Novel synthesis, characterisation and antitumour activity of dimethyl-3,6-di(aryl)-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboxylates

Article abstract of DOI:10.3184/174751914X14001747558135

A series of new dimethyl-3,6-di(aryl)-1,4-dihydro-1,2,4,5-tetrazine-1,4- dicarboxylates were synthesised through an intermolecular cyclisation reaction of methyl N'-(a-chloro-substituted benzylidene) hydrazinecarboxylate and triethylamine. Their 1,4-dihyd

Full text of DOI:10.3184/174751914X14001747558135

368 RESEARCH PAPER
VOL. 38 JUNE, 368–370
JOURNAL OF CHEMICAL RESEARCH 2014
Novel synthesis, characterisation and antitumour activity of
dimethyl-3,6-di(aryl)-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboxylates
Lu-Ping Lva,b*, Xiao-Feng Zhoub, Hai-Bo Shic, Jian-Rong Gaoa and Wei-Xiao Hud
aCollege of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, P.R. China
bLinjiang College, Hangzhou Vocational and Technical College, Hangzhou 310018, P.R. China
cZhejiang Medical College, Zhejiang Ningbo 315100, P.R. China
dCollege of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014, P.R. China
A series of new dimethyl-3,6-di(aryl)-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboxylates were synthesised through an
intermolecular cyclisation reaction of methyl N′-(α-chloro-substituted benzylidene) hydrazinecarboxylate and triethylamine.
Their 1,4-dihydro-s-tetrazine structures were characterised by IR, 1H NMR, MS, elemental analysis. Their antitumour activities
were evaluated in vitro by the SRB method for A-549 cells and the MTT method for P-388 cells. The results show that none of
the compounds had a high activity, but the substituents clearly had an effect on their antitumour activity.
Keywords: synthesis, antitumour activity, 1,4-dihydro-s-tetrazine
1,2,4,5-Tetrazinederivativesareofconsiderableinterestbecause
of their unique role in the construction of various nitrogen
heterocycles.1,2Compounds containing the 1,2,4,5-tetrazine
skeleton have been used as pharmaceuticals.3,4 For example,
3-amino-6-aryl-1,2,4,5-tetrazines showed modest antimalarial
activity and some hexahydro-s-tetrazines have useful analgesic
and anti-inflammatory activity. The antibacterial and
antifungal activities of a series of tetrahydro-s-tetrazines have
also been evaluated.
Previously we have shown that some s-tetrazine derivatives
have good antitumour activity, especially 1,4-dihydro-s-
tetrazine-1,4-dicarboxamide.5–8 In a further effort to find
potential antitumour agents, we have synthesised and evaluated
the biological activity of a series of seven 1,2,4,5-tetrazines.9,10
The chemical structures and synthetic route of the target
compounds are shown in Fig. 1.
by the intramolecular cyclisation reaction of 2a in benzene, a
solvent of low polarity.13
The effect of solvent polarity on the synthesis of the target
compounds was investigated (Table 1).It was found that
2-methoxy-5-phenyl-1,3,4-oxadiazole was obtained by an
intramolecular cyclisation reaction of methyl N′-(α-chloro-
benzylidene)hydrazinecarboxylate and Et3N using low polarity
benzene or toluene as the solvent in –10 °C. A mechanism for
the reaction is proposed in Fig. 2. However, an intermolecular
cyclisation reaction occurred when the solvent was modified
to THF or ethanol which have a higher polarity. The plausible
mechanism for this reaction is shown in Fig. 3. Dimethyl-3,6-
diphenyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboxylates (3a)
waspreparedbyusingTHFassolventandareactiontemperature
of –10 °C. The preparation of the target compounds (3a–g)
including four new compounds is summarised in Table 2.
