Venus water loss is dominated by HCO+ dissociative recombination.

Despite its Earth-like size and source material1,2, Venus is extremely dry3,4, indicating near-total water loss to space by means of hydrogen outflow from an ancient, steam-dominated atmosphere5,6. Such hydrodynamic escape likely removed most of an initial Earth-like 3-km global equivalent layer (GEL) of water but cannot deplete the atmosphere to the observed 3-cm GEL because it shuts down below about 10-100 m GEL5,7. To complete Venus water loss, and to produce the observed bulk atmospheric enrichment in deuterium of about 120 times Earth8,9, nonthermal H escape mechanisms still operating today are required10,11. Early studies identified these as resonant charge exchange12-14, hot oxygen impact15,16 and ion outflow17,18, establishing a consensus view of H escape10,19 that has since received only minimal updates20. Here we show that this consensus omits the most important present-day H loss process, HCO+ dissociative recombination. This process nearly doubles the Venus H escape rate and, consequently, doubles the amount of present-day volcanic water outgassing and/or impactor infall required to maintain a steady-state atmospheric water abundance. These higher loss rates resolve long-standing difficulties in simultaneously explaining the measured abundance and isotope ratio of Venusian water21,22 and would enable faster desiccation in the wake of speculative late ocean scenarios23. Design limitations prevented past Venus missions from measuring both HCO+ and the escaping hydrogen produced by its recombination; future spacecraft measurements are imperative.

5'-Haloacetamido-5'-deoxythymidines: novel inhibitors of thymidylate synthase.

5'-Bromoacetamido-5'-deoxythymidine (BAT), 5'-iodoacetamido-5'-deoxythymidine (IAT), 5'-chloroacetamido-5'-deoxythymidine (CAT) and [14C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were investigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT greater than IAT greater than CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4-8-fold higher than it was in the absence of dUMP. The I50 values for BAT were 1 X 10(-5) M and 1.2 X 10(-6) M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thymidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The Km for the enzyme was 9.2 microM, and the Ki determined for competitive inhibition by BAT was 5.4 microM. Formation of a tight, irreversible complex is inferred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min-1, and the steady-state constant of inactivation, Ki, was estimated to be 6.6 microM. The 5'-haloacetamido-5'-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.

Synthesis of 1-deaza-6-thioguanosine and 1-deaza-6-(methylthio)guanosine.

A synthesis of 1-deaza-6-thioguanosine (8) and 1-deaza-6-(methylthio)guanosine (9) from 2-amino-6-chloro-1-deazapurine (4) is described. The reaction of the N2-acetyl derivative of 4 with 2,3,5-tri-O-acetyl-D-ribofuranosyl chloride in the presence of Linde 4A molecular sieve gave the blocked nucleoside 6. Deacetylation of 6 gave the chloro nucleoside 7 which was treated at high temperature with hydrogen sulfide and methyl mercaptan to give 8 and 9, respectively. The structure of 7 was confirmed by 1H NMR and by conversion to the cyclonucleoside 14. Compound 4 gave a 79% increase in life span in the L1210 mouse leukemia screen.

Analogues of 8-azainosine.

A convenient synthesis of 8-azapurine ribonucleosides substituted at the 6 position with thio, alkylthio, alkoxy, amino, and alkylamino groups is described. The reaction of 6-(methylthio)-8-azapurine (1) with 2,3,5-tri-O-acetyl-D-ribofuranosyl chloride in the presence of Linde AW-500 molecular sieve gave a 2:1 mixture of 2 and 3, respectively. This mixture was rearranged by heating with molecular sieve in refluxing toluene to give a 6:1 mixture of 2 and 3. Treatment of 2 or 3 with the appropriate nucleophiles at room temperature gave 6-substituted 8-azapurine ribonucleosides (7-substituted 2- or 3-beta-D-ribofuranosyl-3H-1,2,3-triazolo[4,5-d]pyrimidines) 4-13. The thione 11 rearranges to N-beta-D-ribofuranosyl[1,2,3]thiadiazolo[5,4-d]pyrimidin-7-amine (14) in the solid state or in solution. All of these compounds were cytotoxic to H.Ep. No. 2 cells in culture except the parent base, 8-aza-6-(methylthio)purine (1) and the 8-isomers (3,12, and 13). Three of these compounds-8azaadenosine (4), 8-aza-6-(methylthio)purine ribonucleoside (5), and 8-aza-6-(methoxy)purine ribonucleoside (7)-showed borderline activity in the leukemia L1210 system. The thiadiazolopyrimidine (14) showed activity at three dose levels.

