Dihydrofuro[3,4-c]pyridinones as Inhibitors of the Cytolytic Effects of the Pore-Forming Glycoprotein Perforin

Dihydrofuro[3,4-c]pyridinones as Inhibitors of the Cytolytic Effects of the Pore-FormingGlycoprotein Perforin

Gersande Lena,† Joseph A. Trapani,‡ Vivien R. Sutton,‡ Annette Ciccone,‡ Kylie A. Browne,‡ Mark J. Smyth,‡

William A. Denny,† and Julie A. Spicer†,*

Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The UniVersity of Auckland,PriVate Bag 92019, Auckland 1142, New Zealand, and Cancer Immunology Program, Peter MacCallum Cancer Centre, St Andrew’s Place,East Melbourne, Victoria 3002, Australia

ReceiVed August 25, 2008

Dihydrofuro[3,4-c]pyridinones are the first class of small molecules reported to inhibit the cytolytic effectsof the lymphocyte toxin perforin. A lead structure was identified from a high throughput screen, and aseries of analogues were designed and prepared to explore structure-activity relationships around the corebicyclic thioxofuropyridinone and pendant furan ring. This resulted in the identification of a submicromolarinhibitor of the perforin-induced lysis of Jurkat T-lymphoma cells.

Introduction

The key cytotoxic effector cells, cytotoxic T lymphocytes(CTLa) and natural killer (NK) cells, perform tumor surveillanceand provide a defense against viral infection and intracellularpathogens. The granule exocytosis pathway used by CTLs andNK cells is crucial for the induction of apoptosis in theelimination of virus-infected or transformed cells.1-3 Two ofthe main components found in the cytotoxic granules are a groupof serine proteases known as granzymes,4 and perforin, acalcium-dependent pore-forming glycoprotein of about 67kDa.5-8 Upon stable conjugation of the CTL or NK cell witha target cell, the polarized release of the granule contents intothe synaptic cleft occurs, ultimately resulting in infiltration ofthe target cell by the granzymes. Perforin is essential for thisprocess,7 due at least in part to its ability, upon binding calcium,to assemble into highly ordered aggregates of 12-18 moleculesthat form trans-membrane pores of 10-20 nm in diameter inthe plasma membrane.1,9 This can allow leakage of cell contentsand, probably more importantly, entry of the granzymes, whichpromote both caspase-dependent and -independent apoptosis.4

This process was further elucidated recently with the publica-tion of the crystal structure of a bacterial protein from Photo-rhabdus luminescens which belongs to the same superfamilyof proteins as perforin.9 Both of these proteins contain themembrane attack complex/perforin (MACPF) domain, and itwas concluded that the presence of this domain in both thebacterial protein Plu-MACPF and perforin suggests the abilityto form a “pre-pore” oligomer, which binds to the cell membraneand upon a conformational change converts into a giant �-barrel-linked channel.

Deficiencies of many of the granule components are toleratedin vivo;1 however, perforin is essential for protective immunesurveillance. In 1994, the generation of perforin-deficient micerevealed a susceptibility to, and failure to clear, many viruses

and other intracellular pathogens as well as the spontaneousdevelopment of B-cell lymphoma as the mice aged.10 Whenperforin-deficient mice were backcrossed with the nonobesediabetes mouse strain, it was found that the incidence ofspontaneous diabetes over a one year period was reduced from77% in a perforin +/+ control to 16% in perforin-deficient mice,with onset of disease markedly delayed.11 In humans, defectsor mutations that impair perforin function result in familialhemophagocytic lymphohistiocytosis syndrome, characterizedby severe anemia and hepatosplenomegaly, fever, and thrombo-cytopenia.12-15 CTL and NK cells have also been implicatedin several autoimmune diseases (e.g., insulin-dependent dia-betes)4,16 as well as therapy-induced conditions (e.g., allograftrejection, graft-versus-host disease).17-19

