Synthesis, Structure Analysis, and Antitumor Evaluation of 3,6-Dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide Derivatives

Article abstract of DOI:10.1002/cmdc.201200109

3,6-Dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide derivatives were synthesized, and their structures were confirmed by single-crystal X-ray diffraction. This reaction yields the 1,4-dicarboxamide derivatives rather than the 1,2-dicarboxamide derivatives. Their invitro antitumor activities were evaluated against SGC-7901, HO-8910, MCF-7, and A-549 cells. The results showed several compounds to be endowed with cytotoxicity in the low micromolar range. One compound (IC₅₀=0.57μM) was further evaluated invivo against an A-549 xenograft in BALB/cA nude mice; it effected 76.4% inhibition of tumor weight through intraperitoneal (i.p.) administration of 40mgkg^-1 body weight. Moreover, its acute toxicity was evaluated, and the i.p. LD₅₀ value was 325mgkg^-1 in mice.

Full text of DOI:10.1002/cmdc.201200109

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DOI: 10.1002/cmdc.201200109
Synthesis, Structure Analysis, and Antitumor Evaluation of 3,6-Dimethyl-
1,2,4,5-tetrazine-1,4-dicarboxamide Derivatives
Guo-Wu Rao,* Yan-Mei Guo, and Wei-Xiao Hu[a]
3,6-Dimethyl-1,2,4,5-tetrazine-1,4-dicarboxamide
derivatives
in the low micromolar range. One compound (IC50 =0.57 mm)
was further evaluated in vivo against an A-549 xenograft in
BALB/cA nude mice; it effected 76.4% inhibition of tumor
weight through intraperitoneal (i.p.) administration of
40 mgkgÀ1 body weight. Moreover, its acute toxicity was eval-
uated, and the i.p. LD50 value was 325 mgkgÀ1 in mice.
were synthesized, and their structures were confirmed by
single-crystal X-ray diffraction. This reaction yields the 1,4-dicar-
boxamide derivatives rather than the 1,2-dicarboxamide deriv-
atives. Their in vitro antitumor activities were evaluated against
SGC-7901, HO-8910, MCF-7, and A-549 cells. The results
showed several compounds to be endowed with cytotoxicity
Tetrazine derivatives have good spectral and physical proper-
ties,[1] and hold high potential as antiviral and antitumor
agents.[2,3] Dihydro-1,2,4,5-tetrazine has four isomers: 1,2-, 1,4-,
1,6-, and 3,6-dihydro-1,2,4,5-tetrazine. There seems to be con-
siderable confusion over the structures of 1,2- and 1,4-dihydro-
1,2,4,5-tetrazine isomers, and the same compound is often for-
mulated as both structures. In most cases, the dihydro struc-
ture, which would be the initial reaction product, is presented,
or the authors have formulated their compounds in the dihy-
dro structure, which appeared to be the most accepted at that
time. Some scientists believe that rearrangement can occur be-
tween the 1,2- and 1,4-dihydro-1,2,4,5-tetrazine isomers.[3]
3,6-Bis(phenylethynyl)-1,2,4,5-tetramethyl-1,2,4,5-tetrazine
had been reported as an antitumor compound.[4] Although no
data on antitumor activities were reported, it was the original
expression that 1,2,4,5-tetrazine derivatives may possess antitu-
mor activity. We attempted to investigate whether modifica-
tions to this original structure could enhance the antitumor ac-
tivities of this compound class.
Scheme 1. Synthesis of compound series 1: a) CH3CHO, H2NNH2·H2O, EtOH,
5–108C, 12 h; b) NaOH, O2, Pd/C, 108C, 35–37 h; c) 1. BTC, DMAP, CHCl3,
reflux, 3 h; 2. HNR1R2, reflux.
Thirteen 1,2,4,5-tetrazine-1,4-dicarboxamide derivatives were
synthesized through a one-pot method by combining 3,6-di-
methyl-1,6-dihydro-1,2,4,5-tetrazine (2) with bis(trichlorometh-
yl) carbonate (BTC) and amine, with 4-dimethylaminopyridine
(DMAP) as catalyst. The synthetic route is shown in Scheme 1.
