Synthesis of the 2-chloro analogues of 3'-deoxyadenosine, 2',3'-dideoxyadenosine, and 2',3'-didehydro-2',3'-dideoxyadenosine as potential antiviral agents.

2-Chloro-3'-deoxyadenosine (2-chlorocordycepin), 2-chloro-2',3'-dideoxyadenosine (2-ClddAdo), and 2-chloro-2',3'-didehydro-2',3'-dideoxyadenosine (2-ClddeAdo) were synthesized from 2-chloroadenosine (2-ClAdo) as candidate antiretroviral agents on the basis that 2-chloro substitution would prevent enzymatic deamination and increase efficacy relative to 2',3'-dideoxyadenosine (ddAdo). Reduction of 2-chloro-5'-(4,4'-dimethoxytrityl)-2',3'-O-thiocarbonyladenosine with n-Bu3SnH, followed by detritylation with AcOH, unexpectedly gave a mixture of 2-chlorocordycepin and 2-chloroadenine. Treatment of the crude n-Bu3SnH reduction product with 1,1'-thiocarbonyldiimidazole, followed by another cycle of n-Bu3SnH reduction and detritylation with silica gel afforded 2-ClddAdo and a byproduct identified as 2-chloro-2',3'-O-methyleneadenosine. Treatment of 2-chloro-5'-O-(4,4'-dimethoxytrityl)-2',3'-thiocarbonyladenosine with 1,3-dimethyl-2-phenyl-1,3,2-diazaphospholidine followed by silica gel detritylation afforded 2-ClddeAdo. 2-ClddAdo and 2-ClddeAdo were tested for activity against human immunodeficiency virus (HIV) in a cultured human T4+ lymphocyte cell line. At a concentration of 100 microM, 2-ClddAdo inhibited reverse transcriptase (RT) production by 97%, while 2',3'-dideoxyadenosine (ddAdo) gave greater than 99% inhibition. In growth assays against uninfected T4+ cells, however, 100 microM 2-ClddAdo gave 23% inhibition while 100 microM ddAdo was nontoxic. At a nontoxic concentration of 20 microM, RT production was 75% inhibited by ddAdo but only 43% inhibited by 2-ClddAdo. Thus, a 2-chloro substituent increased host cell toxicity but decreased antiretroviral activity. The unsaturated analogue 2-ClddeAdo was more cytotoxic than 2-ClddAdo, and antiviral effects could not be measured above 20 microM, where there was only 75% inhibition of RT production. Because of the decreased therapeutic index of 2-ClddeAdo relative to 2-ClddAdo and ddAdo, greater than 90% inhibition of viral protein synthesis at a noncytotoxic concentration could not be achieved. In growth assays with cultured human T and B lymphocytes, 100 microM 2-chlorocordycepin gave 60-70% growth inhibition, while the IC50 against mouse fibroblasts was only 30 microM. The high cytotoxicity of 2-chlorocordycepin precluded consideration of this compound as an antiviral agent.

4-Amino-3-(2,3,5-tri-O-benzyl-beta-D-ribofuranosyl)-5-pyrazole-carb oni trile. Synthesis and conversion into a sugar-blocked 5,7-disubstituted formycin analog.

The title compound, a potential intermediate to protected C-nucleoside analogs related to formycin A, was synthesized via a new route wherein 2,3,5-tri-O-benzyl-1-O-p-nitrophenyl)-D-ribofuranose was converted to 2,5-anhydro-3,4,6-tri-O-benzyl-D-allonic acid, and further transformed into 4-(tert-butyloxycarbonyl)-5-ethoxycarbonyl-3-(2,3,5-tri-O-benzyl-beta-D- ribofuranosyl)pyrazole. After amidation and dehydration to form the 4-(tert-butyloxycarbonyl)-5-pyrazolecarbonitrile, acidolysis followed by a Curtius-type sequence afforded the 4-amino-5-pyrazole-carbonitrile nucleoside. Treatment of the latter with nitrous acid and copper chloride in a Sandmeyer-type reaction gave a diazonitrile rather than a chloronitrile. Attempts to convert either the aminonitrile or the diazonitrile to 5,7-diamino-3-(2,3,5-tri-O-benzyl-beta-D-ribofuranosyl)pyrazolo[4,3-d] pyrimidine (5-aminoformycin A) by condensation with guanidine or N,N-dimethylguanidine were unsuccessful. Condensation of the aminonitrile with carbon disulfide in pyridine provided access to the formycin system in the form of 3-(2,3,5-tri-O-benzyl-beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine-5,7 - dithione.