Compounds 3a–g were evaluated for their antitumour
activity in vitro by the MTT method for P-388 cells and the
SRB methods for A-549 cells. The results are summarised in
Table 3. When the concentration of the compound solution is
10–6 mol L–1, and the inhibition ratio of the solution to cancer cell
growth is more than 50%, the compound is usually considered
to be effective. According to this criterion, it can be seen from
Table 3 that no compound is effective against both P-388 and
Results and discussion
In order to prepare 1a–g, a substituted benzaldehyde was
reacted with methyl hydrazinecarboxylate using methanol as
solvent according to the literature method.11 When preparing
2a–g from 1a–g, the literature method was modified by
using Cl2 as a chlorinating agent instead of using SOCl2.12
2-Methoxy-5-phenyl-1,3,4-oxadiazole (4) was synthesised
Methanol
CHO
NH2NHCOOCH3
CH=N NHCOOCH3
1a–g
X
X
COOCH3
Cl
N
N
Cl2
dry CHCl3
X
Et3N
C=N NHCOOCH3
THF,-10℃
N
N
X
X
COOCH3
2a–g
3a–g
a
b
c
d
e
f
g
X
H
m-Cl
p-CH3
m-NO2
p-Cl
2,4-diCl
p-NO2
Fig. 1 The synthetic route of the target compounds and their chemical structures.
* Correspondent. E-mail: mailofllp@126.com
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JOURNAL OF CHEMICAL RESEARCH 2014 369
Table 1 The effect of solvent polarity on the synthesis of target compounds
Reaction
temperature/°C
Sovlent
Compound
Yield/%
M.p./°C (lit.)
1H NMR
O
OCH3
Benzene
–10
95
49–51 (50–51)14
4.95 (s, 3H), 7.3–7.9 (m, 5H)14
N
N
O
50–52 (50–51)14
90–92 (90–91)15
4.95 (s, 3H), 7.3–7.9 (m, 5H)14
3.50 (s, 3H), 7.27–7.84 (m, 5H)15
OCH3
Toluene
THF
–10
–10
73.3
90.4
N
N
COOCH3
N
N
N
N
COOCH3
COOCH3
N
N
Ethanol
–10
82.1
91–92 (90–91)15
3.58 (s, 3H), 7.24–7.88 (m, 5H)15
N
N
COOCH3
O
Cl
C
O
Et3N
C
N
N
C
OCH3
N
NH
C
OCH3
Benzene,20℃
Intramolecular cyclization
O
OCH3
N
N
4
Fig. 2 Intramolecular cyclisation reaction.
C=N N COOCH3
Cl
H
X
Et3N
C=N N COOCH3
X
THF,-10℃
X
H3COOC
COOCH3
N
N
C
N
N
X
N
N
X
COOCH3
Fig. 3 Intermolecular cyclisation reaction.
Table 3 Inhibition of in vitro tumour cell growth by the compounds 3a–g
Rate of inhibition of P-388
C (tetrazine)/mol L–1
Rate of inhibition of A-549
C (tetrazine)/mol L–1
Compd
Table 2 Synthesis of 3a–g
10–4 10–5 10–6 10–7 10–8 10–4 10–5 10–6 10–7 10–8
Entry
X
M.p./°C
90–92 (90–91)15
200–202
94–96
142–145
113–115 (112–113)15
114–115 (113–114)15
181–183
Yield/%
3a
3b
3c
3d
3e
3f
0
0
15.4 11.9 13.4 12.2 56.2 2.8 6.6 9.8 19.6
10.3 12.7 11.0 7.0 55.4 1.9 0.8 7.0 8.5
3a
H
90.4
43.7
75.5
78.3
86.4
75.8
75.4
3ba
3ca
3da
3e
m-Cl
31.3 17.3 12.6 8.4 9.0 46.6
11.0 11.9 23.0 10.1 9.6 57.7
0
0
0
0
0
0
0
0
0
0
0
p-CH3
m-NO2
p-Cl
17.7
1.1 22.9 13.6 10.4 60.0 2.4
69.3 1.1 11.6
7.6
7.1 57.2 6.7 0.7 3.5 1.3
3g
4.6 0.2 4.7 5.5 6.2 52.9 4.1 4.7 4.8 7.6
3f
3ga
2,4-diCl
p-NO2
Criteria of bioassay evaluation: when the concentration of the compound
solution is 10–6 mol L–1, the inhibition ration of the solution to cancer cell
growth is more than 50%, the compound is considered as to be effective.
aNew coupounds.
