The Synthesis and Antitumor Activity of the Sodium Salt and Copper (II) Complex of N-[(Trimethylamineboryl)-Carbonyl]-L-Phenylalanine Methyl Ester

Sodium N-[(trimethylamineboryl)-carbonyl]-L-phenylalanine 2 and {N-[(trimethylamineboryl)-carbonyl]-L-phenylalanyl- carbxylato}-bis-{N-[(trimethylaminebryl)-carbonyl]-L-phenylalanine} dicopper (II) 3 were successfully synthesized. The agents blocked L1210 leukemic cell DNA and RNA syntheses by inhibiting multiple enzyme activities for nucleic acid synthesis, e.g. PRPP amido transferase, IMP dehydrogenase, DNA polymerase α, thymidine kinase, and TMP kinase. The copper (II) complex 3 demonstrated improved ability to inhibit L1210 partially purified DNA topoisomerase II compared to the parent compound while the sodium salt was inactive at 100 μM.


INTRODUCTION
N-[(Trimethylamineboryl)-carbonyl]-L-phenylalanine methyl ester 1 has been shown to be an effective antineoplastic agent o. Mode of action studies in L1210 cells demonstrated that DNA and RNA syntheses were inhibited. Enzyme activities including PRPP amido transferase, IMP dehydrogenase, and DNA polymerase ct were significantly inhibited by the phenylalanine derivative 1. Preliminary studies with trimethy_lamine carboxyborane have shown that whereas the amine borane derivatives are potent antineoplastic agents 2, metal complexation of these agents with copper, cobalt, chromium, iron and zinc complexes, produced more potent derivatives 3,4. Thus the following study involves comparison of the modes of action of the sodium salt and the copper (II) complex ofN-[(trimethylamineboryl)-carbonyl]-L-phenylalanine in L 1210 cells.

Chemistry
All chemicals were used as received from the manufacturer. Solvents were distilled prior to use. Trimethylamine carboxyborane was provided by Boron Biologicals, Inc. (Raleigh, NC). N-[(Trimethyl,amineboryl)-carbonyl]-L-phenylalanine methyl ester 1 was synthesized according to the method of Sood et al. . All other chemicals used in syntheses were purchased from Aldrich Chemical Company (Milwaukee, WI). A Perkin Elmer 1320 Infrared Spectrophotometer was used for infrared (IR) analyses. IR spectra were obtained as KBr disks or as nujol mulls using sodium chloride plates. A Varian 300 MHz NMR spectrometer was used to generate 1H-NMR spectra. Chemical shifts are relative to the external standard tetramethylsilane (i 0). A Thomas-Hoover capillary melting point apparatus was used to determine melting points, which were uncorrected. Elemental analyses were performed by M-H-W Laboratories (Phoenix, AZ). Silica gel 60F 254 plates (silica gel on aluminum, Aldrich Chemical Company) were used for thin layer chromatography. Synthesis of Sodium N-[(trimethylamineboryl)-carbonyl]-L-phenylalanine (2): N-[(Trimethylamineboryl)-carbonyl]-L-phenylalanine methyl ester 1 (1 g, 3.59 mmol) was suspended in 50 ml distilled water to which 3.6 ml of an 0.986 M aqueous NaOH solution (0.14 g, 3.59 mmol) was slowly added while stirring. After 15 minutes, the reaction appeared to be complete by TLC analysis although small amounts of an insoluble white solid are present. The white solid was removed by vacuum filtration and solvent was removed under reduced pressure leaving a white hygroscopic solid. The white hygroscopic solid was further purified by silica gel column chromatography using one void volume of ethyl acetate, followed by ethyl acetate acetonitrile (1:1) until completed. Combined  Inhibition of various enzyme activities was performed by first preparing the appropriate L1210 cell homogenates or subcellular fractions, 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 taM of compounds 2 and 3 after 60 min incubations. DNA polymerase t activity was determined in cytoplasmic extracts isolated by Eichler et al. 's method l. The polymerase assay DNA polymerase o was described by Sawada et al. 12 with 3H-TTP. Messenger-, ribosomal-and transfer-RNA polymerase enzymes were isolated with different concentrations of ammonium sulfate; individual RNA polymerase activities were determined using 3H -UTP 13,14. Ribonucleoside reductase activity was measured using 14C -CDP with dithioerythritol 15. 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) in the medium of Maley and Ochoa 16. Carbamyl (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. Cell membranes were lysed using a Dounce homogenizer following the addition of Triton X-100 at a volume equivalent to 1/100th the total cell suspension's volume. Trypan blue staining of nuclei was used to determine complete cell lysis microscopically. An equal volume of buffer containing 1.75 M sucrose was added to the cell suspension and mixed by gently swirling. After mixing, the total volume was loaded on a sucrose cushion [1.4 M sucrose, 20 mM potassium phosphate (pH 7.5), 5 mM MgCI2, mM dithiothreitol (DTT), 0.1 mM EDTA, and mM PMSF] and centrifuged at 18,000 rpm for 45 min at 4C. The sucrose cushion was removed via vacuum aspiration and the remaining pellet was resuspended in buffer containing 20 mM potassium phosphate (pH 7.5), 2 mM MgCI2, mM dithiothreitol (DTT), 0.1 mM EDTA, and mM PMSF, mM [3-mercaptoethanol, 10% glycerol, and 100 mM NaC1. The suspension was incubated at 4C for 30 min. The process was repeated with buffers containing increasing concentrations of NaC1 up to 400 mM. The supernatants containing enzyme activity were used for assays. The effects of compounds 2 and 3 on isolated DNA topoisomerase II activity was determined by the method of Miller Vol. 5, No. 1, 1998 The