The intermediate raw material of compound 2 was prepared
according to published methods.[5,6] All compounds were char-
acterized by IR and NMR spectroscopy as well as mass spec-
trometry, and the results are summarized in Table 1. Their
structures were further confirmed by single-crystal X-ray dif-
fraction.
Figure 1. X-ray crystal structure of compound 1d.
the C2ÀN1i [1.409(3) ꢀ], N1iÀN2i [1.417(2) ꢀ], C2iÀN1
[1.409(3) ꢀ], and N1ÀN2 [1.417(2) ꢀ] bond lengths correspond
to typical single bonds in compound 1d. Therefore, the tetra-
zine ring is the 1,4-dihydro structure with the N-substituted
groups at the 1,4-positions and not the 1,2-positions; the com-
pound is 3,6-dimethyl-N1,N4-diisopropyl-1,2,4,5-tetrazine-1,4-di-
carboxamide (1d), rather than 3,6-dimethyl-N1,N2-diisopropyl-
1,2,4,5-tetrazine-1,2-dicarboxamide (3d). Therefore the prod-
ucts of the reaction of compound 2, BTC, and amine bear the
1,4-dicarboxamide structure rather than that of 1,2-dicarboxa-
mide.
The single-crystal structure of compound 1d was deter-
mined by X-ray crystallography, and its structure is illustrated
in Figure 1. The N2=C2 [1.271(2) ꢀ] and N2i=C2i [1.271(2) ꢀ]
bonds correspond to typical C=N double-bond lengths, and
[a] Dr. G.-W. Rao, Y.-M. Guo, Prof. Dr. W.-X. Hu
College of Pharmaceutical Science
Zhejiang University of Technology, Hangzhou 310014 (P.R. China)
E-mail: rgw@zjut.edu.cn
The in vitro antitumor activities of these compounds were
evaluated against the growth of human gastric cancer SGC-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200109.
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Table 1. Synthesis of compound 1.
Compd
NR1R2
Time [h]
Yield [%]
mp [8C]
1H NMR: d [ppm][a]
1a
1b
20
15
11.5
2.8
201–202
154–155
2.36 (s, 6H), 2.89 (d, J=5.0 Hz, 6H), 6.47 (s, 2H)
1.20 (t, J=7.0 Hz, 6H), 2.36 (s, 6H), 3.29–3.35 (m, 4H), 6.48 (s, 2H)
0.96 (t, J=7.5 Hz, 6H), 1.57-1.61 (m, 4H), 2.37 (s, 6H), 3.25 (q, J=7.5,
6.3 Hz, 4H), 6.52 (d, J=5.5 Hz, 2H)
1c
1d
1e
1 f
38
13
40
18
48
21.3
35.1
46.1
16.8
1.6
120–122
169–170
112–113
134–135
109–111
1.21 (d, J=6.5 Hz, 12H), 2.36 (s, 6H), 3.95–3.99 (m, 2H), 6.29 (d,
J=8.0 Hz, 2H)
0.95 (t, J=7.3 Hz, 6H), 1.36–1.40 (m, CH2), 1.55 (t, J=7.3 Hz, 4H),
2.36 (s, 6H), 3.26–3.31 (m, 4H), 6.50 (s, 2H)
0.95 (d, J=7.0 Hz, 12H), 1.79–1.87 (m, 2H), 2.37 (s, 6H), 3.12 (t,
J=6.8 Hz, 4H), 6.59 (t, J=5.8 Hz, 2H)
0.93 (t, J=7.5 Hz, 6H), 1.18 (d, J=5.5 Hz, 6H), 1.49–1.57 (m, 4H),
2.36 (s, 6H), 3.76–3.82 (m, 2H), 6.27 (d, J=8.5 Hz, 2H)
1g
0.92 (t, J=7.5 Hz, 12H), 1.42–1.49 (m, 4H), 1.55–1.64 (m, 4H), 2.36
(s, 6H), 3.63–3.68 (m, 2H), 6.23 (d, J=9.0 Hz, 2H)
1h
1i
53
51
7.4
9.0
134–135
146–147[7]
2.35 (s, 6H), 2.47 (s, 6H), 6.92–7.35 (m, 8H), 8.41 (s, 2H)
2.25 (s, 6H), 2.28 (s, 6H), 2.49 (s, 6H), 7.11 (d, J=8.1 Hz, 2H), 7.23
(dd, J=2.2, 5.9 Hz, 2H), 7.31 (d, J=1.9 Hz, 2H), 8.37 (s, 2H)
1j
1k
1l
56
38
46
25.6
26.7
15.7
195–197
256–257
236–238
2.38 (s, 6H), 6.69–6.73 (m, 4H), 7.32–7.35 (m, 4H), 9.06 (s, 2H),
9.26 (s, 2H)
2.40 (s, 6H), 6.48–6.50 (m, 2H), 6.97–6.99 (m, 2H), 7.09 (t, J=8.0 Hz
2H), 7.17 (t, J=2.3 Hz, 2H), 9.14 (s, 2H), 9.40 (s, 2H)
2.42 (s, 6H), 6.80–6.83 (m, 2H), 6.89–6.95 (m, 4H), 7.98 (dd, J=1.2,
6.8 Hz, 2H), 9.02 (s, 2H), 10.16 (s, 2H)
1m
51
5.0
246–248
[a] 400 or 500 MHz, CDCl3 or [D6]DMSO.
