Diethyl Phosphorocyanidate Research Articles

Synthesis and biological activity of the 2-desamino and 2-desamino-2-methyl analogs of aminopterin and methotrexate

The previously undescribed 2-desamino and 2-desamino-2-methyl analogues of aminopterin (AMT) and methotrexate (MTX) were synthesized from 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile. The AMT analogues were obtained via a three-step sequence consisting of condensation with di-tert-butyl N-(4-aminobenzoyl)-L-glutamate, heating with formamidine or acetamidine acetate, and mild acidolysis with trifluoroacetic acid. The MTX analogues were prepared similarly, except that 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile was condensed with 4-(N-methylamino)benzoic acid and the resulting product was annulated with formamidine or acetamidine acetate to obtain the 2-desamino and 2-desamino-2-methyl analogues, respectively, of 4-amino-4-deoxy-N10-methylpteroic acid. Condensation with di-tert-butyl L-glutamate in the presence of diethyl phosphorocyanidate followed by ester cleavage with trifluoroacetic acid was then carried out. Retention of the L configuration in the glutamate moiety during this synthesis was demonstrated by rapid and essentially complete hydrolysis with carboxypeptidase G1 under conditions that likewise cleaved the L enantiomer of MTX but left the D enantiomer unaffected. The 2-desamino and 2-desamino-2-methyl analogues of AMT and MTX inhibited the growth of tumor cells, but were very poor inhibitors of dihydrofolate reductase (DHFR). These unexpected results suggested that activity in intact cells was due to metabolism of the 2-desamino compounds to polyglutamates.

Resolution of amine enantiomers using precolumn derivatization with dansyl- L-proline and reversed-phase liquid chromatography

A derivatization procedure was developed for converting enantiomeric amino acids, amino alcohols and amines into diastereoisomers for resolution by liquid chromatography. Dansyl- L-proline (DLP) was used as a chiral reagent for the precolumn derivatization of many enantiomeric compounds bearing primary and secondary amino groups. The diastereoisomeric amides that were formed in the presence of diethyl phosphorocyanidate can be resolved efficiently on a conventional reversed-phase column. The resolution was optimized by varying the mobile phase. Intramolecular hydrogen bonding of the diastereoisomeric amide is shown to be very important for the efficient resolution of enantiomers. The detection limits for DLP derivatives were 4 × 10 −14−5 × 10 −14 mol.

Rapid determination by high-performance liquid chromatography of free fatty acids released from rat platelets after derivatization with monodansylcadaverine

The release of free fatty acids from rat platelets, triggered by thrombin stimulation, was monitored by high-performance liquid chromatography (HPLC) after precolumn derivatization with monodansylcadaverine (MDC). A rapid filtration procedure was devised for the precise determination of free fatty acids released from aggregated platelets, instead of the conventional method using a stop solution or enzyme reactions. The fatty acids thus collected were derivatized with MDC in the presence of diethyl phosphorocyanidate (DEPC). The simultaneous separation of MDC derivatives of fatty acids was achieved on a reversed-phase TSKgel ODS-80 TM column within 60 min by linear gradient elution, using 0.2 M Tris-HCl buffer (pH 7.8)-methanol (50:50, v/v) and acetonitrile. The MDC derivatives were detected with excitation and emission wavelengths of 340 and 518 nm, respectively. The amounts of liberated fatty acids were in the range from 45.0 pmol for myristoleic acid (C 14:1) to 395.0 pmol for palmitic acid (C 16:0) per 1.9 · 10 7 platelets.

Fluorometric Determination of Carboxylic Acids by High Performance Liquid Chromatography after Derivatization with Monodansyl Cadaverine

Free fatty acids were determined by high performance liquid chromatography after derivatization with monodansyl Cadaverine (MDC) at room temperature for 15min in the presence of diethyl phosphorocyanidate as a coupling reagent. The fluorescence properties (λex340nm, λem 518nm) of MDC-arachidic acid (C20:0) were identical to those of dansyl alanine. The MDC derivatives of saturated and unsaturated fatty acids were completely separated within 50min by gradient elution on a TSK-gel 80TM ODS column. When fluorometrically detected, the detection limits (S/N=3) of fatty acid were below 100fmol. The calibration curves passed through the origin and gave good linearity in the range from 5 to 200pmol. The method was successfully applied to the determination of fatty acids in the rabbit plasma. This method would be quite useful for the analysis of various biological samples for thermolabile carboxylic acids, since it requires no heating procedure.

Solubility and Coupling Reactivity of Protected Peptides in Highly Polar Solvents

Abstract The solubility of protected peptides as well as the coupling rate was quantitatively examined in highly polar solvents. Symmetrical anhydrides in dimethyl sulfoxide (DMSO) containing N,N-dimethylformamide (DMF) or DMF solution of lithium chloride and diethyl phosphorocyanidate with triethylamine in DMSO–DMF were proved to be efficient coupling reagents for solid phase synthesis of hardly soluble peptides.