Statistical Analysis
Data as percentage of control is displayed in tables and figures as the means _+ standard deviations. 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.

RESULTS AND DISCUSSION Chemistry
The sodium salt 2 was quickly formed in the presence of sodium hydroxide. The production of side products was noted which contributed to the lower than expected yield. The copper (II) complex was successfully synthesized using IR-120 cation exchange resin charged with the desired cation. Ion exchange proceeded by elution of a solution of the sodium salt 2 through a column of ion exchange resin. The structures and purity of the compound were confirmed and elemental analysis, melting points, and both 1H-NMR and infrared spectroscopy are reported. Attempts to form complexes by mixing methanolic solutions of carboxyborane sodium salts with methanolic solutions of metal chloride salts were unsuccessful. The synthesis of sodium N-[(trimethylamine-boryl)}-L-phenylalanine using sodium hydroxide provided an adequate yield of 47.4%; however, the appearance of additional reaction products was apparent by TLC. It is possible that the reaction conditions in the presence of sodium hydroxide may be caustic. After the initial reaction, only a slight amount of starting material and a large proportion of product were visible by TLC. Column chromatography proved a greater problem in that two impurities with similar Rf values with respect to the desired product were apparent in the related fractions. It was not possible to remove these impurities by silica gel column Vol. 5, No. 1, 1998 The Synthesis and Antitumor Activity of the Sodium Salt and Copper (II) Complex of N-[(Trimethylamineboryl)-Carbonyl]-L-Phenylalanine Methyl Ester chromatography as more of these impurities were generated with subsequent elutions. The formation of impurities during silica gel chromatography was possibly due to the acidity of the stationary phase which may facilitate protonation and rearrangement of the carboxyborane to boric acid as described by Sood,et al. 36,or hydrolysis of the amide and/or ester linkages in the dipeptide. Initial attempts to synthesize metal complexes of carboxyborane adducts followed the method of Norwood et al. 37,38 in which the methanolic solution of sodium salts were added to methanolic solutions of metal chlorides. When applied to the study, a mixture of impurities were present at each stage of Norwood's procedure and purification was not possible. However, the use of IR-120 cation exchange resin provided a more successful means to synthesis tetrakis-la-{N-{(trimethylamineboryl)-carbonyl-L-phenylalanine-carboxylato}-bis-[N-(trimethylamine-boryl)-carbonyl] L]phenylalanine} dicopper(II) 3 without the need for further purification. The synthesis of other metal complexes using ion exchange resin was not pursued due to the limited amount of starting materials remaining after multiple attempts at metal complexation using the Norwood method. Antitumor Activity Synthesized compounds demonstrated in vitro cytotoxicity primarily in suspended tumor cell lines (e.g, murine L1210 lymphoid leukemia, human Tmolt3 T cell acute lymphoblastic leukemia, and HeLa-S suspended human uterine cervical carcinoma) with variable activity in human solid tumor cell cultures.
Murine L1210 cytotoxicity assays demonstrated good activity for compound 2 (EDs0 2.68 tg/ml) which was more effective than the parent 1 (EDs0 3.34 lag/ml)35. Rat osteogenic sarcoma UMR-106 activity was noted for only compound 3 (EDs0 3.24 lag/ml). Against the growth of Tmolt3 only the parent 1 was active (EDs0 1.31 g/ml). Compounds 2 (EDs0 2.37 lag/ml) and 3 (EDs0 1.91 lag/ml) were active against human HeLa-S suspended cervical carcinoma growth while the parent compound was inactive. In contrast compounds 1 (EDs0 3.38 lag/ml)and 2 (EDs0-1.82 lag/ml) were active against solid HeLa cervical carcinoma growth while compound 3 was inactive. The growth of KB nasopharynx was reduced by compound 1 (EDs0 1.99 ktg/ml) alone. Only compound 3 (EDs0 3.22 lag/ml) effectively reduced SW480 colorectal adenocarcinoma cell growth. Against MB9812 bronchogenic lung growth, compounds 2 (EDs0 1.33 tg/ml) and 3 (EDs0 3.84 lag/ml) demonstrated activity while the parent was inactive. Compound 2 (EDs0 2.97 lag/ml) alone inhibited the growth of lung carcinoma A549, while only compound 3 (EDs0 2.39 g/ml) was effective against A431 epidermoid carcinoma growth. None of the compounds tested were active against the growth of HCT-8 ileum or HS-683 glioma [Table ]. The sodium salt and the copper (II) complex of N-[(trimethylamineboryl)-carbonyl]-L-phenylalanine were effective cytotoxic agents against the growth of a variety of rodent and human tumor cell lines. A wide variability was demonstrated with respect to which of these agents were the most active in a given screen.
