Flow cytometric determination of atypical antigen expression in acute leukemia for the study of minimal residual disease.

The aim of this study was to investigate to which extent acute leukemias could be monitored for residual disease by using atypical antigen combinations as leukemia-related markers. Atypical antigenic features were determined by double color flow cytometry and included coexpression of lymphoid and myeloid related antigens, unphysiological coexpression of immature and mature antigens, and lack of an antigen that is normally expressed during maturation. Atypical immunophenotypes were detected in 35 of 68 patients with AML (51.5%) and 15 of 24 patients with ALL (62.5%). When 12 patients with leukemia-associated markers were again analyzed at relapse, the relevant antigen combinations were retained in 11 of them. The sensitivity of this two color flow cytometric assay as determined in dilution experiments was 1 in 10(3) to 10(4) cells. Follow-up studies of bone marrow samples revealed that, after induction chemotherapy cells with leukemia-associated markers were detectable in several patients at a frequency of 0.5 to 4%, but only patients in whom the cells with atypical antigens never disappeared suffered from relapse. In contrast, patients who became negative for the atypical cells remained in complete remission (median remission duration after the first negative bone marrow assessment by flow cytometry 52 weeks, range 20-102). We conclude that atypical antigen combinations, which are present in a meaningful number of acute leukemias, are a valuable means of monitoring acute leukemia patients during follow-up. This flow cytometric approach can complement other strategies to get a more accurate definition of remission in acute leukemia.     

Role of Adenosine Kinase in the Biological (Antiviral and Anticellular) Activities of Adenosine Analogues

Abstract

A paired adenosine kinase-positive/adenosine kinase-negative cell system is proposed to distinguish those adenosine analogues that need to be phosphorylated to exert their biological effects from those that are mainly targeted at S-adenosyl-L-homocysteine hydrolase.     

Design,Synthesis and Antiviral Activity of α-L-Arabinofuranosyl Derivatives of 2-Substituted-5,6-dichlorobenzimidazoles

Abstract

A number of 2-substituted-5,6-dichloro-l-(α-L-arabinofuranosyl)benzimidazoles have been prepared by condensation of 2-bromo-5,6-dichlorobenzimidazole or 2,5,6-trichlorobenzimidazole with tetra-O-acetyl-L-arabinofuranose. 2-Alkylamino derivatives were prepared by a substitution of the 2-chloro group with the appropriate amines. All target compounds were evaluated for activity against HCMV and HSV-1. The 2-chloro and 2-bromo derivatives showed moderate activity against HCMV at non-cytotoxic concentrations.     

Design and synthesis of acyclic nucleoside analogs with chlorinated imidazo[1,2-a]pyridine bases

A series of acyclic C-nucleoside analogs of 2,6-dichloro- and 2,6,7-trichloroimidazo[1,2-a]pyridine were synthesized and tested for antiviral activity. The appropriate hydroxymethyl-substituted heterocycles were treated successively with thionyl chloride, an appropriate nucleophile, then diisopropylethylamine to obtain the desired acyclic nucleoside analogs. These compounds were evaluated for activity against human cytomegalovirus and herpes simplex virus, type 1. Two of the dichloro analogs, but none of the trichloro analogs demonstrated slight antiviral activity (IC50's = 20-45 microM) at non-cytotoxic concentrations.     

Design, synthesis, and antiviral evaluation of 2-deoxy-D-ribosides of substituted benzimidazoles as potential agents for human cytomegalovirus infections

