Combinatorial Optimization of Isatin-β-Thiosemicarbazones as Anti

Mar 30, 2005 - Chart 1. Isatin Thiosemicarbazones with Previously Reported .... IBT (1), 14 ± 8.7, >454 ± 0, >32 .... (4{10}).33 This was prepared f...
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J. Med. Chem. 2005, 48, 3045-3050

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Combinatorial Optimization of Isatin-β-Thiosemicarbazones as Anti-poxvirus Agents Michael C. Pirrung,*,† Sunil V. Pansare,†,§ Koushik Das Sarma,† Kathy A. Keith,‡ and Earl R. Kern‡ Department of Chemistry, Levine Science Research Center, Box 90317, Duke University, Durham, North Carolina 27708-0317, and Department of Pediatrics, University of Alabama School of Medicine, Birmingham, Alabama 35233 Received October 25, 2004

Novel strategies are required to combat pox virus infections, whether caused by escape of viruses such as monkeypox from indigenous areas or intentional release of smallpox. Anti-smallpox drugs with a unique mode of antiviral action, inhibition of transcription termination, were known but not therapeutically useful. Using a combinatorial method, variants of the basic isatin-β-thiosemicarbazone structure were prepared and examined for cytotoxicity and antiviral activity in vaccinia virus- and cowpox virus-infected human cells. Potent and much more selective N-aminomethyl-isatin-β-thiosemicarbazones were discovered. Introduction The Centers for Disease Control have identified several viral pathogens that could be used as weapons of mass destruction. Smallpox (variola) is one virus with potential for use in bioterrorism.1 In the 1970s, smallpox was essentially eradicated through immunization, and vaccination ceased. Much of the younger population thus does not have immunity to smallpox, and the immune status of individuals who received vaccinations long ago is questionable. High-risk subpopulations have recently been immunized against smallpox, but the timeliness, practicality, effectiveness, and costs of immunization of the broad population are unclear. For example, over 40 million Americans are advised against smallpox immunization because of their health status. Consequently, novel defenses against widespread smallpox infections must be considered.2 A strategic alternative to immunization is anti-smallpox drugs that can be used as the situation warrants, for example in local areas subject to viral infection. Recent developments adapting existing nucleoside antiviral agents toward poxviruses3 have validated drug therapy strategies against smallpox despite their limited past impact.4 Thiosemicarbazones appear to be a structural class with anti-pox virus activity.5 Isatin derivatives such as methisazone (marboran), the β-thiosemicarbazone of N-methyl isatin, have been described as smallpox chemoprophylactic agents.6 Methisazone decreases morbidity and mortality when given to susceptible contacts, but has no direct therapeutic efficacy vs variola and is no longer manufactured as a drug substance. Other isatin derivatives have been reported to inhibit replication of vaccinia virus, which is closely related to variola and is often used as a laboratory model for it.7,8 Activity against other viruses (cytomegalovirus, Moloney leukemia virus) has also been reported for other isatin * Corresponding author. Current address: Department of Chemistry, University of California, Riverside, CA 92521-0403. Fax: 951-8272749. E-mail: [email protected]. † Duke University. ‡ University of Alabama School of Medicine. § Current address: Department of Chemistry, Memorial University of Newfoundland, St. Johns, NL A1B 3X7.

Chart 1. Isatin Thiosemicarbazones with Previously Reported Anti-poxvirus Activity (1, 2), and Template 3 for the Combinatorial Library