The copper (II) complex 3, however, was not necessarily the most effective cytotoxic agent against each cell line. This indicated that metal complexation by the phenylalanine derivative does not necessarily improve cytotoxicity for certain cell lines.  Tables 2 and 3]. The synthesis of L1210 DNA and RNA was significantly inhibited by compounds 2 and 3 in a concentration dependent manner, with maximal inhibition at 100 M. DNA synthesis was reduced 48 and 38% by compounds 2 and 3, respectively, at 100 laM. RNA synthesis was reduced 25 and 69% by compounds 2 and 3, respectively, at 100 laM. Both compounds significantly inhibited DNA polymerase activity at all concentrations employed, with greater than 50% inhibition at 100 t, tM. Compounds 2 and 3 were most effective in inhibiting purine de novo synthesis with a greater than 50% reduction at 25 laM for compound 2 and at 100 laM for compound 3. Both PRPP amido transferase and IMP dehydrogenase activities were reduced greater than 50% at drug concentrations of 50 and 100 M, respectively, by compound 2 and at 50 tM by compound 3 for both activities. These compounds were also able to affect thymidine kinase and TMP kinase activities causing significant reduction in both enzyme activities at 50 laM for compound 2 and greater than 50% reduction in both enzyme activities at 25 laM for compound 3. These compounds did not affect L1210 deoxynucleotide pool levels other than a significant increase in d(GTP) levels of 22% and 23% by compounds 2 and 3, respectively, at 100 tM concentration.
Compounds 2 and 3 also demonstrated the ability to cause moderate L1210 strand scission [ Fig. and 2].
This was indicated by the increase in low molecular weight (fragmented) DNA distributed throughout the alkaline sucrose gradients and a reduction of higher molecular weight double stranded DNA compared to the control. The ct-DNA U.V. absorption did not change after incubation for 24 hours with compounds 2 or 3.
This indicated no change in the helical structure of DNA and that the agents did not chemically interact with the DNA bases. There were no effects on U.V. absorption from 220 to 340 nm after 24 hour incubations with compounds 2 or 3. This suggested that these agents do not alkylate DNA nucleotide bases. There was no change in the thermal denaturation Tm values after 24 h incubation with the drugs, indicating no intercalation between DNA base pairs had occurred. The ct-DNA viscosity studies demonstrated less time was required to move through the reservoirs. Such an increase in flow rate suggested DNA viscosity was reduced by the agents. The ability of compound 2 and 3 to reduce DNA viscosity is consistent with their ability to cause moderate DNA strand scission.
Compounds 1 3 were evaluated for their mode of action as LI:I0 DNA topoisomerase II inhibitors [ Fig. 3].
Only the copper complex 3 demonstrated inhibitory activity at 100 laM concentration. Compound 3 demonstrated concentration dependent inhibitory effects on L1210 topoisomerase II activity as evaluated by densitometry, affording an ICs0 value of 29 tM. In comparison the prototypical topoisomerase II inhibitor VP-16 which exhibited an ICs0 value of 22 laM.
In summary, mode of action studies on compound 2 primarily affected DNA synthesis at 100 tM while compound 3 had a greater effect on RNA synthesis. The de novo purine synthetic pathway appeared to be an important target for both compounds. Compound 2 preferentially reduced IMP dehydrogenase activity at 25 laM. In contrast, compound 3 markedly inhibited thymidine kinase and TMP kinase at 25 l-tM. At higher concentrations both compounds had their greatest effects on the purine pathway regulatory enzymes, PRPP amido transferase and IMP dehydrogenase, suggesting that inhibition of purine de novo synthesis is important in causing cytotoxicity. The magnitude of reduction afforded by compounds 2 and 3 on regulatory enzymes was sufficient to account for the observed reduction in DNA and RNA syntheses. In comparison, the parent compound 1, also significantly reduced DNA and RNA syntheses. In contrast to compounds 2 and 3, however, the parent compound had its greatest inhibitory effects on rRNA polymerase and ribonucleoside reductase activities 34. The moderate effect of these compounds on double stranded DNA strand scission suggested that these compounds may cause DNA fragmentation but there was little evidence that the bases of DNA were the direct target of the agents. Copper (II) complexation to form compound 3 did improve the compound's ability to inhibit L120 DNA topoisomerase II activity compared to the parent compound which should improve the ability to inhibit DNA synthesis and cause cell death.