Isopropyl Moiety Research Articles

NMR structure of the hypothetical protein NMA1147 from Neisseria meningitidis reveals a distinct 5-helix bundle.

NMR structure of the hypothetical protein NMA1147 from Neisseria meningitidis reveals a distinct 5-helix bundle.

NMR structure of the hypothetical protein AQ-1857 encoded by the Y157 gene from Aquifex aeolicus reveals a novel protein fold.

Uniformly (U) 13C, 15N-labeled AQ-1857 was cloned, expressed and purified following standard protocols. Briefly, the full length gene (Y157_AQUAE) from Aquifex aeolicus was cloned into a pET21d (Novagen) derivative, yielding the plasmid pQR6-21. The resulting construct contains eight nonnative residues at the C-terminus (LEHHHHHH) that facilitate protein purification. Escherichia coli BL21 (DE3) pMGK cells, a rare codon enhanced strain, were transformed with pQR6-21, and cultured in MJ minimal medium containing (15NH4)2SO4 and U-13C-glucose as sole nitrogen and carbon sources. U-13C,15N AQ-1857 was purified using a two-step protocol consisting of Ni-NTA affinity (Qiagen) and gel filtration (HiLoad 26/60 Superdex 75, Amersham Biosciences) chromatography. The final yield of purified U-13C, 15N AQ-1857 (> 97% homogeneous by SDS-PAGE; 14.4 kDa by MALDI-TOF mass spectrometry) was about 10 mg/L. In addition, a sample which was U-15N and 5% biosynthetically directed fractionally 13C-labeled was generated for stereospecific assignment of isopropyl methyl groups.8 Two samples of 5%13C,U-15N and U-13C,15N AQ-1857 were prepared at concentrations of 1.0 mM in 95% H2O/5% D2O solution containing 20 mM MES, 100 mM NaCl, 10 mM DTT, 5 mM CaCl2, 0.02% NaN3 at pH 6.5. All NMR data were collected at 20°C on Varian INOVA 600 and 750 spectrometers. The spectra were processed and analyzed using the programs NMRPipe9 and XEASY,10 respectively. Resonance assignments were obtained as described11 using a suite of reduced-dimensionality NMR experiments, including 3D HNNCAHA, HαβCαβ(CO)NHN, HCCH-COSY, and 2D HBCB(CGCD)HD. These data were complemented by conventional12 HNNCACB and HC(C)H TOCSY experiments. Assignments were obtained for 93% of the backbone and 13Cβ, and for 91% of the side chain chemical shifts. Stereospecific assignments were obtained for 44% of the β-methylene groups exhibiting non-degenerate proton chemical shifts, and for all Val and Leu isopropyl moieties. The chemical shifts were deposited in the BioMagResBank (accession code: 5683). Upper distance limit constraints for structure calculations were obtained from 3D 15N-and 13C-resolved [1H,1H]-NOESY12 (Table I). In addition, 3JHNα scalar couplings measured in 3D HNNHA12 yielded ϕ-angle constraints, and backbone dihedral angle constraints were derived from chemical shifts as described13 for residues located in regular secondary structure elements (Table I). Structure calculations were performed using the program DYANA.14 Statistics for the structure determination (Table I) show that a high-quality NMR structure was obtained (Fig. 1). AQ-1857 (PDB ID: 1NWB) contains seven β-strands A to F and two α-helices. A(↓), F(↓) and G(↑) form a 3-stranded, and D(↓), E(↑), B(↑) and C(↓) form a 4-stranded sheet. The two sheets form a “sandwich” being rotated by ∼45 degrees relative to each other (Fig. 2). The segment 40–45 and the C-terminal tail 102–116 are flexibly disordered in solution. The 20 DYANA conformers with the lowest residual DYANA target function chosen to represent the NMR solution structure of AQ-1857 are shown after superposition of the backbone heavy atoms N, Cα and C′ of the regular secondary structure elements for minimal RMSD. A: The novel fold of AQ-1857: ribbon drawing of the DYANA conformer with the lowest residual target function value. The α-helices I and II are shown in red and yellow, the β-strands A to G are in cyan, other polypeptide segments are in grey, and the N- and C-terminal ends of the protein are indicated as ‘N’ and ‘C’. B: Same as in (A), but rotated by 90° about the vertical axis. α-Helix I: residues 14–25, II: 77–79; β-Strand A: 10–12, B: 33–36, C: 52–53, D: 63–65, E: 69–72, F: 84–89, G: 94–99. The NMR structure of AQ-1857 is the first structure representative of the larger HesB family2, 3 of proteins. No meaningful structural homologues were identified using the programs SKAN,15 DALI,16 or CE.17 This finding strongly supports the notion that AQ-1857 possesses a hitherto uncharacterized, novel fold (Fig. 2). Interestingly, the PROSITE consensus pattern for the HesB family spans the flexibly disordered C-terminal tail of the protein. In fact, two of the three cysteinyl residues which have been proposed to be involved in iron-sulfur cluster assembly in members of this family5, 6 are located in this tail (not shown in Fig. 1; the third cysteine is located in position 43 in the loop connecting β-strands 2 and 3). It is thus very likely that the flexibly disordered tail is of functional importance, and it is tempting to speculate that this tail adopts an ordered conformation only upon involvement in Fe-S cluster assembly. This work was supported by the National Institutes of Health (P50 GM62413-01), the National Science Foundation (MCB 00075773 to T.S.; DBI-9904841 to B.H), and the Center for Computational Research at UB.

