Synthesis and Cytotoxicity of Cyanoborane Adducts of N 6 -Benzoyladenine and 6-Triphenylphosphonylpurine

N 6 -Benzoyladenine-cyanoborane (2), and 6-triphenylphosphonylpurine-cyanoborane (3) were selected for investigation of cytotoxicity in murine and human tumor cell lines, effects on human HL-60 leukemic metabolism and DNA strand scission to determine the feasibility of these compounds as clinical antineoplastic agents. Compounds 2 and 3 both showed effective cytotoxicity based on ED50 values less than 4 μg/ml for L1210, P388, HL-60, Tmolt3, HUT-78, HeLa-S3 uterine, ileum HCT-8, and liver Hepe-2. Compound 2 had activity against ovary 1-A9, while compound 3 was only active against prostate PL and glioma UM. Neither compound was active against the growth of lung 549, breast MCF-7, osteosarcoma HSO, melanoma SK2, KB nasopharynx, and THP-1 acute monocytic leukemia. In mode of action studies in human leukemia HL-60 cells, both compounds demonstrated inhibition of DNA and protein syntheses after 60 min at 100 μM. These compounds inhibited RNA synthesis to a lesser extent. The utilization of the DNA template was suppressed by the compounds as determined by inhibition of the activities of DNA polymerase α, m-RNA polymerase, r-RNA polymerase and t-RNA polymerase, which would cause adequate inhibition of the synthesis of both DNA and RNA. Both compounds markedly inhibited dihydrofolate reductase activity, especially in compound 2. The compounds appeared to have caused cross-linking of the DNA strands after 24 hr at 100 μM in HL-60 cells, which was consistent with the observed increased in ct-DNA viscosity after 24 hr at 100 μM. The compounds had no inhibitory effects on DNA topoisomerase I and II activities or DNA-protein linked breaks. Neither compound interacted with the DNA molecule itself through alkylation of the nucleotide bases nor caused DNA interculation between base pairs. Overall, these antineoplastic agents caused reduction of DNA and protein replication, which would lead to killing of cancer cells.

nucleotides have been reported to be even more potent in suppressing the growth of murine and human cancer cells/2/. A common feature of all of these derivatives was that they effectively suppressed DNA synthesis and the activities of enzymes involved in the nucleic acid metabolism. Selected compounds demonstrated DNA strand scission with inhibition of DNA topoisomerase II activity. Based on these previous studies, NCbenzoyladenine-cyanoborane (2), 6-triphenyl-phosphonylpurine-cyanoborane (3) were selected for investigation in human leukemia HL-60 cells for cytotoxicity, effects on metabolic events, and DNA strand scission to determine the feasibility of these compounds as clinical antineoplastic agents.

METHODS
The cyanoborane adducts of the substituted purines were prepared via a Lewis acid exchange reaction/9/, by using excess (e.g., 4 molar equivalents) triphenylphosphine-cyanoborane (1) and the amine (purine) in dry dimethylfonnamide (DMF) at 55-70C under nitrogen atmosphere (Scheme 2). In the Lewis acid exchange reaction, a weakly basic or bulky amine or phosphine, as its substituted borane (C6H5)3P.HB r + NaBH3C N Reflux N2(g) (C6H5)3P:BH2CN + HBr + H 2 Scheme 1 Synthesis of the Lewis acid exchange reagent, triphenylphosphine-cyanobomne (1). adduct (e.g., Ph3P:BH2CN), is exchanged for a more basic or less bulky amine, (e.g., a substituted purine). These boron exchange reactions must be carried out under anhydrous conditions to avoid coordination of the cyanoborane to water and subsequent degradation to boric acid. The Lewis acid exchange reaction is a general route which has also been used in the preparation of other cyanoborane adducts of aliphatic/3, 9/, aromatic /3,8/, and heterocyclic /8,9/ amines, as well as their borane and carboxyborane adducts /9/. Triphenylphosphine-cyanoborane (_1) was prepared as previously reported/9, 10/(Scheme 1) by refluxing Ph3P HBr and NaBH3CN in dry THF under nitrogen atmosphere. The reaction of 6-chloropurine and triphenylphosphine-cyanoborane produced the unexpected product, 6-triphenylphosphonylpurinecyanoborane (). This resulted when the free triphenylphosphine, formed after the Lewis acid exchange, underwent aromatic nucleophilic substitution displacing the chlorine in the 6 position of the purine ring.

Synthesis of Compounds
All chemicals and reagents were obtained from Aldrich Chemical Company (Milwaukee, WI) and used as received except for dry solvents which were dried and distilled using standard procedures/11/. TLC was performed using silica gel 60F 254 plates (silica gel on plastic, Aldrich Chemical Company). Melting points 2O 7bnva C.Scarlett et al.