thiosemicarbazone derivatives (N-methylisatin-β-4′,4′diethylthiosemicarbazone and N-allylisatin-β-4′,4′-diallylthiosemicarbazone).9 The isatin that has been the most studied is isatinβ-thiosemicarbazone (IBT, 1), as an inhibitor of vaccinia virus (Chart 1). Although the precise viral target and mechanism of action of IBT has not been proved, the primary effect of the drug is to enhance viral postreplicative transcription elongation, either directly or by suppressing termination, resulting in the formation of longer-than-normal transcripts.10 Transcripts extended at their 3′-ends and arising from convergent transcriptional promoters increase the intracellular concentration of dsRNA, which in turn stimulates the cellular dsRNAdependent 2-5A pathway that establishes an antiviral state in cells. This ultimately results in the activation of RNAse L, degradation of viral and cellular mRNA and rRNA, and abortion of the virus infection.11,12 IBTtreated cells phenocopy mutations in the viral A18R gene.10,11 The A18R gene product, a DNA helicase, is an RNA release factor, possibly a transcription termination factor, that normally promotes release of RNA at pause sites.13-15 The absence of A18R activity due to mutation results in read-through of pause sites, hence the IBT-like, transcription read-through phenotype. Either the IBT phenocopy or the A18R mutant phenotype can be compensated for by mutations in the viral G2R or J3R genes, which encode presumptive positive transcription elongation factor activities.16-19 While the exact viral target of IBT is unknown, viral mutants that

10.1021/jm049147h CCC: $30.25 © 2005 American Chemical Society Published on Web 03/30/2005

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are either drug dependent or drug resistant have been isolated and mapped to G2R, J3R, and the viral RNA polymerase,20 supporting both the virus specificity of the drug and a transcription-specific mode of action.16,18-21 Inhibition of A18R helicase activity on the viral RNA polymerase is one possible mode of action of IBT. Hence, IBT should exhibit its antiviral activity only in poxvirusinfected cells. Studies by Katz22 have supported this model and reported additional antiviral thiosemicarbazones.23 The isatin thiosemicarbazones therefore constitute lead structures around which a combinatorial library could be constructed. Points for ready diversification include the aromatic ring and N-alkyl substituents. Isatins can be prepared in one step from anilines by the Sandmeyer or Martinet reactions.24 While they are not easily N-alkylated with conventional alkylating agents, they react readily with imonium ions to produce Naminomethyl derivatives.8 The physical (solubility) properties of isatins and their derivatives make them challenging to handle and purify, so we developed25 a solid-phase synthesis of isatin thiosemicarbazones that has proved essential to the efficient preparation of derivatives 3 in pure form; it is also amenable to parallel/pool synthesis. The method relies on a trityl thiosemicarbazone resin that captures isatins from solution. On treatment with either formaldehyde and a secondary amine or an imonium ion (prepared from the aminal and acetyl chloride), the N-aminomethyl isatin is formed. The product can be removed from the support with TFA. Based on the mode of action of IBT, screening of isatin thiosemicarbazone libraries should be cell-based, hence screening cannot be on-bead. We therefore adopted the indexed combinatorial library method, in which organized pools are used in initial screening and the activities of these pools indicate single compounds for further synthesis and testing.26

Pirrung et al.

Chart 2. Isatin Building Blocks 4{a}

Chart 3. Secondary Amine Building Blocks 5{b}

Results The indexed combinatorial library we designed includes two different pool sets, one in which the isatin is held constant and all amines are used, and one in which the amine is held constant and all isatins are used. The isatins were chosen (Chart 2) based on reactivity in the previously developed25 reaction sequence and commercial availability or ready preparation. In particular, it was not possible to efficiently generate Mannich bases from isatins bearing substituents at the 7-position (adjacent to nitrogen), evidently for steric reasons, nor with electron-withdrawing groups at the 5-position. Both cyclic and acyclic and polar and nonpolar amines were chosen (Chart 3). The isatins 4 were prepared from the anilines by Sandmeyer (4{49}) or Martinet reactions (4{10}) and loaded onto trityl thiosemicarbazone resin. To prepare one dimension of the indexed library (Scheme 1), imonium ions were generated from a pool of 10 formaminals of the secondary amines 5 and allowed to react with individual resinbound isatins. The products were cleaved from the support with TFA/triisopropylsilane to yield pools 3{1,b} to 3{10,b}. Ten resins bearing isatins 4 were mixed in equal amounts and treated with imonium ions generated from individual aminals derived from secondary

