Synthesis and characterization of novel 1,2,4-triazine derivatives with antiproliferative activity

Article abstract of DOI:10.1016/j.bmc.2010.01.053

A series of novel small molecules with a 1,2,4-triazine scaffold was obtained according to a recently published and highly efficient synthetic route. Screening for antiproliferative and cytotoxic activity revealed distinct anticancer effects against the human leukemia cell line K-562 combined with a remarkable low cytotoxicity. All compounds were in agreement with the 'rule-of-five' claims by Lipinski and calculated log P_calc values were experimentally confirmed (log P_exp). For the most active compounds, in vitro serum albumin binding was investigated and structure-activity relationships were established.

Full text of DOI:10.1016/j.bmc.2010.01.053

Bioorganic & Medicinal Chemistry 18 (2010) 1816–1821
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterization of novel 1,2,4-triazine derivatives
with antiproliferative activity
b
a
a
Fabian Krauth a, , Hans-Martin Dahse , Hans-Hermann Rüttinger , Petra Frohberg
*
a Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, 06120 Halle, Germany
b Leibniz-Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute (HKI), Beutenbergstraße 11a, D-07745 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel small molecules with a 1,2,4-triazine scaffold was obtained according to a recently pub-
lished and highly efficient synthetic route. Screening for antiproliferative and cytotoxic activity revealed
distinct anticancer effects against the human leukemia cell line K-562 combined with a remarkable low
cytotoxicity. All compounds were in agreement with the ‘rule-of-five’ claims by Lipinski and calculated
log Pcalc values were experimentally confirmed (log Pexp). For the most active compounds, in vitro serum
albumin binding was investigated and structure–activity relationships were established.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 9 November 2009
Revised 18 January 2010
Accepted 20 January 2010
Available online 25 January 2010
Keywords:
1,2,4-triazin-5-ones
Small molecules
Leukemia
K-562
Rule-of-five
log P
Imatinib
Serum albumin binding
1. Introduction
comparison of the experimentally determined log Pexp with the
estimated log Pcalc values calculated based on group contributions
In the past few years, numerous small molecules possessing a
1,2,4-triazine scaffold have been shown to exhibit a great variety
of pharmacological effects. Several reports have been published
on the application of these compounds, such as 5-lipoxygenase
(5-LO) inhibitors,1,2 herbicides, bactericides, fungicides, antimicro-
bials,3,4 and gonadotropin-releasing hormone receptor (GnRH-R)
antagonists.5 Even for fused 1,2,4-triazine compounds not only
antitumoral and antimetastatic activities against a wide range of
cancer cells but also kinase inhibiting activities could be ob-
served.6–10 In 2007, cancer accounted for ꢀ7.9 million death cases
(around 13% of all deaths). Deaths from cancer worldwide are pro-
jected to continue rising, with an estimated 12 million deaths in
2030.11
From our experience with heterocyclic compounds containing
an amidrazone scaffold,12,13 we aimed to synthesize novel 1,2,4-
triazines as efficient anticancer drugs with low cytotoxicity and
good bioavailability properties. In this paper, we present the
straightforward synthesis of eight 1,2,4-triazin-5-ones 3a–h
(Scheme 1), and the indirect determination of log PO/W values (log -
Pexp) by reversed-phase high-performance liquid chromatography
(RP-HPLC) based on the logarithm of capacity factors (log k). The
from fragments of the molecular structure,14,15 was also done.
Antiproliferative and cytotoxic activity of the 1,2,4-triazin-5-ones
3a–h against HUVEC (human umbilical vein endothelial cells), K-
562 (human leukemia cell line), and HeLa cells were determined.
The established anticancer drugs, such as tyrosine kinase inhibitor
imatinib (GleevecÒ) and anthracycline antibiotic doxorubicin
(AdriamycinÒ) were also tested in the same assays for comparison.
As protein binding can affect the distribution of the drugs within
the body and their rates of metabolism and excretion, spectrofluo-
rimetric analysis of the four most active compounds (against K-
562) was conducted to determine the in vitro dissociation con-
stants (Kd) for human serum albumin (HSA) binding. The potential
of the 1,2,4-triazin-5-ones 3a–h as lead structures for anticancer
therapy is also discussed.
2. Results and discussion
2.1. Synthesis of 1,2,4-triazin-5-one compounds
Hydrazonoyl chlorides 1a–b as starting compounds for triazi-
none synthesis were prepared according to known literature pro-
cedures.16,17 Conversion of 1a–b with the respective amines in
dioxane led to amidrazone intermediates 2a–h. One to two hours
of refluxing 2a–h with formaldehyde in the presence of p-toluen-
* Corresponding author. Tel.: +49 345 55 25178; fax: +49 345 55 27026.
