Cytotoxicity and Antineoplastic Activities of Alkylamines and Their Borane Derivatives

The alkylamines and their related boron derivatives demonstrated potent cytotoxicity against the growth of murine and human tissue cultured cells. These agents did not necessarily require the boron atom to possess potent cytotoxic action in certain tumor lines. Their ability to suppress tumor cell growth was based on their inhibition of DNA and protein syntheses. DNA synthesis was reduced because purine synthesis was blocked at the enzyme site of IMP dehydrogenase by the agents. In addition ribonucleotide reductase and nucleoside kinase activities were reduced by the agents which would account for the reduced d[NTP] pools. The DNA template or molecule may be a target of the drugs with regard to binding of the drug to nucleoside bases or intercalaction of the drug between DNA base pairs. Only some Of the agents caused DNA fragmentation with reduced DNA viscosity. These effects would contribute to overall cell death afforded by the agents.


Introduction
Alkylamines and their borane derivatives have shown potent hypolipidemic activity in mice, reducing LDL cholesterol and serum cholesterol and triglyceride levels while elevating HDL cholesterol levels [l,2]. The long chain derivatives, e.g.N.N-dimethyloctadecylamine borane, afforded the most potent hypolipidemic activity [2].
Thus, the purpose of this study is to determine if this correlation exists for a series of alkylamines and their borane derivatives. Antineoplastic and cytotoxic activity will be determined against murine L-1210 lymphoid leukemia, human Tmolt3 leukemia, HeLa-S 3 uterine carcinoma, A549 lung, colorectal adenocarcinoma, KB nasopharynx, osteosarcoma, and glioma.
The mode of action of four representative compounds in the series will be investigated for their effects on L-1210 lymphoid leukemia cell nucleic acid metabolism. percent inhibition of tumor growth on day I0. 6-Meracaptopurine was used as the internal standard. [5] Cytotoxic Activity  were tested for cytotoxic activity by preparing 1 mM solutions of each drug in 0.05% Tween 80/Hz0 by homogenization. The solutions were sterilized by passing them through an Acrodisc filter (45).
The following cell lines were maintained by the literature techniques 4 for murine L-12 i0 [13]. The polymerase assay was that of RNA polymerase enzymes were isolated with different concentrations of ammonium sulfate (Anderson et al., [15] Hall et al. [16] and the individual RNA polymerase activities were determined using 3H-UTP. Ribonucleoside reductase activity was measured with 14C-CDP [17]. The deoxyribonucleotides labeled with 14C-dCDP were separated from 14C-rCDP from the ribonucleotides by TLC on PEI plates. Thymidine, TMP and TDP kinase activities were measured with 3H-thymidine (58.3 mCi/mol) in the medium of Maley and Ochoa [18].
PRPP amidotransferase activity was determined by the method of Spassova et al. [19], and IMP dehydrogenase activity was determined with 14C-IMP (Amersham, Arlington Heights, IL) where XMP was separated on PEI plates (Fisher Scientific) by TLC (Becker et al.[20]).
Carbamyl phosphate synthetase activity was determined by the method of Kalman et al. 21 and citrulline was determined colorimetrically (Archibald, [22]). Aspartate transcarbamylase activity was determined by the method of Kalman et al. [21] and carbamyl aspartate was determined colorimetrically [23].
Thymidylate synthetase activity was analyzed by the method of Kampf et al. [24]. The 3H20 measured was proportional to the amount of TMP formed from (3H)-dUMP. Dihydrofolate reductase activity was determined by the spectrophotometric method of Ho et ai. [25]. Protein was determined for all of the enzymatic assays (Lowry et al. [26]).
DNA Assays Deoxyribonucleoside triphosphates were extracted by the method of Bagnara and Finch [27].
Deoxyribonucleoside triphosphates were determined by the method of Hunting and Henderson [28] with calf thymus DNA, E. coli DNA polymerase I, non-limiting amounts of the three deoxyribonucleoside triphosphates not being assayed, and either 0.4 mCi of (3H-methyl)-dTTP or (5-3H)-dCTP.
