 1437, 1381, 1334,

1323, 1288, 1270, 1243, 1220, 1186, 1162, 1137, 1117,

1068, 1020, 988, 965, 955, 941, 923, 894, 876, 852, 832,

1

N-(2-Triﬂuorophenyl)-2-[(2-oxo-3-(4-methoxyphenyl)-2H-

802, 778, 766, 722, 698, 683, 653, 625, 603; H-NMR

[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]-acetamide

(3j)

(400 MHz) d: 2.42 (s, 3H, 4-CH₃Ph), 4.29 (s, 2H, –S–

CH₂), 6.86 (t, 1H, H-5⁰, 3-CF₃Ph), 7.40 (d, 2H, J = 8.1,

H-3⁰, 5⁰4-CH₃OPh), 7.59 (t, 1H, J = 7.7, H-10), 7.70–7.65

(m, 2H, H-4⁰3-CF₃Ph, H-8), 7.84 (d, 1H, J = 8.3, H-6⁰

3-CF₃Ph), 7.94 (t, 1H, J = 7.7, H-9), 8.14 (s, 1H, H-2⁰

3-CF₃Ph), 8.25 (d, 2H, J = 8.1, H-2⁰, 6⁰4-CH₃Ph), 8.47 (d,

1H, J = 7.9, H-11), 10.83 (s, 1H, –C(O)NH); LC-MS, m/

z = 522 [M?1], 523 [M?2]; Anal. calcd. for

C₂₆H₁₈F₃N₅O₂S: C, 59.88; H, 3.48; N, 13.43; S, 6.15;

Found: C, 59.88; H, 3.48; N, 13.43; S, 6.15.

Yield: Method C, 83.7 %; M.p. 228–230 ꢁC; IR (cm^-1):

3229, 3078, 3038, 3012, 2936, 2844, 1748, 1664, 1591,

1562, 1548, 1536, 1499, 1469, 1455, 1420, 1379, 1336,

1315, 1289, 1272, 1255, 1241, 1205, 1170, 1137, 1118,

1071, 1057, 1028, 994, 982, 954, 941, 903, 879, 839, 813,

771, 723, 701, 684, 651, 643, 622; ¹H-NMR (400 MHz) d:

3.88 (s, 3H, 4-CH₃OPh), 4.32 (s, 2H, –S–CH₂), 7.16 (d, 2H,

J = 8.9, H-3⁰, 5⁰4-CH₃OPh), 7.53–7.45 (m, 2H, H-3⁰, 6⁰

2-CF₃Ph), 7.72–7.66 (m, 2H, H-8, 10), 7.77 (t, 2H, J = 7.3,

H-4⁰, 5⁰2-CF₃Ph), 8.00 (t, 1H, J = 7.7, H-9), 8.37 (d, 2H,

J = 8.9, H-2⁰, 6⁰4-CH₃OPh), 8.50 (d, 1H, J = 7.9, H-11),

10.09 (s, 1H, –C(O)NH); LC-MS, m/z = 538 [M?1], 540

[M?3]; Anal. calcd. for C₂₆H₁₈F₃N₅O₃S: C, 58.10; H,

3.38; N, 13.03; S, 5.97; Found: C, 58.10; H, 3.37; N, 13.03;

S, 5.95.

N-(4-Triﬂuorophenyl)-2-[(2-oxo-3-(4-methylphenyl)-2H-

[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]-acetamide (3m)

Yield: Method A, 83.1 %; Method B, 87.5 %; Method C,

88.2 %; M.p. 241–244 ꢁC; IR (cm^-1): 3332, 3244, 2979,

2897, 1691, 1649, 1589, 1518, 1487, 1404, 1381, 1320, 1289,

1272, 1243, 1183, 1163, 1139, 1107, 1066, 1012, 942, 874,

862, 838, 777, 764, 703, 685, 653, 626; ¹H-NMR (400 MHz)

d: 2.42 (s, 3H, 4-CH₃Ph), 4.29 (s, 2H, –S–CH₂), 7.41 (d, 2H,

J = 8.1, H-3⁰, 5⁰4-CH₃Ph), 7.74–7.65 (m, 4H, H-3⁰, 5⁰

4-CF₃Ph, H-8, 10), 7.87 (d, 2H, J = 8.3, H-2⁰, 6⁰4-CF₃Ph),

7.95 (t, 1H, J = 7.7, H-9), 8.25 (d, 2H, J = 8.1, H-2⁰, 6⁰

4-CH₃Ph), 8.47 (d, 1H, J = 7.9, H-11), 10.85 (s, 1H,

–C(O)NH); LC-MS, m/z = 522 [M?1], 524 [M?3]; Anal.

N-(3-Triﬂuorophenyl)-2-[(2-oxo-3-phenyl-2H-[1,2,4]triaz-

ino[2,3-c]quinazolin-6-yl)thio]acetamide

(3k) Yield:

Method A, 84.2 %; Method B, 90.5 %; Method C, 95.6 %;

M.p. 230–232 ꢁC; IR (cm^-1): 3332, 3206, 3130, 3053,

2978, 2897, 1970, 1688, 1651, 1591, 1565, 1542, 1530,

1495, 1481, 1469, 1434, 1380, 1322, 1288, 1269, 1247,

1236, 1215, 1182, 1163, 1136, 1115, 1097, 1068, 1024,

123

2624

Med Chem Res (2013) 22:2610–2632

calcd. for C₂₆H₁₈F₃N₅O₂S: C, 59.88; H, 3.48; N, 13.43; S,

6.15; Found: C, 58.87; H, 3.48; N, 13.42; S, 6.14.

–C(O)NH); LC-MS, m/z = 396 [M?1], 398 [M?3]; Anal.

calcd. for C₁₉H₁₄FN₅O₂S: C, 57.71; H, 3.57; N, 17.71; S,

8.11; Found: C, 57.72; H, 3.60; N, 17.71; S, 8.12.

N-(2-Fluorophenyl)-2-[(2-oxo-3-(4-methoxyphenyl)-2H-

[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]-acetamide (3n)

Yield: Method A, 77.5 %; Method B, 87.4 %; Method C,

75.9 %; M.p. 229–232 ꢁC; IR (cm^-1): 3333, 3076, 3011,

2960, 2927, 2838, 1702, 1660, 1616, 1587, 1562, 1535, 1508,

1495, 1485, 1468, 1447, 1418, 1365, 1339, 1326, 1303, 1275,

1254, 1193, 1177, 1160, 1139, 1117, 1108, 1076, 1025, 992,

941, 888, 850, 807, 782, 771, 753, 726, 705, 686, 668, 636,

625; ¹H-NMR (400 MHz) d: 3.57 (s, 3H, 4-CH₃OPh), 4.02 (s,

2H, –S–CH₂), 6.88–6.82 (m, 4H, H-3⁰, 5⁰4-CH₃OPh), H-3⁰, 6⁰

2-FPh), 6.98 (t, 1H, H-4⁰2-FPh), 7.37 (t, 1H, J = 7.7, H-10),

7.44 (d, 1H, J = 7.9, H-8), 7.57 (t, 1H, J = 8.1, H-5⁰, 2-FPh),

7.66 (t, 1H, J = 7.7, H-9), 8.07 (d, 2H, J = 8.9, H-2⁰, 6⁰

4-CH₃OPh), 8.17 (d, 1H, J = 7.9, H-11), 9.94 (s, 1H,

–C(O)NH); LC-MS, m/z = 488 [M?1], 489 [M?2]; Anal.

calcd. forC₂₅H₁₈FN₅O₃S:C, 61.59;H, 3.72; N, 14.37;S, 6.58;

Found: C, 61.59; H, 3.73; N, 14.37; S, 6.60.

N-(3-Fluorophenyl)-2-[(2-oxo-3-phenyl-2H-[1,2,4]triazino

[2,3-c]quinazolin-6-yl)thio]acetamide (3q) Yield: Method

A, 85.1 %; Method B, 94.7 %; Method C, 99.0 %; M.p.

228–229 ꢁC; IR (cm^-1): 3305, 3266, 3208, 3145, 3092,

3038, 2963, 2938, 2917, 2888, 2850, 1676, 1647, 1604, 1584,

1549, 1502, 1487, 1469, 1445, 1431, 1391, 1328, 1316, 1284,

1266, 1253, 1183, 1162, 1139, 1117, 1079, 1048, 1022, 1001,

987, 969, 941, 925, 885, 870, 851, 813, 784, 770, 759, 690,

680, 655, 622, 613; ¹H-NMR (400 MHz) d: 4.29 (s, 2H, –S–

CH₂), 6/91 (t, 1H, J = 8.3, H-5⁰3-FPh), 7.44–7.35 (m, 2H,

H-4⁰, 6⁰3-FPh), 7.73–7.53 (m, 6H, H-8, 10; H-2⁰3-FPh; H-3⁰,

4⁰, 5⁰Ph), 7.96 (t, 1H, J = 7.7, H-9), 8.30 (d, 2H, J = 7.7,

H-2⁰, 6⁰Ph), 8.48 (d, 1H, J = 7.9, H-11), 10.89 (s, 1H, –

C(O)NH); ¹³C-NMR (100 MHz) d: 36.74 (–SCH₂), 106.34

(2⁰3-FPh), 110.29 (4⁰3-FPh), 115.44 (6⁰3-FPh), 118.36

(11a), 126.13 (8), 126.82 (10), 128.11 (11), 128.96 (3⁰, 5⁰Ph),

129.83 (2⁰, 6⁰Ph), 130.91 (5⁰3-FPh), 132.11 (1⁰3-FPh),

133.94 (4⁰Ph), 141.31 (3⁰3-FPh), 144.19 (1⁰Ph), 149.72 (9),

151.08 (11b), 154.86 (3), 160.14 (7a), 161.67 (6), 163.59 (2),

166.24 (CONH); LC-MS, m/z = 458 [M?1], 460 [M?3];

Anal. calcd. for C₂₄H₁₆FN₅O₂S: C, 63.01; H, 3.53; N, 15.31;

S, 7.01; Found: C, 63.00; H, 3.52; N, 15.30; S, 6.99.

