Sterol synthesis. Syntheses of 15-oxygenated 5alpha, 14beta-cholest-7-en-3beta-ol derivatives

Treatment of 3beta-benzoyloxy-14alpha, 15alpha-epoxy-5alpha-cholest-7-ene with boron trifluoride-etherate gave, in 43% yield, 3alpha-benzoyloxy-5alpha, 14beta-cholest-7-en-15-one with the unnatural C-D ring juncture. Reduction of the latter compound with lithium aluminum hydride gave 15alpha, 14beta-cholest-7-en-3beta, 15alpha-diol and 5alpha, 14beta-cholest-7-en-3beta, 15beta-diol in 9% and 81% yields, respectively.     

Estimation of impurity profiles of drugs and related materials: part 19: theme with variations. Identification of impurities in 3-oxosteroids

Due to the varied reactions leading to the 3-oxo group in steroids and the reactivity of its environment, a large number of impurities related to this group are formed during the reaction steps and the degradation studies. In this paper the experiences from the authors laboratory with the 3-oxo-related impurities in 19-nor-4-ene-3-oxosteroids (norgestrel, norethisterone, nandrolone, its esters and Nestorone) as well as corticosteroids (prednisolone, mazipredone, etc) are presented. The impurities include saturated 3-ones, 1-ene-3-ones, 5(10)-ene-3-ones, 3-deoxo and 3-ethinyl-3,5-diene derivatives, 6-ene, 8(14)-ene, 6,8(14)-diene, 6-hydroxy (alpha and beta), 10beta-hydroxy and 6-one derivatives in 4-ene-3-oxosteroids and 8(9)-ene, 9(11)-ene, 11alpha-hydroxy, 11-oxo and 4-ene-3-one derivatives in 11beta-hydroxy-1,4-diene-3-oxosteroids. The chromatographic, spectroscopic and hyphenated techniques used in this study include TLC, GC, HPLC with diode array UV detector, GC-MS, LC-MS and NMR methods.     

Developing an effective toxicology/quality assurance partnership. Improving quality, compliance, and cooperation

A novel approach to the synthesis of the orally active estrogen 14 alpha,15 alpha-methylene estradiol (8, J 824) is described, starting with 3-methoxy-estra-1,3,5(10),8,14-pentaen-17 alpha-ol (5). The 14 alpha, 15 alpha-methylene bridge was sonochemically introduced by regioselective and stereoselective Simmons-Smith methylenation of the 14-double bond. Birch reduction of the 8-double bond provided the desired 8 beta-H, 9 alpha-H steroid, whereas ionic hydrogenation afforded the 8 beta-H, 9 beta-H isomer, together with an epimerization of the 17 alpha-hydroxy group. Oxidation of the Birch product yielded the corresponding 17-oxo steroid, which gave the title compound by diborane reduction. For radioimmunoassay development the 6-(O-carboxymethyl)-oximino derivative of 8 was prepared as hapten and the 2-hydroxy derivative of 8 was synthesized as a potential metabolite of 8, and 8 was tritium labeled as well.     

Anti-inflammatory and analgesic effects of cucurbitacins from Wilbrandia ebracteata

The anti-inflammatory and antinociceptive actions of the CH2Cl2 extract and semipurified fraction (F-III) from roots of Wilbrandia ebracteata Cogn. have been investigated in rats and mice. The CH2Cl2 extract (1-10 mg/kg, i.p.; ID50 5 mg/kg) and (3-30 mg/kg, p.o.; ID50 15 mg/kg) inhibited, in a dose-related manner, carrageenan-induced paw edema in rats. The subfraction (F-III) from CH2Cl2 extract and compounds isolated as cucurbitacin B and E also inhibited carrageenan-induced edema. The CH2Cl2 extract and F-III also exhibited significant analgesic action in acetic acid-induced pain in mice. In the formalin test, the CH2Cl2 extract (0.3-10 mg/kg, i.p.) and (3-30 mg/kg, p.o.) caused inhibition of the neurogenic (first phase) and inflammatory phase (second phase) of formalin-induced pain. However, the CH2Cl2 extract was more effective in relation to the second phase than in inhibition of the formalin-induced edema. These findings suggest that CH2Cl2 extract has potent anti-inflammatory and analgesic action and that F-III and cucurbitacin B and E may account for these actions.     

