Dihydropyrimidines 4, 6, and 15, uniquely designed to unambiguously establish structural and conformational determinants for DHP receptor occupation and for modulation of calcium channel function, were prepared and examined for calcium channel modulation. Our results confirm and firmly establish a preference for syn-orientation of an unsymmetrically substituted aryl moiety at the DHP receptor (15d vs 15e). We propose a normal vs capsized DHP boat model to explain structural and conformational requirements for modulation of calcium channel function that requires an obligatory left-hand side alkoxy cis-carbonyl interaction for maximal DHP receptor affinity, the effect of channel function being determined by orientation of the 4-aryl group. Enantiomers having an up-oriented pseudoaxial aryl group (normal DHP boat) will elicit calcium antagonist activity, whereas enantiomers having a down-oriented pseudoaxial aryl group (capsized DHP boat) will elicit calcium agonist activity. Single enantiomers of macrocyclic lactone 15b demonstrate opposite channel activity. Antagonist activity resides in enantiomer 15b-A (S-configuration, left-hand side alkoxy cis-carbonyl with up-oriented pseudoaxial aryl group and normal DHP boat), whereas agonist activity resides in enantiomer 15b-B (R-configuration, left-hand side alkoxy cis-carbonyl with down-oriented pseudoaxial aryl group and capsized DHP boat). Moreover, this model is consistent with and provides a rational explanation of previous literature in this area, most notably the observation of chiral inversion and potency diminution upon replacement of ester by hydrogen in the Bay K 8644 series.

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Macrocyclic amino carboxylate (DOTA and D03A, where DOTA is l,4,7,10-tetrakis(carboxymethyl)-l,4,7,10-

2-Heterosubstituted-4-aryl-1,4-dihydro-6-methyl-5-pyrimidinecar box ylic acid esters 8, which lack the potential CS symmetry of dihydropyridine calcium channel blockers, were prepared and evaluated for biological activity. Biological assays using potassium-depolarized rabbit aorta and radioligand binding techniques showed that some of these compounds are potent mimics of dihydropyridine calcium channel blockers. The combination of a branched ester (e.g. isopropyl, sec-butyl) and an alkylthio group (e.g. SMe) was found to be optimal for biological activity. When compared directly with similarly substituted 2-heteroalkyldihydropyridines 9, dihydropyrimidines 8 were found to be 30-fold less active. The solid-state structure of dihydropyrimidine analogue 8g shows that these compounds can adopt a molecular conformation which is similar to the reported conformation of dihydropyridine calcium channel blockers.

By site-directed mutagenesis, TEM-1 beta-lactamase was altered to contain single amino acid changes of E104K, R164S, and E240K, in addition to double changes of E104K/R164S or R164S/E240K and the triple change of E104K/R164S/E240K. Hydrolysis rates for cephaloridine and benzylpenicillin were lowered at least 1 order of magnitude for all enzymes containing R164S substitutions. All mutant enzymes exhibited increased kcat values for beta-lactam antibiotics containing an aminothiazole oxime side chain. Hydrolysis of ceftazidime was most affected, with kcat values increased 3-4 orders of magnitude in all enzymes with the substituted R164S moiety. Km values decreased for all substrates except ceftazidime in the enzymes with multiple mutations. Aztreonam was most affected, with Km values lowered 23-56-fold in the enzymes bearing multiple mutations. When the crystal structures of aztreonam and related monobactams were studied and projected into an active-site model of the PC1 beta-lactamase, it became apparent that the two lysine residues might serve equivalent roles by interacting with the carboxylate of the aminothiazole oxime side chain. Hydrogen-bonding interactions involving the oxime and N7 of the lysine, particularly Lys-104, may also be important in some antibiotics. Ser-164 apparently serves an indirect role, since it is somewhat distant from the active-site cleft.

To enhance the intrinsic potency of dihydropyrimidine calcium channel blockers, we have modified the structure of previously described 2-heteroalkyl-1,4-dihydropyrimidines 2 to 3-substituted 1,4-dihydropyrimidines 3. Structure-activity studies using potassium-depolarized rabbit aorta show that ortho, meta-disubstituted aryl derivatives are more potent than either ortho- or meta-monosubstituted compounds. While vasorelaxant activity was critically dependent on the size of the C5 ester group, isopropyl ester being the best, a variety of substituents (carbamate, acyl, sulfonyl, alkyl) were tolerated at N3. Our results show dihydropyrimidines 3 are significantly more potent than corresponding 2-heteroalkyl-1,4-dihydropyrimidines 2 and only slightly less potent than similarly substituted 2-heteroalkyl-1,4-dihydropyridines 4 and 5. Whereas dihydropyridine enantiomers usually show 10-15-fold difference in activity, the enantiomers of dihydropyrimidine 3j show more than a 1000-fold difference in activity. These results strengthen the requirement of an enamino ester for binding to the dihydropyridine receptor and indicate a nonspecific role for the N3-substituent.

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