Potential anticancer agents: 5-(N-substituted-aminocarbonyl)- and 5-(N-substituted-aminothiocarbonyl)-5,6,7,8-tetrahydrofolic acids.

5-[[N-[(Ethoxycarbonyl)alkyl]amino]carbonyl] (6-9) and the corresponding aminothiocarbonyl (12-15) derivatives of 5,6,7,8-tetrahydrofolic acid were prepared as multisubstrate analogues of the substrate--cofactor adduct in the reactions catalyzed by the folate-mediated one-carbon transfer reactions. Evaluation in vitro showed that 7 (alkyl = hexyl) was cytotoxic to H.Ep.-2 cells (ED50, 4 microM) but noncytotoxic to proliferating L1210 cells. No activity was observed for 7 against the P388 leukemia in mice.

Preparation and purification of L-(+/-)-5-formyl-5,6,7,8-tetrahydrofolic acid.

Reinvestigation of the conversion of folic acid to leucovorin [L-(+/-)-5-CHO-THF] led to improved methods for the synthesis of this drug, which is suitable for clinical use. Also, methods were developed for the chromatographic and nonchromatographic purification of less pure samples of L-(+/-)-5-CHO-THF.

Synthesis of N-10-methyl-4-thiofolic acid and related compounds.

Compound 21 (N10-methyl-4-thiofolic acid) and related compounds were prepared as potential inhibitors of the cofactor forms of tetrahydrofolate. The preparation of 2-acetylamino-4-(benzylthio)-6-chloro-5-nitropyrimidine (4) provided an intermediate that was allowed to react with methyl p-[(3-aminoacetonyl)methylamino]benzoate oxime (16). The oxime function of the resulting 6-substituted aminopyrimidine 6 was hydrolyzed to give the corresponding acetonylaminopyrimidine 7, which on reductive cyclization gave methyl p-[[[2-amino-4-(benzylthio)-7,8-dihydro-6-pteridinyl]methyl]methylamino]benzoate (9). This dihydropteridine was oxidized with potassium permanganate, and the product was treated successively with sodium hydrosulfide to replace the benzylthio group and with aqueous sodium hydroxide to hydrolyze the ester function to give p-[[(2-amino-3,4-dihydro-4-thioxo-6-pteridinyl)methyl]methylamino]benzoic acid (N10-methyl-4-thiopteroic acid, 12). Another route to 12 involved the interaction of 2,5-diamino-4,6-dichloropyrimidine (15) with 16 to give methyl p-[[(2-amino-4-chloro-7,8-dihydro-6-pteridinyl)methyl]methylamino]benzoate (13). Displacement of the chloro group of 13 with sodium hydrosulfide followed by the simultaneous air oxidation of the dihydropteridine ring and saponification of the ester group gave 12. After protection of the 2-amino and 4-thioxo moieties of 12, the resulting intermediate benzoic acid was coupled with diethyl L-glutamate. The product of this reaction was deblocked to give 21. Methylation of 21 gave the corresponding 4-(methylthio) derivative 22, which on reaction with hydrazine gave the 4-hydrazino analog 23 of methotrexate. Reduction of 12 and 21 with sodium hydrosulfite gave the dihydropteridines 24 and 25, respectively. The title compound was an excellent inhibitor of the growth of Streptococcus faecium ATCC 8043. However, this and related compounds were ineffective inhibitors of dihydrofolic reductase and showed no significant activity in either the KB cell culture screen or against L1210 leukemia cells in mice.

Nucleosides containing chemically reactive groups.

5'-Amino-5'-deoxyinosine (1) and 1-(6-amino-2,5,6-trideoxy-beta-D-erythro-hexofuranosyl)thymine (9) were prepared and substituted on the amino group with chemically reactive functions in an effort to find inhibitors of enzymes that metabolize the corresponding nucleotides. The resulting 5'-substituted methynitrosoureas 3, 11a, and 11b, bromoacetamides 4 and 13, phenyl carbamates 5 and 14, and 4-(fluorosulfonyl)benzamides 6 and 15 were tested for cytotoxicity to H.Ep-2 cells in culture and as inhibitors of incorporation of precursors into nucleic acids of L1210 cells. The inosine derivatives were also evaluated as inhibitors of hypoxanthine phosphoribosyltransferase. Compounds 4, 6 and 13 showed moderate inhibition of formation of nucleic acids, and compound 4 demonstrated significant cytotoxicity (ED50 less than 5 microgram/mL).

