Synthesis and antitumor activity of 3-Methyl-4-oxo-3,4-dihydroimidazo [5,1-d][1,2,3,5]tetrazine-8-carboxylates and -carboxamides

Article abstract of DOI:10.3390/molecules15129427

Seventeen novel 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine- 8-carboxylate and -carboxamide derivatives were synthesized and evaluated for their growth inhibition in seven human solid tumor and a human leukemia HL-60 cell lines. Compound IVa showed more activity than the other compounds and the positive control temozolomide. In the presence of 40 μg/mL of IVa, the survival rate of all tested tumor cells was less than 10%. Esters displayed more potent antitumour activity than amides and temozolomide against HL-60 cells. These compounds also exhibited considerably enhanced water-solubility.

Full text of DOI:10.3390/molecules15129427

Molecules 2010, 15, 9427-9436; doi: 10.3390/molecules15129427
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antitumor Activity of 3-Methyl-4-oxo-3,4-
dihydroimidazo [5,1-d][1,2,3,5]tetrazine-8-carboxylates
and -carboxamides
Dan Liu 1, Jian-Guo Yang 2, Jie Cheng 1 and Lin-Xiang Zhao 1,*
1
Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, Shenyang
Pharmaceutical University, Shenyang 110016, China; E-Mails: sammyld@163.com (D.L.);
spujack2008@126.com (J.C.)
2
Zhejiang Jinhua CONBA Bio-pharm Co. Ltd., Jinhua 321016, China;
E-Mail: yangjg999@163.com (J.-G.Y.)
* Author to whom correspondence should be addressed; E-Mail: zhaolinxiang@syphu.edu.cn
(L.-X.Z.); Tel.: (+86)24-23986420; Fax: (+86)24-23986420.
Received: 4 November 2010; in revised form: 10 December 2010 / Accepted: 14 December 2010 /
Published: 20 December 2010
Abstract: Seventeen novel 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-
carboxylate and -carboxamide derivatives were synthesized and evaluated for their growth
inhibition in seven human solid tumor and a human leukemia HL-60 cell lines. Compound
IVa showed more activity than the other compounds and the positive control
temozolomide. In the presence of 40 g/mL of IVa, the survival rate of all tested tumor
cells was less than 10%. Esters displayed more potent antitumour activity than amides and
temozolomide against HL-60 cells. These compounds also exhibited considerably
enhanced water-solubility.
Keywords: antitumour activity; 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]-
tetrazine-8-carboxylates; 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-
carboxamides; synthesis

Molecules 2010, 15
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1. Introduction
Temozolomide (Temodar, 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbox-
amide), was approved bythe U.S. FDA to treat the patients suffering from glioblastoma and anaplastic
astrocytoma in 1999. In vivo, temozolomide is converted into 5-(3-methyl-1-triazeno)imidazole-4-
carboxamide (MTIC) through chemical degradation without enzymatic catalysis, whereas dacarbazine
requires metabolic activation to generate the active form [1]. MTIC decomposes spontaneously to form
5-aminoimidazole-4-carboxamide (ACI) and a methyldiazonium ion [2], and the latter attacks the
guanine segment of a sequence of three or more guanines on DNA leading to DNA methylation of
(Scheme 1). This DNA damage can be repaired by O-6-methylguanine-DNA methyltransferase
(MGMT) expressed in some tumor cells, which is the primary mechanism of tumor resistance to
alkylating agents, including temozolomide [3]. Currently, combination therapies of temozolomide with
established anticancer drugs, such as cisplatin and irinotecan are being investigated [4,5].
Scheme 1. Metabolic pathways of temozoloimide and dacarbazine.
CONH2
O
O
N
O
H2N
H
N
N
H2N
N
N
N
N
N
CH3
NH2
N
H2O
N
N
CH2
+
CH3
N
NH
MTIC
NH
ACI
Temozoimide
[Enzyme]
CH3
O
H2N
N
N
N
CH3
N
NH
Dacarbazine
We were interested in the imidazotetrazinone skeleton due to its particular metabolism and low
toxicity. In order to improve the antitumor activity and water-solubility, some new temozolomide
hydrochlorides were designed and synthesized and their antitumor activity was assayed. There is a
general agreement that antitumor imidazotetrazinones display powerful activity when the N3 position
is methyl-substituted [6,7], so two series of compounds IIIa - IIIh and IVa - IVi with a methyl group
at the N3 position and esters or amides group at the 8 position were designed and synthesized. The
basic substitutents at the position 8 formed hydrochlorides to improve the stability and solubility in
water. The target compounds could be hydrolyzed in vivo to form 3-methyl-4-oxo-3,4-dihydro-
imidazo[5,1-d][1,2,3,5]tetrazine-8-carboxylic acid, which is the active metabolite of temozolomide and
shows moderate activity against TLX lymphoma in vitro. In this paper, the antitumor activity of the 17
new compounds against seven human solid tumor cell lines (PC-3, HCT-15, T47D, MDA-MB-231,
DU145, HT29, and LNCaP) and a leukemia cell line (HL-60) was tested in vitro. Further antileukemia
studies are also planned.