Phytoalexins from hairy roots of Hyoscyamus albus treated with methyl jasmonate

The treatment of hairy roots of Hyoscyamus albus with copper sulfate (Cu2+) and methyl jasmonate (JAMe) produced several phytoalexins having the vetispyrane skeleton. Lubimin and solavetivone were isolated after treatment with Cu2+. Seven sesquiterpenoid phytoalexins were isolated from the culture medium after treatment with JAMe, including lubimin, solavetivone, 3-hydroxysolavetivone and four new compounds (1-4). Structures of the new compounds were elucidated to be (3R,4S,5R,7S,9R)-3-hydroxy-9-tigloyloxysolavetivone (1), (3R,4S,5R,7S,9R)-3-hydroxy-9-(3-methylbutenoyloxy)-solavetivone (2), (3R,4S,5R,7S,9R)-3-hydroxy-9-isobutanoyloxysolavetivone (3); and (3R,4S,5R,7S,9R)-3,9-dihydroxysolavetivone (4). The induction pattern of phytoalexins in hairy roots treated with JAMe was different in those treated with Cu2+, and co-treatment with JAMe and Cu2+ gave only solavetivone.     

Phenylethanoid glycosides from Digitalis purpurea and Penstemon linarioides with PKCalpha-inhibitory activity

In a continuation of our search for potential tumor inhibitors from plants, it was found that the CH2Cl2-MeOH (1:1) extracts from Digitalis purpurea and Penstemon linarioides both showed PKCalpha-inhibitory bioactivity. Bioassay-directed fractionation of the extract from D. purpurea yielded the new, weakly active phenylethanoid glycoside 2-(3-hydroxy-4-methoxy-phenyl)-ethyl-O-(alpha-L-rhamnosyl)-(1-->3) -O- (alpha-L-rhamnosyl)-(1-->6)-4-O-E-feruloyl-beta-D-glucopy ran oside (1) together with the four known compounds calceolarioside A (2), calceolarioside B (3), forsythiaside (4), and plantainoside D (5). The extract from P. linarioides yielded the three known glycosides leucosceptoside A (6), acteoside (7), and poliumoside (8), together with the iridoid plantarenaloside (9). All of the isolated compounds, except compound 9, showed inhibitory activity against PKCalpha with IC50 values (in microM) of 125 (1), 0.6 (2), 4.6 (3), 1.9 (4), 14.8 (5), 19.0 (6), 9.3 (7), and 24.4 (8).     

Cyclopropane-derived peptidomimetics. Design, synthesis, evaluation, and structure of novel HIV-1 protease inhibitors