To date, the only reported inhibitors of perforin function arecomplex natural products, primarily concanamycin A and otherV-ATPase inhibitors such as bafilomycin A and prodigiosin 25-Cs20 that inhibit cytoplasmic granule acidification.21 Otherreported inhibitors include cytochalasin D (an inhibitor of actinpolymerization), antimycin A, and oligomycin A (inhibitors ofcell respiration) and some protein kinase inhibitors (calphostinC, herbimycin A, staurosporine).22 All of these classes ofinhibitors exert their effects on perforin processing, storage, orrelease rather than by directly inhibiting perforin’s action onthe target cell. In this regard, small-molecule inhibitors ofperforin function are of potential interest as a new class oftherapeutic immunosuppressive agents, but to the best of ourknowledge there have been no reports of such compounds. Wereport here studies on dihydrofuro[3,4-c]pyridinones as small-molecule inhibitors of perforin-induced lysis.

Results and Discussion

Synthesis of Target Compounds. The lead compound (1)was selected from a high throughput screen23,24 of approximately100000 compounds sourced from commercial libraries using a384-well plate format. Compounds were screened (at 20 µM)by incubation with sheep red blood cells, which in the absenceof an inhibitor are lysed by perforin, generating turbidity thatcan be measured by the change in absorbance at 650 nm.

The lead structure 1 (Figure 1) is easily disconnected intotwo subunits: the subunit A being the bicyclic thioxofuropyri-dinone, with subunit B corresponding to the furan ring.Analogues of 1 containing variations of both subunits were

* To whom correspondence should be addressed. Phone: +64 9 3737599.Fax: +64 9 3737502. E-mail: [email protected].

† The University of Auckland;‡ Peter MacCallum Cancer Centrea Abbreviations: CTL, cytotoxic T lymphocyte; NK, natural killer;

MACPF, membrane attack complex/perforin; ROESY, rotational nuclearOverhauser effect; PLO, pneumolysin; FasL, Fas-ligand; TNFR, tumornecrosis factor R; TRAIL, TNF-related apoptosis inducing ligand; SAR,structure-activity relationship.

J. Med. Chem. 2008, 51, 7614–76247614

10.1021/jm801063n CCC: $40.75 2008 American Chemical SocietyPublished on Web 11/14/2008

prepared (Tables 1 and 2). Modifications of subunit A involvedthe preparation of the furopyridinedione (5), the furopyridinone(8), the pyrrolopyridinedione (9), and the thioxopyrrolopyridi-none (11) (Schemes 1 and 2). For the variations of subunit B,a diverse range of aldehydes were either commercially availableor prepared where required. The two subunits can be assembledvia an aldolization-crotonization reaction.

A literature search showed that compounds 1, 12, 14, 26,and 32 had been described previously.25-27 These compoundswere prepared by reaction of 7 (or 5 in the case of compound32) with the relevant aldehydes. The preparation of 7 itself wasbased on chemistry developed during the course of efforts todetermine the structure of vitamin B6,28,29 and we used thismethod, with some minor modifications (Scheme 1) to prepare7 and subsequently an array of 4-oxo and 4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one analogues. Ethyl 2-ethoxy-acetate (2) was reacted with acetone in the presence of sodiumhydride to give ethoxyacetylacetone (3). Standard reactionconditions between 3 and 2-cyanoacetamide produced the2-pyridone derivative 4. The lactonization was performed inconcentrated HCl and followed by reaction with phosphoruspentachloride to afford the R-chloro-pyridine derivative 6.Finally, displacement of the chlorine with thiourea yielded thethioxofuropyridinone 7.

Intermediate 8 was prepared by catalytic hydrogenation ofcompound 6, in accordance with previously reported results.30

The lactam 9 was obtained by reaction of 4 with concentratedsulfuric acid (Scheme 2).31 Preparation of the correspondingthioxo-analogue 11 involved the reaction of 2-thiocyanoaceta-mide with 3 to give the thioxopyridine 10. The lactamizationthen proceeded under the same conditions and afforded 11 inmoderate yields.