E-mail address: fabian.krauth@pharmazie.uni-halle.de (F. Krauth).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.053

F. Krauth et al. / Bioorg. Med. Chem. 18 (2010) 1816–1821
1817
R2
R1
N
N
N
R1NHCO
R2
R1NHCO
N
R2
N
a
b
O
N
H
N
H
R3
R3
Cl
1a-b
2a-h
3a-h
2e, 3e: R2=C6H5; R3=piperidine
2a, 3a: R2=C6H5; R3=(CH3)2N
1a: R2=C6H5
2f, 3f: R2=4-Cl-C6H4; R3=piperidine
2g, 3g: R2=C6H5; R3=morpholine
2h, 3h: R2=4-Cl-C6H4; R3=morpholine
2b, 3b: R2=4-Cl-C6H4; R3=(CH3)2N
2c, 3c: R2=C6H5; R3=pyrrolidine
1b: R2=4-Cl-C6H4
1-3: R1=2-Cl-C6H4
2d, 3d: R2=4-Cl-C6H4; R3=pyrrolidine
Scheme 1. Reagents and conditions: (a) dimethylamine for 2a–b, pyrrolidine for 2c–d, piperidine for 2e–f, morpholine for 2g–h, dioxane, room temperature, 12 h and (b)
H2CO, TsOH, EtOH, 1–2 h, reflux.
sulfonic acid yielded nearly pure 1,2,4-triazin-5-ones 3a–h
As shown in Table 1 the experimental data are in excellent
agreement with the in silico data showing the applicability of the
computational method for this class of compounds. Both reference
compounds for pharmacological testing, imatinib and doxorubi-
cine, were also subjected to MIPC. Most of the triazinone com-
pounds listed in Table 1 have similar log Pcalc values as that of
imatinib, an orally bioavailable drug that does not violate the
‘rule-of-five’. However, for imatinib a protonation at experimental
pH conditions has to be considered in contrast to 3a–h, which is re-
flected in an effective log Pexp value of 1.2.22 Doxorubicin does not
fulfill three out of four claims and is therefore applied
intravenously.
(Scheme 1).18
2.2. Determination of bioavailability parameters
The triazinone compounds 3a–h were subjected to the freely
accessible program MIPC (Molinspiration Property Calculator) to
obtain the Lipinski descriptors for bioavailability estimation.14,15
These parameters describe molecular properties important for
drug pharmacokinetics in the human body, especially their oral
absorption. The rule says, that an oral active drug must not violate
more than one of the following criteria: 65 hydrogen donors
(nOHNH), 610 hydrogen acceptors (nON), MW 6500 Da, log Pcalc
65. All triazinones meet these claims (Table 1). The log PO/W value,
defined as the logarithm of the partition coefficient between n-oct-
anol and water, is an important parameter to reflect a compound’s
hydrophobicity and is therefore crucial for drug absorption and
transport. As expected due to missing hydrogen donor residues
(nOHNH), calculated log Pcalc values of all compounds are in a quite
high but still suitable range. Exclusively compound 3f violated one
of the ‘rule-of-five’ claims with a log Pcalc value of 5.1. The log Pcalc
values were compared with experimental log Pexp values obtained
from the logarithm of the capacity factors (log k) that were mea-
sured by reversed-phase high-performance liquid chromatography
(RP-HPLC) (see Section 4).19,20 The determination of log k derived
from RP-HPLC retention times demonstrates a convenient tech-
nique for estimating log PO/W values and was carried out in this
study to replace the time-consuming log PO/W determination by
the shake-flask method.21
2.3. Pharmacological activities and serum albumin binding
Antiproliferative and cytotoxic activities of compounds 3a–h
were determined as described in previous papers and in the
experimental part (Section 4.4) herein.24–26 Among the eight
1,2,4-triazin-5-ones synthesized, 3a–d showed the highest anti-
proliferative effect on the human leukemia cell line K-562 with a
moderate growth inhibition efficacy on the human umbilical vein
endothelial cell line (HUVEC). Though the most potent compound
3c is obviously less active against K-562 than imatinib, the pre-
ferred drug for treating chronic myeloid leukemia (CML), a compa-
rable low growth inhibiting effect on HUVEC was found.
Cytotoxicity of 3c against HeLa cells is ranging at similar values.
In contrast to doxorubicin, an established but highly cytotoxic drug
for the treatment of acute myeloid leukemia (AML), lymphoma,
sarcoma, and carcinoma, 3c showed a five times lower antiprolifer-
Table 1
Substitution patterns, Lipinski’s ‘rule-of-five’ descriptors, experimental log P values (log Pexp
)
Cl
R2
N
N
N
R2
R3
MW
nONa
nOHNHa
log Pcalc
log Pexp
a
a
O
R3
3a
3b
3c
3d
3e
3f
C6H5
4-Cl–C6H4
C6H5
4-Cl–C6H4
C6H5
4-Cl–C6H4
C6H5
4-Cl–C6H4
—
—
N(CH3)2
N(CH3)2
Pyrrolidine
Pyrrolidine
Piperidine
Piperidine
Morpholine
Morpholine
—
328.80
363.25
354.84
389.29
368.87
403.31
370.84
405.28
493.62
543.52
5
5
5
5
5
5
6
6
8
0
0
0
0
0
0
0
0
2
7
3.5
4.2
3.9
4.6
4.4
5.1
3.3
4.0
3.9
0.57
3.6
4.2
4.0
4.6
4.6
5.0
3.5
3g
3h
4.0
Imatinib
Doxorubicin
1.222
0.7123
—
12
n.d.: not determined.
a
Lipinski descriptors: MW, molecular weight; nON, number of hydrogen acceptors; nOHNH, number of hydrogen donors; log Pcalc, log P values calculated by molinspiration
property calculator for the neutral species; log Pexp, experimentally determined log P values by RP-HPLC.