Molecules 2010, 15
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2. Results and Discussion
2.1. Chemistry
The synthetic route to the 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbox-
ylates IIIa - IIIh and 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carboxamides
IVa - IVi is presented in Scheme 2. The substituents of compounds IIIa - IIIh and IVa - IVi are listed
in Table 1. Commercially available 5-aminoimidazole-4-carboxamide (AIC) was first treated with
sodium nitrite in dilute hydrochloric acid to give the corresponding diazo compound, to which methyl
isocyanate was added dropwise to afford temozoimide [8]. After hydrolysis with sodium nitrite and
sulfuric acid, carboxylic acid I was produced. Acyl chloride II provided by reaction of compound I
and thionyl chloride was reacted with alcohols and amines to yield the target compounds IIIa - IIIh
and IVa – Ivi, respectively.
Scheme 2. Synthetic route to the target compounds.
CONH2
H2NOC
NH2
NH
N
N
H2NOC
N
N
N
a
b
c
N
N
N
N
CH3
N
O
AIC
Temozoimide
O
COOH
N
COCl
N
. HCl
C
X R
N
O
N
O
N
N
N
e
N
N
d
N
N
N
N
N
N
X = O, N
R = alkylamine
CH3
CH3
CH3
O
I
II
IIIa - IIIh
IVa - IVi
Reagents and conditions: (a) NaNO2/HCl, -5 ºC; (b) methyl isocyanate, DMSO, r.t.; (c) NaNO2,
H2SO4, r.t.; (d) SOCl2, DMF, reflux; (e) substituted alcohol or amine, THF, r.t..
2.2. Antiproliferative activities
In the present study, the antiproliferative activity of the synthesized compounds was tested in vitro
on seven human tumor cell lines, including prostate cancer (PC-3, LNCaP and DU-145), breast cancer
(T47D, MDA-MB-231) and colon cancer (HT-29, HCT-15) by the MTT assay. The potential
cytotoxicities of all compounds were determined by measuring the percentage of cell survival, as
summarized in Table 1. The data suggested that at the concentration of 40 g/mL, the amide IVa
displayed powerful inhibition against all seven tested tumor cell lines, whose viabilities were all below
10%. The other compounds and temozolomide showed moderate inhibition against all cell lines.
Among the other compounds derivative IVh showed better inhibition against T47D breast cancer cells
and DU145 prostate cancer cells (cell viability was 32.65% and 32.77% respectively), while the
corresponding results for temozolomide were 62.35% and 70.67%. Compared with the control drug,
compound IVi possessed more potent inhibitory activity against breast cancer cell T47D，the survival
percentage was 34.97% vs. 62.35% of temozolomide. On the whole, the temozolomide analogues
showed higher cytotoxicity against seven tested cancer cell lines when the position 8 of temozolomide
was substituted by a acylamide.

Molecules 2010, 15
Table 1. The substituents and cytotoxicity of the title compounds IIIa - IVi.
9430
Survival percent / %a)
Compd.
X
R
MDA-
MB-231
PC-3
LNCaP
T47D
DU145
HT29
HCT-15
O
O
O
O
O
O
-CH2CH2N(CH3)2 . HCl
-CH2CH2N(C2H5)2 . HCl
-(CH2)3N(CH3)2 . HCl
-(CH2)3N(C2H5)2 . HCl
87.98±0.40 89.37±1.75 66.40±1.07 74.61±1.61 92.60±1.37 93.89±1.28 92.30±0.69
92.10±1.42 81.89±1.45 63.65±1.41 70.36±1.50 92.70±2.05 86.22±1.66 82.00±1.93
114.86±0.52 91.33±0.94 81.13±0.35 76.51±1.03 90.15±1.70 79.92±1.59 95.59±0.49
88.46±0.96 75.95±0.41 62.93±1.40 65.70±0.59 74.97±1.24 70.96±0.92 91.95±1.58
IIIa
IIIb
IIIc
IIId
IIIe
IIIf
-CH(CH3)CH2N(CH3)2 . HCl 86.92±1.41 85.28±0.67 73.06±2.04 73.97±1.31 129.12±1.72 92.62±0.97 88.13±0.77
-CH(CH3)CH2N(C2H5)2 . HCl 94.43±0.75 76.83±1.19 78.84±0.35 80.64±0.94 87.47±1.90 84.55±1.68 93.70±1.54
.
