ONE POT SYNTHESIS OF ARYL THIOPHOSPHORAMIDATE DERIVATIVES OF AZT

ABSTRACT

Novel aryl thiophosphoramidate derivatives of the anti-HIV nucleoside analogue 3′-azido-3′-deoxythymidine (AZT) have been prepared by the thiophosphorochloridate chemistry. These materials were designed to act as membrane-soluble prodrugs of the bioactive free nucleotides.     

Design and synthesis of the 3′-aminocarbonylamino,aminothiocarbonylamino and N-(Hydroxy)guanidinyl derivatives of thymidine as potential anti-HIV agents

The 3′-substituted-3′-deoxythymidines 3a (urea), 3b (thiourea) and 3c [N-(hydroxy)guanidinyl)] were designed based on the known structure-activity correlations for the active anti-HIV agent, 3′-azido-3′-deoxythymidine (AZT). Hydrolysis of 3′-cyanamido-3′-deoxythymidine ( 2 ) with ammonium hydroxide in the presence of hydrogen peroxide afforded the 3′-urea analogue 3a , whereas reaction with hydrogen sulfide in methanol gave the 3′-thiourea derivative 3b. The 3′-[N-(hydroxy)guanidinyl] compound 3c was synthesized by reaction of the 3′-cyanamide 2 with hydroxylamine.     

Synthesis of Novel Bithiophene-Substituted Heterocycles Bearing Carbonitrile Groups

Symmetrical and unsymmetrical bithiophene-substituted heterocycles bearing carbonitriles including imidazo[1,2-a]pyridine, benzimidazole, and pyridine derivatives have been synthesized via different synthetic protocols. The bithiophene bis-imidazo[1,2-a]pyridine derivatives 3a,b were achieved in three steps starting from 2-acetyl-5-bromothiophene. Suzuki coupling reaction of 2a with 5-formylthiophen-2-ylboronic acid forms the formyl derivative 5, which by condensation with 3,4-diaminobenzonitrile in the presence of sodium bisulfite furnishes the unsymmetrical bithiophene derivative6. The bis-benzimidazole derivative 8 was obtained via hexabutylditin-mediated homocoupling of 5-bromothiophene-2-carboxaldehyde, while the benzimidazole derivatives 12a,b were prepared via the formyl derivatives 11a,b, a product of Velsmier formylation reaction of 10a,b. Two synthetic protocols for the aryl/hetaryl-2,2′-bithiophene derivative 14 have also been presented. In addition, the guanyl hydrazones of bithiophenes, 16 and 17, were prepared from bis(tri-n-butylstannyl)-2,2′-bithiophene through a Stille coupling reaction followed by a condensation step.     

Derivatives of 3′-Azidothymidine with 6-Cyanopyridone as Base or as Phosphoramidate Ester and their Antiretroviral Activity

Strongly pairing ethynylpyridone C-nucleosides are attractive surrogates for thymidine in oligonucleotides. Exploratory work on the antiviral activity of 3′-azidothymidine (AZT) derivatives with ethynylpyridone as base had identified strong lipophilicity as a limiting factor. Two strategies are being pursued to overcome this issue. In order to make the base more polar, the ethynyl group has been replaced with a cyano group, leading to a cyanopyridone C-nucleoside, whose eleven-step synthesis is reported here, together with the synthesis of a 3′-azido-2′,3′-dideoxynucleoside derivative. The base pairing with adenine in a DNA duplex was studied by UV melting analysis of a self-complementary hexamer containing the 6-cyano-2′-deoxynucleoside instead of thymidine. A melting point increase of 2 °C compared to the unmodified control was found. The other strategy employs a phosphoramidate prodrug design with less lipophilic amino acid esters. Here, anti-HIV test of the alaninyl and prolinyl methyl esters of AZT gave promising results in cell culture experiments, increasing the selectivity index up to 5.8-fold for the III_B strain and up to 5-fold for the ROD strain of the virus, as compared to the parent nucleoside. These findings help to design the next generation of pyridone C-nucleosides with potential applications as antivirals.     