Total synthesis of avellanins A and B, new fungal metabolites from Hamigera avellanea with pressor effect.

Avellanins A and B (1 and 2), new pressor-active fungal metabolites from Hamigera avellanea, have been conveniently synthesized from methyl anthranilate. This synthetic study has established the absolute stereostructure of avellanin B (2). Coupling of methyl anthranilate with N-protected amino acid derivatives was best carried out by use of ((9-fluorenylmethyl)oxy)carbonyl (Fmoc)-amino acid chloride. Chain elongation for avellanins was accomplished with diethyl phosphorocyanidate, while cyclization was achieved with diphenyl phosphorazidate.

ChemInform Abstract: Syntheses of Cytotoxic Cyclic Peptides of Marine Origin

Lipophilic cyclic peptides from marine invertebrates constitute a growing class of naturally occurring antineoplastic and/or cytotoxic substances. These peptides commonly contain two kinds of unusual amino acids, thiazole and oxazoline amino acids, as their constituents. Our continuing efforts on peptide synthesis using two organophosphorus coupling reagents, diphenyl phosphorazidate (DPPA, (C6H5O) 2P (O) N3) and diethyl phosphorocyanidate (DEPC, (C2H5O) 2P (O) CN), have achieved the syntheses of dolastatin 3 (the proposed structure), ascidiacyclamide, patellamides A, B, and C, ulithiacyclamide, and ulicyclamide. Cytotoxic activity of all of synthesized cyclic peptides and their derivatives was investigated using L1210 murine leukemia cells in culture. Ulithiacyclamide having the oxazoline and disulfide functions showed marked cytotoxicity, ID50=0.04 μg/ml.

Exploitation of new synthetic reagents and their application to the total synthesis of biologically active natural products

Diphenyl phosphorazidate (DPPA), diethyl phosphorocyanidate (DEPC), and trimethylsilyldiazomethane (TMSCHN2) have been introduced as versatile reagents for organic synthesis. Total syntheses of several biologically active natural products containing various different carbon skeletons have been achieved by use of DPPA, DEPC, and reactions developed by our group. Cytotoxic and/or antineoplastic cyclic peptides 1-7 of marine origin have been prepared using DPPA and DEPC as peptide coupling reagents and their cytotoxic activities against L 1210 murine leukemia cells have been explored. Preferred conformation of ascidiacyclamide (4), a representative of cytotoxic cyclic peptides, has been determined by X-ray crystallography and it has been proven that ascidiacyclamide (4) has no ionophoric activity toward alkali metal ions. Synthesis of dolastatin 3 bearing the revised structure 2 has been described. Cytotoxic depsipeptides from a tunicate, didemnins A (11c), B (12c), and their relatives (11b and 12b), have been efficiently prepared by condensation of the key eastern (13) and western (14b and 14a) fragments. The absolute configuration of avellanin B (26b), a fungal cyclic peptide exhibiting a pressor effect, has been determined by its synthesis. Its analog, avellanin A (26a), has been also efficiently prepared. Synthesis of AI-77-B (27), which was isolated from Baccilus pumilus AI-77 and has an interesting antiulcer activity, has been stereoselectively achieved starting from the 6-methylsalicylic acid ester (30), the L-leucinal derivative (31), and D-pyroglutamic acid (36).

シアノヒドリンＯ‐ジエチルりん酸エステル類の有機合成への応用

Diethyl phosphorocyanidate (DEPC) has been extensively utilized as a versatile reagent for cyanation under mild and non-aqueous reaction conditions. This article describes a new method for the synthesis of cyanophosphates by reaction of DEPC with various kinds of carbonyl compounds in the presence of lithium cyanide, and its application in organic synthesis is devided into four items : 1) synthesis of α, β-unsaturated nitriles and arylacetonitriles from aryl ketones via cyanophosphates, 2) synthesis of 3-aryl-4-hydroxybenzonitriles from p-benzoquinone cyanophosphates with aromatic and heteroaromatic compounds, 3) boron trifluoride etherate catalyzed allylic rearrangement of enone cyanophosphates and its application to β-cyanoenones, 4) 2-cyanoindole-3-acetonitriles from 3-indolecarbaldehydes via cyanophosphates and its application to carbazole derivatives by cycloaddition with activated alkynes.

ChemInform Abstract: A New and Facile Synthesis of 2‐Alkenenitriles Using 3‐Buten‐2‐one Cyanohydrin Diethyl Phosphate. Application to (.+‐.)‐(E)‐Nuciferol Synthesis.

AbstractThe diethyl phosphate (I), derived from diethyl phosphorocyanidate and methyl vinyl ketone in the presence of lithium cyanamide, reacts with the organocopper compounds (II) to yield the γ‐coupling products (IIIa).

