Hypolipidemic Activity of Amine-Borane Aducts of Cyclohexylamine and Toluidine in Rodents

The amine-borane adducts of cyclohexylamine and toluidine were observed to be potent hypolipidemic agents in mice, I.P. and rats orally at 8 mg/kg/day lowering both serum cholesterol and triglyceride levels after 14-16 days. These compounds were able to lower tissue lipids including the cholesterol content of the aorta wall. The agents successfully lower VLDL- and LDL-cholesterol content while elevating HDL-cholesterol content significantly. The agents also modulate lipid regulatory enzyme activities in a manner to reduce liver lipid levels. These studies demonstrate that the nitrogen atom does not have to be apart of the aromatic ring as in heterocyclic-amine borane to afford good hypolipidemic activity in rodents.


INTRODUCTION
Previously we have examined a number of amine-cyanoboranes[l] aminecarboxyboranes and their amides and esters [2][3][4][5], di-and triopeptide boranes [6], heterocyclic-amine boranes [7], polyborate salts [8], choline and thiocholine boranes [9], phosphoacetate boranes[10] and deoxyribonucleoside cyanoboranes [ll]. These agents were effective in rodents at 8 mg/kg/day, significantly lowering both serum cholesterol and triglyceride levels after [14][15][16] days. Cholesterol levels were lowered in rat VLDL and LDL fractions while the HDL cholesterol levels were markedly elevated. The mode of action of these derivatives was to acclerate bile cholesterol excretion and alteration of regulatory enzyme activities involved in de novo lipid synthesis. Furthermore, sub-acute toxicity studies in mice indicated no organ specific toxicity and mean survival doses were high [12]. Thus, we have decided to examine a series cyanoand carboxyborane adducts of cyclohexylamine and toluidine for their hypolipidemic activity in rodents. These studies should determine if the nitrogen atom is necessary in the aromatic ring to maintain activity as a hypolipidemic agent in rodents.

Normolipidemic studies
For structure activity studies, CF I male mice (-28g) were administered amine-borane adducts of cyclohexylamine and toluidine in i CMC at 8 mg/kg/day, I.P. Blood samples were obtained on days 9 and 16 between 7"30 and 8"30 a.m. Daily dosing of the agents was between 9"00 and I0"00 a.m. the serum was obtained by centrifuging the blood for i0 min. at 3500 g. The serum cholesterol levels were determined by a modification of the procedure Lic.bermann-Burchard reaction [14]. Serum triglyceride were determined using a commercial kit [Boehringer Mannheim Diagnostics]. Sprague Dawley male rats (-230 g) were administered orally Compounds 7, 9_ or 18 at 8 mg/kg/day, for two weeks. Weekly blood samples were obtained by tail vein bleeding. Animal weight, organ weight and food consumption Control and treated normolipidemic Sprague Dawley male rat(-230g) weights were obtained and expressed as a percentage of the initial body weight (week zero). Food consumption (gm/day/rat) was noted for two weeks for control and treated rats [15]. Tissue lipid levels Normolipidemic Sprague Dawley male rats (-230 g) which were treated orally for two weeks with compound 7, 9_ or 1__8 at 8 mg/kg/day, were sacrificed and tissue samples of the liver, small intestinal mucosa and aorta were removed. A 24 hr fecal sample was also obtained. A i0 homogenate in 0.25 M sucrose + 0.001 M EDTA, pH 7.2, was prepared for each tissue. An aliquot (2 ml) of the homogenate was extracted [16][17] and the number of mg of lipid extracted was weighed. The lipid residue was taken up in methylene chloride and the levels of cholesterol [14], triglycerides neutral lipids [18] and phospholipids [19] were determined. Protein content of the whole homogenate was determined [20]. Serum [23] and adenosine triphosphate dependent citrate lyase activities [24] were determined spectrophotometrically at 540 nm as the hydroxylamate of acetyl coenzyme A formed after 20 min at 37C. Cholesterol-7-hydroxylase activity was determined using [1,2-3H]cholesterol (60 mCi/mmol) [25], and acyl CoA cholesterol acyl transferase activity was determined using [l-14C]oleic acid (56.7 mCi/mmol) [26]. Cholesterol synthesis was measured using [I-14C] acetyl CoA (62 mCi/mmol) and a post-mitochondrial supernatant (9000 g x 20 min) which was incubated for 60 rain at 37C [27]. The [29]. For acetyl coenzyme A carboxylase activity, the enzyme had to be polymerized for 30 rain at 37C and then the assay mixture containing sodium 14C-bicarbonate (41.0 mCi/mmol) was added and incubated for 30 min at 37C with test drugs [30], s__n-Glycerol-3-phosphate acyl transferase activity was determined with s__n-glycerol-3ophosphate [L-2-3H(N)] (7.1 Ci/mmol) with the microsomal fraction of liver homogenates.
The reaction was terminated after 60 rain and the lipids were extracted with chloroform/methanol (2"1) containing i HCI and counted [31].
Phosphatidylate phosphohydrolase activity was measured as inorganic phosphate released after 60 min [32]o The released inorganic phosphate after color development with ascorbic acid and ammonium molybate was quantitated at 820 nm. Hepatic lipoprotein lipase was determined using glycerol-triol4C-palmitate [64 mCi/mol] emulsified with lecithin by the method of Chair et alo [33]. Protein content of the liver homogenates was determined[ 20 Data displayed in Tables 1-5 represent means + standard deviations. The Student's "t" test was applied between control groups and the individual drug treatment groups using the raw data. The analysis of variance (ANOVA) was applied among test drugs and is reported in the text only.