Synthesis of (5′S)‐5′‐C‐Alkyl‐2′‐deoxynucleosides

We describe the synthesis of (5′S)‐5′‐C‐butylthymidine ( 5a ), of the (5′S)‐5′‐C‐butyl‐ and the (5′S)‐5′‐C‐isopentyl derivatives 16a and 16b of 2′‐deoxy‐5‐methylcytidine, as well as of the corresponding cyanoethyl phosphoramidites 9a , b and 14a , b , respectively. Starting from thymidin‐5′‐al 1 , the alkyl chain at C(5′) is introduced via Wittig chemistry to selectively yield the (Z)‐olefin derivatives 3a and 3b (Scheme 2). The secondary OH function at C(5′) is then introduced by epoxidation followed by regioselective reduction of the epoxy derivatives 4a and 4b with diisobutylaluminium hydride. In the latter step, a kinetic resolution of the diastereoisomer mixture 4a and 4b occurs, yielding the alkylated nucleoside 2a and 2b , respectively, with (5′S)‐configuration in high diastereoisomer purity (de=94%). The corresponding 2′‐deoxy‐5‐methylcytidine derivatives are obtained from the protected 5′‐alkylated thymidine derivatives 7a and 7b via known base interconversion processes in excellent yields (Scheme 3). Application of the same strategy to the purine nucleoside 2′‐deoxyadenine to obtain 5′‐C‐butyl‐2′‐deoxyadenosine 25 proved to be difficult due to the sensitivity of the purine base to hydride‐based reducing agents (Scheme 4).     

Cyclocondensation of 3-amino-2-iminonaphtho[1,2-d]-thiazole with α-ketocarboxylic acid derivatives: Synthesis of 2-substituted 3-Oxo-3H-naphtho[1′,2′:4,5]thiazolo-[3,2-b][1,2,4]triazines as potential anti-HIV agents

The cyclocondensation of 3-amino-2-iminonaphtho[1,2-d]thiazole ( 1 ) with a series of α-keto mono- and dicarboxylic acid derivatives 5a-i under different conditions was investigated. When the experiments were performed by refluxing in glacial acetic acid, the corresponding cyclized products, 2-substituted 3-oxo-3H-naphtho[1′,2′:4,5]thiazolo[3,2-b][1,2,4]triazines 4 were obtained in fair to good yields. On the other hand, when the reaction was conducted in boiling ethanol, it gave only the open chain condensates 6 , or in rare cases, together with minor amount of 4 . Since the intermediates 6 exist as mixture of both E- and Z-isomers, cyclization through heating was found insufficient. In any event representative compounds 4b,g,i have been evaluated for anti-HIV activity, but none of them were active.     

Synthesis of representative 10-aryl-, 10-aralkyl- and 10-heteraryl-9H-naphtho[1′,2′:4,5]thiazolo[3,2-b]- [1,2,4]triazin-9-ones as potential anti-HIV agents

To prepare the title compounds, cyclocondensation of 1-amino-2-iminonaphtho[1,2-d]thiazole ( 2 ) with some representative glyoxylic acid derivatives was investigated. Heating 2 with methyl phenylglyoxylate ( 3a ) in methanol afforded only the open chain intermediates 4a,b . However, when this reaction was performed in re-fluxing glacial acetic acid, the expected compound, 10-phenyl-9H-naphtho[1′,2′:4,5]thiazolo[3,2-b][1,2,4]- triazin-9-one ( 5a ) was produced in 27% yield. Similar treatment of 2 with benzyl-, 2-furyl- and 2-thienylgly-oxylic acids 3b-d gave the corresponding 10-benzyl-, 10-(2-furyl)- and 10-(2-thienyl)-9H-naphtho[1′,2′:4,5]thi-azolo[3,2-b][1,2,4]triazin-9-ones 5b-d in 48–67% yields. As by-products, 9-benzoyl- and 9-(2-thenoyl)naphtho-[1′,2′:4,5]thiazolo[3,2-b][1,2,4]triazoles 6a,d were also isolated. Compound 5a was selected for in vitro anti-HIV evaluation but found to be inactive.     

Influence of Chelating Groups on the Luminescence Properties of Europium(III) and Terbium(III) chelates in the 2,2′-bipyridine series

Eight different 2,2′-bipyridine derivatives, i.e. 2, 5, 8, 10, 12, 13, 15 , and 19 (Schemes 1 and 2), were prepared to study the influence of the chelating groups on the luminescence properties of their Eu^III and Tb^III chelates. According to our luminescence results, 2,2′-(methylenenitrilo)bis(acetic acid) as well as (methylenenitrilo)bis-(methylphosphonic acid) in 6- and 6′-position of 2,2′-bipyridine is a suitable group when developing luminescent markers for bioaffinity assays based on time-resolved luminescence measurement.     