7901, human ovarian cancer HO-8910, human breast cancer
MCF-7, and human lung cancer A-549 cell lines by SRB and
MTT assays. Cisplatin (DDP) was introduced as a positive con-
trol in the assay of MCF-7 and A-549 cells. The results are sum-
marized in Table 2 and show that three compounds—1e, 1i,
and 1l—are highly effective against cancer cell lines. IC50
values for compounds 1e and 1l against SGC-7901 cells are
1.34 and 3.81 mm, respectively. IC50 values for compound 1i
against the SGC-7901, HO-8910, MCF-7, and A-549 cell lines are
0.72, 0.90, 0.45, and 0.57 mm, respectively. Compound 1i and
1l display similar cytotoxic activities against the tested cancer
cells.
Compound 1i was profiled for its acute toxicity to Kunming
male and female mice by intraperitoneal (i.p.) administration,
and the results are listed in Table 3. All mice given a single i.p.
injection of compound 1i at a dose of 262.0 mgkgÀ1 body
weight showed no clinical signs or loss of body weight, and
the mortality rate was zero. At 295.0 mgkgÀ1, the mortality
rate was 40% (4/10) and various clinical signs such as loosen-
ing of hair, inanimation, and loss of locomotor activity were
observed post-injection and disappeared slowly on live mice.
Table 2. Anticancer activities against SGC-7901, HO-8910, MCF-7, and A-
549 cell lines in vitro.
Compd
IC50 [mm][a]
HO-8910
SGC-7901
MCF-7
A-549
1a
1b
1c
1d
1e
1f
1g
1h
1i
NT
>100
>100
>100
1.34
>100
>100
>100
0.72
NT
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
279.34
>100
>100
>100
0.45
>100
>100
>100
>100
Table 3. Acute toxicity of compound 1i on Kunming mice.[a]
68.47
Dose
[mgkgÀ1
Males[b]
Females[b]
Clinical
signs
Mortality
LD50
>100
>100
>100
0.57
117.21
NT
]
[mgkgÀ1
]
262.0
295.0
328.0
410.0
512.0
5
5
5
5
5
5
5
5
5
5
Normal
[c]
[c]
[c]
[c]
0
4
8
8
9
325
0.90
NT
>100
1j
1k
1l
NT
>100
3.81
>100
NT
6.56
7.96
5.55
1m
DDP
NT
NT
NT
NT
>100
27.15
57.28
17.74
[a] 14 days post-single-i.p. administration. [b] Number of mice tested in
the group. [c] Loosening of hair; inanimation; loss of locomotor activity;
atrophy of spleen.
[a] NT: not tested.