With five different bicyclic intermediates available, thepreparation of a series of analogues of compound 1 wasundertaken in a multiparallel fashion using a carousel. To asuspension of starting material in ethanol was added each ofthe various aldehydes and a catalytic amount of piperidine(Scheme 3). The aldolization-crotonization reactions wereheated at reflux and monitored by mass spectrometry. Dependingon the reactivity of the aldehydes, the reaction time varied from40 min to 20 h. The products precipitated out directly from thereaction mixture and were simply collected by filtration, rinsed,and dried.

As expected, the reaction between 5, 7, and a selected set ofaldehydes proceeded efficiently to give a series of compoundsappended with both unsubstituted and substituted furan, thiophene,pyrrole, imidazole, phenyl (Table 1), benzofuran, benzothiophene,and indole (Table 2) rings.

The position of the link between subunits A and B wasexplored by reaction of 7 with 3-furaldehyde and thiophene-3-carboxaldehyde to give 3-linked compounds 39 and 40,respectively (Table 1). For targets containing a bicyclic B-subunit, 5 and 7 were reacted with 3-benzofuran-, 3-ben-

zothiophene-, and 3-indolecarboxaldehyde to afford the analo-gous compounds linked through the 3-position (59, 60, 66, Table2).

Within the set of compounds prepared from 7, the use ofcommercially available aldehydes was not always possible.Thus, it is indicated in Tables 1 and 2 when the requiredaldehydes have been prepared. The preparation of 5-phenylth-iophen-3-carboxaldehyde (71, Table 1 footnote) was achievedaccording to known procedures.32 The synthesis of aldehydeswith substituents on the bicyclic aromatic rings (benzofurans72-76, benzothiophenes 77, 78, 83-86, and indoles 79-82 inTable 2) also involved the use of previously reported prepara-tions.33-36 The characterization details of 71-86 are given inthe Supporting Information.

On the basis of the activity shown by compounds in the leadthioxofuropyridinone (7) series, a smaller number of aldehydeswere then reacted with templates 5, 9, and 11. This affordedthe 5-methylfuran-(32), 5-phenylthiophene-(33), and pyrrolo-(34) compounds (Table 1), as well as the 2-benzothiophene-(51) and 3-benzothiophene (65) products (Table 2) in thefuropyridinedione (5) series. The 5-methylfuran-(36, 35) and5-phenylthiophene-(38, 37) compounds were prepared in boththe pyrrolopyridinedione (9) and thioxopyrrolopyridinedione(11) series, respectively (Table 1), whereas in Table 2, onlythe optimal 3-benzothiophene-(69) compound was prepared byreaction with template 11.

The furopyridinone template 8, however, reacted quitedifferently under the same aldolization-crotonization conditionsand none of the desired products could be isolated. The mainproduct of the reaction was characterized by crystallographyand corresponds to ethyl 4-(hydroxymethyl)-6-methylnicoti-nate,37 signifying opening of the lactone under the conditionsdescribed in Scheme 3.

The stereoselectivity of the aldolization-crotonization reac-tion has previously been reported to favor the formation ofZ-stereoisomers.25,31 Our observations for a sample of 12 inDMSO-d6 in an experiment using a rotational nuclear Over-hauser effect (ROESY) experiment are consistent with theseresults. The olefinic proton (δ ) 7.08 ppm) and the pyridineth-ione 7-H (δ ) 7.19 ppm) correlate strongly, indicating theirclose proximity. In addition, no correlation between the pyridi-nethione 7-H and the furan protons was observed (see Support-ing Information for 1H and ROESY NMR spectra). Theseobservations confirmed that 12 had been prepared predominantlyas the Z-isomer. HPLC and LCMS analyses of the finalcompounds (including 12) did show the presence of a minorpeak, the same molecular weight by LCMS, and most likelybeing the E-isomer. Separation of the two isomeric forms of23 by preparative HPLC was carried out, and although difficultdue to low solubility and very similar retention times, signifi-cantly enriched samples were obtained. However, subsequentHPLC analysis of these showed the same composition as beforeseparation, suggesting an interconversion of the two isomers insolution.

