key: cord-252108-04xr5xdl
authors: Havrylyuk, Dmytro; Zimenkovsky, Borys; Vasylenko, Olexandr; Day, Craig W.; Smee, Donald F.; Grellier, Philippe; Lesyk, Roman
title: Synthesis and biological activity evaluation of 5-pyrazoline substituted 4-thiazolidinones
date: 2013-06-06
journal: Eur J Med Chem
DOI: 10.1016/j.ejmech.2013.05.044
sha: 
doc_id: 252108
cord_uid: 04xr5xdl

A series of novel 5-pyrazoline substituted 4-thiazolidinones have been synthesized. Target compounds were evaluated for their anticancer activity in vitro within DTP NCI protocol. Among the tested compounds, the derivatives 4d and 4f were found to be the most active, which demonstrated certain sensitivity profile toward the leukemia subpanel cell lines with GI(50) value ranges of 2.12–4.58 μM (4d) and 1.64–3.20 μM (4f). The screening of antitrypanosomal and antiviral activities of 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1-yl)-thiazolidine-2,4-diones was carried out with the promising influence of the mentioned compounds on Trypanosoma brucei, but minimal effect on SARS coronavirus and influenza types A and B viruses.

The non-condensed heterocyclic systems with thiazolidinone [1] and pyrazoline [2, 3] moieties have emerged as powerful scaffolds in drug design. Among diazole-substituted 4-thiazolidinones highly active anticancer agents have been identified including inhibitors of necroptosis [4] , tumor necrosis factor a [5] and tyrosine phosphatase [6] . Our previous study, based on a hybrid pharmacophore approach, allowed to establish a number of patterns in the structureeactivity relationship (SAR) context for 4-thiazolidinones with a pyrazoline fragment in 2, 3 and 4 positions of the thiazolidone cycle, which possessed antitumor activity [7, 8] .

On the other hand, thiazolidinones and pyrazolines have occupied a unique position in the design and synthesis of novel biologically active agents that exert trypanocidal activity [9e13]. The 2-thioxo-4thiazolidinone-3-acetic acid derivatives were identified as inhibitors of Trypanosoma brucei dolicholphosphate mannose synthase [11] . The 2-hydrazolyl-4-thiazolidinone-5-carboxylic acid derivatives have shown promising activity on the cruzipain protease [12] . The most promising compound in series of aryl-4-oxothiazolylhydrazones was shown to be very active at non-cytotoxic concentrations in in vitro assays against Trypanosoma cruzi cell cultures and exhibited potency comparable with the reference compounds (IC 50 (Y strain) ¼ 0.3 mM) [9] . Among pyrazoline derivatives, some novel compounds have been identified as inhibitors of the trypanosomal cysteine protease cruzain with IC 50 of 40e230 nM [13] .

The antiviral activity of heteryl substituted 4-thiazolidinones is promising. Among thiazoleethiazolidine conjugates [14] and noncondensed derivatives with thiazolidinone and pyridine [15e17] or pyrimidine [18e20] cycles, anti-HIV agents were identified. In addition, this group of compounds was active against hepatitis C virus [21] , Tobacco Mosaic virus [22] and Vesicular stomatitis virus [23] . Previously we also demonstrated the efficiency of certain pyrazolineethiazolidinone conjugates on influenza viruses and SARS coronavirus [24] .

The present work is an extension of our ongoing efforts toward developing promising biologically active agents using a hybrid pharmacophore approach. We made the design (Fig. 1 ) and synthesized hybrid compounds by linking the main structural unit of the 4-thiazolidinone ring system with the pyrazoline, and examined their antitumor, trypanocidal and antiviral activities in vitro. We have found two compounds from 5-pyrazoline substituted 4-thiazolidinones, which possessed a commensurate antitumor activity compared to the pyrazolineethiazolidinone analogous compounds reported previously [7, 8] and evaluated anti-trypanosomal activity and antiviral activity of the synthesized compounds.

The general methods for synthesis of target thiazolidinonee pyrazoline conjugates are depicted in Schemes 1 and 2.