NCH3
CH2CH2N
HCl
O
76.95±0.67 68.62±1.20 53.67±1.69 58.17±0.98 73.98±0.54 75.19±0.74 81.96±1.10
IIIg
O
-CH(CH3)CH2N(CH3)2 . HCl 78.07±1.71 75.03±1.28 53.50±1.22 68.57±0.84 54.61±1.28 66.48±0.91 81.99±0.94
IIIh
IVa
IVb
IVc
IVd
NH
NH
NH
NH
-CH2CH2N(CH3)2 . HCl
-CH2CH2N(C2H5)2 . HCl
-(CH2)3N(CH3)2 . HCl
-(CH2)3N(C2H5)2 . HCl
< 10
< 10
< 10
< 10
< 10
< 10
< 10
63.80±1.87 91.73±1.01 71.70±1.35 58.78±0.86 87.11±0.87 82.59±1.23 126.86±2.90
74.82±1.79 86.17±0.86 78.90±1.05 66.13±1.39 181.76±1.60 84.26±1.20 91.70±1.10
80.20±1.71 78.66±0.60 50.94±0.46 53.14±2.42 175.59±1.54 61.00±3.91 101.83±1.20
.
N
NCH3
IVe
HCl
88.19±1.21 96.05±0.70 96.01±0.76 71.27±1.18 162.14±1.48 81.99±1.34 94.81±0.78
.
HCl
N
NCH2CH3
79.63±0.42 115.79±4.25 67.08±0.75 67.09±1.30 99.92±1.37 76.28±1.15 90.64±0.83
64.24±1.22 73.56±0.87 48.29±1.27 62.58±1.43 54.12±1.70 87.42±0.54 91.90±3.14
64.16±0.76 55.78±0.60 32.65±0.63 54.94±1.12 32.77±2.35 83.06±1.76 88.47±1.40
IVf
IVg
IVh
.
HCl
N(CH3)2
NH
NH
.
N(C2H5)2 HCl
.
HCl
NH
62.89±2.46 59.61±0.87 34.97±0.68 59.39±1.14 52.12±0.68 93.25±0.82 73.44±0.99
IVi
N
-OH
-NH2
70.94±2.75 62.84±5.80 59.22±1.71 70.53±0.45 71.94±1.44 77.08±1.90 97.57±1.04
85.13±2.97 68.51±1.25 62.35±0.54 86.85±1.67 70.67±1.02 85.20±0.93 76.03±1.38
I
Tem
a. Survival percent (%) at 40 µg /mL of tested compounds on seven human tumor cell lines. Data
shown are means ± SD of three independent experiments. Tem = temozolomide
In addition, the activity of partial synthetic compounds against leukemia cell line HL-60 was
monitored by surveying the IC50 values compared with control compound temozolomide. The IC50
values of all tested compounds are listed in Table 2. The growth inhibition activity against HL-60 cell
of compound IIIa, IIIb, IIIc, IIId, IIIe, IIIf, IVf, IVg, was more powerful than that of temozolomide.
The IC50 values were 12.11 mol/mL, 3.24 mol/mL, 3.32 mol/mL, 2.80 mol/mL, 2.63 mol/mL,
3.18 mol/mL, 44.84 mol/mL, 24.95 mol/mL, and >80 mol/mL, respectively, making them
potentially promising candidates for the treatment of leukemia, Further antileukemia studies will
be conducted.
Table 2. Growth inhibition activity of some temozolomide analogues on HL-60 cell.
IC50(mol/mL)a)
12.11±1.21
3.24±0.09
IC50(mol/mL)a)
IC50(mol/mL)a)
24.95±0.17
70.74±2.57
79.50±1.22
Compd.
IIIa
IIIb
Compd.
IVa
IVb
Compd.
IVg
IVh
>80
>80
>80
3.32±0.16
IIIc
IVc
IVi
2.80±0.26
>80
45.47±0.35
>80
IIId
IVd
I
2.63±0.24
3.18±0.07
>80
44.84±0.30
Tem
IIIe
IIIf
IVe
IVf
a. Cells were treated with various concentrations of the tested compounds for 3 days and the cell
growth inhibition was determined using the trypan blue assay. Data shown are means ± SD of three
independent experiments.