Site-directed mutagenesis experiments on the putative deprotonation site of squalene-hopene cyclase from Alicyclobacillus acidocaldarius.

To provide insight into the catalytic mechanism for the final deprotonation reaction of squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius, mutagenesis experiments were conducted for the following ten residues: Thr41, Glu45, Glu93, Arg127, Trp133, Gln262, Pro263, Tyr267, Phe434 and Phe437. An X-ray analysis of SHC has revealed that two types of water molecules ("front water" and "back waters") were involved around the deprotonation site. The results of these mutagenesis experiments allow us to propose the functions of these residues. The two residues of Gln262 and Pro263 probably work to keep away the isopropyl group of the hopanyl cation intermediate from the "front water molecule," that is, to place the "front water" in a favorable position, leading to the minimal production of by-products, i.e., hopanol and hop-21(22)-ene. The five residues of Thr41, Glu45, Glu93, Arg127 and Trp133, by which the hydrogen-bonded network incorporating the "back waters" is constructed, increase the polarization of the "front water" to facilitate proton elimination from the isopropyl moiety of the hopanyl cation, leading to the normal product, hop-22(29)-ene. The three aromatic residues of Tyr267, Phe434 and Phe437 are likely to play an important role in guiding squalene from the enzyme surface to the reaction cavity (substrate channeling) by the strong affinity of their aromatic residues to the squalene substrate.

Biotransformation of a 4α-hydroxylated eudesmane with Exserohilum halodes: Chemo-enzymatic synthesis of cryptomeridiol and 6- epi-colartin derivatives

Microbial transformation of a 4α-hydroxylated eudesmane by Exserohilum halodes has been achieved. Regioselective hydroxylations in both “A” and “B” rings, on C-2 and C-8, were detected, but the main hydroxylating action was directed to the isopropyl moiety and cryptomeridiol analogues were isolated. Semi-synthesis of two sesquiterpenolides, 6- epi-colartin derivatives, were accomplished from the significant metabolites hydroxylated at C-12.

Metabolism of fungicide diethofencarb in grape (Vitis vinifera L.): definitive identification of thiolactic acid conjugated metabolites.