N-(2-Fluorophenyl)-2-[(2-oxo-3-(4-methylphenyl)-2H-[1,2,4]

triazino[2,3-c]quinazolin-6-yl)thio]acetamide (3o) Yield:

Method C, 92.3 %; M.p. 267–270 ꢁC; IR (cm^-1): 3240, 3041,

2988, 1671, 1657, 1597, 1564, 1550, 1534, 1499, 1471, 1451,

1388, 1365, 1337, 1323, 1310, 1287, 1272, 1260, 1241, 1207,

1188, 1163, 1151, 1139, 1125, 1105, 1072, 1031, 993, 977, 940,

903, 867, 835, 805, 792, 780, 769, 756, 731, 704, 684, 639, 627;

¹H-NMR (400 MHz) d:2.42(s, 3H, 4-CH₃Ph), 4.33 (s, 2H, –S–

CH₂), 7.19–7.14 (m, 2H, H-3⁰, 6⁰2-FPh), 7.30 (t, 1H, H-4⁰

2-FPh), 7.41 (d, 2H, J = 8.1, H-3⁰, 5⁰4-CH₃Ph), 7.68 (t, 1H,

J = 7.7, H-10), 7.74 (d, 1H, J = 7.9, H-8), 7.83 (t, 1H, H-5⁰,

2-FPh), 7.97 (t, 1H, J = 7.7, H-9), 8.25 (d, 2H, J = 8.1, H-2⁰,6⁰

4-CH₃Ph), 8.48 (d, 1H, J = 7.9, H-11), 10.24 (s, 1H, –

C(O)NH); LC-MS, m/z = 472 [M?1], 474 [M?3]; Anal.

calcd. for C₂₅H₁₈FN₅O₂S: C, 63.68; H, 3.85; N, 14.85; S, 6.80;

Found: C, 63.68; H, 3.85; N, 14.85; S, 6.80.

N-(3-Fluorophenyl)-2-[(2-oxo-3-(4-methylphenyl)-2H-

[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]acetamide (3r)

Yield: Method C, 84.8 %; M.p. 268–270 ꢁC; IR (cm^-1):

3247, 3067, 3009, 2978, 2919, 2859, 1686, 1650, 1624, 1607,

1587, 1563, 1542, 1518, 1485, 1421, 1387, 1324, 1267, 1243,

1198, 1185, 1155, 1138, 1107, 1075, 1031, 1018, 994, 980,

942, 928, 894, 863, 834, 784, 767, 755, 702, 681, 638, 626,

613; ¹H-NMR (400 MHz) d: 2.42 (s, 3H, 4-CH₃Ph), 4.26 (s,

2H, –S–CH₂), 6.90 (t, 1H, H-5⁰3-FPh), 7.45–7.36 (m, 4H,

H-4⁰, 6⁰3-FPh, H-3⁰, 5⁰4-CH₃Ph), 7.72–7.59 (m, 3H, H-2⁰

3-FPh, H-8, 10), 7.95 (t, 1H, J = 7.7, H-9), 8.25 (d, 2H,

J = 8.1, H-2⁰, 6⁰4-CH₃Ph), 8.47 (d, 1H, J = 7.9, H-11),

10.70 (s, 1H, –C(O)NH); LC-MS, m/z = 472 [M?1], 474

[M?3]; Anal. calcd. for C₂₅H₁₈FN₅O₂S: C, 63.68; H, 3.85; N,

14.85; S, 6.80; Found: C, 63.70; H, 3.87; N, 14.85; S, 6.82.

N-(2-Fluorophenyl)-2-[(2-oxo-3-methyl-2H-[1,2,4]triazi-

no[2,3-c]quinazolin-6-yl)thio]acetamide (3p) Yield: Method

A, 75.3 %; Method B, 81.6 %; Method C, 82.2 %; M.p.

214–215 ꢁC; IR (cm^-1): 3265, 3212, 3123, 3076, 2990,

2922, 2864, 1685, 1665, 1621, 1600, 1584, 1551, 1506, 1488,

1458, 1437, 1384, 1360, 1332, 1301, 1279, 1254, 1206, 1190,

1172, 1156, 1134, 1116, 1038, 951, 895, 873, 853, 810, 765,

697, 684, 666, 640, 631, 612; ¹H-NMR (400 MHz) d: 2.39 (s,

3H, CH₃), 4.29 (s, 2H, –S–CH₂), 7.19–7.14 (m, 2H, H-3⁰, 6⁰

2-FPh), 7.29 (t, 1H, H-4⁰2-FPh), 7.66 (t, 1H, J = 7.7, H-10),

7.72 (d, 1H, J = 7.9, H-8), 7.86 (t, 1H, H-5⁰, 2-FPh), 7.96 (t,

1H, J = 7.7, H-9), 8.46 (d, 1H, J = 7.9, H-11), 10.21 (s, 1H,

N-(4-Fluorophenyl)-2-[(2-oxo-3-(4-methylphenyl)-2H-

[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]acetamide (3s)

Yield: Method A, 85.3 %; Method B, 88.4 %; Method C,

92.3 %; M.p. 236–238 ꢁC; IR (cm^-1): 3266, 3151, 3068,

2972, 2896, 1660, 1613, 1591, 1562, 1548, 1512, 1496, 1469,

1405, 1366, 1338, 1307, 1284, 1269, 1226, 1186, 1166, 1154,

1137, 1106, 1073, 1032, 996, 962, 941, 904, 832, 803, 770,

756, 712, 702, 685, 641, 626, 605; ¹H-NMR (400 MHz) d:

2.42 (s, 3H, 4-CH₃Ph), 4.25 (s, 2H, –S–CH₂), 7.18 (t, 2H,

J = 8.8, H-3⁰, 5⁰4-FPh), 7.41 (d, 2H, J = 8.1, H-3⁰, 5⁰

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2625

4-CH₃Ph), 7.69–7.64 (m, 3H, H-2⁰, 6⁰4-FPh, H-10), 7.71 (d,

1H, J = 7.9, H-8), 7.96 (t, 1H, J = 7.7, H-9), 8.25 (d, 2H,

J = 8.1, H-2⁰, 6⁰4-CH₃Ph), 8.47 (d, 1H, J = 7.9, H-11),

10.53 (s, 1H, –C(O)NH); LC-MS, m/z = 472 [M?1],474

[M?3]; Anal. calcd. for C₂₅H₁₈FN₅O₂S: C, 63.68; H, 3.85;

N, 14.85; S, 6.80; Found: C, 63.67; H, 3.84; N, 14.87; S, 6.81.

J = 7.9, H-11), 10.48 (s, 1H, J = 5.4, –C(O)NH); ¹³C-NMR

(100 MHz) d: 18.19 (–CH₃), 36.69 (–SCH₂), 118.61 (11a),

121.23 (2⁰, 6⁰Ph), 126.04 (8), 126.63 (10), 127.52 (4⁰Ph), 127.98

(11), 129.22 (3⁰, 5⁰Ph), 136.02 (9), 138.43 (1⁰Ph), 144.17 (11b),

151.97 (3), 154.57 (7a), 155.22 (6), 161.00 (2), 166.52 (CONH);

EI-MS, m/z (I_rel, %) = 411 (3.1), 347 (8.3), 285 (99.9), 284

(15.8), 282 (6.6), 280 (7.8), 246 (17.1), 244 (34.8), 243 (11.9), 241

(7.2), 216 (20.2), 215 (18.9), 210 (6.7), 202 (8.9), 171 (14.8), 170

(27.2), 169 (11.5), 161 (12.4), 159 (6.0), 144 (7.7), 140 (19.4), 137

(22.3), 132 (6.1), 128 (17.2), 125 (6.6), 124 (13.3), 118 (5.5), 107

(5.9), 103 (6.6), 102 (13.6), 77 (9.6), 76 (8.4), 47 (5.0); LC-MS, m/

z = 412 [M?1], 414 [M?3], 415 [M?4]; Anal. calcd. for

C₁₉H₁₄ClN₅O₂S: C, 55.41; H, 3.43; Cl, 8.61; N, 17.00; S, 7.78;

Found: C, 55.40; H, 3.43; Cl, 8.60; N, 16.98; S, 7.77.