Synthesis of the 7-Hydroxy Metabolites of Methotrexate and 10-Ethyl-10-Deazaaminopterin

The potent folate antagonists methotrexate and 10-ethyl-10-deazaaminopterin are oxidized in vivo to their 7-hydroxy derivatives. A specific cyanation at the C-7 position of the pteridine ring system using diethyl phosphorocyanidate converted the dimethyl esters of methotrexate and 10-ethyl-10-deazaaminopterin to the corresponding 7-cyano dimethyl ester derivatives, which were hydrolyzed to the 7-hydroxy metabolites.

ChemInform Abstract: Synthesis and Cycloaddition Reaction of 2‐Cyano‐3‐indoleacetonitriles.

AbstractThe formylindoles (I) and the ketones (VII) react with diethyl phosphorocyanidate (II), producing the compounds (III), (V), (VIII), or (IX).

ChemInform Abstract: Allylic Rearrangement of Cyanophosphates. Part 3. Reaction of Acyclic Enone Cyanophosphates.

AbstractThe vinylic ketones (I) and the aldehydes (XIV) are cyanophosphorylated with diethyl phosphorocyanidate (II), producing the allylic phosphates (III) or (XV).

Regiospecific arylation of 1,4-benzoquinone cyanohydrin phosphate: synthesis of 3-aryl-4-hydroxybenzonitriles

Reaction of 1,4-benzoquinones (1a,b) with diethyl phosphorocyanidate and lithium cyanide was found to give the monocyanophosphates (2a,b). When catalysed by BF3·OEt2, the cyanophosphates (2a,b) reacted with aromatic and heteroaromatic compounds (indole, thiophene, and furan derivatives) to give 3-aryl-4-hydroxybenzonitriles. Under the same conditions ethyl 1-methylindole-2-carboxylate and 2,5-dimethylfuran were converted into 6-oxobenzopyrano[3,4-b]indole-2carbonitriles (15a,b) and benzofuran-5-carbonitriles (22a,b) by reaction with the cyanophosphates (2a,b), respectively.

海洋産抗腫よう活性環状ペプチドの合成研究

Lipophilic cyclic peptides from marine invertebrates constitute a growing class of naturally occurring antineoplastic and/or cytotoxic substances. These peptides commonly contain two kinds of unusual amino acids, thiazole and oxazoline amino acids, as their constituents. Our continuing efforts on peptide synthesis using two organophosphorus coupling reagents, diphenyl phosphorazidate (DPPA, (C6H5O) 2P (O) N3) and diethyl phosphorocyanidate (DEPC, (C2H5O) 2P (O) CN), have achieved the syntheses of dolastatin 3 (the proposed structure), ascidiacyclamide, patellamides A, B, and C, ulithiacyclamide, and ulicyclamide. Cytotoxic activity of all of synthesized cyclic peptides and their derivatives was investigated using L1210 murine leukemia cells in culture. Ulithiacyclamide having the oxazoline and disulfide functions showed marked cytotoxicity, ID50=0.04 μg/ml.

A facile synthesis of ulicyclamide

Ulicyclamide ( 1), a cytotoxic cyclic peptide from a marine tunicate, has been efficiently synthesized by the solid-phase method using diethyl phosphorocyanidate for the coupling and trimethylsilyl triflate for the final deprotection.

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.

ChemInform Abstract: Reaction of 3‐Acylindoles with Diethyl Phosphorocyanidate. A Facile Synthesis of 2‐Cyano‐3‐indole‐acetonitriles.

Abstract3‐Acetylindol (I) liefert mit dem Phosphorcyanidat (II) und LiCN das Indol‐Derivat (III) neben wenig (IV) und (V).

Synthesis and cycloaddition reaction of 2-cyano-3-indoleacetonitriles.

The reactions of 3-acylindoles [3-indolecarbaldehyde (1), 3-acetylindole (12), 3-benzoylindole (13), and N-substituted 3-indolecarbaldehydes (20a-d)] with diethyl phosphorocyanidate (DEPC) in the presence of lithium cyanide (LiCN) are described. Treatment of 1 with DEPC and LiCN gave a mixture of (E)- and (Z)-3-cyanomethylene-1-diethylphosphono-2-hydroxyindolines (2 and 3). On the other hand, reaction of 12, 13 and 20a-d with DEPC and LiCN afforded 2-cyano-3-indoleacetonitrile derivatives (16, 19 and 21a-d). Strong base-induced cycloaddition reactions of 2-cyano-1-methyl-3-indoleacetonitrile (21a) with carbon-carbon triple bonds were carried out and gave the corresponding condensation products, 1-amino-4-cyanocarbazoles (22a-e), 6-amino-11-cyanobenzo[b]carbazole (23), pyrido[4, 3-b]carbazole (25), and pyrido[3, 4-b]carbazole (26), in moderate yields.

Total synthesis of ulithiacyclamide, a strong cytotoxic cyclic peptide from marine tunicates

Total synthesis of ulithiacyclamide, a strong cytotoxic cyclic peptide from marine tunicates, has been achieved by the use of diphenyl phosphorazidate(DPPA) and diethyl phosphorocyanidate(DEPC). Two different cyclization processes were applied to the construction of this unique cyclic peptide having a disulfide bridge.