The metabolic fate of diethofencarb (isopropyl 3,4-diethoxycarbanilate) separately labeled with (14)C at the phenyl ring and 2-position of the isopropyl moiety was studied in grape (Vitis vinifera L.). The acetonitrile solution of (14)C-diethofencarb at a rate of 500 g a.i. ha(-)(1) was once applied topically to fruits or leaves at the maturity stage of fruits (PHI 35 days), and the plants were grown in the greenhouse until harvest. In the grape plants, diethofencarb was scarcely translocated to the untreated portion and was degraded more in the fruit as compared to the leaf. For the fruit, diethofencarb primary underwent O-deethylation at the 4-position of the phenyl ring to form the phenolic derivative, isopropyl 3-ethoxy-4-hydroxycarbanilate (0.9% of the total radioactive residue, TRR). This metabolite was successively transformed via conjugation with glucose at the phenolic hydroxy group (8.1-18.1% TRR) or with thiolactic acid at the 5-position of the phenyl ring (1.5-1.7% TRR). The thiolactic acid conjugate was further metabolized mainly to two different types of glucose conjugates at the 4-position of the phenyl ring (8.7-13.5% TRR) and the hydroxy group in the thiolactic acid moiety (6.4-7.3% TRR), as evidenced by (1)H NMR and atmospheric pressure chemical ionization-liquid chromatography-mass spectrometry together with cochromatographies with synthetic standards.

CYP2C8 and CYP3A4 are the principal enzymes involved in the human in vitro biotransformation of the insulin secretagogue repaglinide.

To identify the principal human cytochrome P450 (CYP) enzyme(s) responsible for the human in vitro biotransformation of repaglinide. Previous experiments have identified CYP3A4 as being mainly responsible for the in vitro metabolism of repaglinide, but the results of clinical investigations have suggested that more than one enzyme may be involved in repaglinide biotransformation. [14C]-Repaglinide was incubated with recombinant CYP and with human liver microsomes (HLM) from individual donors in the presence of inhibitory antibodies specific for individual CYP enzymes. Metabolites, measured by high-performance liquid chromatography (HPLC) with on-line radiochemical detection, were identified by liquid chromatography-mass spectrophotometry (LC-MS) and LC-MS coupled on-line to a nuclear magnetic resonance spectrometer (LC-MS-NMR). CYP3A4 and CYP2C8 were found to be responsible for the conversion of repaglinide into its two primary metabolites, M4 (resulting from hydroxylation on the piperidine ring system) and M1 (an aromatic amine). Specific inhibitory monoclonal antibodies against CYP3A4 and CYP2C8 significantly inhibited (> 71%) formation of M4 and M1 in HLM. In a panel of HLM from 12 individual donors formation of M4 and M1 varied from approximately 160-880 pmol min-1 mg-1 protein and from 100-1110 pmol min-1 mg-1 protein, respectively. The major metabolite generated by CYP2C8 was found to be M4. The rate of formation of this metabolite in HLM correlated significantly with paclitaxel 6alpha-hydroxylation (rs = 0.80; P = 0.0029). Two other minor metabolites were also detected. One of them was M1 and the other was repaglinide hydroxylated on the isopropyl moiety (M0-OH). The rate of formation of M4 in CYP2C8 Supersomes was 2.5 pmol min-1 pmol-1 CYP enzyme and only about 0.1 pmol min-1 pmol-1 CYP enzyme in CYP3A4 Supersomes. The major metabolite generated by CYP3A4 was M1. The rate of formation of this metabolite in HLM correlated significantly with testosterone 6beta-hydroxylation (rs = 0.90; P = 0.0002). Three other metabolites were identified, namely, M0-OH, M2 (a dicarboxylic acid formed by oxidative opening of the piperidine ring) and M5. The rate of M1 formation in CYP3A4 Supersomes was 1.6 pmol min-1 pmol-1 CYP enzyme but in CYP2C8 Supersomes it was only approximately 0.4 pmol min-1 pmol-1 CYP enzyme. The results confirm an important role for both CYP3A4 and CYP2C8 in the human in vitro biotransformation of repaglinide. This dual CYP biotransformation may have consequences for the clinical pharmacokinetics and drug-drug interactions involving repaglinide if one CYP pathway has sufficient capacity to compensate if the other is inhibited.