The starting 3,5-diaryl-4,5-dihydropyrazoles synthesized using known methods from appropriate chalcones [25] easily reacted with 5-bromothiazolidine-2,4-dione [26] yielding 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1-yl)-thiazolidine-2,4-dione 1a and 1b. It is known that chemical modification of the N3 position of thiazolidinone cycle has an essential influence on the antitumor activity [27, 28] . Relying on these observations we utilized potassium salt of 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1-yl)-thiazolidine-2,4-dione, generated in situ, in the reactions with 2-chloro-N-arylacetamides. Following the mentioned reaction the new N3substituted non-condensed thiazolidinoneepyrazoline conjugates 2ae2c were synthesized. Based on the Mannich reaction of 1a and 1b with secondary amines the thiazolidinone analogs 3ae3g were obtained (Scheme 1).

Aiming at the detailed elaboration of SAR, especially the influence of the linking group of thiazolidinoneepyrazoline conjugates on the anticancer activity, 5-[2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-2oxoethylidene]-thiazolidine-2,4-diones (4ae4f) were synthesized by the method, described previously [29] . Reaction of 3,5-diaryl-4,5-dihydropyrazoles with (2,4-dioxothiazolidine-5-ylidene)-acetyl chloride [27] afforded in excellent yield and purity the compounds 4ae4f. Following the reaction of generated in situ potassium salts of 4b and 4e with 2-chloro-N-arylacetamides the group of N3substituted 4-thiazolidinones 5ae5e were synthesized (Scheme 2).

The data characterization of synthesized novel heterocyclic substituted thiazolidones are presented in Experimental part. Analytical and spectral data ( 1 H NMR, 13 C NMR) confirmed the structure of the synthesized compounds.

Protons CH 2 eCH of pyrazoline fragment in the 1 H NMR spectra of synthesized compounds showed characteristic patterns of an AMX system. The proton (CH) of thiazolidinone core of 1ae1b, 2ae2c and 3ae3g showed the broad singlet at d w5.59e5.99 and the protons of the methylene group (CH 2 CO) of 2ae2c and 5ae5e appeared as a broad singlet at d w4.44e4.49 ppm. In the 1 H NMR spectra of the 1ae1b and 4ae4f NH proton of thiazolidinone cycle the broad singlet at dw12.20e12.72 was found.

Synthesized derivatives 1a, 1b, 2a, 3ae3d, 3f, 4a, 4d, 4e, 4f and 5d were selected by National Cancer Institute (NCI, Bethesda USA) Developmental Therapeutic Program (DTP) and evaluated at the concentration of 10 À5 M toward a panel of approximately sixty cancer cell lines (http://dtp.nci.nih.gov). The human tumor cell lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, central nervous system, ovarian, renal, prostate and breast cancers. Primary anticancer assays were performed according to the NCI protocol as described elsewhere [30e33]. The compounds were added at a single concentration and the cell cultures were incubated for 48 h. The end point determinations were made with a protein binding dye, sulforhodamine B (SRB). The results for each compound are reported as the percent growth (GP%) of treated cells when compared to untreated control cells ( Table 1 ). The range of percent growth shows the lowest and the highest percent growth found among the different cancer cell lines. The most active compounds 4d and 4f were found to be effective against 12 and 18 cell lines, respectively, compound 4a was found to be moderately effective against few cell lines, while the other compounds (1a, 1b, 2a, 3ae3d, 3f, 4e, 5d) did not show any activity (Table 1) .

Finally, compounds 4d and 4f were selected for an advanced assay against a panel of approximately sixty tumor cell lines at 10fold dilutions of five concentrations (100 mM, 10 mM, 1 mM, 0.1 mM and 0.01 mM) [30e33] . The percentage of growth was evaluated spectrophotometrically versus controls not treated with test agents after 48-h exposure and using SRB protein assay to estimate cell viability or growth. Doseeresponse parameters were calculated for each cell line: GI 50 e molar concentration of the compound that inhibits 50% net cell growth; and TGI e molar concentration of the compound leading to the total inhibition. Furthermore, a mean graph midpoints (MG_MID) were calculated for each of the parameters, giving an average activity parameter over all cell lines for the tested compound. For the MG_MID calculation, insensitive cell lines were included with the highest concentration tested ( Table 2 ).