1818
F. Krauth et al. / Bioorg. Med. Chem. 18 (2010) 1816–1821
ative activity against K-562, but a 22 times lower cytotoxicity on
HeLa cells (Table 2). These results suggest that there is little corre-
lation between cytotoxicity and antiproliferative activity for the
1,2,4-triazin-5-ones. If one compares the effect of R3 on the anti-
proliferative activity against K-562 cells, an optimum exists for
the pyrrolidine moiety. 4-Chloro substitution on the phenyl ring
R2 not only raises the log P values, but lowers the effect on K-
562. Interestingly the existence of the chlorine atom in 3b and
3d seems to raise the growth inhibiting effect on HUVEC cells. An
introduction of a morpholine group leads to a distinct decrease
of any activity. Considering the estimated new cases (44,790 men
and woman) and deaths (21,870 men and woman) from leukemia
in the United States in 2009,27 and the proceeding emerge of resis-
tances against chemotherapeutic agents (multidrug resistance),
there is a demand for novel, more effective anticancer agents. Tak-
ing 1,2,4-triazin-5-one as lead structure for the development of
less toxic and selective anti-leukemia drugs, further chemical mod-
ifications in the substitution patterns of the aromatic groups are
envisioned for optimization of pharmacological activity. An intro-
duction of hydrogen donor groups may be considered as well.
For 3a–d, their binding to human serum albumin (HSA), the
most abundant protein (35–50 mg mLꢁ1) in plasma, was studied
using an existing spectrofluorimetric in vitro method.28–31 Exempl-
arily, Figure 1A shows the spectra of the intrinsic fluorescence
Figure 1. (A) Fluorimetric emission spectra of HSA in the absence and presence of
3c (concentrations are indicated). (B) Reduction of fluorescence (% Quenching) of
HSA as a function of 3c concentration. The solid line presents the best fit of the
quadratic binding curve.
binding of the 1,2,4-triazin-5-ones, it is also necessary to investi-
quenching of 2.0 lM HSA (1.3 mg/10 mL) in PBS (phosphate
gate binding to
a1-acid glycoprotein (AGP), the second important
buffered saline) buffer (pH 7.4) in the presence of varying concen-
trations of 3c. The spectra exhibit a concentration dependent
quenching of intrinsic HSA fluorescence emission. Figure 1B dem-
onstrates the percentual reduction in fluorescence (% Quenching)
of HSA at 332 nm as a function of compound concentration. The
data were fitted to a quadratic binding equation and the average
plasma protein. Additionally, further lead optimization is neces-
sary to identify significantly more potent analogs in this series.
4. Experimental
4.1. Chemicals
Kd value was found to be 2.46 0.51
lM. With a Kd of 78.64
6.71 M imatinib showed a weak affinity towards HSA, which is
l
Human serum albumin (essentially fatty acid free, prepared
from fraction V albumin, Sigma–Aldrich, Taufkirchen, Germany),
reference standards for log P determination and solvents for re-
versed-phase high-performance liquid chromatography (RP-HPLC)
were of the highest available purity (Sigma–Aldrich, Taufkirchen,
Germany). All chemicals and solvents for synthesis were of reagent
grade and used without further purification. Water was purified
with a Direct-Q5 water purification system (Millipore, Eschborn,
Germany).
not surprising if one considers the well-known relationship
between lipophilicity (log Pexp) and HSA binding.28
3. Conclusions
A series of eight 1,2,4-triazin-5-ones was synthesized, which
exhibit distinct antiproliferative activities against the chronic mye-
loid leukemia (CML) cell line K-562 combined with a low cytotox-
icity. Calculated molecular properties, such as the logarithm of
partition coefficients (log Pcalc), were experimentally confirmed.
The described molecular characteristics are in agreement with
the Lipinski’s ‘rule-of-five’ claims for orally bioactive drugs. There-
fore we are planning to conduct kinase inhibition assays for com-
pounds 3a–d. Serum albumin binding for the most active
compounds 3a–d was determined revealing Kd values in the range
4.2. Synthesis
4.2.1. General
Melting points were determined on a Boëtius hot-stage appara-
tus. Elemental analyses were performed by Leco Microlab, Inc.,
and determined values are within 0.4% of theory. NMR spectra were
recorded on a Gemini 2000 operating at 399.96 MHz for 1H NMR and
between 2.5 and 10.2 lM. To gain a deeper inside of the plasma
Table 2
Antiproliferative (GI50) and cytotoxic (CC50) effects of triazinones 3a–h and Kd values of triazinone compounds 3a–d and imatinib for the reduction of the intrinsic fluorescence of
HSA
Compound
HUVEC GI50
(lg/mL)
K-562 GI50
(lg/mL)
HeLa CC50
(
lg/mL)
Kd (lM)
3a
3b
3c
3d
3e
3f
34.5 2.8
16.1 1.4
40.9 4.9
18.9 1.5
24.7 2.1
>50
5.8 0.4
11.0 0.7
5.2 0.4
8.6 0.6
42.0 5.5
>50
39.5 7.6
39.4 5.2
44.3 5.8
>50
26.0 2.3
>50
7.96 5.71
10.21 5.50
2.46 0.51
2.77 2.06
n.d.
n.d.