Reactive 5'-substituted thymidine derivatives as potential inhibitors of nucleotide biosynthesis.

Fourteen derivatives of thymidine substituted at the 5'-position with haloacetamido (2-4), 2- and 3-bromopropionamido (5 and 6), bromoacetoxy (7), O-mesylglycolamido (8), bromo- and chloro-N-methylacetamido (10 and 11), bromomethanesulfonamido (12), ethyloxamido (13), 4- and 3-(fluorosulfonyl)benzamido (14 and 15), and (phenoxycarbonyl)amino (16) groups have been synthesized and evaluated as potential inhibitors of enzymes that metabolize purine and pyrimidine nucleosides. Rates of reaction of these nucleosides with mercaptoethanol at pH 7 were compared and related to biological activity. Compounds 2, 3, and 7 were cytotoxic to H.Ep.-2 and L1210 cells in culture and 5'-(bromo- and iodoacetamido)-5'-deoxythymidine (2 and 3) showed good activity against P388 leukemia in mice.

Reactive 5'-substituted 2',5'-dideoxyuridine derivatives as potential inhibitors of nucleotide biosynthesis.

5'-(Bromoacetamido)-2',5'-dideoxyuridine (3) and derivatives (8, 10, 12, and 14) substituted at the 5-position with bromo, iodo, fluoro, and ethyl groups have been synthesized as potential inhibitors of enzymes that metabolize pyrimidine nucleosides. Also prepared were 2',5'-dideoxyuridine derivatives (4-6) substituted at the 5'-position with 2-bromopropionamido, iodoacetamido, and 4-(fluorosulfonyl)benzamido groups. Compounds 3, 5, 8, 12, and 14 were examined for effect on macromolecular synthesis in L1210 leukemia cells in culture and compared with 5'-(bromoacetamido)-5'-deoxythymidine (1, BAT), a compound with demonstrated cytotoxicity and activity in vivo against P388 murine leukemia. Compounds 3, 8, 12, and 14 inhibited DNA synthesis without significant inhibition of RNA synthesis, and protein synthesis was affected less than DNA synthesis. Compounds 3, 5, 6, 8, 10, 12, and 14 were cytotoxic to H.Ep.-2 and L1210 cells in culture, and 3, 5, 8, and 12 showed activity in the P388 mouse leukemia screen.

Synthesis of potential anticancer agents. Pyrido[4,3-b][1,4]oxazines and pyrido[4,3-b][1,4]thiazines.

Hydrolysis of the chloro group of ethyl (6-amino-4-chloro-5-nitropyridin-2-yl)carbamate (3) with formic acid gave the corresponding 4-hydroxypyridine 4. Catalytic hydrogenation of the nitro group of 4 gave the 5-amino-4-hydroxypyridine 5, which was reacted with alpha-halo ketones in acetic acid at room temperature to give a series of 3- and 2,3-substituted ethyl (5-amino-2H-pyrido[4,3-b][1,4]oxazin-7-yl)carbamates 8. Treatment of 8 with hot concentrated hydrochloric acid regenerated the pyridine synthon 5. In the reaction of 3 with thioacetate, the product underwent hydrolysis and air-oxidation to give the corresponding disulfide 6. Simultaneous reduction of both the nitro group and disulfide linkage of 6 gave the 5-amino-4-mercaptopyridine 7, which was reacted with alpha-halo ketones either in acetic acid at room temperature or in a mixture of ethanol and water at reflux to give a series of 3-, 2,3-, and 2,2,3-substituted ethyl (5-amino-2H-pyridol[4,3-b][1,4]thiazin-7-yl)carbamates 9. The effects of these pyridooxazines and pyridothiazines upon the proliferation and the mitotic index of cultured L1210 cells and upon the survival of mice bearing P388 leukemia were determined.

Potential inhibitors of nucleotide biosynthesis. 2. Halomethyl ketone derivatives of pyrimidine nucleosides.

Several halomethyl ketone derivatives of pyrimidine nucleosides have been prepared for evaluation as cytotoxic agents. The first series are 1-(8-halo-2,5,6,8- tetradeoxy -beta-D-erythro-oct-7 - ulofuranosyl )thymines (7-9), whereas the second type are halo derivatives of acetophenone (12-14 and 16). These compounds are cytotoxic, and one (13) showed activity against the P388 leukemia in vivo.