Enantioselective synthesis of benzylic stereocentres via Claisen rearrangement of enantiomerically pure allylic alcohols: preparation of ( R)- and ( S)-3-methyl-2-phenylbutylamine

The Johnson–Claisen rearrangement of enantiopure allylic alcohols in triethylorthopropionate is the key step for the preparation of chiral molecules with benzylic stereogenic carbon atoms bearing an isopropyl moiety. The synthetic procedure is applied to the preparation of ( R)- and ( S)-3-methyl-2-phenylbutylamine.

Biosynthetic studies on a myxobacterial antibiotic, cystothiazole A: biosynthetic precursors of the carbon skeleton.

Biosynthetic studies on cystothiazole A (1), a beta-methoxyacrylate-type antifungal compound from a myxobacterium, were performed by feeding the producing organism, Cystobacter fuscus, with stable-isotope-labeled compounds including [2-(13)C]acetate, [1,2-(13)C2]acetate, [1-(13)C]propionate, L-[1-(13)C]serine, L-[methyl-(13)C]methionine, and DL-valine-d8. The polyketide moiety of 1 was found to be derived from acetate and propionate, the bithiazole moiety from L-serine, the O-methyl groups from the S-methyl group of L-methionine, and the isopropyl moiety from L-valine, which should be the metabolic precursor of isobutyryl-CoA.

Oxidation, reduction, and methylation of carnosic acid by Nocardia.

Preparative-scale incubations with Nocardia sp. NRRL 5646 were conducted to produce new derivatives of the abietane diterpene chemoprotectant and antioxidant carnosic acid (1). Reductive biotransformation of the C-20 carboxylic acid functional group followed by biological methylation at the C-11 phenol afforded 4. Oxidative cyclization of 1 to carnosol 5 followed by dihydroxylation at the isopropyl moiety afforded 6. Metabolites 4 and 6 are new carnosic acid derivatives whose structures were confirmed by mass spectrometry and NMR spectroscopic analysis. The radical quenching properties of 4-6 using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical-scavenging assay showed activities similar to that of mixed tocopherols and carnosic acid.

Chemical-microbiological synthesis of cryptomeridiol derivatives by Gliocladium roseum: semisynthesis of 11-hydroxyeudesmanolides.

Biotransformations of 4alpha- and 4beta-hydroxyeudesmane derivatives by the filamentous fungus Gliocladium roseum were achieved. Hydroxylation at C-11 was the main action of this microorganism, producing new cryptomeridiol (12 and 14) and 4-epi-cryptomeridiol derivatives (6 and 7), respectively, in good yields. The biotransformation activity of G. roseum toward 4beta-hydroxyeudesmane was focused on the isopropyl moiety, but more scattered on the 4alpha-hydroxylated derivative, acting in both the "A" and "B" rings and the isopropyl group of the molecule. Semisyntheses of 11-hydroxyeudesmanolides from the isolated 11,12-dihydroxylated metabolites were also accomplished and used in assigning the stereochemistry of hydroxylation.

Products of aqueous chlorination of bisphenol A and their estrogenic activity.