Scheme 1. Preparation of Pools in the First Dimension of the Library

Isatin-β-Thiosemicarbazones as Anti-poxvirus Agents

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 3047 Table 2. Efficacy in Inhibition of Cowpox Virus Plaque Formation (cowpox Brighton) and Cytotoxicity of IBT Derivatives 3{a,b} in HFF Cells Compared with Other Known Anti-poxvirus agentsa compound

EC50 (µM) ( SEM

CC50 (µM) ( SEM

SI

3{8,1} 3{8,4} 3{8,9} 3{8,10} CDV IBT (1) methisazone (2)

6.2 ( 1.6 29 ( 12 11 ( 13 6.0 ( 2.9 38 ( 1.9 60 ( 10 14 ( 0.3

>244 ( 0 >235 ( 0 201 ( 23 181 ( 67 >317 ( 0 >454 ( 0 >427 ( 0

>39 >8.1 18 30 >8.3 >7.6 >31

a Values are the mean of two or more assays ( standard deviation; CC50 is the half-maximal cytotoxic concentration; SI is CC50/EC50.

Figure 1. Results of screening of isatin-β-thiosemicarbazone pools (the identity of the fixed compound in each pool is indicated by numbers below the figure) against vacciniainfected HFF cells. Inhibition of cytopathic effect is given as (mean EC50, µg/mL)-1, meaning that more potent compounds show taller bars. Table 1. Efficacy in Inhibition of Vaccinia Virus Plaque Formation (vaccinia Copenhagen) and Cytotoxicity of IBT Derivatives 3{a,b} in HFF Cells Compared with Other Known Anti-poxvirus Agentsa compound

EC50 (µM) ( SEM

CC50 (µM) ( SEM

SI

3{8,1} 3{8,4} 3{8,9} 3{8,10} CDV IBT (1) methisazone (2)

1.0 ( 0.8 6.8 ( 8.7 0.8 ( 0.1 0.6 ( 0.5 27 ( 7.9 14 ( 8.7 3.3 ( 3.2

>244 ( 0 >235 ( 0 201 ( 23 181 ( 67 >317 ( 0 >454 ( 0 >427 ( 0

>244 >35 251 302 >12 >32 >129

a Values are the mean of two or more assays ( standard deviation; CC50 is the half-maximal cytotoxic concentration; SI is CC50/EC50.

amines 5. Cleavage from the support yielded pools 3{a,1} to 3{a,10}, the second dimension of the library. All pools were characterized by HPLC and FAB MS to establish the presence of each imputed product in approximately equimolar amounts. Screening was performed on these twenty pools for inhibition of cytopathic effects in vaccinia-infected HFF tissue culture cells. The potencies shown in Figure 1 identified 5-bromoisatin as a key pharmacophore for antiviral activity in this library. Secondary amine 5{4} was found in the most active amine pool, though its contributions were not as dominating as the isatin 4{8}. Analysis of these screening results by statistical criteria described earlier26a validated these hits. Four compounds based on the most potent isatin 4{8} were selected for solid-phase synthesis as singles. These compounds were examined for inhibition of plaque formation in vaccinia-infected and in cowpox-infected HFF cells. Additionally, to determine the selectivity index (SI),3 their cytotoxicities were measured in HFF cells using neutral-red uptake. For comparison, other agents with reported activity against poxviruses were subjected to the same assays. Results are summarized in Tables 1 and 2. It is interesting that the most potent and selective compounds [3{8,9} and 3{8,10}] include acyclic amines, as contrasted with previously identified cyclic amines. There is also not a direct relationship between the most potent pool (3{a,4}) and the most potent singles. This observation is consistent with a