The stability of 1 and 60 in DMSO was investigated. Threeseparate studies were conducted; samples of the two compoundswere stored at room temperature or -20 °C, as well as a thirdsample, which was frozen and defrosted for each HPLC analysis.This last sample is an attempt to replicate conditions that arecommonly used for stock solutions in biological testing. Eachof the three samples was then subjected to HPLC analysis on aweekly basis over four weeks. Results indicate that bothcompounds gradually convert to the other geometrical isomer,as after four weeks, 12%, 13%, and 10% of the E-isomer was

Figure 1

Inhibitors of the Pore-Forming Protein Perforin Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7615

observed for compound 1 and 11%, 11%, and 8% for compound60 at room temperature, -20 °C, and freeze-thaw, respectively(see Figures 3 and 4 in Supporting Information for data).

Subunit B as a Monocyclic Substituent (Table 1). Thecompounds were evaluated for inhibition of the lytic activityof perforin on Jurkat T lymphoma cells using the release of51Cr as a measure of cell lysis24 (see Experimental Section fordetails). The results, as the concentration of drug to reduce thelevel of perforin-induced lysis to 50% of controls (IC50), aregiven in Tables 1 and 2.

The lead compound (1) showed significant inhibitory activityin this assay, with an IC50 of 2.7 µM. Compounds 12-41 werethen explored for the nature of the appended ring (subunit B).Removal of the furanyl 5-methyl group from 1 to give 12resulted in decreased potency (IC50 4.4 µM), as did replacementof the 5-methylfuran with a 5-phenylfuran group (13; IC50 4.0µM). Because of concern about the mutagenic potential of thefuran (compounds 1 and 12 were positive in the AmesSalmonella typhimurium TA100 tester strain38), compounds14-16 explored replacement of the furan with thiophene. Allof these compounds were active in the lysis assay, with the bestbeing the 5-phenyl analogue (16; IC50 3.4 µM), although nonewere as potent as the 5-methylfuran 1. The thiophene 14 andthe pyrrole 17, however, both tested negative in the Ames assay.

Compounds 17-19 employed various 5-membered nitrogenheterocycles (pyrrole, imidazole, and N-methylpyrrole, respec-tively) as furan replacements, but none of these compounds wereactive. A series of compounds with a benzene ring bearing arange of electron-donating and -withdrawing substituents at ageometrically similar 4-position were also prepared (20-31).No activity was observed for any of these analogues irrespectiveof substitution. A limited number of other compounds withortho- or meta- substitution were synthesized but also had noactivity (data not shown). These data in general suggest thedesirability of a strong H-bond acceptor, but not a donor, inthe appended ring.

Compounds 32-34 study the replacement of the thioamidesulfur by oxygen. For the 5-methylfuran pair (32/1), this resultedin loss of activity, while for the pyrrole pair (34/17), bothcompounds were inactive. Interestingly, for the 5-phenylth-iophene pairing (33/16), only a slight loss of activity wasobserved (IC50s 5.0 and 3.4 µM). Compounds 35-38 replacedthe potentially metabolically liable lactone moiety with a lactam.One compound (35) was too unstable to test reliably, but both36 and its lactone counterpart 32 were inactive and the pairs37/16 and 38/33 clearly demonstrate the lack of utility of thelactam. Finally, compounds 39-41 explored alternative linkinggeometry to the appended heterocyclic ring; comparison of pairs

Table 1. Structures and Inhibitory Activities of Dihydrofuro[3,4-c]pyridinones with a Monocyclic Substituent

compd linka X Y Zb R2 IC50 (µM)c

1 2 S O O Me 2.712 2 S O O H 4.413 2 S O O Ph 4.014 2 S O S H 8.615 2 S O S Me 7.216 2 S O S Ph 3.417 2 S O NH H >2018 2 S O NH (3-aza)d >2019 2 S O NMe H >2020 2 S O CHdCH H >2021 2 S O CHdCH 4-F >2022 2 S O CHdCH 4-Cl >2023 2 S O CHdCH 4-Bre