The tested compounds showed inhibition activity (GI 50 < 10 mM) against 47 from 55 (4d) and 56 from 59 (4f) human tumor cells with average GI 50 /TGI values of 7.02 mM/38.07 mM (4d) and 4.38 mM/ 50.99 mM (4f) ( Table 2) . With regard to the sensitivity against some individual cell lines among several subpanel, the compounds 4d and 4f demonstrated a certain sensitivity profile toward the leukemia subpanel tumor cell lines with GI 50 values range of 2.12e4.58 mM (4d) and 1.64e3.20 mM (4f) ( Table 3 ).

The SAR study revealed that: (1) the level of antitumor activity of active thiazolidinones with pyrazoline fragment in 5 position (4d and 4f) is compatible with effectivity levels of heteryl substituted thiazolidones, described previously [34e42]; (2) conjugation of pyrazoline and thiazolidinone cycles using oxomethylidene linking group (4f) allowed us to increase the activity, in comparison with the structurally related conjugate representative 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1-yl)-thiazolidine-2,4-dione 1b; (3) introduction of the substituents in 3N-position of thiazolidine fragment did not have significant influence on the antitumor activity.

NCI's COMPARE algorithm [30e33] allows to assume biochemical mechanisms of action of the novel compounds on the basis of their in vitro activity profiles when comparing with those of standard agents. We performed COMPARE computations for the compounds 4d and 4f against the NCI "Standard Agents" database at the GI 50 and TGI levels (Table 4 ). However, obtained Pearson correlation coefficients (PCC) did not allow to distinguish cytotoxicity mechanism of tested compounds with high probability. The compound 4d showed the highest correlation at the GI 50 level with dihydroorotate dehydrogenase inhibitor brequinar (PCC ¼ 0.651) and compound 4f e with maytansine (RNA/DNA antimetabolite, PCC ¼ 0.636).

Antiviral activity of 1a, 1b, 2ae2c, 3d and 3e was determined against SARS coronavirus (SARS CoV) and influenza types A and B viruses (Flu A, Flu B). The obtained results are summarized in Table 5 .

Although antiviral activity was evident, virus inhibition occurred at or near the cytotoxic concentration. The compounds showed insignificant activities against the four strains of influenza virus with the range levels of selectivity index from 1.0 to 2.1. Compound 2a had moderate activity against the duck strain of influenza A with a 50% effective concentration (EC 50 ) of 21.78 mM and selective index (SI) of >16.3; but did not have significant activity against other influenza strains. The majority of the compounds showed no activity against SARS CoV. The positive control compounds ribavirin, oseltamivir carboxylate, and M128533 were active as expected in the assays.

The compounds 1a, 2b and 2c were selected in advanced in vitro assay against Trypanosoma brucei brucei (Tbb) and Trypanosoma brucei gambiense (Tbg). The doseeresponse curves with drug concentrations ranging from 10 mg/ml to 0.625 mg/ml are depicted on The results showed a moderated activity of compounds (Table 6) on both parasite strains, namely IC 50 (Tbb) ¼ 5.43e13.87 mM and IC 50 (Tbg) ¼ 2.53e6.66 mM.

In the present paper new 4-thiazolidinone based conjugates with pyrazoline moiety at 5 position are described. Antitumor activity assay of thirteen synthesized compounds allowed us to identify highly active thiazolidinoneepyrazoline hybrids 4d and 4f, which demonstrated certain sensitivity profile toward the leukemia subpanel tumor cell lines with GI 50 values range of 2.12e4.58 mM (4d) 

The starting 3,5-diaryl-4,5-dihydro-1H-pyrazole [25] , 5-bromothiazolidine-2,4-dione [26] , and (2,4-dioxothiazolidine-5-ylidene)-acetyl chloride [27] were obtained according to the Table 1 Anticancer screening data at the concentration of 10 mM. methods described previously. Preparation of compounds 4ae4d and 4f was described in our previous report [29] . 

A suspension of compound 1a or 1b (3 mmol) and potassium hydroxide (3 mmol) was stirred at r.t. during 5 min, later appropriate 2-chloro-N-arylacetamide (3.3 mmol) was added and the mixture was refluxed for 5 h in EtOH (10 ml). Obtained powders were filtered off, washed with ethanol and recrystallized with DMF:ethanol (1:2) mixtures. 