3g
>50
>50
>50
n.d.
3h
45.6 5.6
10.9 1.2
0.132
>50
>50
38.8 1.4
2.0 0.8
n.d.
78.64 6.71
n.d.
Imatinib
Doxorubicin
0.1 (6.7 ꢂ 10ꢁ3
)
1.0 0.6
GI50, concentration which inhibits cell proliferation by 50% compared to control; CC50, concentration which is toxic for 50% of the cells compared to control (4 parallels per
concentration); Kd, dissociation constant; n.d.: not determined.

F. Krauth et al. / Bioorg. Med. Chem. 18 (2010) 1816–1821
1819
at 100.6 MHz for 13C NMR spectra in DMSO-d6 which was also used
as internal standard. Chemical shifts are given in d units and refer to
the center of the signal. EI-mass spectra were obtained with an AMD
402 mass spectrometer (AMD Intectra) at 70 eV. Reactions were
monitored by TLC (Silica gel 60 F254, Merck) using chloroform/ether
(7:3, v/v) and heptane/ethylacetate (3:1, v/v) and compounds were
detected with ultraviolet light (254 nm). Compounds 1a, 2a, 2b, 2e,
2g,17 1b,16 and 3e18 were obtained by published procedures.
4.2.3.1. 2,3,4,5-Tetrahydro-4-(2-chlorophenyl)-6-dimethylamino-
2-phenyl-1,2,4-triazin-5-one 3a. dY = 7.63–6.86 (9H, Ph), 5.21
(2H, NCH2N), 2.90 (6H, s, 2CH3). dC = 152.4 (1C, C@O), 147.6 (1C,
C@N), 145.0–114.5 (12C, Ph), 63.5 (1C, NCH2N), 39.5 (2C, 2CH3).
m/z (EI-MS) calcd for C17H17ClN4O 328; found 328 (M+, 100), 161
(78), 118 (74).
4.2.3.2. 2,3,4,5-Tetrahydro-4-(2-chlorophenyl)-2-(4-chlorophenyl)-
6-dimethylamino-1,2,4-triazin-5-one 3b. Employing 3.5 g of 2b
(10 mmol) in the procedure described above gave 3b (1.3 g, 37%) as
yellow crystals. Mp: 125–128 °C; dY = 7.63–7.28 (8H, Ph), 5.23 (2H,
NCH2N), 2.91 (6H, s, 2CH3). dC = 152.3 (1C, C@O), 147.9 (1C, C@N),
143.9–115.9 (12C, Ph), 63.3 (1C, NCH2N), 39.4 (2C, 2CH3). m/z (EI-
MS) calcd for C17H16Cl2N4O 362; found 362 (M+, 100), 195 (99), 125
(91).
4.2.2. Representative procedure for the synthesis of compounds
2
A solution of 1a (3.0 g, 10 mmol) in dioxane (ꢀ40 cm3) was
added dropwise to pyrrolidine (1.4 g, 20 mmol) in a few cm3 of
dioxane. After stirring at ambient temperature for at least 12 h,
the mixture was poured into cold water (300 cm3). The solid was
collected, washed with water, and dried. Recrystallization from
ethanol gave 2c (2.5 g, 81%) as beige crystals. Mp: 101–106 °C.
4.2.3.3. 2,3,4,5-Tetrahydro-4-(2-chlorophenyl)-2-phenyl-6-(pyrr-
olidin-1-yl)-1,2,4-triazin-5-one 3c. Employing 3.4 g of 2c
(10 mmol) in the procedure described above gave 3c (2.1 g, 61%)
as yellow crystals. Mp: 119–123 °C; dY = 7.62–6.82 (9H, Ph), 5.21
(2H, NCH2N), 3.49 (4H, t, J = 6 Hz, CH2NCH2), 1.86 (4H, m,
J = 6 Hz, 2CH2); dC = 153.1 (1C, C@O), 145.4 (1C, C@N), 145.4–
114.4 (12C, Ph), 63.9 (1C, NCH2N), 48.1 (2C, CH2NCH2), 24.6 (2C,
2CH2). m/z (EI-MS) calcd for C19H19ClN4O 354; found 354 (M+,
100).