Nitrosoureido nucleosides as potential inhibitors of nucleotide biosynthesis.

Several nitrosoureido nucleosides (3a, 3b, 5a, 7a, 7c, and 10a) designed as inhibitors of enzymes that metabolize pyrimidine nucleotides have been prepared and their chemical and biological properties studied. The methylnitrosoureas 3a and 3b were not significantly cytotoxic to H.Ep.-2 and L1210 cells in vitro but showed moderate activity in the P388 mouse leukemia screen (79% ILS for 3a and 56% ILS for 3b). The (chloroethyl)nitrosoureas 7a and 7c inhibited proliferation of L1210 cells, were cytotoxic to H.Ep.-2 cells, and demonstrated good activity against P388 in vivo (135% ILS with one 30-day survivor for 7a and 191% ILS with two 30-day survivors for 7c). Overnight exposure of L1210 cells to 7a and 7c resulted in cell enlargement accompanied by cell lysis. Macromolecular synthesis in enlarged cells, particularly RNA and protein synthesis, was markedly increased relative to that in untreated control cells. The half-lives of each of the nitrosoureas in pH 7 buffer was determined and compared with biological activity.

1,2-Dihydropyrido[3,4-b]pyrazines: structure-activity relationships.

Certain derivatives containing the 1,2-dihydropyrido[3,4-b]pyrazine (1-deaza-7,8-dihydropteridine) ring system are active against experimental neoplasms in mice. The mechanism of action of these agents has been attributed to the accumulation of cells at mitosis. Identification of the structural features that are necessary for activity was accomplished by evaluation of modified 1-deazapteridines and ring and ring-opened analogues. Relative to ethyl 4-amino-1-deaza-7,8-dihydro-6-[(N-methylanilino)methyl]pteridine-2-carbamate (11) and the corresponding 6-phenyl compound (12), no antitumor activity was observed with 7,8-dihydropteridines, 3-deaza-7,8-dihydropteridines, and the corresponding heteroaromatic compounds. Also, activity was diminished or destroyed when 1-deaza-7,8-dihydropteridines were oxidized to 1-deazapteridines or reduced to 1-deaza-5,6,7,8-tetrahydropteridines. In addition, replacement of the 4-amino group with other substituents destroyed activity. The presence of a 6-substituent containing an aryl group appeared to be necessary for activity, which was increased when a methyl group was substituted at the 7-position.

New anticancer agents: synthesis of 1,2-dihydropyrido[3,4-b]pyrazines (1-deaza-7,8-dihydropteridines).

Reaction of alpha-aminoacetophenone oximes (2) with ethyl 6-amino-4-chloro-5-nitropyridine-2-carbamate (1) gave ethyl 6-amino-5-nitro-4-[(2-oxo-2-phenylethyl)amino]pyridine-2-carbamate oximes (3), which were hydrolyzed under acidic conditions to give the corresponding ketones (4). Related pyridines substituted with a keto side chain were prepared from 1 and 1,3-diaminopropanone oximes and by oxidation of the side-chain hydroxy group of ethyl 6-amino-4- [[3-(N-methyl-N-phenylamino)-2-hydroxypropyl]amino]-5-nitropyridine-7- carbamates (6). Catalytic hydrogenation of the nitro group of 4 over Raney nickel in a large volume of ethanol gave the 1-deaza-7,8-dihydropteridines (7). Several of the oximes 3 were successfully hydrogenated to give 7 directly. The resulting 1-deaza-7,8-dihydropteridines showed potent cytotoxicity against cultured L1210 cells and significant anticancer activity against lymphocytic leukemia P-388 in mice. These biological activities are attributed to the accumulation of cells at mitosis.

Analogues of methotrexate.

Analogues of methotrexate (MTX) were prepared by alkylation of side-chain precursors with 6-(bromomethyl)-2,4-pteridinediamine followed, where necessary, by saponification of the intermediate esters and, in two cases, by electrophilic substitution reactions in the pyridine ring portion of 3-deazamethotrexate. Effects of the various modifications on their ability to inhibit dihydrofolate reductase, cytotoxicity, and activity against L1210 leukemia in mice were examined in light of recent findings concerning active transport of MTX and related compounds and the binding features of the MTX-dihydrofolate reductase complex.

Synthesis of pseudo cofactor analogues as potential inhibitors of the folate enzymes.