Monensin is obligatory for the cytotoxic action of a disulfide linked methotrexate-anti-transferrin receptor conjugate.

Methotrexate (MTX) in the form of a gamma-cysteinylglycine derivative was disulfide linked to a monoclonal antibody reactive with the human transferrin receptor to give an antibody-MTX conjugate (anti-TfR-MTX). Antibody directed delivery of MTX to cell surface receptors was readily detected by flow cytometry using an anti-MTX antibody plus a secondary fluorescent antibody probe. Despite the presence of ample drug on the cell membrane, the conjugate alone was not cytotoxic over the course of several days. Expression of specific toxic activity, however, was obtained in conjunction with the carboxylic ionophore monensin, in whose presence anti-TfR-MTX displayed an IC50 of 8 X 10(-8) M. These results suggest that the ionophore causes antibody-drug conjugate to bypass the normal transferrin receptor cyclic pathway, allowing sufficient drug to reach, bind to, and inactivate intracellular dihydrofolate reductase.

Methotrexate analogues. 30. Dihydrofolate reductase inhibition and in vitro tumor cell growth inhibition by N epsilon-(haloacetyl)-L-lysine and N delta-(haloacetyl)-L-ornithine analogues and an acivicin analogue of methotrexate.

Analogues of methotrexate (MTX) with strong alkylating activity were prepared by replacing the L-glutamate side chain with N omega-haloacetyl derivatives of L-lysine and L-ornithine. Haloacetylation was accomplished in 30-40% yield by reaction of the preformed L-lysine and L-ornithine analogues of MTX with p-nitrophenyl bromoacetate or chloroacetate in aqueous sodium bicarbonate at room temperature. All four haloacetamides were potent inhibitors in spectrophotometric assays measuring noncovalent binding to purified dihydrofolate reductase (DHFR) from L1210 cells. In experiments designed to measure time-dependent inactivation of DHFR from L1210 cells and Candida albicans, the N epsilon-(bromoacetyl)-L-lysine and N delta-(bromoacetyl)-L-ornithine analogues gave results consistent with covalent binding, whereas N epsilon- and N delta-chloroacetyl analogues did not. The N delta-(bromoacetyl)-L-ornithine analogue appeared to be the more reactive one toward both enzymes. Amino acid analysis of acid hydrolysates of the L1210 enzyme following incubation with the bromoacetamides failed to demonstrate the presence of a carboxymethylated residue, suggesting that alkylation had perhaps formed an acid-labile bond. In growth inhibition assays with L1210 cultured murine leukemia cells, the four haloacetamides were all more potent than their nonacylated precursors but less potent than MTX. The greater than 40,000-fold MTX-resistant mutant cell line L1210/R81 was only partly cross-resistant to the haloacetamides. An analogue of MTX with acivicin replacing glutamate was a potent inhibitor of DHFR from chicken liver and L1210 cells but was 200 times less potent than MTX against L1210 cells in culture.

Synthesis and biologic activity of new side-chain-altered methotrexate and aminopterin analogs with dual inhibitory action against dihydrofolate reductase and folylpolyglutamate synthetase.