4.2.2.1. N-(2-Chlorophenyl)-2-phenylhydrazono-2-(pyrrolidin-
1-yl)acetamide 2c. dY = 9.66 (1H, s, CONH), 9.47 (1H, s, NNH),
8.22–6.83 (9H, Ph), 3.29 (4H, t, J = 6 Hz, CH2NCH2), 1.86 (4H, m,
J = 6 Hz, 2CH2); dC = 160.5 (1C, C@O), 138.9 (1C, C@N), 145.1–
113.9 (12C, Ph), 49.2 (2C, CH2NCH2), 25.9 (2C, 2CH2). m/z (EI-MS)
calcd for C18H19ClN4O 342; found 342 (M+, 100%).
4.2.2.2. N-(2-Chlorophenyl)-2-(4-chlorophenyl)-hydrazono-2-
(pyrrolidin-1-yl)-acetamide 2d. Employing 3.4 g of 1b
(10 mmol) in the procedure described above gave 2d (2.6 g, 86%)
as beige crystals. Mp: 113–115 °C. dY = 9.63 (1H, s, CONH), 9.49
(1H, s, NNH), 8.13–6.87 (8H, Ph), 3.28 (4H, t, J = 6 Hz, CH2NCH2),
1.86 (4H, m, J = 6 Hz, 2CH2); dC = 159.9 (1C, C@O), 138.9 (1C,
C@N), 143.7–113.9 (12C, Ph), 48.6 (2C, CH2NCH2), 25.2 (2C,
2CH2). m/z (EI-MS) calcd for C18H18Cl2N4O 376; found 376 (M+,
100%), 221 (41).
4.2.3.4. 2,3,4,5-Tetrahydro-4-(2-chlorophenyl)-2-(4-chlorophenyl)-
6-(pyrrolidin-1-yl)-1,2,4-triazin-5-one 3d. Employing 3.8 g of
2d (10 mmol) in the procedure described above gave 3d (2.2 g,
58%) as yellow crystals. Mp: 134–136 °C; dY = 7.62–7.25 (8H, Ph),
5.23 (2H, NCH2N), 3.49 (4H, t, J = 6 Hz, CH2NCH2), 1.85 (4H, s,
2CH2); dC = 153.7 (1C, C@O), 146.2 (1C, C@N), 144.9–116.4 (12C,
Ph), 64.3 (1C, NCH2N), 48.9 (2C, CH2NCH2), 25.3 (2C; 2CH2). m/z
(EI-MS) calcd for C19H18Cl2N4O 388; found 388 (M+, 100), 221 (64).
4.2.2.3. N-(2-Chlorophenyl)-2-(4-chlorophenyl)-hydrazono-2-
(piperidin-1-yl)acetamide 2f. Employing 3.4 g of 1b (10 mmol)
and 1.6 g piperidine (20 mmol) in the procedure described above
gave 2f (3.5 g, 92%) as pale yellow crystals. Mp: 138–143 °C.
dY = 9.51 (1H, s, CONH), 9.34 (1H, s, NNH), 8.12–7.10 (8H, Ph),
3.00 (4H, t, J = 6 Hz, CH2NCH2), 1.65 (4H, m, J = 6 Hz, 2CH2), 1.52
(2H, m, J = 6 Hz, CH2); dC = 159.9 (1C, C@O), 138.9 (1C, C@N),
142.7–115.1 (12C, Ph), 48.4 (2C, CH2NCH2), 25.2 (2C, 2CH2), 23.9
(1C, CH2). m/z (EI-MS) calcd for C19H20Cl2N4O 390; found 390
(M+, 100%), 84 (89), 235 (49).
4.2.3.5. 2,3,4,5-Tetrahydro-4-(2-chlorophenyl)-2-(4-chlorophenyl)-
6-(piperidin-1-yl)-1,2,4-triazin-5-one 3f. Employing 3.4 g of 2f
(10 mmol) in the procedure described above gave 3f (2.5 g, 63%)
as yellow crystals. Mp: 197–198 °C; dY = 7.60–7.26 (8H, Ph), 5.21
(2H, NCH2N), 3.25 (4H, t, J = 6 Hz, CH2NCH2), 1.57 (4H, m,
J = 5 Hz, 2CH2); dC = 152.1 (1C, C@O), 147.4 (1C, C@N), 143.7–
116.0 (12C, Ph), 63.0 (1C, NCH2N), 48.0 (2C, CH2NCH2), 24.6 (2C,
2CH2), 23.9 (1C, CH2). m/z (EI-MS) calcd for C20H20Cl2N4O 402;
found 402 (M+, 100), 235 (74).
4.2.3.6. 2,3,4,5-Tetrahydro-4-(2-chlorophenyl)-2-phenyl-6-(mor-
pholin-4-yl)-1,2,4-triazin-5-one 3g. Employing 3.6 g of 2g
(10 mmol) in the procedure described above gave 3g (2.3 g, 62%)
as yellow crystals. Mp: 153–155 °C; dY = 7.64–6.88 (9H, Ph), 5.26
(2H, NCH2N), 3.72 (4H, t, J = 4.7 Hz, CH2OCH2), 3.27 (4H, s,
CH2NCH2); dC = 152.0 (1C, C@O), 145.9 (1C, C@N), 144.7–114.6
(12C, Ph), 66.4 (2C, CH2OCH2), 63.1 (1C, NCH2N), 47.6 (2C,
CH2NCH2). m/z (EI-MS) calcd for C19H19ClN4O2 370; found 370
(M+, 100).