Toward establishing the general efficacy of using trisubstituted cyclopropanes as peptide mimics to stabilize extended peptide structures, the cyclopropanes 20a-d were incorporated as replacements into 9-13, which are analogues of the known HIV-1 protease inhibitors 14 and 15. The syntheses of 20a-d commenced with the Rh2[5(S)-MEPY]4-catalyzed cyclization of the allylic diazoesters 16a-d to give the cyclopropyl lactones 17a-d in high enantiomeric excess. Opening of the lactone moiety using the Weinreb protocol and straightforward refunctionalization of the intermediate amides 18a-d gave 20a-d. A similar sequence of reactions was used to prepare the N-methyl-2-pyridyl analogue 28. Coupling of 20a-d and 28 with the known diamino diol 22 delivered 9-13. Pseudopeptides 9-12 were found to be competitive inhibitors of wild-type HIV-1 protease in biological assays having Kis of 0.31-0.35 nM for 9, 0.16-0.21 nM for 10, 0.47 nM for 11, and 0.17 nM for 12; these inhibitors were thus approximately equipotent to the known inhibitor 14(IC50 = 0.22 nM) from which they were derived. On the other hand 13 (Ki = 80 nM) was a weaker inhibitor than its analogue 15 (Ki = 0.11 nM). The solution structures of 9 and 10 were analyzed by NMR spectroscopy and simulated annealing procedures that included restraints derived from homo- and heteronuclear coupling constants and NOEs; because of the molecular symmetry of9 and 10, a special protocol to treat the NOE data was used. The final structure was checked by restrained and free molecular dynamic calculations using an explicit DMSO solvent box. The preferred solution conformations of 9 and 10 are extended structures that closely resemble the three-dimensional structure of 10 bound to HIV-1 protease as determined by X-ray crystallographic analysis of the complex. This work convincingly demonstrates that extended structures of peptides may be stabilized by the presence of substituted cyclopropanes that serve as peptide replacements. Moreover, the linear structure enforced in solution by the two cyclopropane rings in the pseudopeptides 9-12 appears to correspond closely to the biologically active conformation of the more flexible inhibitors 14 and 15. The present work, which is a combination of medicinal, structural, and quantum chemistry, thus clearly establishes that cyclopropanes may be used as structural constraints to reduce the flexibility of linear pseudopeptides and to help enforce the biologically active conformation of such ligands in solution.     

Synthesis and anti-HIV-1 activity of a series of 1-alkoxy-5-alkyl-6-(arylthio)uracils

A series of 1-alkoxy-5-alkyl-6-(arylthio)uracils was synthesized and tested for their ability to inhibit HIV-1 replication. Treatment of 2-alkyl-3,3-bis(methylthio)acryloyl chlorides (5a-e) with AgOCN in benzene followed by reaction of the resulting isocyanates 6a-e with an appropriate alkoxyamine gave N-alkoxy-N'-((2-alkyl-3,3-bis(methylthio)acryloyl)ureas (10a-z) in good to excellent yields. Cyclization of 10a-z in AcOH containing a catalytic amount of p-TsOH produced 1-alkoxy-5-alkyl-6-(methylthio)uracils (11a-z). Oxidation of 11a-z with 3-chloroperoxybenzoic acid in CH2Cl2 resulted in high yields of 1-alkoxy-5-alkyl-6-(methylsulfonyl)uracils (12a-x and 12z) and 1-(benzyloxy)-6-(methylsulfinyl)thymine (12y), which were subsequently reacted with an appropriate arenethiol in ethanolic NaOH solution to afford 1-alkoxy-5-alkyl-6-(arylthio)uracils (14-49). Substitution at the 3- and 5-positions of the C-6-(phenylthio) ring by two methyl groups significantly increased its original anti-HIV-1 activity (EC50: 6-((3,5-dimethylphenyl)thio)-5-isopropyl-1-propoxyuracil (18), 0.064 microM; 6-((3,5-dimethylphenyl)thio)-1-(3-hydroxypropoxy)-5-isopropyluracil++ + (23), 0.19 microM). Among the various alkoxy substituents at the N-1, the propoxy group was the most beneficial for improving the anti-HIV-1 activity. The 1-propoxy derivative 18 proved to be the most potent inhibitor of HIV-1 replication, followed by the 1-(3-hydroxypropoxy) derivative 23. Introduction of an isopropyl group at C-5 of the uracil base also remarkably enhanced the activity. When compound 18 was incubated with a rat liver homogenate preparation, no metabolite was observed, thus confirming the metabolic stability of the N-O bond in these 1-alkoxyuracils.     