(2b). Yield 68%, mp 220e222 C. 1 

Anti-trypanosomal activity of 5-(3-naphthalen-2-yl-5-aryl-4,5-dihydropyrazol-1yl)-thiazolidine-2,4-dione derivatives (1a, 2b, 2c). 137.9, 133.9, 133.3, 133.2, 130.1, 129.3,  128.9, 128.7, 128.6, 128.2, 127.6, 127.2, 127.1, 123.6 A solution of (2,4-dioxothiazolidin-5-ylidene)-acetyl chloride (3 mmol) in 5 ml of dioxane was added to a mixture of appropriate 3,5-diaryl-4,5-dihydro-1H-pyrazole (3 mmol) and triethylamine (3 mmol) in 5 ml of dioxane and later was heated to 70e80 C during 15 min, cooled and poured water (50 ml). Obtained powder was filtered off, washed with water and recrystallized with DMF:ethanol (1:2) mixtures. Results for each tested compound were reported as the percent of growth of the treated cells when compared to the untreated control cells. The percentage growth was evaluated spectrophotometrically versus controls not treated with test agents. The cytotoxic and/or growth inhibitory effects of the most active selected compounds were tested in vitro against the full panel of human tumor cell lines at concentrations ranging from 10 À4 to 10 À8 M. 48-h continuous drug exposure protocol was followed and an SRB protein assay was used to estimate cell viability or growth.

Using absorbance measurements [time zero (Tz), control growth in the absence of drug (C), and test growth in the presence of drug (Ti)], the percentage growth was calculated for each drug concentration. Percentage growth inhibition was calculated as: ½ðTi À TzÞ=ðC À TzÞ Â 100 for concentrations for which Ti ! Tz; ½ðTi À TzÞ=Tz  100 for concentrations for which Ti < Tz:

Dose response parameters (GI 50 , TGI) were calculated for each compound. Growth inhibition of 50% (GI 50 ) was calculated from [(Ti À Tz)/(C À Tz)] Â 100 ¼ 50, which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB staining) as compared to the net protein increase seen in the control cells. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti ¼ Tz. Values were calculated for each of these parameters if the level of activity was reached; however, if the effect was not reached or was excessive, the value for that parameter was expressed as more or less than the maximum or minimum concentration tested. The lowest values were obtained with the most sensitive cell lines. Compounds having GI 50 values 100 mM were declared to be active.

Primary antiviral assay was performed on a respiratory viruses panel (Flu A (H1N1), Flu A (H3N2), Flu A (H5N1), Flu B, SARS CoV) [43] . Compounds were diluted to 20 mg/ml in DMSO then eight half-log dilutions were prepared in MEM solution with 50 mg/ml gentamicin. Each dilution was added to 5 wells of a 96-well plate with 80e100% confluent cells, and three wells of each dilution were then infected with the test virus using a multiplicity of infection of <0.006 CCID50 per cell for each virus. Two wells remained uninfected as toxicity controls. A known active compound was run in parallel as a control. After cytopathic effect (CPE) was observed microscopically, plates were stained with neutral red dye for approximately 2 h, then supernatant dye was removed from the wells and the incorporated dye was extracted in 50:50 Sorensen citrate buffer/ethanol and read on a spectrophotometer at 540 nm. The optical density of test wells was converted to percent of cell and virus controls, then the concentration of test compound required to inhibit viral CPE by 50% (EC 50 ) was calculated by regression analysis. The concentration of compound that would cause 50% cytotoxicity in the uninfected cells was similarly calculated (CC 50 ). EC 50 and CC 50 were presented in mM. The selective index (SI) is the CC 50 divided by EC 50 .