Introduction of C-Sulfonate Groups into Disaccharide Derivatives

To prepare C-sulfonate derivatives of disaccharides two different strategies were followed. Thus 6- and 6′-C-sulfocellobiosides 4 and 10–12 were prepared starting from a suitably protected cellobioside. The 6′-C-sulfoaminocellobioside 18 was prepared by construction of the molecule through a glycosylation reaction. In both cases, the synthetic pathway involves regioselective tosylation, introduction of a sulfur atom by nucleophilic displacement with potassium thioacetate and oxidation with hydrogen peroxide.     

Nucleotide,XVIII. Synthese und Eigenschaften von (tert-Butyldimethylsilyl)guanosinen,Guanosin-3′-phosphotriestern und Guanosin-haltigen Oligoncleotiden

Nucleotides, XVIII. Synthesis and Properties of (tert-Butyldimethylsilyl)guanosines, Guanosine-3′-Phosphotriesters and Guanosine-containing Oligonucleotides Silylation of N²-benzoyl- (1) and N²-isobutyrylguanosin (2) by tert-butyldimethylsilyl chloride led to the various mono-, di- and tri-O-tert-butyldimethylsilyl derivatives 3–15 . The synthesis of 2′- (24–31) and 3′-phosphotriesters ( 16–23 and 32 ) could be achieved by phosphorylations of partially protected guanosines. The guanosine-3′-phosphodiester 33 and the 5′-OH-guanosine-3′-phosphotriester 34 are used in condensation reactions as 5′- and 3′-terminal components, respectively, to form dinucleoside mono- ( 39 and 40 ) and diphosphates (41–48) in relatively good yields. The various products were characterized by elemental analyses, ¹H-, ¹³C-, and ³¹P-NMR spectra as well as UV and CD spectra.     

Oligonucleotides Containing Novel 4′‐C‐ or 3′‐C‐(Aminoalkyl)‐Branched Thymidines

The synthesis of four novel 3′‐C‐branched and 4′‐C‐branched nucleosides and their transformation into the corresponding 3′‐O‐phosphoramidite building blocks for automated oligonucleotide synthesis is reported. The 4′‐C‐branched key intermediate 11 was synthesized by a convergent strategy and converted to its 2′‐O‐methyl and 2′‐deoxy‐2′‐fluoro derivatives, leading to the preparation of novel oligonucleotide analogues containing 4′‐C‐(aminomethyl)‐2′‐O‐methyl monomer X and 4′‐C‐(aminomethyl)‐2′‐deoxy‐2′‐fluoro monomer Y (Schemes 2 and 3). In general, increased binding affinity towards complementary single‐stranded DNA and RNA was obtained with these analogues compared to the unmodified references (Table 1). The presence of monomer X or monomer Y in a 2′‐O‐methyl‐RNA oligonucleotide had a negative effect on the binding affinity of the 2′‐O‐methyl‐RNA oligonucleotide towards DNA and RNA. Starting from the 3′‐C‐allyl derivative 28 , 3′‐C‐(3‐aminopropyl)‐protected nucleosides and 3′‐O‐phosphoramidite derivatives were synthesized, leading to novel oligonucleotide analogues containing 3′‐C‐(3‐aminopropyl)thymidine monomer Z or the corresponding 3′‐C‐(3‐aminopropyl)‐2′‐O,5‐dimethyluridine monomer W (Schemes 4 and 5). Incorporation of the 2′‐deoxy monomer Z induced no significant changes in the binding affinity towards DNA but decreased binding affinity towards RNA, while the 2′‐O‐methyl monomer Z induced decreased binding affinity towards DNA as well as RNA complements (Table 2).     

Vier neue Metabolite von Giberella zeae: 5-Formyl-zearalenon, 7′-Dehydrozearalenon, 8′-Hydroxy- und 8′-epi-Hydroxy-zearalenon

From cultures of Gibberella zeae ergosterol, zearalenone ( 1 ) and hitherto unknown minor metabolites, i.e.. 5-formyl-zearalenone ( 6 ), 7′-dehydrozearalenone ( 8 ), 8′-hydroxyzearalenone ( 9 ) and 8′-epi-hydroxyzearalenone ( 11 ) were isolated. The production of zearalenone and its congeners proved to be very strongly dependent on the conditions of culture. 5-Formyl-zearalenone ( 6 ) as well as 3-formylzearalenone ( 4 ) were synthesized from zearalenone ( 1 ) and characterized by the di-O-methyl derivatives 7 and 5 respectively. The structure of 7′- dehydrozearalenone ( 8 ) was deduced from spectral data. The di-O-methyl derivatives 10 and 12 respectively of the two corresponding epimeric 8′-hydroxy derivatives 9 and 11 yielded the same β-diketone 13 . The keto-enol equilibrium of 13 was studied.     