Two acute monocytic leukemia (AML-M5a) cell lines (MOLM-13 and MOLM-14) with interclonal phenotypic heterogeneity showing MLL-AF9 fusion resulting from an occult chromosome insertion, ins(11;9)(q23;p22p23)

We describe two new human leukemia cell lines, MOLM-13 and MOLM-14, established from the peripheral blood of a patient at relapse of acute monocytic leukemia, FAB M5a, which had evolved from myelodysplastic syndrome (MDS). Both cell lines express monocyte-specific esterase (MSE) and MLL-AF9 fusion mRNA. Gene fusion is associated with a minute chromosomal insertion, ins(11;9)(q23;p22p23). MOLM-13 and MOLM-14 are the first cell lines with, and represent the third reported case of, MLL gene rearrangement arising via chromosomal insertion. Both cell lines carry trisomy 8 which was also present during the MDS phase, as well as the most frequent trisomies associated with t(9;11), ie, +6, +13, +19 variously present in different subclones. Despite having these features in common, differences in antigen expression were noted between the two cell lines: that of MOLM-13 being CD34+, CD13-, CD14-, CD15+, CD33+; whereas MOLM-14 was CD4+, CD13+, CD14+, CD15+, CD33+. Differentiation to macrophage-like morphology could be induced in both cell lines after stimulation with INF-gamma alone, or in combination with TNF-alpha, which treatment also induced or upregulated, expression of certain myelomonocyte-associated antigens, including CD13, CD14, CD15, CD64, CD65 and CD87. Together, these data confirm that both cell lines are likely to be novel in vitro models for studying monocytic differentiation and leukemogenesis.     

Soluble interleukin-2 receptor levels in lupus nephritis

Soluble interleukin-2 receptor (IL-2R) levels were measured and correlated prospectively with clinical, histologic and serologic findings over a 9-month period in 62 lupus patients. Initially, 39 patients had clinical nephritis and 23 patients did not have nephritis. The 62 lupus patients has significantly higher IL-2R than 15 normal controls, most of this difference attributable to patients with nephritis. During lupus nephritis flare 9 of 10 patients showed significant elevations of IL-2R while only 6 of the 10 patients showed either elevation of anti-DNA antibody or decrease in CH50. During disease remission or stable clinical activity changes in IL-2R levels paralleled changes in anti-DNA antibody and CH50. Nephritis patients with cellular proliferative histology had significantly higher IL-2R levels than those with membranous or mesangial nephropathy. IL-2R correlated strongly with histologic activity and chronicity indices, IgG and C3 deposition whereas anti-DNA antibody and CH50 levels did not. IL-2R levels did not correlate with serum creatinine suggesting that elevations of IL-2R were not simply due to decreased clearance. These observations suggest that serum IL-2R level is a useful marker of disease activity in lupus nephritis and may serve as a helpful adjunct in management of this disorder.     

Highly regio- and stereoselective acylboration, acylsilation, and acylstannation of allenes catalyzed by phosphine-free palladium complexes: an efficient route to a new class of 2-acylallylmetal reagents

A new method for the synthesis of substituted 2-acylallylmetal reagents in a highly regio- and stereoselective fashion involving a three-component assembly of allenes, acyl chlorides, and bimetallic reagents (B-B, Si-Si, and Sn-Sn) catalyzed by phosphine-free palladium complexes is described. Treatment of various allenes (CR(2)R(3)=C=CH(2)) with acyl chlorides (R(1)COCl) and bispinacolatodiboron in the presence of PdCl(2)(CH(3)CN)(2) in toluene at 80 degrees C gave 2-acylallylboronates in moderate to good yields. The acylsilation of allenes with acid chlorides and hexamethyldisilane (5) proceeded successfully in the presence of Pd(dba)(2) in CH(3)CN affording the corresponding allylsilanes (CR(2)R(3)=C(COR(1))CH(2)SiMe(3)) in good to moderate yields. Several chloroformates (R(4)OCOCl) also react with 1,1-dimethylallene (2a) and 5 to afford allylsilanes (CR(2)R(3)=C(COOR(4))CH(2)SiMe(3)) in 66-70% yields. Acylstannation of allenes could also be achieved by slow addition of hexabutylditin (10) to the reaction mixture of acyl chloride (or chloroformate) and allene 2a in CH(3)CN in the presence of Pd(dba)(2) at 60 degrees C; the corresponding 2-substituted allylstannanes were isolated in moderate to good yields. The above catalytic reactions are completely regioselective and highly stereoselective. A mechanism is proposed to account for the catalytic reactions and the stereochemistry.     