N-(2-Chlorophenyl)-2-[(2-oxo-3-(4-methoxyphenyl)-2H-

[1,2,4]triazino[2,3-c]quinazolin-6-yl)thio]-acetamide (3t)

Yield: Method C, 66.1 %; M.p. 249–252 ꢁC; IR (cm^-1): 3542,

3408, 3249, 3213, 3164, 3114, 3069, 3040, 3014, 2962, 2916,

2835, 1695, 1641, 1601, 1577, 1561, 1541, 1514, 1487, 1471,

1459, 1440, 1380, 1343, 1323, 1304, 1287, 1275, 1254, 1199,

1177, 1142, 1118, 1080, 1057, 1021, 995, 960, 944, 904, 842,

814, 801, 783, 767, 725, 701, 690, 637, 627; ¹H-NMR

(400 MHz) d: 3.88 (s, 3H, 4-CH₃OPh), 4.35 (s, 2H, –S–CH₂),

7.16 (d, 2H, J = 8.9, H-3⁰, 5⁰4-CH₃OPh), 7.21 (t, 1H, J = 7.4,

H-5⁰2-ClPh), 7.33 (t, 1H, J = 7.4, H-4⁰2-ClPh), 7.52 (d, 1H,

J = 8.1, H-3⁰2-ClPh), 7.69 (t, H-10, J = 7.7, H-10), 7.75 (d,

1H, J = 7.9, H-8), 7.79 (d, 1H, J = 8.1, H-6⁰2-ClPh), 7.98 (t,

1H, J = 7.7, H-9), 8.39 (d, 2H, J = 8.9, H-2⁰, 6⁰4-CH₃OPh),

8.49 (d, 1H, J = 7.9, H-11), 9.99 (s, 1H, –C(O)NH); LC-MS,

m/z = 504 [M?1], 505 [M?2], 506 [M?3]; Anal. calcd. for

C₂₅H₁₈ClN₅O₃S: C, 63.68; H, 3.85; N, 14.85; S, 6.80; Found:

C, 63.68; H, 3.85; N, 14.87; S, 6.81.

N-(4-Chlorophenyl)-2-[(2-oxo-3-phenyl-2H-[1,2,4]triazino

[2,3-c]quinazolin-6-yl)thio]acetamide

(3w) Yield:

Method A, 75.3 %; Method B, 92.4 %; Method C, 91.8 %;

M.p. 247–249 ꢁC; IR (cm^-1): 3626, 3254, 3064, 2973,

2887, 1660, 1587, 1555, 1524, 1505, 1485, 1467, 1443,

1391, 1368, 1331, 1310, 1285, 1266, 1228, 1181, 1158,

1134, 1089, 1075, 1046, 1011, 1001, 988, 970, 935, 901,

878, 841, 825, 809, 781, 771, 753, 737, 704, 685, 649; ¹H-

NMR (400 MHz) d: 4.27 (s, 2H, –S–CH₂), 7.40 (d, 2H,

J = 8.7, H-3⁰, 5⁰, 4-ClPh), 7.65–7.59 (m, H-3⁰, 4⁰, 5⁰Ph),

7.73–7.66 (m, 4H, H-8, 10, 2⁰, 6⁰4-ClPh), 7.97 (t, 1H,

J3 = 7.7, J4 = 1.5, H-9), 8.30 (d, 2H, J = 7.7, H-2⁰, 6⁰

Ph), 8.49 (d, 1H, J = 7.9, H-11), 10.62 (s, 1H, –C(O)NH);

LC-MS, m/z = 474 [M?1], 476 [M?3; Anal. calcd. for

C₂₄H₁₆ClN₅O₂S: C, 60.82; H, 3.40; Cl, 7.48; N, 14.78; S,

6.77; Found: C, 60.84; H, 3.43; Cl, 7.47; N, 14.80; S, 6.78.

N-(3-Chlorophenyl)-2-[(2-oxo-3-phenyl-2H-[1,2,4]triazi-

no[2,3-c]quinazolin-6-yl)thio]acetamide (3u) Yield: Method

A, 84.3 %; Method B, 93.2 %; Method C, 84.4 %; M.p.

258–260 ꢁC; IR (cm^-1): 3304, 3269, 3191, 3127, 3090, 3068,

3008, 2958, 2903, 1770, 1694, 1652, 1592, 1565, 1546, 1500,

1482, 1443, 1418, 1404, 1382, 1343, 1331, 1314, 1289, 1265,

1248, 1235, 1192, 1167, 1143, 1097, 1076, 1032, 1020, 1002,

989, 964, 942, 905, 865, 849, 812, 782, 770, 755, 728, 689,

682, 655, 638, 613; ¹H-NMR (400 MHz) d: 4.27 (s, 2H, –S–

CH₂), 7.15(d, 1H, H-4⁰3-ClPh), 7.38(t, 1H, J = 8.1, 3-ClPh),

7.53 (d, 1H, J = 7.6, H-6⁰3-ClPh), 7.65–7.59 (m, 3H, H-3⁰, 4⁰,

5⁰Ph), 7.72–7.67 (m, 2H, H-8, 10), 7.85 (s, 1H, H-2⁰3-ClPh),

7.97 (t, 1H, J = 7.7, H-9), 8.30 (d, 2H, J = 7.7, H-2⁰, 6⁰Ph),

8.49(d,1H, J = 7.9,H-11), 10.68(s, 1H, –C(O)NH);LC-MS,

m/z = 473 [M], 475 [M?2], 476 [M?3]; Anal. calcd. for

C₂₄H₁₆ClN₅O₂S:C,60.82;H,3.40;Cl, 7.48;N,14.78;S,6.77;

Found: C, 60.83; H, 3.43; Cl, 7.49; N, 14.80; S, 6.79.

N-(4-Chlorophenyl)-2-[(2-oxo-3-(4-methylphenyl)-2H-[1,

2,4]triazino[2,3-c]quinazolin-6-yl)thio]acetamide

(3x)

Yield: Method C, 81.9 %; M.p. 267–268ꢁC; IR (cm^-1): 3248,

3115, 3063, 2975, 2898, 1659, 1626, 1589, 1561, 1548, 1518,

1492, 1469, 1396, 1366, 1333, 1307, 1283, 1268, 1239, 1185,

1161, 1137, 1094, 1073, 1033, 1012, 996, 984, 963, 940, 904,

856, 831, 769, 737, 711, 702, 684, 639, 625; ¹H-NMR

(400 MHz) d: 2.42 (s, 3H, 4-CH₃Ph), 4.26 (s, 2H, –S–CH₂),

7.42–7.38 (m, 4H, H-3⁰, 5⁰4-ClPh, H-3⁰, 5⁰4-CH₃Ph),

7.71–7.65 (m, 4H, H-8, 10; H-2⁰, 6⁰4-ClPh), 7.95 (t, 1H,

J = 7.7, H-9), 8.25 (d, 2H, J = 8.3, H-2⁰, 6⁰4-CH₃Ph), 8.47

(d, 1H, J = 7.9, H-11), 10.63 (s, 1H, –C(O)NH); LC-MS, m/

z = 488 [M?1], 489 [M?2], 490 [M?3]; Anal. calcd. for

C₂₅H₁₈ClN₅O₂S:C,61.54;H,3.72;Cl, 7.27;N,14.35;S,6.57;

Found: C, 61.54; H, 3.72; Cl, 7.27; N, 14.35; S, 6.57.

N-(4-Chlorophenyl)-2-[(2-oxo-3-methyl-2H-[1,2,4]triazino

[2,3-c]quinazolin-6-yl)thio]acetamide (3v) Yield: Method

C, 87.1 %; M.p. 273–275 ꢁC; IR (cm^-1): 3253, 3187, 2985,

2916, 1657, 1597, 1581, 1557, 1503, 1490, 1466, 1433, 1395,

1361, 1318, 1286, 1260, 1243, 1226, 1205, 1157, 1133, 1094,

1045, 1010, 982, 953, 863, 824, 769, 741, 698, 686, 644, 631, 606;

¹H-NMR (400 MHz) d: 2.41 (s, 3H, CH₃), 4.23 (s, 2H, –S–CH₂),

7.38 (d, 2H, J = 8.7, H-3⁰, 5⁰, 4-ClPh), 7.69–7.64 (m, 4H, H-8,

10, 2⁰, 6⁰, 4-ClPh), 7.94 (t, 1H, J = 7.7, H-9), 8.46 (d, 1H,

N-(2-Nitrophenyl)-2-[(2-oxo-3-(4-methylphenyl)-2H-[1,2,

4]triazino[2,3-c]quinazolin-6-yl)thio]acetamide (3y) Yield:

Method A, 79.8 %; Method B, 76.3 %; Method C, 85.3 %;

M.p. 228–229 ꢁC; IR (cm^-1): 3247, 3118, 3076, 3035, 2976,

2918, 2855, 1658, 1626, 1597, 1567, 1551, 1530, 1512, 1502,

123

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Med Chem Res (2013) 22:2610–2632

1486, 1469, 1439, 1383, 1362, 1337, 1308, 1287, 1270, 1243,

1207, 1186, 1164, 1138, 1108, 1039, 1020, 993, 978, 941, 904,

872, 834, 780, 769, 751, 723, 703, 683, 640, 626, 605; ¹H-NMR

(400 MHz) d: 2.56 (s, 3H, 4-CH₃Ph), 3.51 (s, 2H, –S–CH₂),

6.66–6.59 (m, 3H, H-3⁰, 5⁰4-CH₃Ph), H-4⁰2-NO₂Ph),

6.98–6.88 (m, 2H, H-8, 10), 7.04–6.99 (m, 2H, H-3⁰, 5⁰

2-NO₂Ph), 7.23–7.16 (m, 2H, H-6⁰2-NO₂Ph, H-9), 7.47 (d, 2H,

J = 7.5, H-2⁰, 6⁰4-CH₃Ph), 7.70 (d, 1H, J = 7.9, H-11), 10.00

(s, 1H, –C(O)NH); LC-MS, m/z = 499 [M?1]; Anal. calcd. for

C₂₅H₁₈N₆O₄S: C, 60.23; H, 3.64; N, 16.86; S, 6.43; Found: C,

60.25; H, 3.66; N, 16.87; S, 6.44.

The precipitate was ﬁltered, dried and recrystallized from

the dioxane-water (1:1).