Bloodstream forms of T. brucei brucei strain 90-13 and T. brucei gambiense Feo strain were cultured in HMI9 medium supplemented with 10% FCS at 37 C under an atmosphere of 5% CO 2 [44] . In all experiments, log-phase parasite cultures were harvested by centrifugation at 3000Âg and immediately used. Drug assays were based on the conversion of a redox-sensitive dye (resazurin) to a fluorescent product by viable cells as previously described [45] . Drug stock solutions were prepared in pure DMSO. T. brucei bloodstream forms (10 5 cells/ml) were cultured in 96-well plates either in the absence or in the presence of different concentrations of inhibitors in a final volume of 200 ml. After a 72-h incubation, resazurin solution was added in each well at the final concentration of 45 mM and fluorescence was measured at 530 nm and 590 nm absorbance after a further 4-h incubation. The percentage of inhibition of parasite growth rate was calculated by comparing the fluorescence of parasites maintained in the presence of drug to that of in the absence of drug. DMSO was used as control. Concentration inhibiting 50% of parasite growth (IC 50 ) was determined from the doseeresponse curve with a drug concentrations ranging from 10 mg/ml to 0.625 mg/ml and presented in mM. IC 50 value is the mean þ/À the standard deviation of three independent experiments.

Yield 82%, mp 268e270 C. 1 H NMR (400 MHz, DMSO-d 6 þ CCl 4 ): d 12.71 (br. s, 1H, NH), 8.27 (s, 1H, arom), 7.95e8.11 (m, 4H, arom), 7.88 (s, 1H, CH), 7.56e7.60 (m, 2H, arom)

5-diaryl-4,5-dihydropyrazol-1-yl)-2-oxoethylidene]-2,4-dioxothiazolidin-3-yl}-N-arylacetamides (5ae5e) A suspension of compound 4b or 4f (3 mmol) and potassium hydroxide (3 mmol) was stirred at r.t. during 5 min, later appropriate 2-chloro-N-arylacetamide (3.3 mmol) was added and the mixture was refluxed for 5 h in EtOH (10 ml). Obtained powders were filtered off

3-phenyl-4,5-dihydropyrazol-1-yl]-2-oxoethylidene}-2,4-dioxothiazolidin-3-yl)-N-p-tolylacetamide (5a)

04 (d, 2H, J ¼ 7.9 Hz, arom), 5.70 (dd, 1H, CH 2 CH

DMSO-d 6 þ CCl 4 ): d 10.04 (s, 1H, NH), 8.02 (s

4 -d ioxoth iazolidin-3-yl )-N-(4-chlorophenyl)-acetamide (5c). Yield 79%, mp 289e290 C. 1 H NMR (400 MHz, DMSO-d 6 þ CCl 4 ): d 10.51 (s, 1H, NH), 7.98 (s, 1H, COCH), 7.83e7.86 (m, 2H, arom), 7.48e7.58 (m, 5H, arom), 7.34e7.42 (m, 3H, arom)

-{2-[5-(4-methoxyphenyl)-3-naphthalen-2-yl-4,5-dihydropyrazol-1-yl]-2-oxoethylidene}-2,4-dioxothiazolidin-3-yl)-acetamide (5d). Yield 86%, mp 268e269 C

46 (d, 2H, J ¼ 6.4 Hz, arom), 7.21 (d, 2H, J ¼ 6.4 Hz, arom), 6.90 (br. s, 3H, arom), 5.68 (dd, 1H, CH 2 CH

4-Thiazolidones: centenarian history, current status and perspectives for modern organic and medicinal chemistry

Biological activities of pyrazoline derivatives e a recent development

Recent advances in the therapeutic applications of pyrazolines

Structureeactivity relationship study of a novel necroptosis inhibitor

Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-a

2-Thiazolylimino/heteroarylimino-5-arylidene-4-thiazolidinones as new agents with SHP-2 inhibitory action

Synthesis of novel thiazolone-based compounds containing pyrazoline moiety and evaluation of their anticancer activity

Synthesis of new 4-azolidinones with 3,5-diaryl-4,5-dihydropyrazole moiety and evaluation of their antitumor activity in vitro

Synthesis, cruzain docking, and in vitro studies of aryl-4-oxothiazolylhydrazones against Trypanosoma cruzi

Synthesis and structureeactivity relationships of parasiticidal thiosemicarbazone cysteine protease inhibitors against plasmodium falciparum, Trypanosoma brucei, and Trypanosoma cruzi