To assess the estrogenic activity potentially stemming from bisphenol A (BPA) in drinking water, APCI/LC/MS and NMR were used to identify the products of its aqueous chlorination under the following conditions: 500 microg/L bisphenol A and 1.46 mg/L sodium hypochlorite (pH 7.5) at 25 degrees C. The 13 products (4-chloro-BPA; 2,6'-dichloro-BPA; 2,6-dichloro-BPA; 2,2',6'-trichloro-BPA; 2,2',6,6'-tetrachloro-BPA; trichlorophenol; 4-isopropyl-2'-hydroxylphenol; and six kinds of polychlorinated phenoxyphenols (PCPPs)) were found in the chlorinated BPA solution. Three main pathways are proposed: (1) chlorine-substitution reactions on the aromatic ring, followed by dehydration to form the chlorine-substituted BPA, (2) chlorine substitution reactions followed by cleavage of the alpha-C on the isopropyl moiety with positive partial charge and beta'-C on the benzene moiety with a negative partial charge to form trichlorophenol and 4-isopropyl-2'-hydroxylphenol, and (3) the formation of PCPPs. Especially for pathway 2, the reaction mechanism was clarified based on semiempirical quantum mechanical calculations. The reaction proceeded by attack of the OH and Cl (from HOCl) on the alpha-C on the isopropyl moiety with a positive partial charge and on the beta'-C with a negative partial charge on the benzene moiety. The activation energies forthe HOCl/4-chloro-BPA and 2,2',6,6'-tetrachloro-BPA reactions were 0.14 and 0.15 kcal/mol, respectively. Finally, the estrogenic activity of the aqueous chlorinated BPA solution was assessed by an estrogen receptor binding assay and a yeast two-hybrid system. It was found that the binding affinity of the chlorinated aqueous BPA at 60 min was 24 times that before chlorination. The transcriptional activation-induced by products were detected by a yeast two-hybrid system based on the ligand-dependent interaction of two proteins, a human ER and a coactivator, suggesting that the chlorinated BPA solution elicits an ability to mimic the effect of the estrogen hormone.

ISOPROTURON DEGRADATION AS AFFECTED BY THE GROWTH OF TWO ALGAL SPECIES AT DIFFERENT CONCENTRATIONS AND pH VALUES

Metabolism of [14C-u-phenyl]isoproturon [3-(4-isopropylphenyl)-1,1-dimethylurea] by two soil and freshwater microorganisms, green alga Chlorella kesslerei and cyanobacterium Anabaena inaequalis, was studied as a function of pH, pesticide concentration, and incubation time. Metabolized isoproturon, in the media, ranged from 0% (Chlorella at pH 5.5 after 1 d) to 22% (Anabaena at pH 5.5 after 10 d). Twenty-five percent faster degradation of isoproturon by Anabaena occurred at pH 5.5 versus pH 7.5, when measured over 10 d. Increased 14C incorporation into tissue, with time and at lower pH, was due mainly to bioaccumulation of [14C]isoproturon and/or its metabolites in the cells. Metabolic degradation resulted in four identifiable (by TLC) metabolites. Based on this, a degradation pathway is proposed, involving mono- and di-N-demethylation, hydroxylation of the isopropyl moiety, and hydrolysis to 4-isopropylaniline. Similarity in the metabolites produced suggests that the enzyme systems responsible for metabolizing isoproturon are almost identical in both photosynthetic micro-algae.

Binding site of acyl moiety in ester hydrolysis by Candida rugosa lipase

The sizes of the large, medium, and small substituent recognition sites (L, M, and S pockets, respectively) in Candida rugosa lipase (CRL) were roughly estimated by measuring the specific hydrolytic activity against several p-nitrophenyl esters. These relative sizes were assessed as L pocket>phenyl, ethyl>M pocket>methyl>S pocket>hydrogen. The hydrolysis of a series of p-nitrophenyl esters of ω-substituted fatty acids was also examined. In this series, p-nitrophenyl esters having one methylene between the ester–carbonyl carbon and cyclohexyl, phenyl, or isopropyl moiety largely demonstrated decreases in hydrolytic activity.

Structural determination of metabolites of S-1153, a new, potent, non-nucleoside, anti-HIV agent in rat liver microsomes

1. S-1153, a non-nucleoside agent that is under development in the USA as a new antiHIV agent, has potent antiviral activity based on the inhibition of reverse transcriptase. 2. S-1153wasincubatedwithratliver microsomes andNADPH,andsevenmetabolites were formed. The main metabolites were identified as the S -oxide, N -oxide and sulphone of S-1153. 3. Two other minor metabolites were assumed to be S-1153 hydroxylated on the isopropyl moiety. 4. Our findings confirmed the existence of at least three oxidative metabolic pathways of S-1153.

Structural analogues of the calanolide anti-HIV agents. Modification of the trans-10,11-dimethyldihydropyran-12-ol ring (ring C).