Synthesis of s-3-indolemethyl derivatives of 5′-deoxy-5′-thioadenosine

The S-3-(1-methylindole)methyl and S-3-(1,2-dimethylindole)methyl derivatives of 5′-deoxy-5′-thioadenosine have been prepared by reaction of the appropriate 3-indolemethylthioacetate with 5′-deoxy-5′-chloro-adenosine in basic media. 5′-Deoxy-5′-(3-indolemethylthio)adenosines unsubstituted at the indolic nitrogen, cannot be prepared via this route due to facile conversion of the precursor 3-indolemethylthiol derivative to the corresponding 3,3′-diindolemethyl sulfide.     

Solid‐Phase Synthesis of Dipeptide‐Conjugated Nucleosides and Their Interaction with RNA

Dipeptide‐conjugated nucleosides were efficiently synthesized from the intermediates of 3′‐amino‐3′‐deoxy‐nucleosides by using the solid‐phase synthetic strategy with HOBt/HBTU (1‐hydroxy‐1H‐benzotriazole/2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluoroborate) as the coupling reagents (Schemes 1–3). CD Spectra and thermal melting studies showed that the synthesized hydrophobic dipeptide? thymidine and ? uridine derivatives 8a – 8d, 13a – d , and 18 had a mild affinity with the polyA?polyU duplex and could induce the change of RNA conformation. The results also implied that the interaction of conjugates with RNA might be related to the sugar pucker conformation of the nucleoside.     

Nucleosides. Part LXI. A Simple Procedure for the Monomethylation of Protected and Unprotected Ribonucleosides in the 2′-O- and 3′-O-Position Using Diazomethane and the Catalyst Stannous Chloride

Intensive studies on the diazomethane methylation of the common ribonucleosides uridine, cytidine, adenosine, and guanosine and its derivatives were performed to obtain preferentially the 2′-O-methyl isomers. Methylation of 5′-O-(monomethoxytrityl)-N²-(4-nitrophenyl)ethoxycarbonyl-O⁶-[2-(4-nitrophenyl)ethyl]-guanosine ( 1 ) with diazomethane resulted in an almost quantitative yield of the 2′- and 3′-O-methyl isomers which could be separated by simple silica-gel flash chromatography (Scheme 1). Adenosine, cytidine, and uridine were methylated with diazomethane with and without protection of the 5′ -O-position by a mono- or dimethoxytrityl group and the aglycone moiety of adenosine and cytidine by the 2-(4-nitrophenyl)ethoxycarbonyl (npeoc) group (Schemes 2–4). Attempts to increase the formation of the 2′-O-methyl isomer as much as possible were based upon various solvents, temperatures, catalysts, and concentration of the catalysts during the methylation reaction.     

An Unexpected (3→2)‐Hydride Shift in Phyllocladane (=13β‐Kaurane) Diterpenoids and in Related Trimethyl‐Substituted Bi‐ and Tricyclic Compounds

The conversion of 2α,3α‐dioxy‐substituted phyllocladane derivatives into the corresponding 3‐ketone proceeds in an unexpected manner: Depending on the reaction conditions, the corresponding 3β‐hydroxy‐substituted compound is formed almost quantitatively, or the desired ketone can be isolated directly (see preceding paper). The reaction mechanism is now disclosed to be a stereospecific C(3)→C(2)‐hydride shift by investigating the reactions of the synthesized (±)‐trans‐decalin‐type (trans‐1,5,5‐trimethylbicyclo[4.4.0]decanes) and (±)‐podocarpane‐type (trans‐1,2,3,4,4a,9,10,10a‐octahydro‐1,1,4a‐trimethylphenanthrenes) model compounds 25 and 35 and of their D‐labeled isomers 25′ and 35′ (Scheme 6). The latter afforded the corresponding 3β‐hydroxy (2β‐D)‐derivatives 38 and 39 as well as the (2β‐D)‐3‐ketones of the general type 5b′ (e.g., 36′ ), thus evidencing a suprafacial (C3)→C(2)‐deuteride shift. This reaction mechanism seems to be a general feature of such 3α,4α‐dioxy‐substituted 1,5,5‐trimethylbicyclo[4.4.0]decane congeners.     