Replacement of the glutamic acid (Glu) moiety in methotrexate (MTX) and aminopterin (AMT) by 2-amino-4-phosphonobutyric acid (APBA) and ornithine (Orn) has been found to give analogs that retain the ability to inhibit dihydrofolate reductase (DHFR) while also displaying high activity against folylpolyglutamate synthetase (FPGS). One of these compounds, the Orn analog of AMT, is the most potent FPGS inhibitor we have found to date. A model to account for the fact that side-chain analogs containing a basic and those containing an acidic terminal group can both competitively inhibit FPGS is proposed. According to this model, binding may involve interaction of an acidic terminal group on the inhibitor with a positively charged active-site residue to which the gamma-carboxyl of the folate-antifolate substrate normally binds. It may also involve the interaction of a basic terminal group on the inhibitor with a different active-site residue which is negatively rather than positively charged and to which the alpha-amino group of the incoming Glu cosubstrate must bind before an amide bond to the gamma-carboxyl of the folate-antifolate can form. The 2 oppositely charged active-site residues assumed to take part in this binding are probably situated near each other and at approximately the same distance from the pteridine-binding site. The ability of compounds to inhibit both DHFR and FPGS makes it possible in principle for such compounds to kill cells via a "self-potentiation" mechanism in which inhibition of tetrahydrofolate synthesis is complemented by interference with the subsequent conversion of tetrahydrofolates to their polyglutamate conjugates. Possible exploitation of this mechanism to overcome MTX resistance is considered.

Methotrexate analogues. 26. Inhibition of dihydrofolate reductase and folylpolyglutamate synthetase activity and in vitro tumor cell growth by methotrexate and aminopterin analogues containing a basic amino acid side chain.

Analogues of the antitumor antifolate methotrexate (MTX) were synthesized in which the glutamate (Glu) moiety was replaced by ornithine (Orn), 2,4-diaminobutyric acid (Dab), or 2,3-diaminopropionic acid (Dap). An aminopterin (AMT) analogue with Orn in place of Glu was also synthesized. The MTX analogues were obtained by reaction of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and N omega-Boc-alpha,omega-diaminoalkanoic acids in the presence of diethyl phosphorocyanidate, followed by deprotection with trifluoroacetic acid (TFA) or by reaction of p-nitrophenyl-mAPA and N omega-Boc-alpha,omega-diaminoalkanoic acids and subsequent treatment with TFA. The AMT analogue (APA-Orn) was synthesized by reaction of p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate with silylated N delta-Boc-L-ornithine in DMF at 55 degrees C for 3 days (45% yield), saponification (83%), and TFA cleavage (89%). APA-Orn was a potent inhibitor of both dihydrofolate reductase (DHFR) from L1210 mouse leukemia (IC50 = 0.072 microM) and partly purified folylpolyglutamate synthetase (FPGS) from mouse liver (Ki = 0.15 +/- 0.06 microM). The MTX analogue (mAPA-Orn) was likewise active against both enzymes, with an IC50 of 0.160 microM for DHFR and a Ki of 20.4 +/- 7.7 microM for FPGS inhibition. The other MTX analogues and the previously reported lysine derivative (mAPA-Lys) showed DHFR affinity similar to that of mAPA-Orn but lacked activity as FPGS inhibitors. The positively charged amino group appears to be detrimental to cellular uptake, as evidenced by the low cytotoxicity of these compounds (IC50 = 0.40-2.4 microM) in comparison with MTX and AMT (IC50 = 0.002 microM) against wild-type L1210 cells. On the other hand, mAPA-Orn and APA-Orn were both more potent than the corresponding Glu derivatives MTX and AMT against L1210/R81 cells, suggesting that in these MTX-resistant cells there may occur a "self-potentiation" process involving enhanced antifolate activity via interference with the polyglutamylation of reduced folates. APA-Orn is the most potent dual inhibitor of DHFR and FPGS discovered to date, but its effectiveness as a therapeutic agent may require some form of prodrug modification to neutralize the terminal amino group of the side chain.

Improved synthesis of 2'-deoxyformycin A and studies of its in vitro activity against mouse lymphoma of T-cell origin.