Reaction of 5,6,7,8-tetrahydrofolic acid (THF,7) with phosgene, thiophosgene, and cyanogen bromide gave the bridged derivatives, 5,10-(CO)-THF (8), 5,10-(CS)-THF (9), and 5,10-(C = NH)-THF (11), respectively. Catalytic hydrogenation of 10-(chloroacetyl)folic acid (2) gave 5,10-(CH2CO)-THF (12). A similar reaction with 10-(3-chloropropionyl)folic acid (3) gave 10-(ClCH2CH2CO)-THF (14) rather than 5,10-(CH2CH2CO)-THF (13). In the catalytic hydrogenation of 10-ethoxalylfolic acid (5), the initial product 10-(EtO2CCO)-THF (22) rearranged readily to give 5-(EtO2CCO)-THF (21). Acylation of THF with chloroacetyl chloride gave a N5,N10-diacylated product (18 or 19), which could not be converted to 5,10-COCH2)-THF (17). Reductive alkylation of THF with glyoxylic acid and 5-hydroxypentanal, respectively, gave 5-(HO2CCH2)-THF (24) and 5-[HO(CH2)5]-THF (25). Reductive dialkylation of THF with formaldehyde gave 5,10-(CH3)2-THF (27), whereas glyoxal gave 5,10-CH2CH2)-THF (10). Also, both folic acid and 5-(CHO)-THF were reductively alkylated with formaldehyde to give 10-methylfolic acid (6) and 5-(CHO)-10-(CH3)-THF (28), respectively. These compounds were tested as inhibitors of the enzymes involved in folate metabolism and for activity against lymphocytic leukemia P388 in mice.

Phosphonate analogs of carbocyclic nucleotides.

Cyclopentadiene was converted in six steps to the key intermediate (+/-)-(1 alpha,2 beta,4 alpha)-4-amino-2-(benzyloxy)cyclopentanol (10), which in turn was converted to the carbocyclic nucleoside analogs 14 and 19 by standard procedures developed in these laboratories. Compounds 14 and 19 were then further converted to the target phosphonates 1b and 2b by modification of literature procedures. The phosphonate 1b was 40-fold more cytotoxic to HEp-2 cells than its parent, CDG, presumably after conversion to the diphosphoryl phosphonate.

Structure-based design of inhibitors of purine nucleoside phosphorylase. 4. A study of phosphate mimics.

9-(3,3-Dimethyl-5-phosphonopentyl)guanine was synthesized and found to be a potent inhibitor of purine nucleoside phosphorylase (PNP) (IC50 = 44 nM). A number of other functional end groups were investigated as phosphate mimics attached to the 9-position of guanine by this same alkyl side chain, which provided a sensitive method for the detection of any interaction of these groups with the phosphate binding site of PNP. Both the sulfonic acid (compound 13) and the carboxylic acid (compound 15) end groups interact significantly with the phosphate binding site, but in different ways, as determined by X-ray crystallographic analysis of the complexes. The sulfonic acid of 13, which binds about one-fourth as tightly as the phosphonate 12, binds in the phosphate subsite much like the phosphonic acid. The carboxylic acid, the interaction of which is much weaker, turns away from the center of the phosphate binding site to form hydrogen bonds with Ser 200 and Met 219. Thus, the only phosphate mimics that bind like phosphate itself are themselves highly ionic, probably with limited ability to penetrate cell membranes.

Thrombocytopenia and hemorrhages in veal calves infected with bovine viral diarrhea virus.

The relationship between bovine viral diarrhea virus (BVDV) infection and thrombocytopenia was studied in 18 veal calves experimentally infected with BVDV. All calves were free of BVDV, and 13 calves were free of serum neutralizing antibodies to BVDV before virus inoculation. Calves were inoculated at approximately 10 days of age, and platelet counts were monitored over a period of several weeks. Ten additional calves housed in close proximity were kept as uninoculated controls. A profound decrease in platelet counts by 3 to 11 days after inoculation was seen in all calves that had neutralizing antibody titers less than 1:32 before infection. Severe thrombocytopenia (less than 5,000 platelets/microliter) was seen in 12 calves, 11 of which also developed hemorrhages. Necropsy findings in 3 severely thrombocytopenic calves that died included multiple hemorrhages throughout the body. Calves that recovered had increased platelet counts, and in most instances, a corresponding increase in neutralizing antibody titers to BVDV. At 11 days after inoculation, BVDV was detected on platelets by use of immunofluorescence, but evidence of surface-bound immunoglobulin was not found. The results suggest that a nonimmunoglobulin-mediated method of platelet destruction or sequestration develops as a sequela to BVDV infection.