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were obtained on a Thomas-Hoover Uni-melt apparatus (capillary method), and were uncorrected. IR spectra were obtained on a Perkin-Elmer 1600 FTIR spectrometer in a potassium chloride liquid cell in CHCI3 or CDC13. NMR spectra were obtained on a 300 MHz Bruker Avance FT-NMR spectrometer using tetramethylsilane as an external standard for H and laC spectra and BF3:OEt2 for IB spectra (6 0 ppm).
Preparation of Triphenylphosphine-cyanoborane (1_): To a mixture of 4.02 g (11.7 retool) of triphenylphosphine hydrobromide and 40 mL of dry THF was added 0.93 g (14.8 mmol) of sodium cyanoborohydride. The suspension was stirred under N2 (g) at reflux for 10 hr. The mixture was cooled to RT, filtered and the solid washed with THF. The filtrate and washings were combined and the solvents were removed under reduced pressure. The white solid was washed with cold water, then cold ethyl ether. After air drying, 3 Preparation of N6-Benzoyladenine-cyanoborane (2) To a solution of 10.01 g (33.23 retool, 4 Eq.) of triphenylphosphine-cyanoborane in 30 mL of dry DMF was added 2.03 g (8.49 mmol) of N-benzoyladenine. The solution was stirred under N2 (g) at 70C for 10 days. The solution was cooled to RT, filtered and the solid washed with methanol. The filtrate and washings were combined and silica gel was added until all of the liquid was adsorbed. The solvents were removed under reduced pressure. The product was purified by column chromatography on silica gel using dichloromethane:methanol (95:5, Rf 0.69). A partial yield/12/of 0.0858 g (0.309 retool, 3.6%) of tan solid To a mixture of 15.76 g (52.34 retool, 4 Eq.) of triphenylphosphine-cyanoborane in 30 mL of dry DMF was added 2.02 g (13.1 mmol) of 6-chloropurine. The mixture dissolved upon heating and was stirred under N2 (g) at 70C for 10 days. The solution was cooled to RT and the resulting suspension was filtered and the solid washed with methanol. The filtrate and washings were combined and silica gel was added until all of the liquid was adsorbed. The solvents were removed under reduced pressure. The product was purified by

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Normal fibroblasts 1788 were also used to test cytotoxicity of the agents. The NCI protocol was used to assess the cytotoxicity of the test compounds and standards in each cell line. Values for cytotoxicity were expressed as EDs0 lag/ml, i.e. the concentration of the compound inhibiting 50% of cell growth. EDs0 values were determined by the trypan blue exclusion technique /13/. A value of less than 4 lag/ml was required for significant activity of growth inhibition. Solid tumor cytotoxicity was determined utilizing crystal violet/MeOH and read at 580 nm (Molecular Devices)/14/.

Incorporation Studies
Incorporation of labeled precursors into 3H-DNA, 3H-RNA and all-protein for 10 6 HL-60 leukemia cells was obtained/15/using a concentration range of 25, 50 and 100 gM of the test agents 2 and 3 over a 60 min incubation. The incorporation of 4C-glycine (53.0 mCi/mmol) into purines /16/ and the incorporation of 4Cformate (53.0 mCi/mmol) into pyrimidines /17/ was determined in a similar manner.