The identification of high and low risk groups for colorectal cancer using rectal mucosal crypt cell production rate (CCPR)

Rectal mucosal proliferation was measured in 116 individuals using the metaphase arrest technique crypt cell production rate (CCPR). CCPR was found to be significantly elevated in individuals with adenomas (n = 42, CCPR = 13 cc c-1h-1, range 7-25 Cl 10-15) compared with normals (n = 21, CCPR = 10 cc c-1h-1 range 5-24 Cl 7-11, Mann-Whitney P = 0.001 z = 3.2). Mucosal proliferation was increased among individuals who were undergoing adenoma follow up but in whom no further adenomas were found (n = 37 CCPR = 12 range 5-26 cc c-1h-1 Cl 10-14) compared to controls (Mann-Whitney P = 0.01 z = 2.4) Proliferation in vegetarians i.e. low risk (n = 16) was similar to controls. Measurement of proliferative indices in rectal mucosa by the stathmokinetic technique CCPR can discriminate between high and low risk groups for colorectal cancer.     

Enantioselectivity of some 1-(benzofuran-2-yl)-1-(1-H-imidazol-1-yl) alkanes as inhibitors of P450Arom

The low stereospecificity of the enantiomers of 1-[(benzofuran-2-yl)-4-chlorophenylmethyl]imidazole (6, R=H, R'=4'-Cl) and the corresponding 4-fluoro compound as inhibitors of aromatase (P450Arom) has been explored using 1-(5,7-dichlorobenzofuran-2-yl)-1-(1H-imidaz-1-yl)ethane (7, R1=R2=Cl, R=CH3), -propane (7, R1=R2=Cl, R=C2H5), and the corresponding 5,7-dibromo compounds resolved as their dibenzoyl-D (or -L) tartrates. Low enantioselectivity ratios of 4.8 (5,7-diCl) and 12.6 (5,7-diBr) were shown for the ethanes. The values for the corresponding propanes were 8.3 and 5.2, respectively, and for these compounds the stereoselectivity was reversed.     

Tachykinin peptides affect differently the second messenger pathways after binding to CHO-expressed human NK-1 receptors

The human NK-1 receptor transfected in Chinese hamster ovary (CHO) cells was studied with use of different tachykinin analogs: Substance P, [Pro9]SP, [Sar9, Met(O2)11]SP, [Gly9 psi (CH2CH2) Leu10]SP, Ac-Arg-septide, septide, [Gly9 psi (CH2CH2) Gly10]SP, NKA, [pGlu6]SP(6-11) and [Lys5]NKA(4-10). Binding experiments with [3H][Pro9]SP discriminated two classes of peptides with either high affinity (K iota in the nanomolar range) for the human NK-1 receptor or with low affinity (K iota in the micromolar range); this second group of peptides included NKA and [pGlu6]SP(6-11). In spite of these differences, both peptide families evoked potent stimulation of phosphatidylinositol hydrolysis (EC50 in the nanomolar range). In contrast, only NK-1 agonists, with high affinity, stimulated with great potency cyclic AMP formation (EC50 from 8 to 50 nM), whereas the second family of peptides were only weak agonists (EC50 in the micromolar range). RP 67580, CP 96345 and GR 94800, a NK-2 antagonist, were either competitive or uncompetitive inhibitors of inositol phosphates or cyclic AMP formations induced by [Pro9]SP, septide or NKA, independently of the agonist or the response studied. Thus, NKA, the presumed NK-2 endogenous peptide that may be co-released with SP, and the enzymatically produced C-terminal fragment of SP, [pGlu6]SP(6-11), may trigger specific pharmacological responses via the NK-1 receptor, at nanomolar concentrations, and thus regulate the action of SP at the NK-1 receptor.     

