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

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.


Introduction
Temozolomide (Temodar, 3-methyl-4-oxo-3,4-dihydroimidazo [5,1-d] [1,2,3,5]tetrazine-8-carboxamide), 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-4carboxamide (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]. 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-dihydroimidazo [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.

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. In addition, the activity of partial synthetic compounds against leukemia cell line HL-60 was monitored by surveying the IC 50 values compared with control compound temozolomide. The IC 50 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 IC 50 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. 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 CH 3 COOH-CH 3 COONa 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.
a. Peak area ratio of synthesized compounds to temozolomide at pH 3.6. A 1 : the peak area of synthesized compound. A 0 : the peak area of temozolomide. Data shown are means ± SD of three independent experiments.

General
1 H-NMR spectra were recorded on a BRUKER ARX-300 instrument in (CD 3 ) 2 SO solution with Me 4 Si 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]. [5,1-d] [1,2,3,5]tetrazine-8-acyl chloride (II). A mixture of compound I (2.0 g, 0.01 mol), SOCl 2 (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.

Biological activity assays
MTT assay: 10 3 cells were seeded in RPMI1640 in each well of a 96-well plate and were allowed to adhere and spread at 37 ºC, 5% CO 2 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 96well plate. Growth inhibition was determined as compared to untreated cells (%).
Trypan Blue assay: Cells were seeded at a density of 1 × 10 5 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 (IC 50 ) which inhibited half of the cell growth was calculated.

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.

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.