broad understanding of pool screening.27 Additionally, cidofovir (CDV), which has attracted significant interest as an anti-poxvirus agent based on its activity against vaccinia,3 is over 30-fold less potent and far less selective than the best isatin-β-thiosemicarbazone Mannich base. Lower potency, leading to lower selectivity, is seen against cowpox, but the structure-activity trends are similar. The compound of most interest from this library is 3{8,10}. Discussion This isatin-β-thiosemicarbazone combinatorial library has provided compounds with significantly enhanced potency over IBT in cell-based activity against vaccinia, and with somewhat enhanced potency against cowpox. Solid-phase synthesis was crucial to the convenient preparation of targets in the IBT class. The mode of action of compounds 3 is presumed to be the same unique mode of action as IBT, which necessitated a cellular assay. These studies suggest further investigation of compounds based on the 5-bromoisatin lead structure, which will be reported in due course. Experimental Section 5-Methoxyisatin (4{4}).28 This compound was prepared from p-anisidine (6.1 g, 0.05 mol), chloral hydrate (9.0 g, 0.05 mol), Na2SO4 (130 g), hydroxylamine hydrochloride (11 g, 0.16 mol), and HCl (4.3 mL, 0.05 mol) by adapting the literature procedure. Yield 5.1 g (58%). 5,6-Dimethoxyisatin (4{5}).29 This compound was prepared from 4-aminoveratrole (7.7 g, 0.05 mol), chloral hydrate (9.0 g, 0.05 mol), Na2SO4 (130 g), hydroxylamine hydrochloride (11 g, 0.16 mol), and HCl (4.3 mL, 0.05 mol) by adapting the literature procedure. Yield 6.0 g (58%). 5,6-Dimethylisatin (4{6}) and 4,5-Dimethylisatin (4{7}).30 These compounds were prepared from 3,4-dimethylaniline (6.1 g, 0.05 mol), chloral hydrate (9.0 g, 0.05 mol), Na2SO4 (130 g), hydroxylamine hydrochloride (11 g, 0.16 mol), and HCl (4.3 mL, 0.05 mol) by adapting the literature procedure. The isomers were separated as follows: the crude product was dissolved in excess aqueous NaOH (10%). The solution was filtered and acidified to pH 5 with glacial acetic acid. The 4,5dimethylisatin precipitated as an amorphous, orange solid. Yield 1.5 g (17%). The filtrate obtained after removing the precipitated isatin was acidified to pH < 1 with concentrated HCl. The 5,6-dimethylisatin precipitated as a crystalline red solid. Yield 3.0 g (34%). 5-Bromo-6-methylisatin (4{8}) and 4-Methyl-5-bromoisatin.31 These compounds were prepared from 3-methyl-4bromoaniline (5 g, 0.03 mol), chloral hydrate (4.8 g, 0.03 mol), Na2SO4 (65 g), hydroxylamine hydrochloride (7.0 g, 0.16 mol), and HCl (3.4 mL, 0.04 mol) by adapting the literature procedure. The isomers were separated as follows: the crude product was dissolved in excess aqueous 10% NaOH (130 mL).