Molecules 2010, 15
9431
As ring-opening of temozolomide and its derivatives can occur under basic or neutral conditions [8],
the stability under different acidic conditions (pH 6.5, pH 6.0, pH 4.5, pH 3.6) was determined by high
performance liquid chromatography (HPLC). After 20 min, more than 97% of all tested compounds
remained intact at pH 3.6, whereas more than 20% of the compounds were decomposed under other
pH conditions. The improvement of water-solubility of the synthesized compounds compared with
temozolomide was estimated by calculating the HPLC peak area ratios in CH3COOH-CH3COONa
buffer with pH 3.6. As shown in Table 3, the solubility of all compounds had increased from 10-fold to
100-fold. The solubility of compound IIIa – IIIh, IVa – IVf was increased at least 60-fold compared
to that of temozolomide.
Table 3. The relative solubility of target compounds IIIa – IVi compared with temozolomide.
a)
a)
a)
Compd.
IIIa
IIIb
A1/A0
Compd.
IIIg
IIIh
A1/A0
Compd.
IVe
IVf
A1/A0
85±3
83±2
35±3
88±2
97±4
78±2
107±3
98±2
76±2
IIIc
IVa
IVg
IIId
93±2
IVb
64±4
IVh
IVi
36±4
13±3
103±3
91±3
77±3
62±3
IIIe
IIIf
IVc
IVd
a.
Peak area ratio of synthesized compounds to temozolomide at pH 3.6. A1: the peak area of
synthesized compound. A0: the peak area of temozolomide. Data shown are means ± SD of three
independent experiments.
3. Experimental
3.1. General
1H-NMR spectra were recorded on a BRUKER ARX-300 instrument in (CD3)2SO solution with
Me4Si as internal standard. MS were determined on Shimadzu GCMS QP-1000 mass spectrometer.
HR-MS were obtained on Finnigan MAT-711 mass spectrometer in EI mode. Infrared spectra were
recorded on a BRUKER IFS-55 FTIR spectrometer. The purity, stability and water-solubility were
calculated in a N300 chromatographic workstation equipped with a Hitachi L-2400 UV detector and
Hitachi pump-L-2130. Optical rotation was recorded on Pekin-Elmer 241 instrument. Melting points
were determined on a Yanaco melting point apparatus and are uncorrected. Unless specified otherwise,
all reagents and solvents were used as supplied by the manufacturer. Temozolomide and compound I
were synthesized according to the literature [8]. Disubstituted aminoalkyl alcohols and disubstituted
aminoalkyl amines were synthesized according to the literature [9,10].
3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-acyl chloride (II). A mixture of
compound I (2.0 g, 0.01 mol), SOCl2 (20 mL) and DMF (2 drops) was refluxed 2.5 h, then evaporated
under reduced pressure. Toluene (10 mL) was added, and the solution was again evaporated to dryness
to give II as a light yellow powder (1.9 g, 97.4%); m.p. 142-143 ºC.
General procedure for the synthesis of target compounds IIIa-IIIh, IVa-Ivi. The appropriate
disubstituted aminoalkyl alcohol or disubstituted aminoalkyl amine (0.00256 mol) was added dropwise
at room temperature to a solution of II (0.5 g, 0.00256 mol) in anhydrous THF (20 mL). The mixture

Molecules 2010, 15
9432
was reacted 3 h, and the precipitate that appeared was filtered and washed with anhydrous THF, ether,
and recrystalled from acetone-methanol to give the title compounds.
[2-(N,N-Dimethylamino)]ethyl 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8 carb-
oxylate hydrochloride (IIIa). Yield 54.5%; m.p.: 208-209 ºC; purity: 98.1%; IR (KBr): 3445, 2974,
1
2723, 1774, 1741, 1619, 1465, 1230 cm-1; MS [M+] (m/z): 266; H-NMR (DMSO-d6): 2.89 (s, 6H,
N(CH3)2), 3.54 (t, 2H, CH2CH2N), 3.90 (s, 3H, 3-CH3), 4.73 (t, 2H, OCH2- CH2), 8.93 (s, 1H, 6-CH),
10.74 (s, 1H, HCl); HR-MS: m/z calcd. for C10H14N6O3, (M+) 266.1127. Found: 266.1130.
[2-(N,N-Diethylamino)]ethyl
3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carb-
oxylate hydrochloride (IIIb). Yield 53.2%; m.p.: 208-209 ºC; purity: 97.9%; IR (KBr): 3432, 3038,
1
2951, 2593, 1758, 1728, 1247 cm-1; MS [M+] (m/z): 294; H-NMR (DMSO-d6): 1.25 (t, J = 7.2 Hz,
6H, N(CH2CH3)2), 3.29 (q, J = 7.2 Hz, 4H, N(CH2CH3)2), 3.54 (t, 2H, OCH2CH2N), 3.90 (s, 3H, CH3),
4.73 (t, 2H, OCH2CH2), 8.93 (s, 1H, 6-CH), 10.57 (brs, 1H, HCl); HR-MS: m/z calcd. for C12H18N6O3,
(M+) 294.1440. Found: 294.1428.