N-Benzyl-2-[(3-methyl-2-oxo-2H-[1,2,4]triazino[2,3-c]quinaz-

oline-6-yl)thio]acetamides (4a) Yield: Method A, 70.2 %;

Method B, 73.5 %; M.p. 216–218 ꢁC; IR (cm^-1): 3284, 3063,

2981, 2914, 1660, 1650, 1581, 1557, 1503, 1468, 1453, 1434,

1396, 1363, 1323, 1306, 1283, 1260, 1221, 1179, 1132, 1102,

1067, 1045, 1031, 953, 880, 858, 773, 734, 695, 687, 632, 606;

¹H-NMR (400 MHz) d: 2.39 (s, 3H, CH₃), 4.08 (s, 2H, –S–

CH₂), 4.34(d, 2H, J = 5.8, –CH₂Ph), 7.29–7.20 (m, 5H, H-2⁰,

3⁰, 4⁰, 5⁰, 6⁰, –CH₂Ph), 7.70–7.64 (m, 2H, H-8, 10), 7.96 (t, 1H,

J = 7.7, H-9), 8.46 (d, 1H, J = 7.9, H-11), 8.81 (t, 1H,

J = 5.6, –C(O)NH); ¹³C-NMR (100 MHz) d: 18.19 (–CH₃),

35.50 (–SCH₂), 43.04 (–N–CH₂–), 118.53 (11a), 125.99 (8),

126.85 (10), 127.60 (2⁰, 6⁰Ph), 127.22 (4⁰Ph), 127.95 (11),

128.67 (3⁰, 5⁰Ph), 135.94 (9), 139.65 (1⁰Ph), 144.20 (3),

151.98 (11b), 154.66 (7a), 155.16 (6), 160.99 (2), 167.25

(CONH); EI-MS, m/z (I_rel, %) = 391 (4.0), 286 (7.0), 246

(7.1), 245 (50.1), 244 (8.4), 243 (38.7), 217 (17.0), 216 (9.2),

215 (5.9), 204 (49.3), 203 (11.2), 202 (7.1), 201 (5.2), 188

(5.0), 172 (6.2), 171 (23.1), 170 (27.4), 148 (23.2), 146 (10.2),

145 (12.6), 144 (8.6), 143 (9.1), 142 (7.5), 119 (9.0), 118

(15.4), 116 (6.9), 115 (10.4), 107 (7.1), 106 (7.9), 105 (9.0),

104 (14.7), 102 (9.0), 91 (99.9), 90 (17.6), 88 (5.4), 79 (10.6),

78 (9.7), 77 (9.6), 65 (9.3), 63 (5.6), 56 (9.3), 55 (5.0), 51 (8.9),

46(9.4);LC-MS, m/z = 392[M?1], 394[M?3];Anal.calcd.

for C₂₀H₁₇N₅O₂S: C, 61.37;H, 4.38; N, 17.89; S, 8.19; Found:

C, 61.39; H, 4.38; N, 17.91; S, 8.21.

N-(2-Carboxyphenyl)-2-[(2-oxo-3-(4-methylphenyl)-2H-[1,

2,4]triazino[2,3-c]quinazolin-6-yl)thio]-acetamide

(3z)

Yield: Method C, 82.4 %; M.p. 223–226 ꢁC; IR (cm^-1):

3317, 2974, 2875, 2822, 2562, 1701, 1670, 1598, 1567,

1554, 1534, 1503, 1470, 1451, 1411, 1376, 1341, 1326,

1311, 1260, 1240, 1185, 1161, 1136, 1106, 1038, 1022,

995, 955, 940, 925, 900, 873, 845, 830, 807, 793, 756, 702,

1

683, 661, 641, 627, 611; H-NMR (400 MHz) d: 2.43 (s,

3H, 4-CH₃Ph), 4.26 (s, 2H, –S–CH2), 6.87 (t, 1H, H-4⁰

2-COOHPh), 7.16 (t, 1H, H- 5⁰23-COOHPh), 7.42 (d, 2H,

H-3⁰, 5⁰4-CH₃Ph), 7.57–7.52 (m, 2H, H-8, 10), 7.72 (t, 1H,

H-9), 8.12 (d, 1H, H-6⁰2-COOHPh), 8.26 (d, 2H, H-2⁰, 6⁰

4-CH₃Ph), 8.51–8.45 (m, 2H, H-3⁰2-COOHPh, H-11),

11.77 (s, 2H, –C(O)NH, COOH); LC-MS, m/z = 498

[M?1], 499 [M?2], 500 [M?3]; Anal. calcd. for

C₂₆H₁₉N₅O₄S: C, 62.77; H, 3.85; N, 14.08; S, 6.44; Found:

C, 62.76; H, 3.84; N, 14.07; S, 6.43.

N-Benzyl-2-[(3-phenyl-2-oxo-2H-[1,2,4]triazino[2,3-c]qui-

nazoline-6-yl)thio]acetamides (4b) Yield: Method A,

93.7 %; Method B, 94.3 %; M.p. 238–240 ꢁC; IR (cm^-1):

3267, 3076, 3057, 3031, 2982, 2919, 2870, 1669, 1649, 1589,

1554, 1506, 1486, 1469, 1453, 1383, 1367, 1334, 1311, 1285,

1267, 1242, 1228, 1215, 1182, 1162, 1136, 1103, 1069, 1029,

1002, 988, 938, 884, 846, 810, 785, 772, 743, 687, 652, 620;

¹H-NMR (400 MHz) d: 4.12 (s, 2H, –S–CH₂), 4.36 (d, 2H,

J = 5.8, –CH₂Ph), 7.29–7.20 (m, 5H, H-2⁰, 3⁰, 4⁰, 5⁰, 6⁰, –

CH₂Ph), 7.65–7.58 (m, 3H, H-3⁰,4⁰, 5⁰Ph), 7.73–7.68 (m, 2H,

H-8, 10), 7.99 (t, 1H, J = 7.7, H-9), 8.29 (d, 2H, J = 6.4,

H-2⁰, 5⁰Ph), 8.51 (d, 1H, J = 7.9, H-11), 8.84 (t, 1H, J = 5.0,

-C(O)NH); LC-MS, m/z = 454 [M?1], 456 [M?3]; Anal.

calcd. for C₂₅H₁₉N₅O₂S: C, 66.21; H, 4.22; N, 15.44; S, 7.07;

Found: C, 66.21; H, 4.22; N, 15.44; S, 7.07.

General procedure for synthesis of N-(R-benzyl)-2-[(3-R-2-

oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-

yl)thio]acetamides (4a–4o)

Method A N-(R-benzyl)-2-chloroacetamides (0.011 mol)

were added to a suspension of potassium salts of 3-R-6-

thio-6,7-dihydro-2H-[1,2,4]triazino[2,3-c]quinazolin-2-

ones (2a, 2b) (0.01 mol) in 20 ml of propanol-2, water-

propanol-2 (1:2), or dioxane and reﬂuxed for 60–90 min.

The mixture was cooled and poured into water. The pre-

cipitate was ﬁltered, dried and recrystallized from the

dioxan–water (1:1).

Method B N,N⁰-CDI (1.95 g, 0.011 mol) was added to a

solution of proper acid (1a, 1b) (0.01 mol) in 10 ml of

anhydrous dioxane or DMFA and heated at the water bath

at 60–80 ꢁC for 1 h with calcium chloride tube. The proper

benzylamine (0.01 mol) was added with stirring to the

resulting mixture and reﬂuxed for 3 h. The mixture was

poured into water, and neutralized to pH 6–7 by acetic acid.

N-(4-Methylbenzyl)-2-[(3-methyl-2-oxo-2H-[1,2,4]triazino

[2,3-c]quinazoline-6-yl)thio]acetamides (4c) Yield: Method

A, 58.0 %; Method B, 79.2 %; M.p. 214–216 ꢁC; IR (cm^-1):

3271, 3099, 3062, 3049, 3020, 3006, 2986, 2915, 2866, 1659,

1644, 1599, 1580, 1556, 1503, 1465, 1434, 1392, 1362, 1333,

1319, 1304, 1284, 1260, 1207, 1178, 1131, 1101, 1079, 1045,

1022, 1009, 954, 876, 859, 841, 807, 769, 725, 702, 684, 669,

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Med Chem Res (2013) 22:2610–2632

2627

631, 605; ¹H-NMR (400 MHz) d: 2.25 (s, 3H, 4-CH₃Ph), 2.39

(s, 3H, CH₃), 4.06 (s, 2H, –S–CH₂), 4.29 (d, 2H, J = 5.6, –

CH₂Ph), 7.04 (d, 2H, J = 7.5, H-3⁰, 5⁰4-CH₃Ph), 7.15 (d, 2H,

J = 7.5, H-2⁰, 6⁰4-CH₃Ph), 7.62 (d, 1H, J = 7.9, H-8), 7.67 (t,

1H, J = 7.7, H-10), 7.95 (t, 1H, J = 7.7, H-9), 8.45 (d, 1H,

J = 7.9, H-11), 8.75 (t, 1H, J = 5.4, –C(O)NH); LC-MS, m/

z = 406 [M?1], 407[M?2]; Anal. calcd. for C₂₁H₁₉N₅O₂S:C,

62.21; H, 4.72; N, 17.27; S, 7.91; Found: C, 62.21; H, 4.72; N,

17.27; S, 7.91.

C₂₁H₁₉N₅O₃S: C, 59.84; H, 4.54; N, 16.62; S, 7.61; Found:

C, 59.84; H, 4.54; N, 16.62; S, 7.61.