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The solution was filtered from much undissolved solids and acidified to pH 5 with glacial acetic acid. The 4-methyl-5bromoisatin precipitated as an amorphous, orange solid. Yield 1.6 g (22%). The filtrate obtained after removing the precipitated isatin was acidified to pH < 1 with concentrated HCl. The 5-bromo-6-methylisatin precipitated as a crystalline orange solid. Yield 1.1 g (15%). The low yields may be due to the poor solubility of the isatin in aqueous NaOH. 1,5,6,7-Tetrahydro-1-aza-s-indacene-2,3-dione (4{9}).32 This was prepared from 5-aminoindan (6.7 g, 0.05 mol), chloral hydrate (9.0 g, 0.05 mol), Na2SO4 (130 g), hydroxylamine hydrochloride (11 g, 0.16 mol), and HCl (4.3 mL, 0.05 mol) by adapting the literature procedure. The crude product is a 2.3/1 mixture of linear/angular isomers. The crude product was dissolved in excess aqueous NaOH (10%). The solution was filtered and acidified to pH 5 with glacial acetic acid. The linear isatin precipitated as a red solid. Yield 2.4 g (26%). The angular isatin could not be precipitated effectively. Note that in this case the linear isatin is precipitated with glacial acetic acid. 5-Methylthioisatin (4{10}).33 This was prepared from 4-methylthioaniline (1.0 g, 7.2 mmol), diethyl ketomalonate (1.2 mL, 7.9 mmol), and acetic acid by adaptation of the literature procedure to give the target compound as a red crystalline solid. Yield 190 mg (14%). Trityl Thiosemicarbazide Resin. To a suspension of the trityl isothiocyanate resin (2.0 g, 1.9 mmol) in anhydrous THF (8 mL) was added a solution of 1 M hydrazine in THF (6 mL, 6 mmol). The mixture was shaken at ambient temperature for 8 h. The resin was filtered, washed with THF (5 × 20 mL), and dried under reduced pressure (2.0 g, 97% loading based on the weight of the resin). Synthesis of Isatin Derivatives. General Procedures. The trityl thiosemicarbazide resin was suspended in a solution of the isatin in DMF. The mixture was shaken at ambient temperature for 20-24 h and filtered. Unreacted isatin was recovered from the filtrate. The functionalized resin was washed successively with DMF, THF, and dichloromethane (3 × 10 mL/g resin) and dried to constant weight. Conversion of the resin-bound isatin thiosemicarbazone to the aminal derivative was achieved by one of the following procedures. Method A. A mixture of resin-bound isatin, 37% aqueous formaldehyde (5 mL/g resin), and a secondary amine (10 mmol/g resin) in DMF was shaken at room temperature for 24 h. The mixture was filtered, and the resin was washed with THF. The resin was shaken with DMSO for 15 min, filtered, washed with THF and dichloromethane (7 mL/g resin), and dried to constant weight. Method B. A solution of the 1,1′-methylenebisamine (5 mmol/g resin) in dichloromethane (7 mL/g resin) was treated with acetyl chloride (5 mmol/g resin) (30 min, RT) to generate the imonium salt. This suspension was added to a suspension of resin-bound isatin in dichloromethane. The mixture was shaken at ambient temperature for 3-5 h and filtered. The resin was washed several times with dichloromethane and dried to constant weight. Cleavage from Resin of Isatin Derivatives (3). The dried resin was suspended in a solution of triisopropylsilane (TIPS) in dichloromethane (10%, 7.5 mL/g resin) for 10 min and an equal volume of a solution of trifluoroacetic acid in dichloromethane (30%) was added dropwise. The mixture was shaken for 2-3 min, and all volatiles were removed under reduced pressure. The residue was suspended in dichloromethane, and the mixture was passed through a plug of CeliteTM (3 g/g resin) soaked with saturated aq Na2CO3 (2.5 mL/g Celite). The dichloromethane solution obtained was dried (Na2SO4) and evaporated to give isatin derivatives 3 that were ∼85% pure by HPLC. Further purification could be achieved by recrystallization. An improved procedure was developed for purification of the IBT derivatives since publication of our original paper.25 After cleavage from the resins, solvent was removed and 1 mL of MeCN was added to the residue. The resulting solution was washed 3 × 1 mL hexanes followed by passing through a Celite/Na2CO3 pad.