[3-(N,N-Dimethylamino)]propyl 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carb-
oxylate hydrochloride (IIIc). Yield 54.3%; m.p.: 224-225 ºC; purity: 98.5%; IR (KBr): 3461, 3074,
1
2960, 2599, 1750, 1723, 1462, 1248 cm-1; MS [M+] (m/z): 280; H-NMR (DMSO-d6): 2.16 (m, 2H,
CH2CH2CH2), 2.79 (s, 6H, N(CH3)2), 3.23 (t, J = 7.4 Hz, 2H, CH2CH2N), 3.89 (s, 3H, 3-CH3), 4.42 (t,
J = 6.1 Hz, 2H, OCH2CH2), 8.89 (s, 1H, 6-CH); HR-MS: m/z calcd. for C11H16N6O3, (M+) 280.1284.
Found: 280.1281.
[3-(N,N-Diethylamino)]propyl 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carb-
oxylate hydrochloride (IIId). Yield 56.8%, m.p.: 204-205 ºC, purity: 98.6%, IR (KBr): 3426, 3038,
1
2954, 2596, 1749, 1728, 1458, 1248 cm-1; MS [M+] (m/z): 308. H-NMR (DMSO-d6): 1.22 (t,
J = 7.3 Hz, 6H, N(CH2CH3)2), 2.13 (m, 2H, CH2CH2CH2), 3.11 (q, J = 7.3 Hz, 4H, N(CH2CH3)2), 3.19
(t, 2H, CH2CH2N), 3.87 (s, 3H, 3-CH3), 4.42 (t, J = 5.9 Hz, 2H, OCH2CH2), 8.88 (s, 1H, 6-CH), 10.45
(s, 1H, HCl). HR-MS: m/z calcd. for C13H20N6O3, (M+) 308.1597. Found: 308.1608.
[1-Methyl-2-(N,N-dimethylamino)]ethyl 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-
8-carboxylate hydrochloride (IIIe). Yield 68.5%; m.p.: 202-203 ºC; purity: 99.0%; IR (KBr): 3504,
3127, 2956, 2632, 1749, 1733, 1459, 1243 cm-1; MS [M+] (m/z): 280; 1H-NMR (DMSO-d6): 1.39 (d,
J = 6.3 Hz, 3H, OCHCH3), 2.86 (s, 6H, N(CH3)2), 3.47 (d, J = 8.5 Hz, 2H, CHCH2N), 3.89 (s, 3H, 3-
CH3), 5.54 (m, 1H, OCHCH3), 8.90 (s, 1H, 6-CH); HR-MS: m/z calcd. for C11H16N6O3, (M+)
280.1284. Found: 280.1281.
[1-Methyl-2-(N,N-diethylamino)]ethyl 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-
carboxylate hydrochloride (IIIf). Yield 68.3%; m.p.: 194-195 ºC; purity: 98.5%; IR (KBr): 3443,
3130, 2975, 2471, 1750, 1728, 1458, 1243 cm-1; MS [M+] (m/z): 308; 1H-NMR (DMSO-d6): 1.26 (q,
J = 7.3 Hz, 6H, CH2CH3), 1.41 (d, J = 6.2 Hz, 3H, OCHCH3), 3.21 (q, J = 7.3 Hz, 4H, CH2CH3), 3.50
(d, J = 4.6 Hz, 2H, CHCH2N), 3.89 (s, 3H, 3-CH3), 5.50 (m, 1H, OCHCH3), 8.89 (s, 1H, 6-CH), 10.08
(s, 1H, HCl); HR-MS: m/z calcd. for C13H20N6O3, (M+) 308.1597. Found: 308.1591.

Molecules 2010, 15
9433
[2-(4-Methylpiperazine)-1-]ethyl 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carb-
oxylate hydrochloride (IIIg). Yield 68.5%; m.p.: 183-185 ºC; purity: 97.8%; IR (KBr): 3426, 3121,
2957, 2585, 1743, 1720, 1463, 1249 cm-1; MS [M+] (m/z): 321; 1H-NMR (DMSO-d6): 3.89 (s, 3H, 3-
CH3), 4.46 (t, 2H, OCH2CH2), 8.86 (s, 1H, 6-CH), 10.21 (brs, 1H, HCl); HR-MS: m/z calcd. for
C13H19N7O3, (M+) 321.1549. Found: 321.1544.
