Hiroki Kumamoto

Anti-Human Immunodeficiency Virus Type 1 Activity and Resistance Profile of 2′,3′-Didehydro-3′-Deoxy-4′-Ethynylthymidine In Vitro

ABSTRACT 2′,3′-Didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T) has been identified as a novel nucleoside analog with potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity and weak cytotoxicity in cell cultures. 4′-Ed4T proved to be 5- to 10-fold more active than its structurally related compound, stavudine (d4T). However, the drug resistance profile of 4′-Ed4T was different from those of d4T and other existing HIV-1 nucleoside reverse transcriptase inhibitors (NRTIs). Approximately 6- to 11-fold decreases in susceptibility to 4′-Ed4T were observed for HIV-1 carrying NRTI-associated mutations (D67N, K70R, T215F, and K219Q) or the lamivudine (3TC)-resistant mutation M184V. In contrast, the susceptibility of the virus carrying the K65R mutation or the multidrug-resistant mutation with the Q151M complex (A62V, V75I, F77L, F116Y, and Q151M) was not altered. Furthermore, the activity of 4′-Ed4T appeared to be enhanced in the presence of K103N, a major nonnucleoside reverse transcriptase inhibitor-resistant mutation. Although 4′-Ed4T was 4.5- to 17.5-fold less active against multidrug-resistant clinical isolates than against a reference strain isolated from a treatment-naïve patient, it was still inhibitory to these isolates at low concentrations. Analysis of 4′-Ed4T-resistant HIV-1 obtained through in vitro selection revealed that the virus was also resistant to 3TC and had two amino acid mutations (P119S and T165A) in addition to the M184V mutation. Since 4′-Ed4T has increased anti-HIV-1 activity, decreased cytotoxicity, and a different resistance profile, it should be considered for further development as a new member of NRTIs.

A versatile intermediate for the synthesis of 3′-substituted 2′,3′-didehydro-2′,3′-dideoxyadenosine (d4A): preparation of 3′-C-stannyl-d4A via radical-mediated desulfonylative stannylation

Abstract A method is described for the introduction of a tributylstannyl group to the 3′-position of 2′,3′-didehydro-2′,3′-dideoxyadenosine ( 2 : d4A). Transalkoxylation of Bu3SnOMe with d4A and subsequent anionic O→C stannyl migration gave 3′-C-tributylstannyl-d4A ( 5 ), but only in a low yield. An alternative route involves several reactions starting from 9-[2,3-anhydro-5-O-(tert-butyldimethylsilyl)-β- d -ribofuranosyl]-N6-pivaloyladenine ( 14 ): ring opening with NaSPh, oxidation of the 3′-C-phenylthio group, removal of the N6-pivaloyl group, 2′-O-mesylation, elimination of methanesulfonic acid, and tin radical-mediated substitution of the 3′-C-benzenesulfonyl group. The overall yield of this approach was 55% from 14 . The synthetic utility of 5′-O-(tert-butyldimethylsilyl)-3′-C-tributylstannyl-d4A ( 18 ) thus obtained was briefly exemplified by the preparation of some 3′-substituted analogues ( 19–23 : I, Br, Ph, vinyl, and phenylethynyl).

Lithiation-mediated CC silyl and stannyl migrations observed in 6-chloro-9-(β-d-ribofuranosyl)purine

Lithiation of 9-[2,3,5-tris-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]-6-chloropurine (1) with LTMP (lithium 2,2,6,6-tetramethylpiperidide) and a subsequent reaction with either Me3SiCl or Bu3SnCl was found to effect C-2-substitution, as a result of “silyl or stannyl migration” from C-8 to C-2 position. Transformation of the resulting 2-stannyl derivative (8) to 2-substituted adenosines was also examined briefly.

Synthesis of (±)-4′-Ethynyl and 4′-Cyano Carbocyclic Analogues of Stavudine (d4T)

The synthesis of (±)-4′-ethynyl (8) and 4′-cyano (9) carbocyclic analogues of the anti-HIV agent stavudine (5, d4T) is reported. The carbocyclic unit (16) was constructed from readily available β-keto ester 10. The ethynyl or cyano group of 8 and 9 were prepared, after the introduction of thymine base to 16, by manipulation of the ester function. Evaluation of the anti-HIV activity of 8 and 9 was also carried out. This work was financially supported in part by grants from the Japan Health Sciences Foundation (SA14718 to H. T.), Japan Society for the Promotion of Science (KAKENHI No. 15790075 to H. K., No. 15590100 to K. H., and No. 15590020 to H. T.), and NIH USA (RO1 AI 38204 to Y.-C. C.).

Synthesis of 2-Alkynylcordycepins and Evaluation of Their Vasodilating Activity

Abstract Based on the recently developed lithiation-mediated stannyl migration of 6-chloropurine derivatives, 2-iodocordycepin was prepared from cordycepin. The reaction of this compound with terminal alkynes was carried out to synthesize a series of 2-alkynyl derivatives. The vasodilating effect of these compounds was evaluated.