Analytical samples were prepared by dissolving 10-20 mg of the compounds in a minimum volume of DMSO and then diluting to ∼0.5 mL with MeOH. These were purified by semiprep HPLC (9.4 mm × 25 cm ZORBAX ODS column, 30100% MeCN/40 min, 5 mL/min flow rate). 3{8,1}. 1H NMR (CDCl3, 300 MHz): δ 7.72 (1H, s, C4H), 7.50 (1H, br s, NH), 6.94 (1H, s, C7H), 6.48 (2H, br s, NH2), 4.43 (2H, s, NCH2N), 2.55 (4H, m, NCH2), 2.46 (3H, s, C6CH3), 1.56 (4H, br s, CH2), 1.42 (2H, m, CH2). Anal. (C16H20BrN5OS): C, H, N. 3{8,4}. 1H NMR (CDCl3, 300 MHz): δ 7.72 (1H, s, C4H), 7.50 (1H, br s, NH), 6.92 (1H, s, C7H), 6.48 (2H, br s, NH2), 4.47 (2H, s, NCH2N), 2.65 (4H, m, NCH2), 2.46 (3H, s, C6CH3), 2.43 (4H, m, NCH2), 2.27 (3H, s, NCH3). Anal. (C16H21BrN6OS): C, H, N. 3{8,9}. 1H NMR (CDCl3, 300 MHz): δ 7.65 (1H, s, C4H), 7.31 (5H, br s, C6H5), 6.54 (1H, s, C7H), 4.48 (2H, s, NCH2N), 3.68 (2H, s, HCH2Ph), 2.69 (2H, m, CH2), 2.34 (3H, s, C6CH3), 1.15 (3H, t, J ) 6.0 Hz, CH3). Anal. (C20H22BrN5OS): C, H, N. 3{8,10}. 1H NMR (CDCl3, 300 MHz): δ 7.72 (1H, s, C4H), 7.50 (1H, br s, NH), 7.07 (1H, s, C7H), 6.48 (2H, br s, NH2), 4.56 (2H, s, NCH2N), 2.57 (2H, t, J ) 6.0 Hz, NCH2), 2.47 (5H, m, C6CH3 + NCH2), 0.98 (2H, m, CH2), 0.82 (3H, t, J ) 6.0 Hz, CH3), 0.61 (1H, m, CH), 0.55 (2H, m, 2CH), 0.14 (2H, m, 2CH). Anal. (C18H24BrN5OS): C, H, N. Virus Pools, Media, and Cells. Vaccinia virus (VV) strain Copenhagen and cowpox virus (CV) strain Brighton stock pools prepared in Vero cells were obtained from Dr. John Huggins of USAMRIID, Frederick, MD, and were diluted to prepare working stocks. Human foreskin fibroblast (HFF) cells were prepared as primary cultures from freshly obtained newborn human foreskins as soon as possible after circumcision. Culture medium was Eagle’s minimal essential medium (MEM) containing 10% fetal bovine serum (FBS) and standard concentrations of L-glutamine, penicillin, and gentamicin. Efficacy. Plaque Reduction Assay for VV and CV. Two days prior to use, HFF cells were plated into six-well plates and incubated at 37 °C with 5% CO2 and 90% humidity. On the day of assay, the drug was made up at twice the desired concentration in 2× MEM with 2% FBS and diluted serially 1:5 in 2× MEM to provide final concentrations of drug ranging from 100 to 0.032 µg/mL. The virus to be used was diluted in MEM containing 10% FBS to a desired concentration which would give 20-30 plaques per well. The media was then aspirated from the wells and 0.2 mL of virus added to each well in triplicate with 0.2 mL of media being added to drug and cell control wells. The plates were incubated for 1 h with shaking every 15 min. After the incubation period, an equal amount of 1% agarose was added to an equal volume of each drug dilution. The drug/agarose mixture was added to each well in 2 mL volumes and the plates incubated for 3 d, after which the cells were stained with 0.02% solution of neutral red in PBS. After a 5-6 h incubation period, the stain was aspirated and plaques were counted using a stereomicroscope at 10× magnification. The MacSynergy II, version 1 computer program was used to calculate the 50% effective concentration (EC50) value. Cytotoxicity. Neutral Red Uptake Assay. HFF cells were plated into 96-well plates 24 h prior to assay at a concentration of 2.5 × 104 cells per well. After 24 h, the media was aspirated and 125 µL of each drug concentration in MEM with 2% FBS was added to the first row of wells and then diluted serially 1:5 using the Beckman BioMek Liquid Handling System. Final drug concentrations ranged from 100 to 0.032 µg/mL. The plates were incubated for 7 d in a CO2 incubator at 37 °C. After incubation, the media/drug was aspirated and 200 µL/well of 0.01% neutral red in PBS was added and incubated for 1 h. The dye was aspirated and the cells were washed with PBS using a Nunc Plate Washer. After removing the PBS, 200 µL/well of 50% EtOH/1% glacial acetic acid (in H2O) was added. The plates were placed on a rotary shaker for 15 min and the optical densities read at 540 nm on a Bio-tek plate reader. The 50% cytotoxic concentration of drug (CC50) was calculated using the software indicated previously.