Stereoselective glycosylation of 2,5,6-trichlorobenzimidazole (1b), 2-bromo-5,6-dichlorobenzimidazole (1c), 5,6-dichlorobenzimidazole (1d), 5,6-dichlorobenzimidazole-2-thione (1e), 5,6-dichloro-2-(methylthio)benzimidazole (1f), 2-(benzylthio)-5,6-dichlorobenzimidazole (1g), and 2-chloro-5,6-dimethylbenzimidazole (1h) with 2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranosyl chloride was achieved to give the desired beta nucleosides 2b-h. Subsequent deprotection afforded the corresponding free beta-D-2-deoxyribosides 3b-h. The 2-methoxy derivative 3i was synthesized by the treatment of 2b with methanolic sodium methoxide. Displacement of the 2-chloro group of 2b with lithium azide followed by a removal of the protective groups gave the 2-azido-5,6-dichlorobenzimidazole derivative (5). The 2-amino derivative (6) was obtained by hydrogenolysis of 5 over Raney nickel. 5,6-Dichloro-2-isopropylamino-1-(2-deoxy-beta-D-erythro- pentofuranosyl)benzimidazole (10) was prepared using 2'-deoxyuridine (7), N-deoxyribofuranosyl transferase and 1d followed by functionalization of the C2 position. Antiviral evaluation of target compounds established that compounds 3b and 3c were active against human cytomegalovirus (HCMV) at non-cytotoxic concentrations. The activity of these 2-deoxy ribosides, however, was less than the activity of the parent riboside, 2,5,6-trichloro-1-beta-D-ribofuranosylbenzimidazole (TCRB). Compared to TCRB, 3b and 3c were somewhat more cytotoxic and active against herpes simplex virus type 1. Compounds 3d-i with other substituents in the 2-position were inactive against both viruses and non-cytotoxic. In contrast, compounds with amine substituents in the 2-position (5, 6, 10) were active against HCMV albeit less so than TCRB. These results establish that 2-deoxy-D-ribosyl benzimidazoles are less active against the DNA virus HCMV than are the corresponding D-ribosides.     

Phosphonate analogues of cyclopropavir phosphates and their E-isomers. Synthesis and antiviral activity

Z- and E-Phosphonate analogues 12 and 13 derived from cyclopropavir and the corresponding cyclic phosphonates 14 and 15 were synthesized and their antiviral activity was investigated. The 2,2-bis(hydroxymethylmethylenecyclopropane acetate (17) was transformed to tetrahydropyranyl acetate 18. Deacetylation gave intermediate 19 which was converted to bromide 20. Alkylation with diisopropyl methylphosphonate afforded after protecting group exchange (21 to 22) acetylated phosphonate intermediate 22. Addition of bromine gave the dibromo derivative 16 which was used in the alkylation–elimination procedure with 2-amino-6-chloropurine to give Z- and E-isomers 23 and 24. Hydrolytic dechlorination coupled with removal of all protecting groups gave the guanine phosphonates 12 and 13. Cyclization afforded the cyclic phosphonates 14 and 15. Z-Phosphonate 12 was a potent and non-cytotoxic inhibitor of human and murine cytomegalovirus (HCMV and MCMV) with EC₅₀ 2.2–2.7 and 0.13 μM, respectively. It was also an effective agent against Epstein-Barr virus (EBV, EC₅₀ 3.1 μM). The cyclic phosphonate 14 inhibited HCMV (EC₅₀ 2.4–11.5 μM) and MCMV (EC₅₀ 0.4 μM) but it was ineffective against EBV. Both phosphonates 12 and 14 were as active against two HCMV Towne strains with mutations in UL97 as they were against wild-type HCMV thereby circumventing resistance due to such mutations. Z-Phosphonate 12 was a moderate inhibitor of replication of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) but it was a potent agent against varicella zoster virus (VZV, EC₅₀ 2.9 μM). The cyclic phosphonate 14 lacked significant potency against these viruses. E-isomers 13 and 15 were devoid of antiviral activity.     

Synthesis and biological activation of an ethylene glycol-linked amino acid conjugate of cyclic cidofovir

Cidofovir (HPMPC) is a broad-spectrum anti-viral agent whose potential, particularly in biodefense scenarios, is limited by its low oral bioavailability. Two prodrugs (3 and 4) created by conjugating ethylene glycol-linked amino acids (L-Val, L-Phe) with the cyclic form of cidofovir (cHPMPC) via a P-O ester bond were synthesized and their pH-dependent stability (3 and 4), potential for in vivo reconversion to drug (3), and oral bioavailability (3) were evaluated. The prodrugs were stable in buffer between pH 3 and 5, but underwent rapid hydrolysis in liver (t(1/2) = 3.7 min), intestinal (t(1/2) = 12.5 min), and Caco-2 cell homogenates (t(1/2) = 20.2 min). In vivo (rat), prodrug 3 was >90% reconverted to cHPMPC. The prodrug was 4x more active than ganciclovir (IC50 value, 0.68 microM vs 3.0 microM) in a HCMV plaque reduction assay. However, its oral bioavailability in a rat model was similar to the parent drug. The contrast between the promising activation properties and unenhanced transport of the prodrug is briefly discussed.     