Investigations on the synthesis and properties of N-phenyl derivatives of 1,4-dioxo(1,4,5-trioxo)-1,2,3,4-tetra(1,2,3,4,5,6-hexa) hydropyrido-[3,4-d]pyridazines and allied compounds

2-(1-Piperidino)- and 2-(4-methyl-1-piperazinyl)-6-methyl-3,4-pyridinedicarboximides (1, 2) react with N-phenylhydrazine yielding N-phenylamino-3,4-pyridinedicarboximides (7, 8). The same reaction with 1,6-dimethyl-2-oxo-1,2-dihydro- and 2-chloro-6-methyl-3,4-pyridinedicarboximides (3, 17) gives the salts of the corresponding N-phenylpyridopyridazines with phenylhydrazine (13, 18), which transform into N-phenylaminoimides (14, 19) during boiling in 80% acetic acid. Compounds 7, 8 and 14 isomerize to the corresponding 2-phenyl-1,4-dioxo(1,4,5-trioxo)-1,2,3,4-tetra(1,2,3,4,5,6-hexa) hydropyrido[3,4-d]pyridazines (9, 10, 15) under the influence of heating in alcoholic solution of C2H5ONa or CH3ONa. Only in the case of imide 19 are 2- and 3-phenyl isomers (20 and 21) formed under these conditions. Some of the obtained compounds were pharmacologically active.     

Identification of novel metabolites of butadiene monoepoxide in rats and mice

Differences in the metabolism of 1,3-butadiene (Bd) in rats and mice may account for the observed species difference in carcinogenicity. Previous studies of the metabolic fate of Bd have identified epoxide formation as a key metabolic transformation which gives 1, 2-epoxy-3-butene (BMO), although some evidence of aldehyde metabolites is reported. In this study, male Sprague-Dawley rats and male B6C3F1 mice received single doses of [4-14C]BMO at 1, 5, 20, and 50 mg/kg of body weight (0.014, 0.071, 0.286, and 0.714 mmol/kg of body weight). Analysis of urinary metabolites indicated that both species preferentially metabolize BMO by direct reaction with GSH when given by ip administration. The excretion of (R)-2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene (IIa), 1-(N-acetyl-L-cystein-S-yl)-2-(S)-hydroxybut-3-ene (IIb), 1-(N-acetyl-L-cystein-S-yl)-2-(R)-hydroxybut-3-ene (IIc), and (S)-2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene (IId) accounted for 48-64% of urinary radioactivity in rats and 46-54% in mice. The metabolites originating from the R-stereoisomer of BMO (IIc and IId) predominated over those arising from the S-stereoisomer (IIa and IIb) in both species. IIc was formed preferentially in mice and IId in rats. The corresponding mercaptoacetic acids, S-(1-hydroxybut-3-en-2-yl)mercaptoacetic acid (IIf) and S-(2-hydroxybut-3-en-1-yl)mercaptoacetic acid (IIg), were identified only in mouse urine (ca. 20% of the recovered radioactivity). 4-(N-Acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (Ia), a metabolite derived from hydrolysis of BMO, accounted for 10-17% of the radioactivity in rat and 6-10% in mouse urine. 4-(N-Acetyl-L-cystein-S-yl)-2-hydroxybutanoic acid (Ib), 3-(N-acetyl-L-cystein-S-yl)propan-1-ol (Ic), and 3-(N-acetyl-L-cystein-S-yl)propanoic acid (Id), also derived from the hydrolysis of BMO, were only present in the rat. Metabolites of 1,2,3,4-diepoxybutane (DEB) were not detected after administration of BMO in rat or mouse urine. This study showed both quantitative and qualitative differences in the metabolism of BMO with varying doses and between species. The data aid in the safety evaluation of Bd and contribute to the interpretation of mathematical models developed for quantitative risk assessment and extrapolation of animals to humans.     