Enzyme assays
Studies for the inhibition of various enzyme activities were performed by first preparing the appropriate HL-60 leukemia cell homogenates or subcellular fraction, then adding the drug to be tested during the enzyme assay. For the concentration response studies, inhibition of enzyme activity was determined at 25,50 and 100 gM of compounds 2 and 3, after 60 rain incubations. DNA polymerase c activity was determined in cytoplasmic isolated extracts [18]. The polymerase activity for was determined with H-TTP /19/.
Messenger-, ribosomal-and transfer-RNA polymerase enzymes were isolated with different concentrations of ammonium sulfate; individual RNA polymerase activities were determined using 3H-UTP /20.21/. Ribonucleoside reductase activity was measured using 4C-CDP with dithioerythritol /22/. The deoxyribonucleotides 14C-dCDP were separated from the ribonucleotides by TLC on PEI plates. Thymidine, TMP and TDP kinase activities were determined using 3H-thymidine (58.3 mCi/mmol) / ct-DNA studies After deoxyribonucleoside triphosphates were extracted/32/, levels were determined by the method of Hunting and Henderson/33/with calf thymus DNA, E. coli DNA polymerase l, non-liniting amounts of the t'ol. 9. Nos. [1][2]2002 Synthesis and C)'totoxiciO, qfQ:anoborane Adducts" three deoxyribonucleoside triphosphates not being assayed, and either 0.4 mCi of (3H-methyl)-dTTP or (5-3H)-dCTP. The effects of compounds 2 and 3 on DNA strand scission was determined by the methods of tCi thymidine hnethyl-3H, 84.0 Ci/mmol/for 24 hr at 37C. HL-60 cells (107) were harvested and then centrifuged at 600 g X 10 min in PBS. They were later washed and suspended in ml of PBS. Lysis buffer (0.5 ml; 0.5 M NaOH, 0.02 M EDTA, 0.01% Triton X-100 and 2.5% sucrose) was layered onto a 5-20% alkaline-sucrose gradient (5 ml; 0.3 M NaOH, 0.7 KC1 and 0.01 M EDTA); this was followed by 0.2 ml of the cell preparation. After the gradient was incubated for 2.5 hr at room temperature, it was centrifuged at 12,000 RPM at 20C for 60 min (Beckman rotor SW60). Fractions (0.2 ml) were collected from the bottom of the gradient, neutralized with 0.2 ml of 0.3 N HC1, and measured for radioactivity. Thermal 30 mg/ml bovine serum albumin, mM ATP, 10 mM MgCI2 and 150 mM KCI. After 30 rain incubation at 37 C the reaction was terminated with 1% SDS and mg/ml proteinase K (v/v). After an additional hour of incubation, aliquots were applied to a 0.8% agarose TBE gel (v/v) containing 0.5 mg/ml ethidium bromide and 1% SDS (w/v). Following overnight electrophoresis at 30 v (constant), the gel was destained and photographed using a U.V-transilluminator and Polaroid film. Topoisomerase activity inhibition was assayed by a similar method. The enzyme reaction consisted of test drugs, 0.5 units of human topoisomerase [TopoGen, Inc., Columbus, OH], 0.5 lag of supercoiled PBR322 DNA in 50 mM Tris-HCl, pH 8.0, 100 mM KCI, 10raM MgC12, 2 mM 2-mercaptoethanol, 30 lug/ml nuclease-free BSA.

Statistic Analysis
Data is displayed in tables and figures as the means + standard deviations of the mean expressed as a percentage of the control value. N is the number of samples per group. The Student's "t"-test was used to determine the probable level of significance (p) between test samples and control samples.
Compound 2 was examined for its mode of action in HL-60 leukemia cells (Table 2). DNA and RNA synthesis after 60 minutes was slightly inhibited by 35% and 25% at 100 laM. Protein synthesis after 60 minutes at 100 laM inhibited 55% at 100 .tM. Utilization of the DNA template showed that the agent inhibited DNA polymerase ct activity by 50% at 100 tM,.mRNA polymerase 41%, rRNA polymerase 37%, and tRNA polymerase 52%. A number of enzyme activities were slightly reduced but were not significantly different from the control. Ribonucleotide reductase activity after 60 minutes was inhibited only 12%, while de novo purine synthesis was inhibited 18%. Compound 2 mildly suppressed PRPP amido transferase activity at 100 IuM by only 6% with an 11% reduction of IMP dehydrogenase activity. Carbamyl phosphate synthase and aspartate transcarbanylase activities were slightly inhibited 14% and 31%. While thymidylate synthase and thymidine kinase activities were increased by 1% and 35%, TMP and TDP kinase was slightly inhibited 22% and 31%. Dihydrofolate reductase activity was markedly inhibited 85%. Studies with ct-DNA showed that compound 2 had no effect on ct-DNA ultraviolet absorption between 220 and 340nm. HL-60 DNA strand scission studies after 24h incubation at 100 laM revealed that compound 2 caused DNA cross-linking ( Figure 1). This was consistent with the increase in ct-DNA viscosity after 24 hr at 100 pM.
Deoxyribonucleotide levels were all slightly reduced after 60 min incubation at 100 laM.
Compound 3 was also examined for its mode of action in HL-60 leukemia cells (Table 3). DNA and RNA synthesis after 60 minutes was slightly inhibited 35% and 10% at 1001aM. Protein synthesis after 60 minutes at 100 laM was inhibited 48% at 100tM. Utilization of the DNA template was moderately inhibited at 100 pM with inhibition of DNA polymerase e activity 20%, mRNA polymerase activity 44%, rRNA polymerase activity 40%, and tRNA polymerase activity 39%. Ribonucleotide reductase activity was inhibited only 25%, while de novo purine synthesis was inhibited 35% after 60 rain. Synthesis and Q,totoxiciv oj'C'yanoborane Adducts Synthesis and C)totoxicit.y of C,yanoborane Adducts transferase activity at 100 laM by only 12% with a 15% reduction of IMP dehydrogenase activity. Carbamyl phosphate synthase activity showed an increase of 14%, while aspartate transcarbanylase activity was inhibited 42%. Only thymidylate synthase activity' was markedly suppressed 76%, with thymidine kinase activity marginally inhibited 28%, TMP kinase activity 43% and TDP kinase activity 46%. Dihydrofolate reductase activity was suppressed 63%. Studies with ct-DNA showed that compound 3 had no effect on ct-DNA ultraviolet absorption between 220 and 340nm. HL-60 DNA strand scission after 24h incubation at 100 7"alrcl C.Scarlett et al.
A4etal...Based Drugs laM revealed that compound 3 caused DNA cross linking (Figure 1) which was consistent with the observed increased in ct-DNA viscosity after 24 hr at 100 gM. Deoxyribonucleotide pools were slightly redtced afier 60 min incubation with agents at 100 laM. Human topoisomerase and II activity was not inhibited by compounds 2 or 3 at 100 laM.