Fluoroanalogues of anti-cytomegalovirus agent cyclopropavir: synthesis and antiviral activity of (E)- and (Z)-9-{[2,2-bis(hydroxymethyl)-3-fluorocyclopropylidene]methyl}-adenines and guanines

Synthesis of fluorinated cyclopropavir analogues 13a, 13b, 14a, and 14b is described starting from alkene 15. Addition of carbene derived from dibromofluoromethane gave bromofluoro cyclopropane 16. Reduction (compound 17) followed by desilylation gave intermediate 18, which was transformed to 2-nitrophenylselenenyl derivative 19. Oxidation to selenoxide 20 was followed by beta-elimination to afford methylenecyclopropane 21. Addition of bromine provided compound 22 for alkylation-elimination of adenine and 2-amino-6-chloropurine. The resultant E,Z isomeric mixtures of methylenecyclopropanes 23a + 24a and 23c + 24c were resolved and the individual isomers were deprotected to give adenine analogues 13a and 14a as well as compounds 13c and 14c. Hydrolytic dechlorination of 13c and 14c furnished guanine analogues 13b and 14b. The only significant antiviral effects were observed with analogue 13a against HCMV and 14a against VZV in cytopathic inhibition assays.     

An unusual presentation of a malignant jejunal tumor and a different management strategy

BACKGROUND: Malignant small bowel tumors are very rare and leiomyosarcoma accounts for less than 15% of the cases. Management of these tumors is challenging in view of nonspecific symptoms, unusual presentation and high incidence of metastasis. In this case report, an unusual presentation of jejunal sarcoma and management of liver metastasis with radiofrequency ablation (RFA) is discussed. CASE PRESENTATION: A 45-year-old male presented with anemia and features of small bowel obstruction. Operative findings revealed a mass lesion in jejunum with intussusception of proximal loop. Resection of bowel mass was performed. Histopathological findings were suggestive of leiomyosarcoma. After 3-years of follow-up, the patient developed recurrence in infracolic omentum and a liver metastasis. The omental mass was resected and liver lesion was managed with radiofrequency ablation. CONCLUSION: Jejunal leiomyosarcoma is a rare variety of malignant small bowel tumor and a clinical presentation with intussusception is unusual. We suggest that an aggressive management approach using a combination of surgery and a newer technique like RFA can be attempted in patients with limited metastatic spread to liver to prolong the long-term survival in a subset of patients.     

Synthesis of 3-aminoimidazo[4,5-c]pyrazole nucleoside via the N-N bond formation strategy as a [5:5] fused analog of adenosine

3-Amino-6-(beta-D-ribofuranosyl)imidazo[4,5-c]pyrazole (2) was synthesized via an N-N bond formation strategy by a mononuclear heterocyclic rearrangement (MHR). A series of 5-amino-1-(5-O-tert-butyldimethylsilysilyl-2,3-O-isopropylidene-beta-D-ribofuranosyl)-4-(1,2,4-oxadiazol-3-yl)imidazoles (6a-d), with different substituents at the 5-position of the 1,2,4-oxadiazole, were synthesized from 5-amino-1-(beta-D-ribofuranosyl)imidazole-4-carboxamide (AICA Ribose, 3). It was found that 5-amino-1-(5-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-beta-D-ribofuranosyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)imidazole (6a) underwent the MHR with sodium hydride in DMF or DMSO to afford the corresponding 3-acetamidoimidazo[4,5-c]pyrazole nucleoside(s) (7b and/or 7a) in good yields. A direct removal of the acetyl group from 3-acetamidoimidazo[4,5-c]pyrazoles under numerous conditions was unsuccessful. Subsequent protecting group manipulations afforded the desired 3-amino-6-(beta-D-ribofuranosyl)imidazo[4,5-c]pyrazole (2) as a 5:5 fused analog of adenosine (1).