N-(2-Methoxybenzyl)-2-[(3-phenyl-2-oxo-2H-[1,2,4]triazi-

no[2,3-c]quinazoline-6-yl)thio]acetamides

(4f) Yield:

Method A, 93.1 %; M.p. 222–223 ꢁC; IR (cm^-1): 3277,

3075, 3035, 2993, 2923, 2838, 1668, 1643, 1584, 1555,

1505, 1489, 1466, 1443, 1421, 1389, 1365, 1338, 1310,

1282, 1268, 1241, 1226, 1189, 1160, 1135, 1114, 1102,

1080, 1062, 1049, 1024, 1001, 989, 964, 938, 887, 877,

849, 812, 781, 767, 747, 707, 690, 654, 628, 620; ¹H-NMR

(400 MHz) d: 3.78 (s, 3H, 2-CH₃OPh), 4.13 (s, 2H, –S–

CH₂), 4.30 (d, 2H, J = 5.6, –CH₂Ph), 6.75 (t, 1H, J = 7.3,

H-5⁰2-CH₃OPh), 6.96 (d, 1H, J = 8.3, H-3⁰2-CH₃OPh),

7.23–7.15 (m, 2H, H-4⁰, 6⁰, 2-CH₃OPh), 7.66–7.57 (m, 3H,

H-3⁰, 4⁰, 5⁰Ph), 7.76–7.68 (m, 2H, H-8, 10), 8.00 (t, 1H,

J = 7.7, H-9), 8.29 (d, 2H, J = 7.7, H-2⁰, 6⁰Ph), 8.51 (d,

1H, J = 7.9, H-11), 8.65 (t, 1H, J = 5.1, –C(O)NH); LC-

MS, m/z = 484 [M?1], 486 [M?3]; Anal. calcd. for

C₂₆H₂₁N₅O₃S: C, 64.58; H, 4.38; N, 14.48; S, 6.63; Found:

C, 64.60; H, 4.40; N, 14.48; S, 6.65.

N-(4-Methylbenzyl)-2-[(3-phenyl-2-oxo-2H-[1,2,4]triazino

[2,3-c]quinazoline-6-yl)thio]acetamides (4d) Yield: Method

A, 90.9 %; M.p. 242–244 ꢁC; IR (cm^-1): 3270, 3069, 3022,

2984, 2917, 2863, 1674, 1641, 1589, 1553, 1509, 1488, 1465,

1446, 1395, 1368, 1333, 1311, 1284, 1271, 1240, 1222, 1185,

1172, 1160, 1134, 1102, 1072, 1019, 1002, 986, 938, 881, 839,

810, 782, 772, 752, 724, 705, 687, 651, 623; ¹H-NMR

(400 MHz) d: 2.25 (s, 3H, 4-CH₃Ph), 4.09 (s, 2H, –S–CH₂),

4.30 (d, 2H, J = 5.6, –CH₂Ph), 7.05 (d, 2H, J = 7.5, H-3⁰, 5⁰

4-CH₃Ph), 7.16 (d, 2H, J = 7.5, H-2⁰, 6⁰4-CH₃Ph), 7.67–7.58

(m, 4H, H-8, H-3⁰, 4⁰, 5⁰Ph), 7.70 (t, 1H, J = 7.7, 10), 7.97 (t,

1H, J = 7.7, H-9), 8.29 (d, 2H, J = 7.3, H-2⁰, 6⁰Ph), 8.49 (d,

1H, J = 7.9, H-11), 8.79 (t, 1H, J = 5.4, –C(O)NH); EI-MS,

m/z(I_rel, %) = 468(4.9), 467(31.6), 319(5.7),308(12.9),307

(56.7), 305 (7.6), 305 (19.5), 290 (8.9), 289 (6.9), 261 (6.0),

260 (5.7), 244 (14.0), 243 (25.7), 217 (9.8), 216 (35.6), 213

(6.6), 205 (10.1), 204 (58.6), 202 (8.8), 200 (16.1), 188 (8.2),

186 (7.4), 185 (6.2), 174 (11.2), 172 (7.4), 170 (15.4), 162

(12.4), 161 (7.9), 149 (25.1), 146 (15.9), 144 (21.0), 134 (9.1),

132 (35.7), 130 (7.7), 126 (5.7), 121 (8.4), 120 (12.7), 119

(16.9), 118 (22.8), 117 (11.9), 116 (8.9), 106 (15.8), 105

(99.9), 103 (28.4), 91 (12.1), 90 (11.8), 88 (9.5), 87 (5.2), 77

(5.7), 73 (5.1), 62 (5.3), 55 (5.1), 44 (11.6), 43 (11.9); LC-MS,

m/z = 468 [M?1], 470 [M?3]; Anal. calcd. for

C₂₆H₂₁N₅O₂S: C, 66.79; H, 4.53; N, 14.98; S, 6.86; Found: C,

66.81; H, 4.53; N, 15.01; S, 6.88.

N-(4-Methoxybenzyl)-2-[(3-methyl-2-oxo-2H-[1,2,4]triazi-

(4g) Yield:

no[2,3-c]quinazoline-6-yl)thio]acetamides

Method A, 65.3 %; Method B, 89.5 %; M.p. 216–218 ꢁC; IR

(cm^-1): 3267, 3117, 3065, 2986, 2953, 2920, 2863, 2829,

1660, 1645, 1598, 1580, 1556, 1503, 1463, 1434, 1393, 1362,

1334, 1321, 1302, 1285, 1260, 1247, 1209, 1181, 1172, 1131,

1108, 1074, 1043, 1016, 1006, 954, 874, 841, 818, 770, 731,

684, 670, 632, 605; ¹H-NMR (400 MHz) d: 2.39 (s, 3H, CH₃),

3.71 (s, 3H, 4-CH₃OPh), 4.04 (s, 2H, –S–CH₂), 4.26 (d, 2H,

J = 5.6, –CH₂Ph), 6.79 (d, 2H, J = 8.3, H-3⁰, 5⁰4-CH₃OPh),

7.18 (d, 2H, J = 8.3, H-2⁰, 6⁰4-CH₃OPh), 7.61 (d, 1H,

J = 7.9, H-8), 7..67 (t, 1H, J = 7.7, H-10), 7.94 (t, 1H,

J = 7.7, H-9), 8.45 (d, 1H, J = 7.9, H-11), 8.73 (t, 1H,

J = 5.4, –C(O)NH); ¹³C-NMR (100 MHz) d: 18.19 (–CH₃),

35.55 (–SCH₂), 42.53 (–N–CH₂–), 55.52 (–OCH₃), 114.08

(3⁰, 5⁰4-CH₃OPh), 118.50 (11a), 125.94 (8), 126.86 (10),

127.92 (11), 129.01 (2⁰, 6⁰4-CH₃OPh), 131.59 (1⁰

4-CH₃OPh), 135.86(9), 144.17(3), 151.96(11b), 154.63(7a),

155.16 (6), 158.66 (4⁰4-CH₃OPh), 160.99 (2), 167.09

(CONH); LC-MS, m/z = 422 [M?1], 424 [M?3]; Anal.

calcd. for C₂₁H₁₉N₅O₃S: C, 59.84; H, 4.54; N, 16.62; S, 7.61;

Found: C, 59.84; H, 4.54; N, 16.62; S, 7.61.

N-(2-Methoxybenzyl)-2-[(3-methyl-2-oxo-2H-[1,2,4]triazi-

no[2,3-c]quinazoline-6-yl)thio]acetamides

(4e) Yield:

Method A, 65.3 %; Method B, 93.4 %; M.p. 227–229 ꢁC; IR

(cm^-1): 3294, 3073, 3005, 2967, 2940, 2918, 1651, 1605,

1585, 1562, 1508, 1489, 1461, 1421, 1390, 1357, 1321, 1287,

1266, 1244, 1222, 1167, 1137, 1113, 1046, 1025, 957, 890,

1

855, 811, 775, 743, 717, 700, 686, 632, 616; H-NMR

(400 MHz) d: 2.39 (s, 3H, CH₃), 3.78 (s, 3H, 2-CH₃OPh),

4.09 (s, 2H, –S–CH₂), 4.29 (d, 2H, J = 5.6, –CH₂Ph), 6.75 (t,

1H, J = 7.3, H-5⁰2-CH₃OPh), 6.96 (d, 1H, J = 7.9, H-3⁰

2-CH₃OPh), 7.23–7.15 (m, 2H, H-4⁰, 6⁰, 2-CH₃OPh),

7.72–7.65 (m, 2H, H-8, 10), 7.98 (t, 1H, J = 7.7, H-9), 8.47

(d, 1H, J = 7.9, H-11), 8.61 (t, 1H, J = 5.6, –C(O)NH); LC-

MS, m/z = 422 [M?1], 424 [M?3]; Anal. calcd. for

N-(4-Methoxybenzyl)-2-[(3-phenyl-2-oxo-2H-[1,2,4]triazi-

no[2,3-c]quinazoline-6-yl)thio]acetamides (4h) Yield:

Method A, 95.1 %; Method B, 96.8 %; M.p. 227–229 ꢁC; IR

(cm^-1): 3270, 3074, 3009, 2974, 2904, 2866, 2836, 1649,

1588, 1554, 1504, 1487, 1467, 1443, 1392, 1365, 1337, 1313,

1302, 1284, 1250, 1217, 1181, 1172, 1136, 1102, 1073, 1030,

123

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Med Chem Res (2013) 22:2610–2632

1017, 987, 966, 940, 879, 843, 825, 810, 780, 770, 753, 724,

689, 652; ¹H-NMR (400 MHz) d: 3.71 (s, 3H, 2-CH₃OPh),

4.09 (s, 2H, –S–CH₂), 4.28 (d, 2H, J = 5.6, –CH₂Ph), 6.79 (t,

2H, J = 8.3, H-3⁰, 5⁰4-CH₃OPh), 7.19 (d, 2H, J = 8.3, H-2⁰,

6⁰4-CH₃OPh), 7.66–7.58 (m, 4H, H-8, H-3⁰, 4⁰, 5⁰Ph), 7.69

(t, 1H, J = 7.7, 10), 7.96 (t, 1H, J = 7.7, H-9), 8.29 (d, 2H,

J = 7.3, H-2⁰, 6⁰Ph), 8.49 (d, 1H, J = 7.9, H-11), 8.76 (t,

1H, J = 5.6, –C(O)NH); LC-MS, m/z = 484 [M?1], 486

[M?3]; Anal. calcd. for C₂₆H₂₁N₅O₃S: C, 64.58; H, 4.38; N,

14.48; S, 6.63; Found: C, 64.55; H, 4.35; N, 14.47; S, 6.60.