24 2 S O CHdCH 4-Me >2025 2 S O CHdCH 4-CF3 >2026 2 S O CHdCH 4-OMe >2027 2 S O CHdCH 4-OCF3 >2028 2 S O CHdCH 4-SMe >2029 2 S O CHdCH 4-NMe2 >2030 2 S O CHdCH 4-NHAc >2031 2 S O CHdCH 4-Ph >2032 2 O O O Me >2033 2 O O S Ph 5.034 2 O O NH H >2035 2 S NH O Me unstable36 2 O NH O Me >2037 2 S NH S Ph >2038 2 O NH S Ph >2039 3 S O O H >2040 3 S O S H >2041 3 S O S Phf >20

a Position of link to substituent. b Templates 5, 7, 9, or 11 were reacted with commercially available aldehydes. c Testing was carried out over a range ofdoses, with the IC50 being equal to the concentration at which 50% inhibition of the lysis of Jurkat cells by perforin was observed, as measured by 51Crrelease. Values are the average of at least two independent IC50 determinations. d Appended ring is 2-imidazolyl. e Used for preparative HPLC separation ofE- and Z-isomers but not tested. f The phenyl substituent is at the 5-position, relative to connection at the 3-position of the thiophene. This compound resultsfrom reaction of 7 with novel aldehyde 71; see Supporting Information for preparation details and characterization of 71.

7616 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 Lena et al.

39/12, 40/14, and 41/16 shows convincingly that this geometryis not favorable.

Subunit B as a Bicyclic Substituent (Table 2). The impactof replacing the furan of 12 with a bicyclic substituent such as

benzofuran (42-47), benzothiophene (48-51), and indole(52-58) is investigated in Table 2. Comparison of the 2-furan(12) and 2-thiophene (14) with the 2-benzofuran (42) and2-benzothiophene (48), respectively, saw a drop of potency inboth cases (12/42; IC50 4.4/8.2 µM and 14/48; IC50 8.6/9.5 µM).More interesting, however, was the indole (52), which possessedunexpectedly good activity (IC50 1.3 µM), especially given thatthe corresponding 2-pyrrole 17 was inactive (IC50 >20 µM).

Tolerance for substitution on the benzene ring of thebenzofurans, benzothiophenes, and indoles was probed via the

Table 2. Structures and Inhibitory Activities of Dihydrofuro[3,4-c]pyridinones with a Bicyclic Substituent

compd linka X Y Z R3 starting aldehydeb IC50 (µM)c

42 2 S O O H 8.243 2 S O O 4-OMe 72 6.744 2 S O O 5-OMe 73 2.445 2 S O O 6-OMe 74 7.246 2 S O O 7-OMe 75 3.047 2 S O O 5-OMe, 6-OBn 76 5.148 2 S O S H 9.549 2 S O S 5-OMe 77 13.150 2 S O S 6-OMe 78 5.451 2 O O S H 4.652 2 S O NH H 1.353 2 S O NH 5-OMe 4.654 2 S O NH 6-OMe 79 3.155 2 S O NH 5-OMe, 6-OBn 80 >2056 2 S O NH 5-F, 6-OMe 81 6.357 2 S O NH 5-OBn 82 >2058 2 S O NMe H >2059 3 S O O H >2060 3 S O S H 0.9761 3 S O S 4-OMe 83 3.862 3 S O S 5-OMe 84 5.163 3 S O S 6-OMe 85 4.764 3 S O S 5-Br 86 7.565 3 O O S H 8.566 3 S O NH H >2067 3 S O NMe H 4.368 3 O O NMe H >2069 3 S NH S H >2070 7d S O NH H 4.1

a Position of link to substituent. b Aldehydes which were reacted with 5, 7, 9, or 11 were commercially available unless stated otherwise; see SupportingInformation for preparation details and characterization of new aldehydes 72-86. c Testing was carried out over a range of doses, with the IC50 being equalto the concentration at which 50% inhibition of the lysis of Jurkat cells by perforin was observed, as measured by 51Cr release. Values are the average ofat least two independent IC50 determinations. d Connection is at the 7-position of the indole.