4.2.2.4. N-(2-Chlorophenyl)-2-(4-chlorophenyl)-hydrazono-2-
(morpholin-4-yl)acetamide 2h. Employing 3.4 g of 1b (10 mmol)
and 1.7 g morpholine (20 mmol) in the procedure described above
gave 2h (3.2 g, 81%) as pale yellow crystals. Mp: 135–142 °C.
dY = 9.64 (1H, s, NNH), 9.56 (1H, s, CONH), 8.14–7.07 (8H, Ph),
3.77 (4H, t, J = 4.5 Hz, CH2OCH2), 3.08 (4H, t, J = 4.5 Hz, CH2NCH2);
dC = 159.9 (1C, C@O), 137.3 (1C, C@N), 142.7–115.4 (12C, Ph), 66.0
(2C, CH2OCH2), 47.5 (2C, CH2NCH2). m/z (EI-MS) calcd for
C18H18Cl2N4O2 392; found 392 (M+, 25%), 237 (100), 125 (51),
266 (48).
4.2.3.7. 2,3,4,5-Tetrahydro-4-(2-chlorophenyl)-2-(4-chlorophenyl)-
6-(morpholin-4-yl)-1,2,4-triazin-5-one 3h. Employing 3.9 g of
2h (10 mmol) in the procedure described above gave 3h (1.6 g,
39%) as yellow crystals. Mp: 186–188 °C; dY = 7.63–7.30 (8H, Ph),
5.27 (2H, NCH2N), 3.71 (4H, t, J = 4.7 Hz, CH2OCH2), 3.28 (4H, s,
CH2NCH2); dC = 151.9 (1C, C@O), 146.3 (1C, C@N), 143.6–116.1
(12C, Ph), 65.4 (2C, CH2OCH2), 62.9 (1C, NCH2N), 47.5 (2C,
CH2NCH2). m/z (EI-MS) calcd for C19H18Cl2N4O2 404; found 404
(M+, 100), 237 (59).
4.2.3. Representative procedure for the synthesis of compounds
3
3.2 g of 2a (10 mmol), a 37%-solution of formaldehyde (1.5 cm3,
20 mmol), and p-toluenesulfonic acid (0.1 g) were refluxed in eth-
anol (ꢀ50 cm3) until the starting amidrazone was converted com-
pletely. The mixture was cooled to room temperature and the
solvent was evaporated. The solid was collected and recrystallized
from ethanol yielding 3a (1.4 g, 42%) as yellow crystals. Mp: 108–
110 °C.

1820
F. Krauth et al. / Bioorg. Med. Chem. 18 (2010) 1816–1821
4.3. Determination of log Pexp by RP-HPLC
The HPLC system consisted of a PU-980 Intelligent HPLC Pump, a
UV-975 Intelligent UV–vis Detector set at 210 nm and an 851-AS
Intelligent Autosampler (Jasco, Groß-Umstadt, Germany). The col-
umn was a LiChroCARTÒ 125-4 with LiChrospherÒ 100 RP-18 end-
capped material (5 lm) (Merck, Darmstadt, Germany). As eluent
for 3,4-dihydro-2H-1,2,4-triazin-5-ones a mixture of methanol-
PBS buffer (75/25 (v/v)) pH 7.4 was used. The flow rate of the mobile
phase was 1 mL/min. The sample volume injected was 15
lL (concd
ꢀ3 M/L). The dead time (t0) was measured by injection of the unre-
l
tained compound formamide dissolved in mobile phase. Lacking
comparable data for compounds 3a–h a more general calibration
according to an OECD protocol was established using the following
reference compounds: benzophenone (log PO/W: 3.2), diphenyl-
amine (log PO/W: 3.4), benzyl benzoate (log PO/W: 4.0), n-butylben-
zene (log PO/W: 4.6), and bibenzyl (log PO/W: 4.8) perfectly covering
the previously calculated log Pcalc value range between 3.5 and 4.0.
Retention times (tR) of the reference compounds were deter-
mined and according to the equation:
Figure 2. Calibration curve for determination of log Pexp values by plotting known
log PO/W values of different reference compounds (Table 3) versus their log k values;
y = 2.74 + 2.07x; r = 0.9920.
k ¼ ðtR ꢁ t0Þ=t0
the capacity factor (k) was calculated which is directly related to
log PO/W (Table 3).
A correlation curve of log PO/W versus log k was obtained by
least-squares linear regression (Fig. 2) and log Pexp values of triaz-
inone compounds were deviated from the linear equation of the
graph (Table 4).19,20
Table 4
Retention times (tR) and the calculated logarithms of capacity factors (log k) of the
1,2,4-triazin-5-ones
Compound
tR (min)a
log k
3a
3b
3c
3d
3e
3f
3.97
6.09
5.80
9.82
8.87
14.4
3.53
5.21
0.43
0.34
0.64
0.91
0.86
1.09
0.36
0.58
4.4. Pharmacology
4.4.1. Cells and culture conditions
Cells of HUVEC (ATCC CRL-1730), K-562 (DSM ACC 10), and
HeLa (DSM ACC 57) were cultured in DMEM (CAMBREX 12-
614F), RPMI 1640 (CAMBREX 12-167F), and RPMI 1640 (CAMBREX
12-167F), respectively. All cells were grown in the appropriate cell
culture medium supplemented with 10 mL/L ultraglutamine 1
3g
3h
a
Retention times were determined in triplicate and the calculation of log k was
done with the arithmetic mean values.