1467, 1455, 1434, 1391, 1363, 1331, 1283, 1260, 1237, 1220,

1179, 1131, 1103, 1077, 1045, 1018, 954, 877, 859, 840, 770,

752, 686, 670, 631, 605; ¹H-NMR (400 MHz) d: 2.39 (s, 3H,

CH₃), 4.07 (s, 2H, –S–CH₂), 4.37 (d, 2H, J = 5.6, –CH₂Ph),

7.02 (t, 1H, J = 7.5, H-5⁰, 2-FPh), 7.15 (t, 1H, J = 9.3, H-3⁰,

2-FPh), 7.27 (t, 1H, J = 6.4, H-6⁰, 2-FPh), 7.34 (t, 1H, J = 7.5,

H-4⁰, 2-FPh), 7.69-7.64 (m, 2H, H-8, 10), 7.96 (t, 1H, J = 7.7,

H-9), 8.46 (d, 1H, J = 7.9, H-11), 8.81 (t, 1H, J = 5.4, –

C(O)NH); LC-MS, m/z = 410 [M?1], 412 [M?3]; Anal.

calcd. for C₂₀H₁₆FN₅O₂S: C, 58.67; H, 3.94; N, 17.10; S, 7.83;

Found: C, 58.65; H, 3.92; N, 17.10; S, 7.81.

N-(3-Triﬂuoromethylbenzyl)-2-[(3-methyl-2-oxo-2H-[1,2,

4]triazino[2,3-c]quinazoline-6-yl)thio]acetamides

(4i)

Yield: Method A, 59.8 %; M.p. 218–220 ꢁC; IR (cm^-1): 3274,

3078, 2977, 2906, 2864, 1655, 1598, 1582, 1558, 1502, 1468,

1392, 1362, 1335, 1320, 1283, 1261, 1221, 1199, 1184, 1169,

1152, 1117, 1100, 1072, 1046, 1024, 1003, 954, 929, 892, 871,

807, 770, 752, 736, 701, 687, 664, 631, 606; ¹H-NMR (400 MHz)

d:2.39(s,3H,CH₃), 4.08 (s, 2H, –S–CH₂), 4.43 (d, 2H, J = 5.6, –

CH₂Ph), 7.45 (t, 1H, J = 7.7, H-10), 7.61–7.55 (m, 4, H-8, H-2⁰,

4⁰, 6⁰3-CF₃Ph), 7.66 (t, 1H, J = 7.5, H-5⁰3-CF₃Ph), 7.91 (t, 1H,

J = 7.7, H-9), 8.46 (d, 1H, J = 7.9, H-11), 8.91 (t, 1H, J = 5.6, –

C(O)NH);LC-MS, m/z = 460 [M?1], 462 [M?3]; Anal. calcd.

for C₂₁H₁₆F₃N₅O₂S: C, 54.90; H, 3.51; N, 15.24; S, 6.98; Found:

C, 54.90; H, 3.51; N, 15.24; S, 6.98.

N-(2-Fluorobenzyl)-2-[(3-phenyl-2-oxo-2H-[1,2,4]triazino

[2,3-c]quinazoline-6-yl)thio]acetamides (4l) Yield: Method

A, 91.2 %; M.p. 246–248 ꢁC; IR (cm^-1): 3283, 3069, 2982,

1669, 1652, 1587, 1563, 1552, 1506, 1487, 1467, 1442,

1432, 1389, 1369, 1340, 1312, 1283, 1266, 1244, 1231,

1168, 1157, 1138, 1101, 1075, 1022, 988, 939, 884, 841,

829, 810, 784, 765, 751, 707, 687, 652, 618; ¹H-NMR

(400 MHz) d: 4.12 (s, 2H, –S–CH₂), 4.39 (d, 2H, J = 5.4, –

CH₂Ph), 7.02 (t, 1H, J = 7.3, H-5⁰, 2-FPh), 7.16 (t, 1H,

J = 9.3, H-3⁰, 2-FPh), 7.27 (t, 1H, J = 6.4, H-6⁰, 2-FPh),

7.35 (t, 1H, J = 7.5, H-4⁰, 2-FPh), 7.65–7.58 (m, 3H, H-3⁰,

4⁰, 5⁰Ph), 7.73–7.67 (m, 2H, H-8, 10), 7.99 (t, 1H, J = 7.7,

H-9), 8.25 (d, 2H, J = 7.1, H-2⁰, 6⁰Ph), 8.49 (d, 1H,

J = 7.9, H-11), 8.84 (t, 1H, J = 5.4, –C(O)NH); ¹³C-NMR

(100 MHz) d: 35.43 (–SCH₂), 36.96 (–N–CH₂–, J = 4.2),

116.37 (3⁰, J²= 21.4, 2-FPh), 118.30 (11a), 124.67 (8),

126.10 (1⁰, J³= 5.2, 2-FPh), 126.27 (10), 128.10 (4⁰, Ph),

129.41 (5⁰, J³= 8.5, 2-FPh), 129.96 (3⁰, 5⁰Ph), 129.97 (6⁰,

J³= 4.5, 2-FPh), 129.83 (2⁰, 6⁰Ph), 130.97 (1⁰Ph), 132.09

(4⁰, J = 6.5, 2-FPh), 136.02 (9), 144.20 (3), 149.65 (2⁰,

J = 3.5, 2-FPh), 151.07 (11b), 154.95 (7a), 160.11 (6),

161.18 (2), 163.29 (2⁰,2-FPh), 167.43 (CONH); LC-MS, m/

z = 472 [M?1], 474 [M?3]; Anal. calcd. for

C₂₅H₁₈FN₅O₂S: C, 63.68; H, 3.85; N, 14.85; S, 6.80;

Found: C, 63.68; H, 3.85; N, 14.85; S, 6.80.

N-(3-Triﬂuoromethylbenzyl)-2-[(3-phenyl-2-oxo-2H-[1,2,

4]triazino[2,3-c]quinazoline-6-yl)thio]acetamides

(4j)

Yield: Method A, 99.9 %; M.p. 227–229 ꢁC; IR (cm^-1): 3267,

3072, 1667, 1648, 1589, 1564, 1550, 1505, 1487, 1468, 1445,

1372, 1329, 1313, 1285, 1269, 1222, 1200, 1180, 1158, 1135,

1110, 1073, 1031, 1002, 990, 938, 901, 877, 847, 811, 798, 783,

766, 755, 689, 653; ¹H-NMR (400 MHz) d: 4.12 (s, 2H, –S–

CH2), 4.45 (d, 2H, J = 5.6, –CH₂Ph), 7.46 (t, 1H, J = 7.7,

H-10), 7.65–7/54 (m, 7H, H-8, H-2⁰, 4⁰, 6⁰3-CF₃Ph, H-3⁰, 4⁰, 5⁰

Ph), 7.69 (t, 1H, J = 7.5, H-5⁰3-CF₃Ph), 7.93 (t, 1H, J = 7.7,

H-9), 8.29 (d, 2H, J = 7.7, H-2⁰, 6⁰Ph), 8.48 (d, 1H, J = 7.9,

H-11), 8.97 (t, 1H, J = 5.6, –C(O)NH); ¹³C-NMR (100 MHz)

d: 35.47 (–SCH₂), 42.70 (–N–CH₂–), 118.24 (11a), 123.61

(CF₃), 124.02 (2⁰3-CF₃Ph), 124.20 (4⁰3-CF₃Ph), 125.31 (CF₃),

125.77 (8), 126.06 (3⁰3-CF₃Ph), 126.85 (10), 128.07 (11),

129.75 (4⁰Ph), 128.95 (3 ⁰, 5⁰Ph), 129.35 (CF₃), 129.60 (CF₃),

129.82 (2⁰, 6⁰Ph), 132.06 (6⁰3-CF₃Ph), 132.09 (5⁰3-CF₃Ph),

131.84 (1⁰Ph), 135.91 (9), 141.25 (1⁰3-CF₃Ph), 144.17 (3),

149.66 (11b), 151.03 (7a), 154.86 (6), 160.07 (2), 167.59

(CONH); LC-MS, m/z = 522 [M?1], 524 [M?3]; Anal. calcd.

for C₂₆H₁₈F₃N₅O₂S: C, 59.88; H, 3.48; N, 13.43; S, 6.15;

Found: C, 59.89; H, 3.50; N, 13.44; S, 6.17.