DISCUSSION
N6-Benzoyladenine-cyanoborane (2), and 6-triphenylphosphonylpurine-cyanoborane (3) proved to be cytotoxic in suspended cancer cells. Surprisingly these compounds were also cytotoxic in solid liver Hepe-2 and ileum HCT-8 carcinoma. In mode of action studies in human leukenic HL-60 cells, both compounds demonstrated inhibition of DNA and protein syntheses after 60 rain at 100 gM. These compounds inhibited RNA synthesis to a lesser extent. The utilization of the DNA template was suppressed by the compounds as determined by inhibition of the activities of DNA polymerase ct, m-RNA polymerase, r-RNA polymerase and t-RNA ploymerase which would cause adequate inhibition of the synthesis of both DNA and RNA. Because the d[NTP] pool levels were slightly reduced after 60 rain further inhibition of DNA synthesis would occur. Both compounds remarkably inhibited dihydrofolate reductase activity, especially compound 2. This would cause the reduction of the one carbon transfer for purine and pyrimidine syntheses/2/. However, the de novo synthesis of purine and pyrimidines was only.marginally affected by the compounds as were their regulatory enzyme activities /2/. Ribonucleotide reductase activity was moderately inhibited which would reduce the amount of ribonucleotide converted to deoxyribonucleotides for DNA synthesis. The reduction of TMP and TDP kinase activities would further reduced thymidine nucleotides levels demonstrated significantly by compound 3. Both compounds appeared to have caused cross-linking of the DNA strands after 24 hr at 100 laM in HL-60 cells, which was consistent with the observed increased in ct-DNA viscosity after 24 hr at 100 gM and lack of inhibition of DNA topoisomerase and II activities with no DNA-protein linked breaks. Neither compounds interacted with the DNA molecule itself through alkylation of the nucleotide bases nor caused DNA interculation between base pairs. Previously studied thymidine, inosine, cytidine, guanosine, and arbinoside cyanborane nucleotides have demonstrated a similar pattern of cytotoxicity on the growth of suspended murine and human tumor cells and solid human tumors. Those nucleoside and nucleotide cyanboranes inhibited DNA and protein synthesis, with a select few of the derivatives reducing RNA synthesis after hr/2/. Mutliple targets of the cyanboranes in DNA synthesis were demonstrated by the compounds. For the nucleoside cyanboranes the major sites of inhibition were IMP dehydrogenase and PRPP amido tranferase activities, suppressing de novo purine synthesis of Tnolh leukemia cells /2/. In contrast, the de novo synthesis of purine, pyrimidine and their regulatory enzyme activities were only marginally suppressed by the current compounds. Although similar nucleoside cyanoboranes inhibited dihydrofolate reductase activity, the current compounds were more potent. The boranated nucleosides cause a reduction of thymidylate synthase activity whereas only compound 3 decreased activity while compound 2 increased activity. However, both types of compounds inhibited TMP and TDP kinase activity and marginally reduced d[NTP] pools. Some of the nucleoside cyanoboranes caused DNA strand scission [thymidine] whereas others ribose and arabinoside] caused DNA cross-linking as the current compounds. However, none of the cyanboranes targeted the DNA molecule itself. I'ol. 9, Nos. I-2. 2002 Synthesis am.t Qtotoxicity of Cyanoborane Adducts CONCLUSION N-Benzoyladenine-cyanoborane (2) and 6-triphenylphosphonylpurine-cyanoborane (3) have been proven to be effective antineoplastic agents in their overall reduction of DNA and protein replication in respect to killing cancer cells. The inhibition of dihydrofolate reductase activity and/or thymidylate synthetase adds to the overall inhibition of DNA and protein synthesis. Even though both compounds showed DNA crosslinking, neither compound interacted with the DNA molecule itself through alkylation of the nucleotide bases nor caused DNA intercalation between base pairs. Sufficient activity was demonstrated by these cyanoborane derivatives to warrant further investigation as potential antineoplastic for clinical use.