7-Amino-3-(2'-deoxy-beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine (2'-deoxyformycin A) was synthesized from formycin A by a sequence consisting of (i) 3',5'-cyclosilylation with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, (ii) 2'-acylation with phenoxythiocarbonyl chloride and 4-(N,N-dimethylamino)pyridine, (iii) N-trimethylsilylation with hexamethyldisilazane, (iv) reduction of the 2'-O-phenoxythiocarbonyl group with tri-n-butyltin hydride, and (v) desilylation with tetra-n-butylammonium fluoride. 2'-Deoxyformycin A was a potent inhibitor of the in vitro growth of S49 lymphoma, a murine tumor of T-cell origin. The IC50 of 2'-deoxyformycin A against S49 cells was 10-15 microM, whereas that of 2'-deoxyadenosine (dAdo) under the same conditions (72-h incubation in medium containing heat-inactivated horse serum) was 180 microM. In the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to block intracellular adenosine deaminase (ADA) activity, 2'-deoxyformycin A and dAdo both gave IC50's of 5-10 microM. When assayed against a mutant S49 subline lacking adenosine kinase (AK) or a subline with a combined deletion of AK and deoxycytidine kinase (dCK), 2'-deoxyformycin A in combination with 10 microM EHNA was inactive at concentrations of up to 50 microM. Similar lack of activity against kinase-deficient cells was shown by formycin A. Thus, phosphorylation of 2'-deoxyformycin A appears to be required for biological activity and is probably catalyzed by AK rather than dCK. 2'-Deoxyformycin A and related 2'-deoxyribo-C-nucleoside analogues of the purine type may be of interest as potential T-cell specific cytotoxic agents.

Synthesis and biologic activity of a C-ring analogue of vitamin D3: biologic and protein binding properties of 11 alpha-hydroxyvitamin D3.

The influence of C-ring substituents on the biologic activity and protein binding properties of vitamin D3 has not been systematically investigated. To this end, we dehydrogenated cholesta-5,7-dien-3 beta-ol (1) to the 5,7,9(11)-triene. After protection of the 5,7-diene with a 4-phenyl-1,2,4- triazoline -3,5-dione Diels -Alder adduct, oxidation of the unprotected 9(11)-olefin gave epoxide 5. Reverse Diels -Alder and reductive ring opening of epoxide 5 gave cholesta-5,7-diene-3 beta, 11 alpha-diol (6). Photolysis of 6 to the previtamin followed by thermal rearrangement resulted in 11 alpha-hydroxyvitamin D3 (8). We found that vitamin 8 increased calcium transport at a dose of 500 pmol/rat but failed to increase bone calcium mobilization at a dose as high as 50 000 pmol/rat. Under the same conditions, corresponding doses of vitamin D3 and 25-hydroxyvitamin D3 increased bone calcium mobilization and intestinal calcium transport. The new vitamin analogue, 8, was slightly less efficient (B-50 = 6.8 X 10(-8) M) than 25-hydroxyvitamin D3, 24(R),25-dihydroxyvitamin D3, and 25-(S), 26-dihydroxyvitamin D3 (7.1 X 10(-9) M, 7.7 X 10(-9) M, and 7.9 X 10(-9) M, respectively) in displacing radiolabeled 25-hydroxyvitamin D3 from rat plasma vitamin D binding protein. On the other hand, vitamin analogue 8 showed significantly greater binding efficiency than 1 alpha-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and vitamin D3 (B-50 = 2.5 X 10(-6) M, 9.84 X 10(-8) M, and 5.46 X 10(-7) M, respectively), under these same conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and biological activity of certain 1,2,3-triazole carboxamide nucleosides related to bredinin and pyrazofurin.

5-Hydroxy-1-(beta-D-ribofuranosyl)-1,2,3-triazole-4-carboxamide (II) has been prepared by direct glycosylation of the trimethylsilyl derivative of 5-hydroxy-1,2,3-triazole-4-carboxamide (IV). The use of trimethylsilyltrifluoromethane sulfonate as a catalyst and 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose in acetonitrile gave a 95% yield of a 1:1 mixture of V and VI as blocked nucleosides which were readily separated on a silica gel column. The synthesis of II was also achieved by the cycloaddition of 2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl azide with ethyl malonamate. II shows significant antiviral activity against herpes and measles virus in vitro.