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370 JOURNAL OF CHEMICAL RESEARCH 2014
(2.0), 58 (18.1). Anal. calcd for C18H14N6O8 (MW=442.34): C, 48.87; H,
3.19; N, 19.00; found: C, 48.98; H, 3.21; N, 19.17%.
A-549 cells. However, in the series 3a–g, when their substituent
X is m-NO2 or p-Cl and the concentration of the compound
solution is 10–6 mol L–1, it seems that the groups (p-Cl, m-NO2)
could produce activity against P-388.
Dimethyl-3,6-di(p-chloro-phenyl)-1,4-dihydro-1,2,4,5-
tetrazine-1,4-dicarboxylate (3e): Following the method used for
3a, with triethylamine (2.0 g, 20.0 mmol) in 25 mL of THF, and 3.0 g
(12.1 mmol) of 2e in 75 mL THF and recrystallisation from absolute
ethanol twice gave 3e (2.2 g, 86.4%) as white crystals. M.p. 112–114 °C.
IR (KBr, cm–1): 2970 (CH3), 1760 (C=O), 1610m (C=N), 1336s (ring).
1H NMR (CDCl3) δ (ppm) 7.45–7.89 (m,4H, ArH),4.25 (s,3H). MS (m/z,
%): 210 M-210, 100), 168 (7.2), 140 (6.9), 112 (5.0), 102 (3.9), 75 (13.4),
59 (4.0). Anal. calcd for C18H14Cl2N4O4 (MW=421.23): C, 51.32; H,
3.35; N, 13.30; found: C, 51.37; H, 3.21; N, 13.23%.
Dimethyl-3,6-di(2,4-dichloro-phenyl)-1,4-dihydro-1,2,4,5-
tetrazine-1,4-dicarboxylate (3f): Following the method used for 3a,
with triethylamine (1.6 g, 17.2 mmol) in 25 mL of THF, and 3.0 g
(10.7 mmol) of 2f in 75 mL THF and recrystallisation from absolute
ethanol twice gave 3f (2.6 g, 75.8%) as white crystals. M.p. 114–115 °C.
IR (KBr, cm–1): 2978s (CH3), 1774m (C=O), 1599m (C=N), 1370
(ring).1H NMR (CDCl3δppm): 7.38–7.86 (m, 3H, ArH),4.26 (s,3H). MS
m/z (%): 244 (M-244, 100), 201 (10.9), 175 (33.4), 159 (11.1), 136 (12.3),
75 (10.5), 59 (23.3). Anal. calcd for C18H12Cl4N4O4 (MW=490.12): C,
44.11; H, 2.47; N, 11.43; found: C, 44.13; H, 2.46; N, 11.65%.
Experimental
Compounds 1a–g were synthesised by the literature method.11
The synthesis of compounds 2a–g were carried out by modified
literature method.12
Compound
4 was synthesised via intramolecular cyclisation
reaction of 2a by the literature method.13 Solvents and reagents
were commercially available without further purification. Melting
points were determined on a XRC-1 apparatus and are uncorrected.
IR spectra were recorded with a KBr discs on a Nicolet FI-IR-170
instrument. 1H NMR spectra were run on a Bruker AC400 (400 MHZ).
spectrometer using TMS as internal standard and CDCl3 as the
solvent. Mass spectra were obtained on a HP5989A spectrometer at an
ionising voltage of 70ev by electron impact. Elemental analyses were
performed on a Carlo ERBA-1106 instrument.