(-)-[1-Methyl-2-(N,N-dimethylamino)]ethyl 3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5] tet-
25
razine-8-carboxylate hydrochloride (IIIh). Yield 75.1%; m.p.: 208-209 ºC; []D = -14.7º; purity:
1
99.1%; IR (KBr): 3430, 3127, 2958, 2629, 1749, 1720, 1461, 1245 cm-1; MS [M+] (m/z): 280; H-
NMR (DMSO-d6): 1.38 (d, J = 6.3 Hz, 3H, OCHCH3), 2.86 (s, 6H, N(CH3)2), 3.49 (d, J = 8.6 Hz,
2H, CHCH2N), 3.89 (s, 3H, 3-CH3), 5.54 (m, 1H, OCHCH3), 8.89 (s, 1H, 6-CH), 10.36 (brs, 1H, HCl);
HR-MS: m/z calcd. for C11H16N6O3, (M+) 280.1284. Found: 280.1297.
3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-[N-(2-dimethylamino-)ethyl] carbox-
amide hydrochloride (IVa). Yield 75.3%; m.p.: 180-181 ºC; purity: 97.6%; IR (KBr): 3387, 3113,
1
2678, 1747, 1659, 1575, 1460, 1205 cm-1; MS [M+] (m/z): 265; H-NMR (DMSO-d6): 2.82 (s, 6H,
N(CH3)2), 3.39 (t, J = 6.0 Hz, 2H, CH2CH2N), 3.68 (t, J = 6.0 Hz, 2H, CNHCH2CH2), 3.88 (s, 3H, 3-
CH3), 8.81 (t, J = 5.6 Hz, 1H, CONH), 8.89 (s, 1H, 6-CH), 10.09 (brs, 1H, HCl); HR-MS: m/z calcd.
for C10H15N7O2, (M+) 265.1287. Found: 265.1303.
3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-[N-(2-diethylamino)ethyl]carboxamide
hydrochloride (IVb). Yield 63.2%; m.p.: 118-119 ºC; purity: 97.7%; IR (KBr): 3460, 3234, 3102,
1
2940, 2643, 1739, 1656, 1255 cm-1; MS [M+] (m/z): 293; H-NMR (DMSO-d6): 1.22 (t, J = 7.2 Hz,
6H, CH2CH3), 3.66 (m, 2H, CONHCH2CH2), 3.87 (s, 3H, 3-CH3), 8.82 (t, J = 5.7 Hz, 1H, CONH),
8.90 (s, 1H, 6-CH); HR-MS: m/z calcd. for C12H19N7O2, (M+) 293.1600. Found: 293.1633.
3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-[N-(3-dimethylamino)propyl] carbox-
amide hydrochloride (IVc). Yield 67.4%; m.p.: 117-119 ºC; purity: 97.5%; IR (KBr): 3409, 3266,
1
2959, 2644, 1756, 1647, 1576, 1258 cm-1; MS [M+] (m/z): 279; H-NMR (DMSO-d6): 1.92 (m, 2H,
CH2CH2CH2), 2.75 (s, 6H, N(CH3)2), 3.07 (t, J = 7.7 Hz, 2H, CH2CH2N), 3.38 (t, J = 6.4 Hz, 2H,
CONHCH2), 3.87 (s, 3H, 3-CH3), 8.72 (t, 1H, CONH), 8.87 (s, 1H, 6-CH), 10.06 (brs, 1H, HCl); HR-
MS: m/z calcd. for C11H17N7O2, (M+) 279.1444. Found: 279.1430.
3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-[N-(3-diethylamino)propyl]
carbox-
amide hydrochloride (IVd). Yield 65.1%; m.p.: 182-183 ºC; purity: 98.0%; IR (KBr): 3442, 3252,
1
2949, 2648, 1733, 1652, 1246 cm-1; MS [M+] (m/z): 307; H-NMR (DMSO-d6): 1.19 (t,
J = 7.2 Hz, 6H, CH2CH3), 1.92 (m, 2H, CH2CH2CH2), 3.39 (t, J = 5.8 Hz, 2H, CONHCH2), 3.87 (s,
3H, 3-CH3), 8.73 (t, 1H, CONH), 8.87 (s, 1H, 6-CH), 10.06 (brs, 1H, HCl); HR-MS: m/z calcd. for
C13H21N7O2, (M+) 307.1757. Found: 307.1758.