(CAMBREX 17-605E/U1), 500 lL/L gentamicin sulfate (CAMBREX
17-518Z), and 10% heat inactivated fetal bovine serum (PAA A15-
144) at 37 °C in high density polyethylene flasks (NUNC 156340).
carried out carefully on the subconfluent monolayers of HeLa cells
after the preincubation time.
4.4.2. Antiproliferative assay
The test substances were dissolved in DMSO before being di-
luted in DMEM. The adherent cells were harvested at the logarith-
mic growth phase after soft trypsinization, using 0.25% trypsin in
PBS containing 0.02% EDTA (Biochrom KG L2163; Biochrom, Berlin,
Germany). For each experiment approximately 10,000 cells were
seeded with 0.1 mL culture medium per well of the 96-well micro-
plates (NUNC 167008).
4.4.4. Condition of incubation
The cells were incubated with dilutions of the test substances
for 72 h at 37 °C in a humidified atmosphere and 5% CO2.
4.4.5. Method of evaluation
To estimate the influence of chemical compounds on cell prolif-
eration of K-562, we determined the numbers of viable cells pres-
ent in multi-well plates via CellTiter-Blue1 assay. It uses the
indicator dye resazurin to measure the metabolic capacity of cells
as the indicator of cell viability. Viable cells of untreated control re-
tain the ability to reduce resazurin into resorufin, which is highly
fluorescent. Non-viable cells rapidly lose metabolic capacity, do
not reduce the indicator dye, and thus do not generate a fluores-
cent signal. Under our experimental conditions, the signal from
the CellTiter-Blue1 reagent is proportional to the number of viable
cells. The adherent HUVEC and HeLa cells were fixed by glutaralde-
hyde and stained with a 0.05% solution of methylene blue for
15 min. After gently washing, the stain was eluted with 0.2 mL of
0.33 N HCl in the wells. The optical densities were measured at
660 nm in SUNRISE microplate reader (TECAN, Switzerland). The
GI50 and CC50 values were defined as the value at the intersection
of the dose response curve with the 50% line, compared to un-
treated control. These comparisons of the different values were
performed with the software Magellan (TECAN).
4.4.3. Cytotoxic assay
For the cytotoxic assay, HeLa cells were 48 h pre-incubated
without the test substances. The dilutions of the compounds were
Table 3
Retention times (tR), calculated logarithms of capacity factors (log k) and log PO/W
values of the reference compounds
a
Reference
tR (min)
log k
log PO/W
Benzophenone
Diphenylamine
Benzyl benzoate
n-Butylbenzene
Bibenzyl
2.97
3.42
4.89
10.63
11.12
1.08
0.24
0.34
0.55
0.95
0.97
—
3.2
3.4
4.0
4.6
4.8
—
Formamide (t0)
a
Retention times were determined in triplicate and the calculation of log k was
done with the arithmetic mean values.

F. Krauth et al. / Bioorg. Med. Chem. 18 (2010) 1816–1821
1821
6. Sztanke, K.; Pasternak, K.; Sztanke, M.; Kandefer-Szerszen, M.; Koziol, A. E.;
Dybala, I. Bioorg. Med. Chem. Lett. 2009, 19, 5095.
7. Dallloul, H. M.; El-Hossain, A. R. M.; Shorafa, H. Z.; El-Shorafa, A. Z. ARKIVOC
2008, 207.
4.5. Spectrofluorimetric determination of serum albumin
binding
8. Sztanke, K.; Pasternak, K.; Rzymowska, J.; Sztanke, M.; Kandefer-Szerszen, M.
Eur. J. Med. Chem. 2008, 43, 1085.
9. Hunt, J. T.; Mitt, T.; Borzilleri, R.; Gullo-Brown, J.; Fargnoli, J.; Fink, B.; Han, W.
C.; Mortillo, S.; Vite, G.; Wautlet, B.; Wong, T.; Yu, C.; Zheng, X.; Bhide, R. J. Med.
Chem. 2004, 47, 4054.
All fluorescence measurements were performed at room temper-
ature on a Perkin Elmer MPF-44 Fluorescence Spectrophotometer
equipped with a Xenon lamp, 4 mm excitation and emission slits.
