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The design, synthesis, biochemical, and biological evaluation of a novel series of 5-thia-2,6-diamino-4(3H)-oxopyrimidine inhibitors of glycinamide ribonucleotide transformylase (GART) are described. The compounds were designed using the X-ray crystal structure of human GART. The monocyclic 5-thiapyrimidinones were synthesized by coupling an alkyl thiol with 5-bromo-2, 6-diamino-4(3H)-pyrimidinone, 20. The bicyclic compounds were prepared in both racemic and diastereomerically pure forms using two distinct synthetic routes. The compounds were found to have human GART KiS ranging from 30 microM to 2 nM. The compounds inhibited the growth of both L1210 and CCRF-CEM cells in culture with potencies down to the low nanomolar range and were found to be selective for the de novo purine biosynthesis pathway. The most potent inhibitors had 2,5-disubstituted thiophene rings attached to the glutamate moiety. Placement of a methyl substituent at the 4-position of the thiophene ring to give compounds 10, 18, and 19 resulted in inhibitors with significantly decreased mFBP affinity.

To develop novel lipophilic thymidylate synthase (TS) inhibitors, the X-ray structure of Escherichia coli TS in ternary complex with FdUMP and the inhibitor 10-propargyl-5,8-dideazafolic acid (CB3717) was used as a basis for structure-based design. A total of 31 novel lipophilic TS inhibitors, lacking a glutamate residue, were synthesized; 26 of them had in common a N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylaniline+ ++ structure in which the aniline was appropriately substituted with simple lipophilic substituents either in position 3 or 4, or in both. Compounds were tested for their inhibition of E. coli TS and human TS and also for their inhibition of the growth in tissue culture of a murine leukemia, a human leukemia, and a thymidine kinase-deficient human adenocarcinoma. The crystal structures of five inhibitors complexed with E. coli TS were determined. Five main conclusions are drawn from this study. (i) A 3-substituent such as CF(3), iodo, or ethynyl enhances binding by up to 1 order of magnitude and in the case of CF(3) was proven to fill a nearby pocket in the enzyme. (ii) A simple strongly electron-withdrawing substituent such as NO(2) or CF(3)SO(2) in the 4-position enhances binding by 2 orders of magnitude; it is hypothesized that the transannular dipole so induced interacts favorably with the protein. (iii) Attempts to combine the enhancements of i and ii in the same molecule were generally unsuccessful (iv) A 4-C(6)H(5)SO(2) substituent provided both electron withdrawal and a van der Waal's interaction of the phenyl group with a hydrophobic surface at the mouth of the active site. The inhibition (K(is) = 12 nM) of human TS by this compound, 7n, showed that C(6)H(5)SO(2) provided virtually as much binding affinity as the CO-glutamate which it had replaced. (v) The series of compounds were poorly water soluble, and also the potent TS inhibition shown by several of them did not translate into good cytotoxicity. Compounds with large cyclic groups linked to position 4 by an SO or SO(2) group did, however, have IC(50)'s in the range 1-5 microM. Of these, 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylamino )phenyl phenyl sulfone, 7n, had IC(50)'s of about 1 microM and was chosen for further elaboration.

The synthesis and biological evaluation of N-[4-(2-trans-[([2,6-diamino-4(3H)-oxopyrimidin-5-yl]methyl)thio]cyclobutyl)benzoyl]-L-glutamic acid (1) is reported. Compound 1 is a potent dihydrofolate reductase (DHFR) inhibitor (Kj = 12 nM) with excellent in vitro cell culture growth inhibition (L1210, IC50 = 29 nM). Protection experiments showed that the cell growth inhibitory activity was due to DHFR inhibition. The key step in the synthesis was the coupling of a cyclobutylmethylthiol with the 5-bromo-2,6-diamino-4-oxopyrimidine 8.

The design, synthesis, and biochemical and biological evaluations of a novel series of 2,6-diaminobenz[cd]indole-containing inhibitors of human thymidylate synthase (TS) are described. The compounds are characterized by having either a pyridine or pyridazine ring in place of the (phenylsulfonyl)morpholinyl group of the known inhibitor N6-[4-(morpholinosulfonyl)benzyl]-N6-methyl-2,6-diaminobenz[ cd]indole glucuronate (i). Active compounds from this series showed human TS inhibition constants below the 10 nM level and were potent, selective submicromolar antitumor agents in cell culture. The compounds were synthesized by reductive alkylation of a substituted 6-aminobenz[cd]indole or reductive cyclization of a substituted 1-cyano-8-nitronaphthalene.