RESULTS
Selected carboxyapd cyanoborane adducts of cyclohexylamine and toluidine were shown to be significantly active in the structure activity study in mice at 8 mg/kg/day. In comparison to the standards lovastatin and clofibrate, compounds i_, 2, 5, 7_, 8, 9, I__Q0, 1_/3, 1__6, 1_/7, and 1__8 lowered serum cholesterol by 23% at 8 mg/kg/day on day 16. Serum triglyceride level on day 16 were reduced by compound 7_, 8, 9, 1_/7 and 1__8 by 27% at 8 mg/kg/day I oP. Three compounds 7, _9 and 1__8 were selected as being representative compounds to further study in rats their hypolipidemic action [ Table 2]. In rats at 8 mg/kg/day, compound 9 lowered serum cholesterol levels 32% after 14 days administration orally, whereas compounds _7 and 1__8 caused at 24-25% reduction. Nevertheless, these compounds were more potent Vol. 2, No. 4,1995 Hypolipidemic Activity of Amine-Borane Aducts of Cyciohexylamine and Toluidine in Rodents   Table 3], liver, small intestinal mucosa and aorta lipid content was reduced. The triglyceride content was reduced more consistently than the cholesterol content in all three tissues.
In the aorta tissue neutral lipids and phospholipid content was also reduced after 14 days treatment. Protein and cholesterol content of the aorta tissue was reduced after treatment with compound 9. Total lipid excretion into the feces was not affected by treatment with the compounds. Fecal triglycerides were elevated significantly by all three compounds; ho.ever, phospholipids excretion was reduced [ Table 3]. Examination of the lipid content of the rat lipoproteins after 14 days administration showed that all three compounds lowered cholesterol content in the chylomicron and the LDL fraction. The HDL cholesterol content was significantly elevated 2 to 3 fold [ Table 4] by all three compounds. Triglyceride content was elevated in the chylomicron fraction after treatment with compounds 9_ and 1__8. Triglyceride content was elevated in the VLDL fraction after treatment with compound 7. HDL triglyceride content was elevated by treatment with compounds 7 and 9. Neutral lipid content of the lipoprotein fractions was not affected by any of the compounds. Phospholipid content of VLDL fraction was reduced by all three compounds while phospholipid content of LDI fraction was Vol. 2, No. 4, 1995 Hypolipidemic Activity of Amine-Borane Aducts of Cyclohexylamine and Toluidine in Rodents