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Dead mice had obvious atrophy of the spleen, and living mice
had no clear lesions of internal organs. At 328.0 mgkgÀ1, the
same clinical signs were observed, and eight of the treated
mice died within nine days post-injection. At the high dose of
512.0 mgkgÀ1, identical clinical signs were observed, and nine
of the treated mice were dead within seven days post-injec-
tion. From the mortality data of all tested animals, the i.p. LD50
value (325 mgkgÀ1) for compound 1i was calculated according
to the method of Austen and Brocklehurst.[8]
Table 4. Antitumor efficacy of compound 1i against A-549 xenografts in
nude mice.[a]
Compd
Dose
[mgkgÀ1
No.
mice[b]
Tumor
weight [g]
Tumor weight
inhibition [%][c]
]
Control
1i
1i
–
12
6
6
6
6
1.82
0.92
0.53
0.43
0.23
–
10
20
40
12
49.5[d]
70.9[d]
76.4[d]
87.4[d]
1i
Docetaxel
Based on these antitumor data in vitro and acute toxicity
data, we further evaluated the in vivo antitumor activity of
compound 1i against A-549 xenografts in BALB/cA nude mice
in comparison with docetaxel as positive control. Compound
1i was administered at three doses (10, 20, and 40 mgkgÀ1
i.p.) and docetaxel at 12 mgkgÀ1 i.p.; the results are document-
ed in Figures 2–4 and Table 4. Figure 2 shows the effects of
compound 1i on tumor growth in nude mice presented as
tumor volume (Vtum) over time. A statistically significant dose-
[a] During 14 days post-xenograft; compounds administered i.p. [b] All fe-
males. [c] Percentage of tumor-weight inhibition versus control. [d] p<
0.01 versus vehicle-treated control group.
Figure 3. Effects of compound 1i on tumor growth in BALB/cA nude mice.
The results are presented as nude mice weight (NMW) over time.
Figure 2. Effects of compound 1i on tumor growth in BALB/cA nude mice.
The results are presented as tumor volume (Vtum) over time.
methyl-1,2,4,5-tetrazine-1,4-dicarboxamide derivatives may
have potential antitumor activities.
dependent decrease in Vtum was observed with increasing
doses of compound 1i. With respect to tumor weight, the re-
sults presented in Table 4 show that compound 1i, adminis-
tered at 40 mgkgÀ1 i.p., exhibited excellent efficacy against A-
549 xenografts in BALB/cA nude mice by 76.4%, slightly lower
than docetaxel (87.4%) administered at 12 mgkgÀ1. Regarding
body weight and toxicity, there was no statistical difference be-
tween average total body weights in mice treated with com-
pound 1i and control, whereas significant body weight loss
was continuously observed beginning at day 4 after treating
with docetaxel (Figure 3). The results are presented as the pic-
ture of the curative effect of compound 1i against A-549 xeno-
grafts in BALB/cA nude mice (Figure 4). The present study indi-
cated that compound 1i could be effective in the treatment of
human lung cancer.
Experimental Section
Synthesis of compound 2: Fresh CH3CHO (51 mL) was placed in
a 250 mL flask equipped with a mechanical stirrer. At À28C, 80%
H2NNH2·H2O (55 mL) and EtOH (14 mL) were then added dropwise
with stirring. The reaction mixture was then maintained at 5–108C
for 12 h. The mixture was then filtered off with suction, and the
white product of 3,6-dimethyl-hexahydro-1,2,4,5-tetrazine was ob-
tained. 3,6-Dimethylhexahydro-1,2,4,5-tetrazine was placed in
a 500 mL four-inner flask equipped with a mechanical stirrer. A so-
lution of NaOH (374 mL, 3.3%) and 5% Pd/C (1.80 g) was added
with stirring, and O2 gas was passed through the mixture at 108C
for 35–37 h. The reaction mixture was filtered off with suction, sa-
turated with NH4Cl, extracted with CH2Cl2, and dried with Na2SO4.
After removal of the drying agent, the yellow solution was concen-
trated carefully at atmospheric pressure to dryness to afford com-
pound 2 as a bright-yellow solid (17.39 g, 34.2%, according to
CH3CHO); mp: 107–1088C (Ref. [5]: 107–1088C).