The design, synthesis, biochemical, and biological evaluation of a novel series of 5-thia-2,6-diamino-4(3H)-oxopyrimidine inhibitors of glycinamide ribonucleotide transformylase (GART) are described. The compounds were designed using the X-ray crystal structure of human GART. The monocyclic 5-thiapyrimidinones were synthesized by coupling an alkyl thiol with 5-bromo-2, 6-diamino-4(3H)-pyrimidinone, 20. The bicyclic compounds were prepared in both racemic and diastereomerically pure forms using two distinct synthetic routes. The compounds were found to have human GART KiS ranging from 30 microM to 2 nM. The compounds inhibited the growth of both L1210 and CCRF-CEM cells in culture with potencies down to the low nanomolar range and were found to be selective for the de novo purine biosynthesis pathway. The most potent inhibitors had 2,5-disubstituted thiophene rings attached to the glutamate moiety. Placement of a methyl substituent at the 4-position of the thiophene ring to give compounds 10, 18, and 19 resulted in inhibitors with significantly decreased mFBP affinity.

The X-ray crystal-structure-based design, synthesis, and biological activity of a novel family of benz[cd]indole-containing inhibitors of thymidylate synthase (TS) are described. The structure-activity of the lead compound was studied by conceptually dividing the molecule into four regions and independently optimizing the substituents for each region. Combination of favored substituents for each region led to inhibitors with Ki's against the human enzyme in the range of 10-20 nM. Thymidine shift experiments suggested that the cytotoxic properties of the best enzyme inhibitors were due to TS targeting in cells. The inhibitors were synthesized from substituted 6-aminobenz[cd]indol-2(1H)-ones by alkylation with both a simple alkyl group and a substituted benzylic portion. The 2,6-diaminobenz[cd]indoles were prepared from the corresponding lactams by conversion to the thiolactam, alkylation to the methylated thiolactam, and then displacement with a substituted or unsubstituted amine.

Results of an experimental search for new macroscopic forces with Yukawa range between 5 and 500 microns are presented. The experiment uses 1 kHz mechanical oscillators as test masses with a stiff conducting shield between them to suppress backgrounds. No signal is observed above the instrumental thermal noise after 22 hours of integration time. These results provide the strongest limits to date between 10 and 100 microns, improve on previous limits by as much as three orders of magnitude, and rule out half of the remaining parameter space for predictions of string-inspired models with low-energy supersymmetry breaking. New forces of four times gravitational strength or greater are excluded at the 95% confidence level for interaction ranges between 200 and 500 microns., 25 Pages, 7 Figures: Minor Corrections

An approach to the total synthesis of the 14-membered macrolide antibiotic aglycon chlorothricolide is presented. Key steps from the two halves to (±)-19,20-Dihydro-24-O methylchlorothricolide, methyl ester, ethyl carbonate include initial esterification across the C1 and C25 carbons followed by macrodilactonization across the C17 and C14 carbons. Ester enolate Claisen rearrangement and subsequent decarboxylation afforded the intact lactone. Functionalization of the top half is explored. The ester enolate Claisen rearrangement of propionate derivatives of resolved 1-(t-butyldimethylsilyl)-trans-2-butene-1-ol is reported. Subsequent protiodesilation of the reduced and protected Claisen products resulted in the formation of 2,3-dimethyl-4-pentenyl ethers. Thus, the secondary α-silyl alcohol has functioned as a chiral primary alcohol equivalent. The Claisen rearrangement of vinyl ethyl derivatives of 5-tert-butyl-1-(hydroxymethyl)-1-cyclohexene is reported. The standard allyl vinyl ether conditions as well as the triethyl orthoacetate and ester enolate variants of the Claisen rearrangement all resulted in the formation of cis(axial)-4-tert-butylcyclohexyl-substituted systems. Thus, in sterically unbiased cases, this [3.3] sigmatropic process results in the axial attachment of the side chain in a cyclohexyl system.