Dimethyl-3,6-diphenyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-
dicarboxylate (3a): Triethylamine (2.04 g, 20.0 mmol) and dried
THF (30 mL) were mixed and cooled to –10 °C. Compound 2a
Dimethyl-3,6-di(4-nitro-phenyl)-1,4-dihydro-1,2,4,5-tetrazine-
1,4-dicarboxylate (3g): Following the method used for 3a, with 0.8 g
(7.9 mmol) of triethylamine in 20 mL of THF, and 0.3 g (1.2 mmol)
of 2g in 10 mL THF and recrystallisation from absolute ethanol/
ethyl acetate twice gave 3g (0.2 g, 75.4%) as yellow crystals. M.p.
181–183 °C. IR (KBr, cm–1): 983s (CH3), 1778 (C=O), 1613m (C=N),
1370s (ring).1H NMR (CDCl3δppm) 7.69–8.77 (m,4H, ArH), 4.29
(s,3H).MS m/z (%): 221 (M-221, 100), 191 (2.6), 177 (1.4), 151 (1.4), 120
(3.5), 92 (2.5), 75 (4.9), 59 (22.3), 50 (1.5). Anal. calcd for C18H14N6O8
(MW=442.34): C, 48.87; H, 3.19; N, 19.00; found: C, 48.84; H, 3.09; N,
19.05%.
(methyl
N′-(α-chloro-benzylidene)hydrazinecarboxylate)
(2.8 g,
13.2 mmol) in THF (25 mL) was added dropwise into the reaction
vessel with stirring. After the addition was complete, the mixture
was heated to 20–25 °C and kept stirring at this temperature for 3 h,
using TLC (n-hexane:ethyl acetate=7:3). After the reaction, the
precipitate (triethylamine hydrochloride) was filtered. The filtrate
was concentrated to remove the solvent. The residue was triturated
with absolute ethanol (6 mL) to afford a white precipitate. The solid
was filtered and recrystallised from absolute ethanol/water to obtain
2.1 g product, yield 90%. M.p. 90–92 °C. IR (KBr, cm–1): 2974s (CH3),
1772m (C=O), 1585m (C=N), 1356s (ring).1H NMR (CDCl3) ppm:
7.24–7.84 (m, 5H, ArH), 3.50 (s, 3H). MS (m/z, %).176 (M-176, 100),
149 (4.0), 104 (40.4), 77 (65.7), 63 (6.3), 51 (30.5), 43 (3.8).Anal. calcd
for C18H14C12N4O4 (MW=352.36): C, 61.36; H, 4.58; N, 15.90; found:
C, 61.30; H, 4.86; N, 16.24%.
Dimethyl-3,6-di(2-chlorophenyl)-1,4-dihydro-1,2,4,5-tetrazine-
1,4-dicarboxylates (3b): Following the method used for 3a with
triethylamine (4.46 g, 44.0 mmol) in 30 mL of THF and 7.0 g
(28.3 mmol) of 2b in 20 mL of THF and recrystallisation from absolute
ethanol/water twice gave 3b (2.6 g, 43.7%) as white crystals. M.p.
91–93 °C. IR (KBr, cm–1): 2983s (CH3), 1782m (C=O), 1634m (C=N),
1336s (ring), 1064 (Ar–Cl).1H NMR (CDCl3) δ (ppm) 7.27–7.81 (m, 4H,
ArH), 3.55 (s, 3H). MS (m/z, %): 421 (M+, 2.7), 210 (100), 166 (57.1), 139
(36.6), 111 (4.2), 75 (14.3), 63 (5.9), 50 (8.9), 43 (20.8). Anal. calcd for
C18H14Cl2N4O4 (MW=421.23): C, 51.32; H, 3.35; N, 13.30; found: C,
51.45; H, 3.50; N, 13.64%.
We are indebted to the Department of Education of Zhejiang
Province (No. Y201224849) and Hangzhou Vocational and
Technical College for financial support and National Centre
for Drug screening, Shanghai, P.R. China for evaluation of
antitumour activities.
Received 4 March 2014; accepted 25 April 2014
Paper 1402505 doi: 10.3184/174751914X14001747558135
Published online: 10 June 2014
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