1-(3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-oyl)-4-methyl piperazine hydro-
chloride (IVe). Yield 71.8%; m.p.: 189-190 ºC; purity: 97.9%; IR (KBr): 3434, 3059, 2943, 2453,
1
1758, 1646 cm-1; MS [M+] (m/z): 277; H-NMR (DMSO-d6): 2.75 (s, 3H, NCH3), 3.21 (m, 8H,

Molecules 2010, 15
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N(CH2CH2)2N), 3.85 (s, 3H, 3-CH3), 8.88 (s, 1H, 6-CH); HR-MS: m/z calcd. for C11H15N7O2, (M+)
277.1287. Found: 277.1287.
1-(3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-oyl)-4-ethyl
piperazine
hydro-
chloride (IVf). Yield 70.8%; m.p.: 200-201 ºC; purity: 98.3%; IR (KBr): 3435, 3119, 2361, 1746,
1
1656, 1558, 1460, 1242 cm-1; MS [M+] (m/z): 291; H-NMR (DMSO-d6): 1.26 (t, J = 7.1 Hz, 3H,
CH2CH3), 3.87 (s, 3H, 3-CH3), 8.89 (s, 1H, 6-CH); HR-MS: m/z calcd. for C12H17N7O2, (M+)
291.1444. Found: 291.1453.
3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-[N-(p-dimethylamino)phenyl] carbox-
amide hydrochloride (IVg). Yield 70.7%; m.p.: 209-210 ºC; purity: 98.1%; IR (KBr): 3347, 3054,
1
2208, 1758, 1679, 1608 cm-1; MS [M+] (m/z): 313; H-NMR (DMSO-d6): 3.04 (s, 6H, N(CH3)2 ),
3.89 (s, 3H, 3-CH3 ), 7.33 (brs, 2H, 3’-, 5’-CH (Ph) ), 7.89 (brs, 2H, 2’-, 6’-CH(Ph) ), 8.96 ( s, 1H, 6-
CH ), 10.45 ( brs, 1H, HCl ); HR-MS: m/z calcd. for C14H15N7O2, (M+) 313.1287. Found: 313.1268.
3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-[N-(p-diethylamino)phenyl]
carbox-
amide hydrochloride (IVh). Yield 67.8%; m.p.: 215-216 ºC; purity: 97.7%; IR (KBr): 3362, 3119,
1
2309, 1748, 1690, 1606, 1573, 1251 cm-1; MS [M+] (m/z): 341; H-NMR (DMSO-d6): 1.04 (t,
J = 6.9 Hz, 6H, (CH2CH3)2), 3.42 (q, J = 6.9 Hz, 4H, (CH2CH3)2), 3.90 (s, 3H, 3-CH3), 7.72 (brs, 2H,
3’-, 5’-CH Ph), 8.10 (brs, 2H, 2’-, 6’-CHPh), 8.98 (s, 1H, 6-CH), 10.74 (brs, 1H, HCl), 11.91 (brs, 1H,
CONH); HR-MS: m/z calcd. for C16H19N7O2, (M+) 341.1600. Found: 341.1608.
3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-(N-pyridine-2-)carboxamide
hydro-
chloride (IVi). Yield 65.4%; m.p.: 217-218 ºC; purity: 98.5%; IR (KBr): 3358, 3096, 1737, 1642,
1
1619, 1569, 1239 cm-1; MS [M+] (m/z): 271; H-NMR (DMSO-d6): 3.89 (s, 3H, 3-CH3), 7.25 (t,
J = 7.0 Hz, 1H, Pyr-5-H), 7.95 (t, J = 7.0 Hz, 1H, Pyr-4-H), 8.24 (d, J = 8.3 Hz, 1H, Pyr-6-H), 8.41, (d,
J = 5.0 Hz, 1H, Pyr-3-H), 8.79 (s, 1H, CONH), 8.97 (s, 1H, 6-CH), 10.14 (s, 1H, HCl); HR-MS: m/z
calcd. for C11H9N7O2, (M+) 271.0818. Found: 271.0811.
3.2. Biological activity assays
MTT assay: 103 cells were seeded in RPMI1640 in each well of a 96-well plate and were allowed to
adhere and spread at 37 ºC, 5% CO2 for 24 h. The compounds with the concentration of 40 g/mL were
then added and incubated for 4 d. Fifty μL of 2 mg/mL MTT solution was added per well and the
cultures were continued for an additional 4 h. The medium was removed by aspiration. The cells were
dissolved in 200 μL DMSO and vibrated for 10 min. Absorbance at 540 nm was measured in the 96-
well plate. Growth inhibition was determined as compared to untreated cells (%).
Trypan Blue assay: Cells were seeded at a density of 1 × 105 cells/mL and incubated with various
concentrations of the tested compounds for 3 days. Total cell number including trypan blue staining
positive and negative cells in each group was counted. The cell growth inhibition ability was
calculated and expressed as the ratio of the cell number in treated group to that of untreated group. The
concentration (IC50) which inhibited half of the cell growth was calculated.