Intrinsic tryptophan fluorescence emission spectra were recorded
at a wavelength of 290 nm. HSA was dissolved in PBS buffer at a con-
centration of 5.0 lM. Stock solutions of 3a–d were prepared in
DMSO at concentrations of 0.1 mM, 1 mM, and 10 mM. For titra-
tions, a series of increasing concentrations of compounds 3a–d in
PBS buffered HSA solution (final concentration 2 lM) was prepared
and incubated for 30 min at ambient temperature. After that, the
fluorescence emission was recorded. The reduction in fluorescence
(% Quenching) was plotted against total compound concentration (
Fig. 1B). Determination of Kd was realized using the quadratic bind-
ing equation for fluorescence quenching as described before and the
Marquardt algorithm in conjunction with a nonlinear least-squares
curve fitting routine (Microcal Origin 6.0).30
10. Abdel-Rahman, R. M. Pharmazie 2001, 56, 18.
11. WHO, http://www.who.int/cancer/en/index.html.
12. Frohberg, P.; Nuhn, P. Heterocycles 1996, 43, 2549.
13. Drutkowski, G.; Donner, C.; Schulze, I.; Frohberg, P. Tetrahedron 2002, 58, 5317.
14. Molinspiration Cheminformatics, http://www.molinspiration.com, accessed on
July 30, 2009.
15. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
1997, 23, 3.
16. Frohberg, P.; Drutkowski, G.; Wagner, C. Eur. J. Org. Chem. 2002, 10, 1654.
17. Frohberg, P.; Wagner, C.; Meier, R.; Sippl, W. Tetrahedron 2006, 62, 6050.
18. Krauth, F.; Friedemann, R.; Rüttinger, H.-H.; Frohberg, P. ARKIVOC 2009, 150.
19. Paschke, A.; Neitzel, P. L.; Walther, W.; Schüürmann, G. J. Chem. Eng. Data 2004,
49, 1639.
20. OECD Guideline for Testing of Chemicals. 117 Partition Coefficients (n-octanol/
water), HPLC Method, OECD, Paris, 1996.
21. OECD Guideline for Testing of Chemicals. 107 Partition Coefficients (n-octanol/
water), Shake Flask Method, OECD, Paris, 1996.
22. Velpandian, T.; Mathur, R.; Agarwal, N. K.; Arora, B.; Kumar, L.; Gupta, S. K. J.
Chromatogr., B 2004, 804, 431.
Acknowledgments
23. Formariz, T. P.; Sarmento, V. H. V.; Silva-Junior, A. A.; Scarpa, M. V.; Santilli, C.
V.; Oliveira, A. G. Colloids Surf., B: Biointerfaces 2006, 51, 54.
Ms. Antje Peters, Ms. Cindy Knigge, Ms. Anne-Kathrin Langer,
and Mr. Stefan Kneisel are acknowledged for helpful assistance in
determining experimental log Pexp data and Dr. Andrea Sinz for
critical reading of the manuscript.
ˇ
24. Dolezal, R.; Waisser, K.; Petrlíková, E.; Kuneš, J.; Kubicová, L.; Machácek, M.;
Kaustová, J.; Dahse, H.-M. Arch. Pharm. Chem. Life Sci. 2009, 342, 113.
25. Laube, T.; Bernet, A.; Dahse, H.-M.; Jacobsen, I. D.; Seifert, K. Bioorg. Med. Chem.
2009, 17, 1422.
26. Dahse, H.-M.; Schlegel, B.; Gräfe, U. Pharmazie 2001, 56, 489.
27. NCI, http://www.nci.nih.gov/cancertopics/types/leukemia.
28. Thorarensen, A.; Li, J.; Wakefield, B. D.; Romero, D. L.; Marotti, K. R.; Sweeney,
M. T.; Zurenko, G. E.; Sarver, R. W. Bioorg. Med. Chem. Lett. 2007, 17, 3113.
29. Zsila, F.; Fitos, I.; Bikádi, Z.; Simonyi, M.; Jackson, H. L.; Lockwood, S. F. Bioorg.
Med. Chem. Lett. 2004, 14, 5357.
30. Parikh, H. H.; McElwain, K.; Balasubramanian, V.; Leung, W.; Wong, D.; Morris,
M. E.; Ramanathan, M. Pharm. Res. 2000, 17, 632.
31. Epps, D. E.; Raub, T. J.; Kezdy, F. J. Anal. Biochem. 1995, 227, 342.
32. Damrot, J.; Nübel, T.; Epe, B.; Roos, W. P.; Kaina, B.; Fritz, G. Br. J. Pharmacol.
2006, 149, 988.
References and notes
1. Bhatia, P. A.; Brooks, C. D. W.; Basha, A.; Ratajczyk, J. D.; Gunn, B. P. J. Med.
Chem. 1996, 39, 3938.
2. Kim, K. H.; Martin, Y. C.; Brooks, C. D. W. Quant. Struct.-Act. Relat. 1996, 15, 491.
3. Sztanke, K.; Pasternak, K.; Rajtar, B.; Sztanke, M.; Majek, M.; Polz-Dacewicz, M.
Bioorg. Med. Chem. 2007, 15, 5480.
4. Abdel-Rahman, R. M. Pharmazie 2001, 56, 195.
5. Pontillo, J.; Guo, Z.; Wu, D.; Struthers, R. S.; Chen, C. Bioorg. Med. Chem. Lett.
2005, 15, 4363.