The effects of the compounds on DNA strand scission were determined by the methods of Suzuki et al. [29], Pera et al. [30], and Woynarowski et al. [31].
L-1210 lymphoid leukemia cells were incubated with i0 Ci thymidine methyl-3H, 84.0 Ci/mmol and drug at i00 M for 24 h at 37C. After harvesting the LI210 cells (107), the cells were centrifuged at 600 g x i0 min in PBS, washed and suspended in 1 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 KCI and 0.01 M EDTA) followed by 0.2 ml cell preparation. After incubating 2.5 hr at room temperature, the gradient was centrifuged at 12,000 rpm at 20C for 60 min (Beckman rotor SW60). Fractions (0.2 ml) were collected from the top of the gradient, neutralized with 0.2 ml of 0.3 N HCI, and radioactivity measured.
Thermal calf thymus DNA [ct-DNA] denaturation studies, UV absorption studies and DNA viscosity studies were conducted after incubating compounds 4, 12, 14 and 22 at i00 M in PBS buffer pH 7.2 at 37oC for 24 hr [32]. RESULTS A number of compounds were active in the Ehrlich ascites carcinoma screen compound 4 afforded 77% inhibition, compound 5 caused 86% inhibition and compound 11 caused 87% while compound 14 resulted in 76% inhibition of tumor growth in vivo.
Generally the alkylamine amines were not as potent as the boron derivatives. [ Table i].   DNA polymerase activity was stimulated by compounds 4 and 12 by 52% and 106%, respectively but was inhibited 67% and 53% by compounds 14 and 22. mRNA polymerase activity was suppressed 18% to 23% by the compounds. 104 rRNA polymerase activity was inhibited by all four compounds 10% to 34%. t-RNA polymerase activity was suppressed 2% to 28% after 60 min. incubation. Ribonucleoside reductase activity was suppressed 22% to 34% at i00 M of the agents. Dihydrofolate reductase activity was reduced 16% by compound 4 but the activity was elevated 31%-48% by compounds 14 and 12. De novo synthesis of purine was inhibited approximately 37% to 57% by the four agents at i00 M after 60 rain incubation. However, the activity of the regulatory enzyme PRPP amido-transferase activity was inhibition.
Nevertheless, IMP dehydrogenase activity was markedly reduced by all four agents 38% to 75%.
The first enzyme in the pyrmidine pathway were significantly reduced, i.e. carbamyl phosphate synthetase activity, which was suppressed 87% by compound 22 whereas the other three compounds caused 15% to 24% reduction in activity. Aspartate transcarbamylase activity was reduced 17% by compound 4 and was elevated by compounds 14 and 22. However, thymidylate synthtase activity was unaffected by the agents.
Thymidine kinase activity was reduced 46% to 69%, TMP kinase activity was reduced 21% to 37% and TTP kinase activity was reduced 10% to 35% by the four agents at I00 M.   The alkylamines which contain no boron moiety generally were more effective against the growth of the solid tumor cell cultures. This activity may be due to some type of deteregent activity of these agents being that they are long chain alkyl groups.  activity was of sufficient amount to explain the observed reduction of purine synthesis as well as DNA synthesis.
Other metabolic site which were affected marginally by the agents were m-, r-and t-RNA polymerases, ribonucleoside reductase, carbamyl phosphate synthetase, and nucleoside kinases.
The inhibition of the activities of these enzymes would be additive with regard to inhibiting DNA synthesis and tumor cell death.
The reduction in some of the d[NTP] pools would also lower DNA synthesis. The reduction in these pools probably is due to the inhibition of purine and pyrimidine de novo synthetic pathways for deoxytriphosphate nucleosides as well as ribonucleoside reductase activity for the conversion of ribosenucleotides to deoxyribonucleotides, ct-DNA studies with the agents suggest that there was some type of interaction with the nucleosides of DNA as demonstrated by the decrease in U.V. absorption, decreased Tm values and lower DNA viscosity.
In the L-1210 cells the observed DNA fragmentation would cause cell death and explain the reduction in DNA viscosity. 108