DISCUSSION
These studies have shown that carboxyand cyanoborane adducts of cyclohexylamine and toluidine possessed good hypolipidemic acitivity in rodents. They were approximately equal in their ability to lower serum cholesterol and triglyceride levels in mice at 8 mg/kg/day as the heterocyclic amine boranes. Thus, it appears the nitrogen does not have to be in the aromatic ring to retain good hypolipidemic activity. Like the heterocyclic amine boranes there was no significant difference between carboxyboranes and cyanoboranes as far as their ability to lower serum cholesterol and triglycerides after 16 days administration I.P. in mice. However, it should be noted that compound 7_ did not afford as good activity in rats as it did in mice, although its magnitude of serum lipid reduction was still comparable to the standards lovastatin and clofibrate. This type of species variation has been noted before with boron derivatives [7]. Like many of the boron derivative these new compounds lowered cholesterol, triglyceride and phospholipid content in the major organs. What is most important is the fact that compound 9 was very effective in l.wering cholesterol content within the rat aorta wall after 14 days administration. These adducts were very impressive with regard to their effects on lipoprotein lipid content. An ideal hypolipidemic agent should lower cholesterol content of the VLDL and LDL fraction since these lipoproteins are responsible for conducting cholesterol to the peripheral tissue including the arterial walls, whereas HDL cholesterol content should be high so that cholesterol may be conducted back to the liver for biliary excretion. These derivatives were able to successfully lower VLDL and LDL cholesterol content and elevate HDL cholesterol content. In fact the elevation HDL cholesterol Vol. 2, No. 4, 1995 Hypolipidemic Activity of Amine-Borane Aducts of Cyclohexylamine and Toluidine in Rodents content afforded by these agents is much higher than the standards lovastatin with a 29 increase [34] or gemfibrozil with a i04 increase in rat HDL-cholesterol content [ll]. Supposedly, a clinical agent which lowers LDLand VLDL-cholesterol content but elevates HDL-cholesterol content protects man from mycocardial infarction [ 35]. The mode of action of the derivatives on regulatory enzyme activities involved in lipid metabolism appeared to be at site which regulated cytoplasmic formation of acetyl-CoA for cholesterol and fatty acid synthesis. These agents were not HMG-CoA reductase inhibitors whereas the heterocyclic amine-carboxy-and cyanoboranes were potent inhibitors. The amine-borane adducts of cyclohexylamine and toluidine accelerated cholesterol-7-hydroxylase activity more than the heterocyclic amine boranes. This would suggest that the former compounds probably accelerate the conversion of cholesterol to bile acids theoretically increasing cholesterol clearance from the body. These derivative marginally inhibited, acyl-cholesterol acyl transferase activity which should reduce cholesterol ester storage in tissues. In aorta walls this enzyme plays a role in depositing cholesterol esters in foam cells and increased plaque growth; thus inhibition of its activity should slow the process. It was hoped that the agents would accelerate cholesterol ester hydrolase activity. This enzyme breaks cholesterol esters down to free cholesterol which can be picked up by HDL to be conducted back to the liver via the reverse cholesterol transport mechanism for biliary excetion of cholesterol. This enzyme level is low in man but it is inducable by selective hypolipidemic agents to cause more clearnce of aorta wall lipids [36]. The heterocyclic amine-boranes were able to increase the activity of cholesterol ester hydrolaseo The neutral lipid and triglyceride pathways were inhibited by these derivatives at the site of phophatidylate phosphohydrolase, so that triglyceride can not be formed from phospholipids. The other regular enzyme of the triglyceride pathway, s__n-glycerol-3-phosphate acyl transferase, was marginally inhibited by compounds 9 and 18. These adducts were more potent in this respect than the heterocyclic amine boranes. Since hepatic lipoprotein lipase activity was also inhibited by the amine borane adducts of cyclohexylamine and toluidine, the inhibition of these three enzyme activities should be of a magnitude to lower triglyceride levels in the blood and tissue. Furthermore, some accelerated loss of triglycerides occurred in the feces, although bile excretion of triglycerides is considered to be of a small magnitude under normal conditions.
In conclusion, the amine-borane adducts of cyclohexylamine and toluidine maintain good hypolipidemic activity and achieve alteration in lipid metabolism and clearance which are desirable. Further investigation is warrented to evaluate such derivatives for clinical trails.