Scheme 1a

a Reagents and conditions: (i) NaH, acetone, Et2O, -5 °C-RT; (ii)2-cyanoacetamide, piperidine (cat.), EtOH, 75 °C, 1 h.; (iii) concd HCl,120 °C, 10 h.; (iv) POCl3, PCl5, 115 °C, 0.5 h.; (v) thiourea, 1-BuOH, 110°C, 10 h.; (vi) 10% Pd/C, Et3N, H2, 2.5 h.

Scheme 2a

a Reagents and conditions: (i) concd H2SO4, RT, 3 d.; (ii) 2-thiocy-anoacetamide, piperidine (cat.), EtOH, 75 °C, 1 h.


synthesis of a series of methoxy-substituted compounds. Forthe benzofurans, within the series, the most potent compoundwas the 5-methoxy (44; IC50 2.4 µM), closely followed by the7-methoxy (46; IC50 3.0 µM), whereas for the benzothiophenes,the 6-methoxy (50; IC50 5.4 µM) was superior. In the indoleseries, although the 5-methoxy (53) and 6-methoxy (54)compounds had appreciable activity (IC50s 4.6 and 3.1 µMrespectively), neither were an improvement on the parent indole52 (IC50 1.3 µM).

In a similar fashion to the compounds of Table 1, the positionof connection for subunit B was also explored. In contrast tothe monocyclic compounds (39-41), where a change ofgeometry was disfavored, several of the 3-linked compoundsshowed significant activity, including the N-methylindole 67(IC50 ) 4.3 µM) and in particular, the 3-benzothiophene (60).With an IC50 ) 0.97 µM, this compound was the most potentin both series and additionally, was nonmutagenic in Amestesting. The 4- (61), 5- (62), and 6-methoxy-3-benzothiophenes(63) were all prepared, but none were as potent as theunsubstituted parent compound 60. Perhaps surprisingly, the3-linked indole showed poor activity (66; IC50 ∼ 20 µM), innoticeable contrast to the 2-linked indole 52 (IC50 1.3 µM).

Replacement of the thioamide sulfur with oxygen (X ) S toX ) O) in this series gave variable results. While for the3-benzothiophene (65) and 3-(N-methylindole) (68), a reductionin potency was observed when the respective pairs 65/60 and68/67 are compared (IC50 8.5/0.97 µM and IC50 > 20/4.3 µM);for the 2-linked benzothiophene (48, IC50 9.5 µM), thismodification resulted in an improvement in activity (51, IC50

4.6 µM).Preparation of a single lactam, compound 69 (IC50 > 20 µM)

also confirmed the lack of utility found with this template inthe compounds of Table 1. One compound with a 7-linkedindole substituent (70) was also prepared and showed significantactivity (IC50 4.1 µM), although this has not been exploredfurther at this point in time.

Further Analysis of Compounds with Perforin Inhi-bitory Activity. To determine whether the dihydrofuro[3,4-c]pyridinone class of inhibitors are specific to perforin, or arein fact more general inhibitors of other pore-forming proteins,lead compound 1 was tested for its ability to inhibit the lyticfunction of the pneumococcal toxin pneumolysin (PLO). PLOis a bacterial pore-forming protein, which like perforin, isreleased as soluble monomers before oligomerizing to formactive pores in the target cell membrane. At a concentration of20 µM of compound 1, lysis of sheep red blood cells by PLOwas observed, showing that 1 specifically inhibits perforin butnot the mechanistically related PLO.