N-(4-Fluorobenzyl)-2-[(3-methyl-2-oxo-2H-[1,2,4]triazino

[2,3-c]quinazoline-6-yl)thio]acetamides

(4m) Yield:

Method A, 64.7 %; Method B, 93.7 %; M.p. 225–227 ꢁC;

IR (cm^-1): 3275, 3075, 2980, 2912, 2866, 1658, 1651,

1580, 1556, 1502, 1468, 1434, 1393, 1363, 1332, 1320,

1283, 1260, 1218, 1181, 1153, 1132, 1096, 1077, 1045,

1017, 953, 879, 849, 824, 810, 770, 735, 698, 685, 631,

1

606; H-NMR (400 MHz) d: 2.39 (s, 3H, CH₃), 4.05 (s,

2H, –S–CH2), 4.32 (d, 2H, J = 5.6, –CH2Ph), 7.04 (t, 2H,

J = 8.7, H-3⁰, 5⁰4-FPh), 7.29 (t, 2H, J = 6.7, H-2⁰, 6⁰

4-FPh), 7.61 (d, 1H, J = 7.9, H-8), 7.67 (t, 1H, J = 7.7,

H-10), 7.95 (t, 1H, J = 7.7, H-9), 8.45 (d, 1H, J = 7.9,

H-11), 8.81 (t, 1H, J = 5.4, –C(O)NH); LC-MS, m/

z = 410 [M?1], 412 [M?3]; Anal. calcd. for

N-(2-Fluorobenzyl)-2-[(3-methyl-2-oxo-2H-[1,2,4]triazino

[2,3-c]quinazoline-6-yl)thio]acetamides

(4k) Yield:

Method A, 78.6 %; Method B, 86.9 %; M.p. 220–222 ꢁC; IR

(cm^-1): 3267, 3076, 2979, 2907, 1650, 1598, 1580, 1556, 1501,

123

Med Chem Res (2013) 22:2610–2632

2629

C₂₀H₁₆FN₅O₂S: C, 58.67; H, 3.94; N, 17.10; S, 7.83;

Found: C, 58.68; H, 3.95; N, 17.11; S, 7.83.

7.43 (d, 1H, J = 7.7, H-3⁰2-ClPh), 7.66-7.58 (m, 3H, H-3⁰,

4⁰, 5⁰Ph), 7.77–7.69 (m, 2H, H-8, 10), 8.01 (t, 1H, J = 7.7,

H-9), 8.29 (d, 2H, J = 7.7, H-2⁰, 6⁰Ph), 8.51 (d, 1H,

J = 7.9, H-11), 8.87 (t, 1H, J = 5.6, –C(O)NH); LC-MS,

m/z = 488 [M?1], 490 [M?3], 491 [M?4]; Anal. calcd.

for C₂₅H₁₈ClN₅O₂S: C, 61.54; H, 3.72; Cl, 7.27; N, 14.35;

S, 6.57; Found: C, 61.55; H, 3.73; Cl, 7.28; N, 14.37; S,

6.58.

N-(4-Fluorobenzyl)-2-[(3-phenyl-2-oxo-2H-[1,2,4]triazino

[2,3-c]quinazoline-6-yl)thio]acetamides (4n) Yield: Method

A, 92.3 %; Method B, 95.6 %; M.p. 236–238 ꢁC; IR (cm^-1):

3283, 3065, 2972, 1662, 1640, 1593, 1552, 1504, 1487, 1470,

1443, 1418, 1377, 1340, 1312, 1285, 1270, 1237, 1223, 1185,

1160, 1140, 1100, 1079, 1065, 1030, 1002, 985, 941, 883, 837,

General procedure for synthesis of N-(phenethyl)-2-[(3-R-

2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6-yl)thio]ace

ta-mides (4q, 4r)

1

814, 785, 771, 753, 707, 689, 653; H-NMR (400 MHz) d:

4.10 (s, 2H, –S–CH2), 4.33 (d, 2H, J = 5.6, –CH2Ph), 7.06 (t,

2H, J = 8.5, H-3⁰, 5⁰4-FPh), 7.31 (t, 2H, J = 6.7, H-2⁰, 6⁰

4-FPh), 7.67–7.58 (m, 4H, H-8, H-3⁰, 4⁰, 5⁰Ph), 7.70 (t, 1H,

J = 7.7, 10), 7.98 (t, 1H, J = 7.7, H-9), 8.29 (d, 2H, J = 7.3,

H-2⁰, 6⁰Ph), 8.49 (d, 1H, J = 7.9, H-11), 8.84 (t, 1H, J = 5.6,

–C(O)NH); LC-MS, m/z = 472 [M?1], 474 [M?3]; Anal.

calcd. forC₂₅H₁₈FN₅O₂S:C, 63.68;H, 3.85; N, 14.85;S, 6.80;

Found: C, 63.69; H, 3.85; N, 14.87; S, 6.81.

Method A N-(Phenethyl)-2-chloroacetamides (0.011 mol)

were added to a suspension of potassium salts of 3-R-6-thio-

6,7-dihydro-2H-[1,2,4]triazino[2,3-c]quinazolin-2-ones

(2a–2b) (0.01 mol) in 20 ml of propanol-2, water-propanol-

2 (1:2), or dioxane and reﬂuxed for 60–90 min. The mixture

was cooled and poured into water. The precipitate was ﬁl-

tered, dried and recrystallized from the dioxane–water (1:1).

N-(2-Chlorobenzyl)-2-[(3-methyl-2-oxo-2H-[1,2,4]triazino

[2,3-c]quinazoline-6-yl)thio]acetamides (4o) Yield: Method

A, 64.6 %; Method B, 82.4 %; M.p. 227–228 ꢁC; IR (cm^-1):

3265, 3066, 2975, 2911, 1659, 1646, 1597, 1581, 1557, 1503,

1467, 1435, 1394, 1362, 1320, 1284, 1260, 1220, 1208, 1184,

1131, 1088, 1045, 954, 879, 858, 769, 745, 698, 685, 670,

630, 605; ¹H-NMR (400 MHz) d: 2.39 (s, 3H, CH₃), 4.12 (s,

2H, –S–CH2), 4.39 (d, 2H, J = 5.6, –CH2Ph), 7.13 (t, 1H,

J = 7.7, H-5⁰2-ClPh), 7.25 (t, 1H, J = 7.7, H-4⁰2-ClPh),

7.36 (d, 1H, J = 7.7, H-6⁰2-ClPh), 7.41 (d, 1H, J = 7.7,

H-3⁰2-ClPh), 7.74–7.66 (m, 2H, H-8, 10), 7.99 (t, 1H,

J = 7.7, H-9), 8.48 (d, 1H, J = 7.9, H-11), 8.82 (t, 1H,

J = 5.6, –C(O)NH); ¹³C-NMR (100 MHz) d: 18.21 (–CH₃),

35.35 (–SCH₂), 41.04 (–N–CH₂–), 118.55 (11a), 126.02 (8),

126.81 (10), 127.48 (5⁰2-ClPh), 128.00 (6⁰2-ClPh), 129.07

(11), 129.20 (4⁰2-ClPh), 129.53 (3⁰2-ClPh), 132.38 (2⁰

2-ClPh), 135.99 (9), 136.49 (1⁰2-ClPh), 144.19 (3), 151.98

(11b), 154.68 (7a), 155.18 (6), 160.99 (2), 167.58 (CONH);

LC-MS, m/z = 426 [M?1], 428 [M?3], 429 [M?4]; Anal.

calcd. for C₂₀H₁₆ClN₅O₂S: C, 56.40; H, 3.79; Cl, 8.32; N,

16.44; S, 7.53; Found: C, 56.40; H, 3.79; Cl, 8.32; N, 16.44;

S, 7.54.

Method B N,N⁰-CDI (1.95 g, 0.011 mol) was added to a

solution of proper acid (1a, 1b) (0.01 mol) in 10 ml of anhy-

drous dioxane or DMFA and heated at the water bath at

60–80 ꢁC for 1 h with calcium chloride tube. The proper phen-

ethylamine (0.01 mol) was added with stirring to the resulting

mixture and reﬂuxed for 3 h. The mixture was poured into water,

and neutralized to pH 6–7 by acetic acid. The precipitate was

ﬁltered, dried and recrystallized from the dioxane–water (1:1).

N-Phenethyl-2-[(3-methyl-2-oxo-2H-[1,2,4]triazino[2,3-c]

quinazoline-6-yl)thio]acetamides (4q) Yield: Method A,

74.0 %; Method B, 88.3 %; M.p. 250–252 ꢁC; IR (cm^-1):