In conclusion, compound 1i has relatively low toxicity and
good efficacy in vivo. All these results revealed that 3,6-di-
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gram. Hydrogen atoms were added at calculated positions and re-
fined using a riding model; they were given isotropic displacement
parameters equal to 1.2 (or 1.5 for methyl H atoms) times the
equivalent isotropic displacement parameters of their parent
atoms, and CÀH distances were restrained to 0.96 ꢀ for methyl H
atoms and 0.98 ꢀ for methine H atoms, while NÀH distances were
set at 0.86 ꢀ. C12H22N6O2, Mr =282.36 Da, tetragonal, a=9.759(3),
b=9.759(3), c=16.604(9) ꢀ, U=1581.4(10) ꢀ3, T=298(2) K, space
group: P 43 21 2, Z=4, 1calcd =1.186 gcmÀ3, m(MoKa)=0.085 mmÀ1
,
12003 reflections measured, 1476 unique (Rint =0.0209) which
were used in all calculations. Fine R1 =0.0477, wR (F2)=0.1535 (all
data). All crystallographic details for compound 1d have been de-
posited with the Cambridge Crystallographic Data Centre: CCDC-
855337 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge from The Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
Acknowledgements
The authors are very grateful to the Natural Science Foundation
of Zhejiang Province (No. Y2090985) and the National Natural
Science Foundation of China (Nos. 20802069 and 20272053) for
financial support, and Shanghai Institute of Materia Medica, Chi-
nese Academy of Sciences for evaluation of antitumor activities.
Figure 4. The curative effect of compound 1i against A-549 lung xenografts
in BALB/cA nude mice. Shown are the tumors from six mice per dosing of
compound 1i or docetaxel at day 14 after treatment; the control set com-
prised 12 mice. Animal studies for evaluation of in vivo antitumor activity
were carried out according to guidelines of the Shanghai Institute of Materia
Medica, Chinese Academy of Sciences.
Keywords: antitumor agents · cytotoxicity · tetrazines · X-ray
diffraction
Synthesis of compound 1d: BTC (3.96 g, 13.3 mmol) was dissolved
in CHCl3 (10 mL) with magnetic stirring, and a solution of com-
pound 2 (1.12 g, 10.0 mmol) and DMAP (0.50 g, 4.1 mmol) in CHCl3
(20 mL) was added dropwise with stirring at À128C. The mixture
was then stirred at reflux for 3 h. After the blowing of N2 gas, a so-
lution of iPrNH2 (2.41 g, 40.8 mmol) and CHCl3 (10 mL) was added
dropwise with stirring. The mixture was stirred at reflux for 13 h.
The organic phase was washed with H2O, dried (MgSO4), filtered,
and concentrated in vacuo. The resulting residue was a red solid of
the crude product (1.39 g, 49.3%). The residue was recrystallized
from EtOH to give compound 1d as a white solid (0.99 g, 35.1%).
Rf =0.88 (petroleum ether/EtOAc, 1:1); mp: 169–1708C; 13C NMR
(125 MHz, CDCl3): d=150.9, 148.8, 42.1, 22.9, 18.2 ppm; IR (KBr):
n˜ =3317, 1685, 1634, 1428, 1384, 1132, 1077, 974 cmÀ1; HRMS (ESI):
m/z [M+H]+ calcd for C12H23N6O2: 283.1882, found: 283.1883, [M+
Na]+, calcd for C12H22N6NaO2: 305.1702, found: 305.1703; HPLC:
tR =6.52 min (>99%). Compounds of 1a–n were synthesized in
the same manner.
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Crystal data for compound 1d: A colorless block of dimensions
0.35ꢂ0.33ꢂ0.28 mm3 was used for data collection with a Bruker
SMART CCD area-detector diffractometer with graphite monochro-
mated MoKa radiation (l=0.71073 ꢀ). The structure was solved by
direct method procedures as implemented in the SHELXS97[9] pro-
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Received: February 26, 2012
Revised: April 12, 2012
Published online on && &&, 0000
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Tumor downsizing: A series of tetrazine
derivatives were synthesized and char-
acterized. Their antitumor activity in
vitro showed several compounds to be
endowed with cytotoxicity in the low
micromolar range. The activity of one
compound in vivo and its acute toxicity
were further evaluated. The results re-
vealed that it has relatively low toxicity
and good efficacy.
G.-W. Rao,* Y.-M. Guo, W.-X. Hu
&& – &&
Synthesis, Structure Analysis, and
Antitumor Evaluation of 3,6-Dimethyl-
1,2,4,5-tetrazine-1,4-dicarboxamide
Derivatives
ChemMedChem 0000, 00, 1 – 4
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www.chemmedchem.org
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These are not the final page numbers!