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Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 3049

Acknowledgment. Financial support by NIH grant AI-48521 and contract NO1-AI-85347 is gratefully acknowledged. The assistance of L. LaBean and J. Wessels in administrative support of this work is greatly appreciated.

(17) Latner, D. R.; Thompson, J. M.; Gershon, P. D.; Storrs, C.; Condit, R. C. The Positive Transcription Elongation Factor Activity of the Vaccinia Virus J3 Protein Is Independent From Its (Nucleoside-2′-O-)-Methyltransferase And Poly(A) Polymerase Stimulatory Functions. Virology 2002, 301, 64-80. (18) Latner, D. R.; Xiang, Y.; Lewis, J. I.; Condit, J.; Condit, R. C. The Vaccinia Virus Bifunctional Gene J3 (Nucleoside-2′-O-)Methyltransferase and Poly(A) Polymerase Stimulatory Factor Is Implicated as a Positive Transcription Elongation Factor by Two Genetic Approaches. Virology 2000, 269, 345-55. (19) Black, E. P.; Condit, R. C. Phenotypic Characterization of Mutants in Vaccinia Virus Gene G2R, a Putative Transcription Elongation Factor. J. Virol. 1996, 70, 47-54. (20) Condit, R. C.; Easterly, R.; Pacha, R. F.; Fathi, Z.; Meis, R. J. A Vaccinia Virus Isatin-Beta-Thiosemicarbazone Resistance Mutation Maps in the Viral Gene Encoding the 132-kDa Subunit of RNA Polymerase. Virology 1991, 185, 857-61. (21) Meis, R. J.; Condit, R. C. Genetic and Molecular Biological Characterization of a Vaccinia Virus Gene Which Renders the Virus Dependent on Isatin-beta-thiosemicarbazone (IBT). Virology 1991, 182, 442-54. (22) Katz, E.; Margalith, E.; Winer, B.;Goldblum, N. Synthesis of Vaccinia Virus Polypeptides in the Presence of Isatin-BetaThiosemicarbazone. Antimicrob. Agents Chemother. 1973, 4, 448. Katz, E.; Margalith, E.; Winer, B. The Effect of Isatin Beta Thiosemicarbazone (IBT)-Related Compounds On IBT-Resistant and on IBT-Dependent Mutants of Vaccinia Virus. J. Gen. Virol. 1974, 25, 239-44. Katz, E.; Margalith, E.; Winer, B. An Isatin Beta-Thiosemicarbazone (IBT)-Dependent Mutant of Vaccinia Virus: The Nature of the IBT-Dependent Step. J. Gen. Virol. 1973, 21, 477-84. Katz, E.; Winer, B.; Margalith, E.; Goldblum, N. Isolation and Characterization of an IBT-Dependent Mutant of Vaccinia Virus. J. Gen. Virol. 1973, 19, 161-4. Formation of Vaccinia Virus DNA-Protein Complex in the Presence of Isatin Beta Thiosemicarbazone (IBT). J. Gen. Virol. 1978, 40, 695-9. (23) Katz, E.; Margalith, E.; Winer, B.Structure Activity Relationships of Thiosemicarbazones on Vaccinia Virus and IBT-Dependent Mutant. Antimicrob. Agents Chemother. 1976, 2, 25560. (24) Popp, F. D. Chemistry of Isatin. Adv. Heterocycl. Chem. 1975, 18, 1-58. (25) Pirrung, M. C.; Pansare, S. V. Trityl Isothiocyanate Support for Solid-phase Synthesis. J. Comb. Chem. 2001, 3, 90-96. (26) a. Pirrung, M. C.; Chen, J. Preparation and Screening Against Acetylcholinesterase of a Non-Peptide ‘Indexed’ Combinatorial Library. J. Am. Chem. Soc. 1995, 117, 1240-45. Pirrung, M. C.; Chau, J. H.