Über die absolute Konfiguration der Visnagane

(+)-cis-Khellactone methyl ether ( 4 ) and (?)-trans-khellactone methyl ether ( 6 ) had earlier been assigned the absolute configurations 3′-S; 4′-S and 3′-S; 4′-R, respectively, on the basis of the FREUDENBERG , rule. Both compounds together with their defunctionalised derivatives (?)- 7 and (+)- 8 (=(+)-lomatin), obtained from a mixture of (+)-visnadin ( 1 ) and (+)-samidin ( 2 ), were investigated by the HOREAU method. A conformational analytical study showed that the optical yield should rise in the order 4 < 6 < 7 , 8. This order was found and the α-phenylbutyric acid liberated was always dextrorotatory. The centre 3′ of the khellactones and their derivatives must be R-chiral and not S. Treatment of (?)- 6 with pyridinium perbromide gave (?)-trans-3-bromokhellactone methyl ether ( 11 ) as orthorhombic crystals. The X-ray crystal structure determination was made using the anomalous scattering of the Mo-K α radiation by Br. The result, — centre 3′ R-chiral (fig. g) — showed that the HOREAU method was correct.     

Studies in spiroheterocycles,Part XV. Investigation of the reaction of 3-(2-oxocycloalkylidene)-indol-2-ones with thiourea and urea derivatives

The reaction of 3-(2-oxocycloalkylidene)indol-2-one 1 with thiourea and urea derivatives has been investigated. Reaction of 1 with thiourea and urea in ethanolic potassium hydroxide media leads to the formation of spiro-2-indolinones 2a-f in 40–50% yield and a novel tetracyclic ring system 4,5-cycloalkyl-1,3-diazepino-[4,5-b]indole-2-thione/one 3a-f in 30–35% yield. 3-(2-Oxocyclopentylidene)indol-2-one afforded 5′,6′-cyclopenta-2′-thioxo/ oxospiro[3H-indole-3,4′(3′H)pyrimidin]-2(1H)-ones 2a,b and 3-(2-oxocyclohexylidene)indol-2-one gave 2′,4′a,5′,6′,7′,8′- hexahydro-2′-thioxo/oxospiro[3H-indole-3,4′ (3′H)-quinazolin]-2(1H)-ones 2c-f . Under exactly similar conditions, reaction of 1 with fluorinated phenylthiourea/cyclohexylthiourea/phenylurea gave exclusively spiro products 2g-1 in 60–75% yield. The products have been characterized by elemental analyses, ir pmr. ¹⁹F nmr and mass spectral studies.     

Tuning the Conformation and Color of Conjugated Polyheterocyclic Skeletons by Installing ortho‐Methyl Groups

ortho‐Methyl effects are exploited to tune steric hindrance between side‐chain N,N′‐diaryls and polycyclic dihydrodibenzo[a,c]phenazine, and in turn control the conformations of N,N′‐diphenyl‐dihydrodibenzo[a,c]phenazine (DPAC) and its ortho‐methyl derivatives M x ‐M y (x=0, 1 or 2, y=1 or 2, x and y correlate with the number of methyl groups in the ortho‐positiond of N,N′‐diphenyl). The magnitude of steric hindrance increases as x and y increase, and the V‐shaped dihydrodibenzo[a,c]phenazine skeleton is gradually tuned from a bent (DPAC) to planar ( M2‐M2 ) structure in the ground state. As a result, the relaxation of the excited‐state structure of DPAC and its numerous analogues could be mimicked by model structures M x ‐M y, demonstrating for the first time the the conformation change from bent‐to‐planar and hence a large range of energy‐gap tuning of polycyclic conjugated structures controlled by the steric hindrance.     

Total synthesis of indole and dihydroindole alkaloids. IX. Studies on the synthesis of bisindole alkaloids in the vinblastine-vincristine series. The biogenetic approach.

A detailed study of the reaction of catharanthine N-oxide and vindoline has been carried out employing various conditions. Under optimum conditions, which involve low temperatures and trifluoroacetic anhydride as reagent, 3′, 4′-dehydrovinblastine (XIII, R = COOCH₃), in reasonable yields is essentially the exclusive product. However two additional products, 18′ (epi)- 3′, 4′-dehydrovinblastine (XIV, R = COOCH₃) and 1′-hydroxy- 3′, 4′-dehydrovinblastine (XVI, R = COOCH₃) are also often isolated. The reaction, which follows the course of a Polonovski-type fragmentation process, has been extended to the N-oxide derivatives of dihydrocatharanthine and decarbomethoxycatharanthine to provide again a series of bisindole alkaloid derivatives, also vinblastines. A mechanistic rationale is provided to explain the various results obtained.