Synthesis and biological activity of 3- and 5-amino derivatives of pyridine-2-carboxaldehyde thiosemicarbazone

A series of 3- and 5-alkylamino derivatives, as well as other structurally modified analogues of pyridine-2-carboxaldehyde thiosemicarbazone, have been synthesized and evaluated as inhibitors of CDP reductase activity and for their cytotoxicity in vitro and antineoplastic activity in vivo against the L1210 leukemia. Alkylation of 3- and 5-amino-2-(1,3-dioxolan-2-yl)pyridines (1, 2) resulted in corresponding 3-methylamino, 5-methylamino, 3-allylamino, 5-ethylamino, 5-allylamino, 5-propylamino, and 5-butylamino derivatives (5, 6, and 11-15), which were then condensed with thiosemicarbazide to yield the respective thiosemicarbazones (7, 8, and 16-20). Oxidation of 3,5-dinitro-2-methylpyridine (21) with selenium dioxide, followed by treatment with ethylene glycol and p-toluenesulfonic acid, produced the cyclic ethylene acetal, 23. Oxidation of 2-(1,3-dioxolan-2-yl)-4-methyl-5-nitropyridine (26) with selenium dioxide, followed by sequential treatment with sodium borohydride, methanesulfonyl chloride, and morpholine afforded the morpholinomethyl derivative 30. Catalytic hydrogenation of 23 and 30 with Pd/C yielded the corresponding amino derivatives 24 and 31. Catalytic hydrogenation of 5-cyano-2-methylpyridine (33) with Raney nickel, followed by treatment with acetic anhydride, gave the amide derivative 35. N-Oxidation of 35, followed by rearrangement with acetic anhydride, produced the acetate derivative, 5-[(acetylamino)methyl]-2-(acetoxymethyl)pyridine (37). Repetition of the N-oxidation and rearrangement procedures with compound 37 yielded the diacetate derivative 39. Condensation of compounds 24, 31, and 39 with thiosemicarbazide afforded the respective 3,5-diaminopyridine-, 4-(4-morpholinylmethyl)-5-aminopyridine-, and 5-(aminomethyl)pyridine-2-carboxaldehyde thiosemicarbazones (25, 32, and 40). The most biologically active compounds synthesized were the 5-(methylamino)-, 5-(ethylamino)-, and 5-(allylamino)pyridine-2-carboxaldehyde thiosemicarbazones (8, 17, and 18), which were potent inhibitors of ribonucleotide reductase activity with corresponding IC50 values of 1.3, 1.0, and 1.4 microM and which produced significant prolongation of the survival time of L1210 leukemia-bearing mice, with corresponding optimum % T/C values of 223, 204, and 215 being obtained when administered twice daily for six consecutive days at dosages of 60, 80, and 80 mg/kg, respectively.     

A pathway for biosynthesis of divinyl ether fatty acids in green leaves

[1-14C]alpha-Linolenic acid was incubated with a particulate fraction of homogenate of leaves of the meadow buttercup (Ranunculus acris L.). The main product was a divinyl ether fatty acid, which was identified as 12-[1'(Z),3'(Z)-hexadienyloxy]-9(Z),11(E)-dodecadienoic acid. Addition of glutathione peroxidase and reduced glutathione to incubations of alpha-linolenic acid almost completely suppressed formation of the divinyl ether acid and resulted in the appearance of 13(S)-hydroxy-9(Z), 11(E),15(Z)-octadecatrienoic acid as the main product. This result, together with the finding that 13(S)-hydroperoxy-9(Z), 11(E),15(Z)-octadecatrienoic acid served as an efficient precursor of the divinyl ether fatty acid, indicated that divinyl ether biosynthesis in leaves of R. acris occurred by a two-step pathway involving an omega6-lipoxygenase and a divinyl ether synthase. Incubations of isomeric hydroperoxides derived from alpha-linolenic and linoleic acids with the enzyme preparation from R. acris showed that 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid was transformed into the divinyl ether 12-[1'(Z)-hexenyloxy]-9(Z), 11(E)-dodecadienoic acid. In contrast, neither the 9(S)-hydroperoxides of linoleic or alpha-linolenic acids nor the 13(R)-hydroperoxide of alpha-linolenic acid served as precursors of divinyl ethers.     