-L.; Chen, J. Discovery of a Novel Tetrahydroacridine Acetylcholinesterase Inhibitor through an Indexed Combinatorial Library. Chem. Biol. 1995, 3, 621-26. Pirrung, M. C.; Chau, J. H.-L.; Chen, J. Indexed Combinatorial Libraries: NonOligomeric Chemical Diversity for the Discovery of Novel Enzyme Inhibitors. In Combinatorial Chemistry: A High-Tech Search for New Drug Candidates; Wilson, S. R., Murphy, R., Eds.; John Wiley & Sons: New York, 1996. An, H.; Haly, B. D.; Cook, P. D. Discovery of Novel Pyridinopolyamines With Potent Antimicrobial Activity: Deconvolution of Mixtures Synthesized By Solution-Phase Combinatorial Chemistry. J. Med. Chem. 1998, 41, 706-16. Andrus, M. B.; Turner, T. M.; Sauna, Z. E.; Ambudkar, S. V. The Synthesis and Evaluation of a SolutionPhase Indexed Combinatorial Library of Nonnatural Polyenes for Multidrug Resistance Reversal. J. Org. Chem. 1999, 64, 2978-79. Andrus, M. B.; Turner, T. M.; Sauna, Z. E.; Ambudkar, S. V. The Synthesis and Evaluation of a Solution Phase Indexed Combinatorial Library of Non-Natural Polyenes For Reversal of P-Glycoprotein Mediated Multidrug Resistance. J. Org. Chem. 2000, 65, 4973-83. Boger, D. L.; Dechantsreiter, M. A.; Ishii, T.; Fink, B. E.; Hedrick, M. P. Assessment of Solution-Phase Positional Scanning Libraries Based on Distamycin A for the Discovery of New DNA Binding Agents. Bioorg. Med. Chem. 2000, 8, 2049-57. Boger, D. L.; Lee, J. K.; Goldberg, J.; Jin, Q. Two Comparisons of the Performance of Positional Scanning and Deletion Synthesis for the Identification of Active Constituents In Mixture Combinatorial Libraries. J. Org. Chem. 2000, 65, 1467-74. Teixido J.; Michelotti, E. L.; Tice, C. M. Ruminations Regarding the Design of Small Mixtures for Biological Testing. J. Comb. Chem. 2000, 2, 658-74. Ambroise, Y.; Yaspan, B.; Ginsberg, M. H.; Boger, D. L. Inhibitors of Cell Migration That Inhibit Intracellular Paxillin/alpha4 Binding: A Well-Documented Use of Positional Scanning Libraries. Chem. Biol. 2002, 9, 1219-26. Ding, B.; Taotofa, U.; Orsak, T.; Chadwell, M.; Savage, P. B. Synthesis and Characterization of PeptideCationic Steroid Antibiotic Conjugates. Org. Lett. 2004, 6, 34336. (27) Konings, D. A. M.; Wyatt, J. R.; Ecker, D. J.; Freier, S. M. Deconvolution of Combinatorial Libraries for Drug Discovery: Theoretical Comparison of Pooling Strategies. J. Med. Chem.

Supporting Information Available: Detailed procedures for preparation of some single IBT-Mannich bases. This material is available free on the web from http://pubs.acs.org.

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