(+)-Calanolide A is a potent inhibitor of reverse transcriptase from human immunodeficiency virus type 1 (HIV-1), which was isolated from an extract of Calophyllum lanigerum, along with seven related compounds. In order to examine the structure-activity relationships of the trans-10,11-dimethyldihydropyran-12-ol ring (designated ring C), a series of structural analogues were prepared and evaluated using a whole cell cytopathicity assay (XTT). Removal of the 10-methyl group resulted in decreased activity, with only one epimer exhibiting anti-HIV activity. Substituting the 10-methyl group with an ethyl chain maintained anti-HIV activity, with only a 4-fold reduction in potency relative to racemic calanolide A. Substitution of the 10-methyl group with an isopropyl moiety completely eliminated the anti-HIV activity. Addition of an extra methyl group at either the 10- or 11-position maintained the basic stereochemical features of the parent calanolide system while removing the chirality at the respective carbon, but resulted in decreased activity relative to calanolide A. In all the above examples, analogues containing a cis relationship between the 10- and 11-alkyl moieties were completely devoid of activity. Synthetic intermediates in which the 12-hydroxyl group was in the ketone oxidation state exhibited suppressing anti-HIV activity, with EC50 values only 5-fold less potent than that of calanolide A for both the 10,11-cis (6) and -trans (5) series. These ketones represent the first derivatives in the calanolide series to exhibit anti-HIV activity while not containing a 12-hydroxyl group. Likewise, ketone derivative 6 was the first example of a compound in the calanolide series having a cis relationship between the 10- and 11-methyl groups found to exhibit anti-HIV activity. Analogues which showed anti-HIV activity in the CEM-SS cytoprotection assay were further confirmed to be inhibitors of HIV-1 reverse transcriptase.

Biosynthesis of cyanohydrin glucosides from unnatural nitriles in intact tissue of Passiflora morifolia and Turnera angustifolia

Passiflora morifolia, which under natural conditions contains cyanohydrin glucosides linamarin, lotaustralin and epilotaustralin, converted cyclopentanecarbonitrile, 2-cyclopentenecarbonitrile and 3-methylbutaneitrile into the corresponding cyanohydrin glucosides. Turnera angustifolia, which normally produces glucosides of cyclopentenone cyanohydrin, converted cyclopentanecarbonitrile, 2-methylpropanenitrile and 2-methylbutanenitrile, but not 3-methylbutanenitrile, into the corresponding cyanohydrin glucosides. Mixtures of epimers were produced when these glucosides contained chiral cyanohydrin carbon atoms. Feeding with cyclopentanecarbonitrile resulted in formation of 1-(β- d-glucopyranosyloxy)cyclopentanecarbonitrile, a saturated analogue of deidaclin and tetraphyllin A. Neither plant utilized cyclopropanecarbonitrile as substrate. The experiments demonstrate broad substrate specificity of nitrile hydroxylases present in these plants. A novel glycoside, 2-[6-O-(β- d-xylopyranosyl )-β- d-glucopyranosyloxy]propane (isopropyl primeveroside), was isolated from P. morifolia. The compound represents a rare example of natural isopropyl glycoside; its characterization included assignment of all 1H and 13C NMR signals of the primeverosyl group using two-dimensional NMR methods. Biosynthesis of the isopropyl moiety of the primeveroside is unclear, but the formation of alcohols corresponding to natural cyanohydrins may be a previously unrecognized extension of the cyanohydrin biosynthesis pathway in higher plants.