Human growth hormone-releasing hormone hGHRH(1-29)-NH2: systematic structure-activity relationship studies

Two complete and two partial structure-activity relationship scans of the active fragment of human growth hormone-releasing hormone, [Nle27]-hGHRH(1-29)-NH2, have identified potent agonists in vitro. Single-point replacement of each amino acid by alanine led to the identification of [Ala8]-, [Ala9]-, [Ala15]- (Felix et al. Peptides 1986 1986, 481), [Ala22]-, and [Ala28, Nle27]-hGHRH(1-29)-NH2 as being 2-6 times more potent than hGHRH(1-40)-OH (standard) in vitro. Nearly complete loss of potency was seen for [Ala1], [Ala3], [Ala5], [Ala6], [Ala10], [Ala11], [Ala13], [Ala14], and [Ala23], whereas [Ala16], [Ala18], [Ala24], [Ala25], [Ala26], and [Ala29] yielded equipotent analogues and [Ala7], [Ala12], [Ala17], [Ala20], [Ala21], and [Ala27] gave weak agonists with potencies 15-40% that of the standard. The multiple-alanine-substituted peptides [MeTyr1,Ala15,22,Nle27]-hGHRH(1-29)-NH2 (29) and [MeTyr1,Ala8,9,15,22,28,Nle 27]-hGHRH(1-29)-NH2 (30) released growth hormone 26 and 11 times, respectively, more effectively than the standard in vitro. Individual substitution of the nine most potent peptides identified from the Ala series with the helix promoter alpha-aminoisobutyric acid (Aib) produced similar results, except for [Aib8] (doubling vs [Ala8]), [Aib9] (having vs [Ala9]), and [Aib15] (10-fold decrease vs [Ala15]). A series of cyclic analogues was synthesized having the general formula cyclo(25-29)[MeTyr1,-Ala15,Xaa25,Nle27,Yaa29+ ++]-GHRH(1-29)-NH2, where Xaa and Yaa represent the bridgehead residues of a side-chain cystine or [i-(i + 4)] lactam ring. The ring size, bridgehead amino acid chirality, and side-chain amide bond location were varied in this partial series in an attempt to maximize potency. Application of lactam constraints in the C-terminus of GHRH(1-29)-NH2 identified cyclo(25-29)[MeTyr1,Ala15,DAsp25,Nle27,Orn29+ ++]-hGHRH(1-29)-NH2 (46) as containing the optimum bridging element (19-membered ring) in this region of the molecule. This analogue (46) was 17 times more potent than the standard. Equally effective was an [i-(i + 3)] constraint yielding the 18-membered ring cyclo(25-28)[MeTyr1,Ala15,Glu25,Nle,27Lys28]- hGHRH-(1-29)-NH2 (51) which was 14 times more potent than the standard. A complete [i-(i + 3)] scan of cyclo(i,i + 3)[MeTyr1,Ala15,Glui,Lys(i + 3),Nle27]-hGHRH(1-29)-NH2 was then produced in order to test the effects of a Glu-to-Lys lactam bridge at all points in the peptide. Of the 26 analogues in the series, 11 had diminished potencies of less than 10% that of the agonist standard, 4 were weak agonists (15-40% relative potency), and 4 analogues were equipotent to the standard. The 7 most potent analogues ranged in potency from 3 to 14 times greater than that of the standard and contained the [i-(i + 3)] cycles between residues 4-7, 5-8, 9-12, 16-19, 21-24, 22-25, and 25-28. The combined results from these systematic studies allowed for an analysis of structural features in the native peptide that are important for receptor activation. Reinforcement of the characteristics of amphiphilicity, helicity, and peptide dipolar effects, using recognized medicinal chemistry approaches including introduction of conformational constraints, has resulted in several potent GHRH analogues.