3275, 3077, 3059, 3025, 2975, 2914, 2860, 1659, 1645,

1598, 1582, 1557, 1504, 1467, 1453, 1433, 1400, 1361,

1331, 1286, 1260, 1242, 1222, 1206, 1193, 1178, 1133,

1101, 1045, 1021, 954, 876, 859, 772, 746, 696, 686, 631,

607; ¹H-NMR (400 MHz) d: 2.34 (s, 3H, CH₃), 2.74 (t, 2H,

J = 7.3, –CH₂CH₂Ph), 3.35 (t, 2H, J = 7.3, –CH₂CH₂Ph),

3.98 (s, 2H, –S–CH2), 7.26–7.14 (m, 5H, H-2⁰, 3⁰, 4⁰, 5⁰, 6⁰,

Ph), 7.70–7.64 (m, 2H, H-8, 10), 7.96 (t, 1H, J = 7.7, H-9),

8.36 (t, 1H, J = 5.0, –C(O)NH), 8.47 (d, 1H, J = 7.9,

H-11); ¹³C-NMR (100 MHz) d: 18.19 (–CH₃), 35.48 (–

SCH₂), 35.61 (–N–CH₂CH₂–), 41.13 (–N–CH₂CH₂–),

118.53 (11a), 125.97 (8), 126.54 (10), 126.77 (4⁰Ph),

127.93 (11), 128.74 (2⁰, 6⁰Ph), 129.04 (3⁰, 5⁰Ph), 135.98

(9), 139.77 (1⁰Ph), 144.18 (3), 151.95 (11b), 154.61 (7a),

155.16 (6), 160.99 (2), 167.16 (CONH); EI-MS, m/z (I_rel,

%) = 406 (8.2), 405 (64.5), 403 (11.9), 375 (11.5), 301

(26.4), 287 (8.5), 286 (9.8), 285 (69.6), 280 (8.2), 262

(12.5), 259 (5.4), 254 (6.2), 250 (11.6), 247 (7.6), 245

(15.5), 244 (68.1), 243 (52.1), 227 (6.4), 218 (5.6), 217

N-(2-Chlorobenzyl)-2-[(3-phenyl-2-oxo-2H-[1,2,4]triazino

[2,3-c]quinazoline-6-yl)thio]acetamides (4p) Yield: Method

A, 92.2 % M.p. 245–247 ꢁC; IR (cm^-1): 3277, 3078, 2987,

2920, 1672, 1649, 1589, 1553, 1507, 1488, 1470, 1444,

1426, 1384, 1340, 1313, 1286, 1270, 1243, 1227, 1185,

1161, 1139, 1101, 1076, 1037, 1025, 1002, 989, 968, 939,

882, 846, 811, 799, 784, 760, 705, 686, 652, 619; ¹H-NMR

(400 MHz) d: 4.16 (s, 2H, –S–CH2), 4.41 (d, 2H, J = 5.6,

–CH2Ph), 7.14 (t, 1H, J = 7.7, H-5⁰2-ClPh), 7.26 (t, 1H,

J = 7.7, H-4⁰2-ClPh), 7.38 (d, 1H, J = 7.7, H-6⁰2-ClPh),

123

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Med Chem Res (2013) 22:2610–2632

Cytotoxic activity against malignant human tumour cells

(79.6), 216 (99.9), 213 (5.6), 206 (6.1), 191 (13.1), 188

(32.3), 178 (5.0), 174 (15.1), 171 (27.4), 170 (22.8), 161

(59.9), 160 (6.1), 157 (7.0), 148 (43.2), 144 (5.3), 143

(46.3), 141 (18.9), 137 (6.9), 132 (11.2), 130 (7.0), 129

(39.2), 120 (7.4), 117 (5.3), 111 (6.2), 105 (49.6), 104

(17.5), 102 (18.9), 100 (6.9), 91 (11.4), 90 (12.5); LC-MS,

m/z = 406 [M?1], 408 [M?3]; Anal. calcd. for

C₂₁H₁₉N₅O₂S: C, 62.21; H, 4.72; N, 17.27; S, 7.91; Found:

C, 62.23; H, 4.72; N, 17.28; S, 7.94.

Primary anticancer assay was performed at human tumour

cell lines panel derived from nine neoplastic diseases, in

accordance with the protocol of the Drug Evaluation

Branch, National Cancer Institute, Bethesda (Avendano

and Menendez, 2008; Baxter et al., 2000; Cummings et al.,

2005; Zaharevitz et al.; 2002). Tested compounds were

added to the culture at a single concentration (10^-5M),

and the cultures were incubated for 48 h. End-point

determinations were made using a protein binding dye,

(SRB). Results for each tested compound were reported as

the percent of growth of the treated cells when compared

with the untreated control cells. The percentage growth was

evaluated spectrophotometrically versus controls untreated

with test agents. The cytotoxic and/or growth inhibitory

effects of the most active selected compounds were tested

in vitro against the full panel of about 60 human tumour

cell lines at 10-fold dilutions of ﬁve concentrations ranging

from 10^-4to 10^-8M. A 48-h continuous drug exposure

protocol was followed, and an SRB protein assay was used

to estimate cell viability or growth. Using the seven

absorbance measurements [time zero, (T_z), control growth

in the absence of drug (C), and test growth in the presence

of drug at the ﬁve concentration levels (T_i)], the percentage

growth was calculated at each of the drug concentrations

levels. Percentage growth inhibition was calculated as

N-Phenethyl-2-[(3-phenyl-2-oxo-2H-[1,2,4]triazino[2,3-c]

quinazoline-6-yl)thio]acetamides (4r) Yield: Method A,

89.8 %; Method B, 91.5 %; M.p. 225–227 ꢁC; IR (cm^-1):

3288, 3083, 3026, 2973, 2919, 2851, 1669, 1645, 1589,

1553, 1507, 1488, 1468, 1389, 1369, 1335, 1312, 1286,

1271, 1235, 1196, 1182, 1161, 1135, 1103, 1078, 1031,

1019, 1002, 989, 939, 874, 847, 811, 781, 771, 751, 699,

687, 652, 612; ¹H-NMR (400 MHz) d: 2.75 (t, 2H,

J = 7.3, –CH₂CH₂Ph), 3.36 (t, 2H, J = 7.3, –CH₂CH₂Ph),

4.02 (s, 2H, –S–CH₂), 7.27–7.17 (m, 5H, H-2⁰, 3⁰, 4⁰, 5⁰, 6⁰,

EtPh), 7.65–7.59 (m, 3H, H-3⁰, 4⁰, 5⁰Ph), 7.74–7.67 (m,

2H, H-8, 10), 7.99 (t, 1H, J = 7.7, H-9), 8.29 (d, 2H,

J = 7.7, H-2⁰, 6⁰Ph), 8.40 (t, 1H, J = 5.0, –C(O)NH), 8.50

(d, 1H, J = 7.9, H-11); LC-MS, m/z = 468 [M?1], 470

[M?3]; Anal. calcd. for C₂₆H₂₁N₅O₂S C, 66.79; H, 4.53;

N, 14.98; S, 6.86; Found: C, 66.81; H, 4.53; N, 15.01; S,

6.88.

½ðT_iꢁ T_zÞ=ðC ꢁ T_zÞꢂ

ꢃ 100 for concentrations for which T_iꢄ T_z;

Biological assay

½ðT_iꢁ T_zÞꢂ=T_zꢂ

Bioluminescence inhibition test

ꢃ 100 for concentrations for which T_i\ T_z:

The marine luminescent bacteria, Photobacterium leiognathi

strain Sh1, isolated from the Azov Sea Shrimp, were used for

the bioluminescence analysis (Antipenko et al., 2009). Bacteria

were cultivated on a nutrient environment containing (g/L):

peptone—5, yeast extract—1.5, meat extract—1.5, sodium

chloride—30, pH = 7.4. In the acute action test (inhibiting

luminescence of bacteria), bacteria were diluted with the 3 %

sodium chloride solution up to a concentration of 10⁵cells/mL.

5–50lg/mL of the studied substances suspended inDMSO were

mixed with 1 mLof the dilutedbacterialsuspension. Vials were

incubated for 10 min at 25 ꢁC; then, the intensity of biolumi-

nescence was measured in percent (%) relative to the controls,

which were prepared without the studied compounds. In the

chronic action test (inhibiting growth and luminescence of

bacteria), growth medium was added for potential breeding in a

ratio of 1:50, and the mix was incubated for 16–18 h at 30 ꢁC,

whereupon the intensity of bioluminescence was measured the

same way as in acute action testing. Tetracycline was used as a

reference. The bacterial luminescence was measured on a

Bioluminometer BLM-8801 (‘Science’, Krasnoyarsk, Russia).

Three dose–response parameters were calculated for each

compound. Growth inhibition of 50 % (GI50) was calculated

from [(T_i-T_z)/(C-T_z)] 9 100 = 50, which is the drug con-

centration resulting in a 50 % lower net protein increase in the

treated cells (measured by SRB staining) as compared to the

net protein increase seen in the control cells. The drug con-

centration resulting in total growth inhibition (TGI) was cal-

culated from T_i= T_z. The LC50 (concentration of drug

resulting in a 50 % reduction in the measured protein at the

end of the drugtreatment as compared tothatat the beginning)

indicating a net loss of cells following treatment was calcu-

lated from [(T_i-T_z)/T_z] 9 100 = -50. Values were calcu-

lated 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, respectively, the maximum or minimum

concentration tested. The log GI50, log TGI and log LC50

were then determined, deﬁned as the mean of the logs of the

individual GI50, TGI and LC50 values, respectively. The

lowest values were obtained with the most sensitive cell lines.

123

Med Chem Res (2013) 22:2610–2632

2631

Acknowledgments The authors gratefully acknowledge the ﬁnan-

cial support for this study by Enamine Ltd (Kiev, Ukraine) , and thank

the team of the Drug Synthesis and Chemistry Branch, the National

Cancer Institute, Bethesda, MD, USA, for in vitro evaluation of

anticancer activity.

Boyd MR (1997) The NCI in vitro anticancer drug discovery screen;

concept, implementation and operation. Can Drug Discov Dev

2:23–43

Boyd MR, Paull KD (1995) Some practical considerations and

applications of the National Cancer Institute in vitro anticancer

drug discovery screen. Drug Dev Res 34:91–109

Conﬂict of interest The authors have declared noconﬂict of

interest.

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Article Doi

Novel N-aryl(alkaryl)-2-[(3-R-2-oxo-2H-[1,2,4]triazino[2,3-c]quinazoline-6- yl)thio]acetamides: Synthesis, cytotoxicity, anticancer activity, COMPARE analysis and docking

DOI: 10.1007/s00044-012-0257-x

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