Azulene derivatives as TXA 2/PGH 2 receptor antagonists—II. Synthesis and biological activity of 6-mono- and 6-dihydroxylated-isopropylazulenes

In order to examine the correlation between activity and hydrophilicity of the side chain of sodium 3-[4-(4-chlorobenzenesulfonylamino)butyl]-6-isopropylazulene-1-sulfonate (KT2-962), a non-prostanoid TXA 2/PGH 2 receptor antagonist, one or two hydroxyl groups were introduced into the isopropyl moiety. A series of 6-hydroxylated-isopropylazulenes were synthesized by regioselective oxidation of 6-isopropylazulenes and their in vitro and in vivo antagonistic activities were studied. Both the primary and tertiary alcohols, monohydroxylated derivatives, exhibited potent biological activities comparable to unmodified 6-isopropylazulenes both in vitro and in vivo. In contrast, the activities of 1,2- and 1,3-diols of 6-substituted derivatives, markedly decreased, but recovered by O-isopropylidenation of the dihydroxyl moiety. These findings indicate that the moderate hydrophobicity of substituent at the 6-position of the azulene ring might be required for the activity and the size of the substituent at this position, not so rigid for keeping potent biological activity.

Photochemical behaviour of poly(organophosphazenes). 14. Photooxidation of poly[bis(4-isopropylphenoxy) phosphazene] under accelerated conditions

The photooxidation of poly[bis(4-isopropylphenoxy)phosphazene] under accelerated conditions has been followed by FTIR and UV visible spectroscopic techniques. The main photooxidation products are acetophenone and phenol groups. In addition, acetone vapors have been detected by GC MS combined techniques concomitant with the IR spectral changes in the CH stretching region, suggesting a significant decrease in the isopropyl moieties. The presence of polymeric sequences having phenol groups under our conditions gives origin to further oxidation reactions due to electron transfer or radical recombination or to hydrogen abstraction reactions promoted by unhindered phenoxyl radicals. In addition, the absence of UV visible light, i.e., under thermooxidation reaction at 60° C has demonstrated that phenol groups are the main responsible of secondary oxidation products. The complexity of the photooxidation mechanism in the solid state for this polymer makes it difficult to determine a definitive degradation mechanism under both thermo- and photooxidative conditions.

6-substituted 2,4-diamino-5-methylpyrido[2,3-d]pyrimidines as inhibitors of dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii and as antitumor agents.

The synthesis and biological activity of 15 6-substituted 2,4-diamino-5-methylpyrido[2,3-d]-pyrimidines are reported. These compounds were synthesized in improved yields by modifications of procedures previously reported by us. Specifically, dimethoxyphenyl-substituted compounds with H and CH3 at the N-10 position and trimethoxyphenyl-substituted compounds with N-10 ethyl, isopropyl, and propargyl moieties were synthesized. These compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii, Toxoplasma gondii, and rat liver, and selected analogues were evaluated as inhibitors of the growth of T. gondii and tumor cells in culture. All the compounds showed increased selectivity (vs rat liver DHFR) for T. gondii DHFR compared to trimetrexate. In general, for the trimethoxy-substituted analogues, increasing the size of the N-10 substituent from a methyl group to larger groups resulted in a decrease in selectivity and potency for both P. carinii and T. gondii DHFR. For the dimethoxy-substituted analogues, N-10 methylation in general decreased potency but increased selectivity for T. gondii DHFR. In an attempt to improve the cell penetration of these analogues, the N-10 naphthyl-substituted analogues were also synthesized. These analogues displayed excellent cell penetration and inhibition of T. gondii cells in culture. Further, these analogues were potent inhibitors of the growth of tumor cells in the preclinical in-vitro screening program of the National Cancer Institute with IC50s in the nanomolar range.

Asymmetric Oxidation of Isopropyl Moieties of Aliphatic and Aromatic Hydrocarbons byRhodococcussp. 11B

A 2,5-dimethylhexane (2,5-DMH) assimilating bacterial strain, identified as Rhodococcus sp. and some Mycobacterium species, catalyzed the asymmetric oxidation of the isopropyl moiety of hydrocarbons. The 2,5-DMH-grown Rhodococcus sp. strain 11 B produced (–)-2,5-dimethylhexanoic acid from 2,5-DMH, and (S)-2-phenylpropionic acid and (R)-2-phenyl-l-propanol from cumene. The biodegradability of isoalkanes, which have various branching patterns, using this strain is also discussed.