Molecules 2010, 15
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3.3. Solubility detection
Compound and temozolomide were dissolved in sodium acetate - acetic acid buffer solution (pH 3.6)
to form a supersaturated solution. The suspension was shaken by ultrasonic irradiation, and filtered.
The peak area ratio was calculated by HPLC with UV detection at 254 nm.
4. Conclusions
In summary, the results presented above indicate that 1) temozolomide esters are more effective
than temozolomide amides in inhibiting HL-60 cell growth, 2) amide IVa with 2-dimethylamino-ethyl
chain has the most powerful potential in all tested solid tumor cell lines; 3) the water-solubility of all
synthesized compounds has been improved in various degrees.
Acknowledgements
This work was supported by China International Science and Technology Cooperation Plan
(2009DEA31200) and National Science and Technology Key Specific Project for Significant Creation
of New Drugs (2009ZX09301-012).
References and Notes
1. Stevens, M.F.G.; Hickman, J.A.; Langdon, S.P.; Chubb, D.; Vickers, L.; Stone, R.; Baig, G.;
Goddard, C.; Gibson, N.W.; Slack, J.A.; Newton, C.; Lunt, E.; Fizames, C.; Lavelle, F. Antitumor
Activity and Pharmacokinetics in Mice of 8-Carbamoyl-3-methyl-imidazo[5,1-d]-1,2,3,5-tetrazin-
4(3H)-one (CCRG 81045; M & B 39831), a Novel Drug with Potential as an Alternative to
Dacarbazine. Cancer Res. 1987, 47, 5846-5852.
2. Denny, B.J.; Wheelhouse, R.T.; Stevens, M.F.; Tsang, L.L.; Slack, J.A. NMR and Molecular
Modeling Investigation of the Mechanism of Activation of the Antitumor Drug Temozolomide
and its Interaction with DNA. Biochemistry 1994, 33, 9045-9051.
3. Fukushima, T.; Takeshima, H.; Kataoka, H. Anti-glioma Therapy with Temozolomide and Status
of the DNA-repair Gene MGMT. Anticancer Res. 2009, 29, 4845-4854.
4. Zustovich, F.; Lombardi, G.; Della Puppa, A.; Rotilio, A.; Scienza, R.; Pastorelli, D. A Phase II
Study of Cisplatin and Temozolomide in Heavily Pre-treated Patients with Temozolomide-
refractory High-grade Malignant Glioma. Anticancer Res. 2009, 29, 4275-4279.
5. Casey, D.A.; Wexler, L.H.; Merchant, M.S.; Chou, A.J.; Merola, P.R.; Price, A.P.; Meyers. P.A.
Irinotecan and Temozolomide for Ewing Sarcoma: the Memorial Sloan-Kettering Experience.
Pediatr. Blood Cancer 2009, 53, 1029-1034.
6. Lunt, E.; Newton, C.G.; Smith, C.; Stevens, G.P.; Stevens, M.F.G.; Straw, C.G.; Walsh, R.J.A.;
Warren, P.J.; Fizames, C.; Lavelle, F.; Langdon, S.P.; Vickers, L.M. Antitumor Imidazotetrazines.
14. Synthesis and Antitumor Activity of 6- and 8-Substituted imidazo[5,1-d]-1,2,3,5-tetrazinones
and 8-Substituted pyrazolo[5,1-d]-1,2,3,5-tetrazinones. J. Med. Chem. 1987, 30, 357-366.
7. Clark, A.S.; Deans, B.; Stevens, M.F.; Tisdale, M.J.; Wheelhouse, R.T.; Denny, B.J.; Hartley, J.A.
Antitumor Imidazotetrazines. 32. Synthesis of Novel Imidazotetrazinones and Related Bicyclic

Molecules 2010, 15
9436
Heterocycles to Probe the Mode of Action of the Antitumor Drug Temozolomide. J. Med. Chem.
1995, 38, 1493-1504.
8. Wang, Y.F.; Stevens, M.F.; Chan, T.M.; Dibenedetto, D.; Ding, Z.X.; Gala, D.; Hou, D.;
Kugelman, M.; Leong, W.; Kuo, S.C.; Mas, J.L.; Schumacher, D.P.; Shutts, B.P.; Smith, L.; Zhan,
Z.Y.J.; Thomson, W.T. Antitumor Imidazotetrazines. 35. New Synthetic Routes to the Antitumor
Drug Temozolomide. J. Org. Chem. 1997, 62, 7288-7294.
9. Hartman, W.W. -Diethylaminoethyl Alcohol (Ethanol, 2-diethylamino). Org. Syn. Coll. 1955, II,
193-184.
10. Buckingham, J. Dictionary of Organic Compounds, 6th ed.; Chapman & Hall: New York, NY,
USA, 1996; Volume 6, p. 5423.
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