First small molecular inhibitors of T. brucei dolicholphosphate mannose synthase (DPMS), a validated drug target in African sleeping sickness

Synthesis of 2-hydrazolyl-4-thiazolidinones based on multicomponent reactions and biological evaluation against T. Cruzi

Synthesis and structureeactivity relationship study of potent trypanocidal thio semicarbazone inhibitors of the trypanosomal cysteine protease cruzain

Design, synthesis, and evaluation of 2-aryl-3-heteroaryl-1,3-thiazolidin-4-ones as anti-HIV agents

Synthesis and anti-HIV studies of 2-adamantyl-substituted thiazolidin-4-ones

Discovery of 2,3-diaryl-1,3-thiazolidin-4-ones as potent anti-HIV-1 agents

Design and synthesis of 2-(2,6-dibromophenyl)-3-heteroaryl-1,3-thiazolidin-4-ones as anti-HIV agents

Synthesis and evaluation of 2-(2,6-dihalophenyl)-3-pyrimidinyl-1,3-thiazolidin-4-one analogues as anti-HIV-1 agents

Predicting anti-HIV activity of 1,3,4-thiazolidinone derivatives: 3D-QSAR approach

Design, microwave-assisted synthesis and HIV-RT inhibitory activity of 2-(2,6-dihalophenyl)-3-(4,6-dimethyl-5-(un)substituted-pyrimidin-2-yl)thiazolidin-4-ones

Non-nucleoside inhibitors of the hepatitis C virus NS5B RNA-dependant RNA polymerase: 2-aryl-3-heteroaryl-1,3-thiazolidin-4-one derivatives

Synthesis and biological activity of 4-(3-aryl-4-oxo-2-thioxothiazolidin-5-ylimino)-3-methyl-1-(N,N-disubstituted aminomethyl) pyrazolin-5-ones

Synthesis and antiviral activity of new pyrazole and thiazole derivatives

Synthesis and anticancer and antiviral activities of new 2-pyrazoline-substituted 4-thiazolidinones

Synthesis and antidepressant activities of some 3,5-diphenyl-2-pyrazolines

Synthesis and antihyperglycemic activity of novel 5-(naphthalenylsulfonyl)-2,4-thiazolidinediones

Synthesis and in vitro anticancer activity of 2,4-azolidinedione-acetic acids derivatives

Structureeanticancer activity relationships among 4-azolidinone-3-carboxylic acids derivatives

Synthesis and evaluation of antitumor activity of 5-[2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-2-oxoethylidene

Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines

Some practical considerations and applications of the national cancer institute in vitro anticancer drug discovery screen

Cancer Drug Discovery and Development

The NCI60 human tumour cell line anticancer drug screen

Synthesis and anticancer activity of novel nonfused bicyclic thiazolidinone derivatives, Phosphorus

New 5-arylidene-4-thiazolidinones and their anticancer activity

A facile synthesis and anticancer activity evaluation of spiro

Thiazolidinone motif in anticancer drug discovery. Experience of DH LNMU medicinal chemistry scientific group

Synthesis and anticancer activity evaluation of 4-thiazolidinones containing benzothiazole moiety

Synthesis and anticancer activity of isatin-based pyrazolines and thiazolidines conjugates

Synthesis and antitumor activity of novel 2-thioxo-4-thiazolidinones with benzothiazole moieties

Synthesis of new 4ethiazolidinone-, pyrazoline-, and isatin-based conjugates with promising antitumor activity

Study of molecular mechanisms of proapoptotic action of novel heterocyclic 4-thiazolidone derivatives

In vitro and in vivo assay systems for study of influenza virus inhibitors

Prolyl oligopeptidase of Trypanosoma brucei hydrolyzes native collagen, peptide hormones and is active in the plasma of infected mice

New protein farnesyltransferase inhibitors in the 3-arylthiophene 2-carboxylic acid series: diversification of the aryl moiety by solid-phase synthesis

We are grateful to Dr. V.L. Narayanan from Drug Synthesis and Chemistry Branch, National Cancer Institute, Bethesda, MD, USA, for in vitro evaluation of anticancer activity. Evaluations of compounds for antiviral activity were supported by funds from contract N01-AI-15435 from the Virology Branch, Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.