Radical-mediated furanose ring reconstruction from 2′,3′-seco-uridine

Abstract Starting from 5′- O -trityl-2′,3′- seco -uridine, reconstruction of a furanose structure was carried out by the following sequence of reactions: (1) regioselective introduction of a phenylselenenyl group to the 2′-position of the 2′,3′- seco -uridine, (2) oxidation and subsequent Wittig reaction of the 3′-hydroxyl group and (3) intramolecular radical reaction (5- exo - trig ring closure) leading to 3′- C -carbon-substituted 2′,3′-dideoxyuridine. Also studied is the Pummerer reaction of the 2′-phenylseleno derivative of 2′,3′- seco -uridine. The resulting product, an α-(acyloxy)phenylselenide, also serves as a substrate for the radical cyclization to allow the introduction of a hydroxyl group at the 2′-position of the reconstructed furanose ring.

Nucleotide binding to human UMP‐CMP kinase using fluorescent derivatives − a screening based on affinity for the UMP‐CMP binding site

Methylanthraniloyl derivatives of ATP and CDP were used in vitro as fluorescent probes for the donor‐binding and acceptor‐binding sites of human UMP‐CMP kinase, a nucleoside salvage pathway kinase. Like all NMP kinases, UMP‐CMP kinase binds the phosphodonor, usually ATP, and the NMP at different binding sites. The reaction results from an in‐line phosphotransfer from the donor to the acceptor. The probe for the donor site was displaced by the bisubstrate analogs of the Ap5X series (where X = U, dT, A, G), indicating the broad specificity of the acceptor site. Both CMP and dCMP were competitors for the acceptor site probe. To find antimetabolites for antivirus and anticancer therapies, we have developed a method of screening acyclic phosphonate analogs that is based on the affinity of the acceptor‐binding site of the human UMP‐CMP kinase. Several uracil vinylphosphonate derivatives had affinities for human UMP‐CMP kinase similar to those of dUMP and dCMP and better than that of cidofovir, an acyclic nucleoside phosphonate with a broad spectrum of antiviral activities. The uracil derivatives were inhibitors rather than substrates of human UMP‐CMP kinase. Also, the 5‐halogen‐substituted analogs inhibited the human TMP kinase less efficiently. The broad specificity of the enzyme acceptor‐binding site is in agreement with a large substrate‐binding pocket, as shown by the 2.1 Å crystal structure.

An Intramolecular Anionic Migration of a Stannyl Group from the 6-Position of 1-(2-Deoxy-d-erythro-pent-1-enofuranosyl)uracil to the 2′-Position: Synthesis of 2′-Substituted 1′,2′-Unsaturated Uridines

Abstract Lithiation of 1-[3,5-bis-O-(tert-butyldimethylsilyl)-2-deoxy- d -erythro-pent-1-enofuranosyl)uracil (1) takes place exclusively at the 6-position of the uracil base. The 6-tributylstannyl (or 6-trimethylsilyl) derivative prepared by quenching the C6-lithiated species with Bu3SnCl (or Me3SiCl) was found to undergo an intramolecular anionic migration to the 2′-positon of the furanoid glycal portion. By manipulation of the 2′-stannyl group, 2′-halogeno and 2′-carbon-substituted 1′,2′-unsaturated uridines were prepared for the first time. In contrast to the reported instability of 1 during deprotection, the 2′-substituted analogs synthesized in the present study gave the corresponding free nucleosides uniformly in high yields upon treatment with NH4F in MeOH.

Three 25-NBOMe-type drugs, three other phenethylamine-type drugs (25I-NBMD, RH34, and escaline), eight cathinone derivatives, and a phencyclidine analog MMXE, newly identified in ingredients of drug products before they were sold on the drug market

Twenty-two samples of ingredients of recreational drugs before being sold on the drug market obtained from a dubious drug dealer were analyzed by gas chromatography/mass spectrometry, high-resolution mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The present study showed 15 novel designer drugs, which have not been described in scientific literature. They included three NBOMe drugs 25H-NBOMe, 25D-NBOMe, and 25E-NBOMe, three other phenethylamine-type drugs 25I-NBMD, RH34, and escaline, eight cathinone derivatives 5-DBFPV, 3,4-MDPHP, 3,4-dimethyl-NEB, 3,4-dimethyl-α-ethylaminopentiophenone, 3,4-dimethyl-α-PVP, 4F-α-ethylaminopentiophenone, bk-IVP, and bk-IBP, and a phencyclidine derivative MMXE. In addition to the above novel compounds, known compounds such as 25I-NBOMe, ADB-CHIMINACA, 5F-ADB, and butane-1,4-diol were also identified from some samples. The electron ionization mass spectra, high-resolution data of molecular formulae, and NMR spectra presented in this article seem very useful for forensic toxicologists, who are obliged to identify new psychotropic drugs in any dubious products and/or human specimens.

Stannyl migration from the base to the sugar portion of 1′,2′-unsaturated uridine: the first example of substitution at the 2′-position

Abstract We report that TBDMS-protected 1-(2-deoxy-D- erythro -pent-1-enofuranosyl)-6-(tributyl-stannyl)uracil, when treated with LDA or LTMP, undergoes an anionic stannyl migration to yield the 2′-stannylated product. Optimization of the reaction conditions has disclosed an efficient entry to compounds variously substituted at the 2′-position. Desilylation of these compounds caused no further elimination, and furnished a hitherto unknown series of nucleoside analogues.