Although the granule exocytosis pathway is crucial to theoperation of CTLs and NK cells, they may also exert theircytotoxic effects through alternative death-receptor mediatedpathways.1,4 These pathways are activated by the binding of

Fas-ligand (FasL), TNFR (tumor-necrosis factor R), or TRAIL(TNF-related apoptosis inducing ligand), which trigger caspaseactivation and ultimately cell death. Thus it is important toestablish that the mechanism of action of the dihydrofuro[3,4-c]pyridinones is perforin-specific and does not also operatethrough the death-receptor pathway. In a similar manner to theperforin-induced lysis assay, Jurkat T lymphoma target cellswere labeled with 51Cr, preincubated with 1, then treated witheither anti-Fas agonistic monoclonal antibody CH11 or recom-binant (rec)-TRAIL in order to induce cell death, which wasthen measured by 51Cr release (see Experimental Section forspecific details). It was found that compound 1 does not blockcell death mediated through FasL or TRAIL (Figure 5, Sup-porting Information). This result has therapeutic implicationsbecause oversuppression of cell killing by cytotoxic lymphocytesmay have negative consequences such as interfering with thecapacity to down-regulate lymphocyte numbers following aninfection.4,39

A small subset of inhibitors (1, 16, 44, 51, 52, 60) wereselected on the basis of their activity in the perforin induced-Jurkat lysis assay and evaluated for their ability to inhibitperforin delivered by an intact NK cell line. The inhibitor (20µM final concentration) and medium were coincubated withKHYG-1 NK cells for 30 min at room temperature, 51Cr-labeledK562 leukemia target cells added, and after 4 h cell lysis wasmeasured by 51Cr release. All compounds tested showedinhibition of NK cell killing of the K562 targets, ranging from50-100% (Table 3). To confirm the inhibitory activity exhibitedby the compounds against KHYG-1 NK cells was due toblocking the action of perforin and not nonspecific toxicitytoward the effector cell, the viability of the NK cells in thepresence of the inhibitor was measured 24 h. later. Viable anddead cells were counted and percent viability calculated basedon total cell count (for further details see Experimental Section).Compounds 16, 44, and 52 were clearly toxic to the KHYG-1cells, but importantly, lead compounds 1 and 60 (and also 51)show it is possible to inhibit NK cell activity yet largely retainNK cell viability.

Finally, lead compound 1 was tested to determine whether itretained the ability to inhibit perforin activity in primary humanNK cells isolated from the buffy coats of healthy blood donors.Using 51Cr-labeled K562 target cells, the release of 51Crresulting from cell lysis was blocked by >70%, showing that 1

Scheme 3a

a Reagents and conditions: (i) R1CHO, piperidine (cat.), EtOH, 75 °C,40 min, -20 h.

Table 3. Capacity of Selected Compounds to Inhibit Perforin Deliveredby KHYG-1 NK Cells

compdJurkat

IC50(µM)

inhibition oftarget cell

deatha,b (%)

viability of KHYG-1killer cells 24 hafter exposure tocompoundsc,d (%)

1 2.7 50 7216 3.4 100 044 2.4 100 051 4.6 65 10052 1.3 95 060 0.97 63 88

a Inhibitor (to 20 µM final concentration) and medium were coincubatedwith KHYG-1 human NK leukemia cells, 51Cr-labeled K562 target cellswere added, and target cell lysis measured by 51Cr release. Percent inhibitioncalculated compared to untreated control. b Use of DMSO vehicle as controlresulted in no (0%) inhibition. c KHYG-1 viability was measured after 24 h.by examining uptake/exclusion of the dye Trypan blue. Viable (clear) anddead (blue) cells were counted, and percent viability calculated based ontotal cell count. d Use of DMSO vehicle as control resulted in no effectupon KHYG-1 cell viability (>95%).


is still able to inhibit perforin delivered by intact human NKcells (Figure 6 in Supporting Information).

Conclusions

The dihydrofuro[3,4-c]pyridinones are the first class of smallmolecules reported to inhibit the cytolytic effects of perforin incells. Molecules with this function are of potential use in thetreatment of some autoimmune diseases and therapy-inducedconditions characterized by undesired perforin secretion. Thepreliminary data presented here includes some relatively potentcompounds (most notably the 2-indole 52 and the 3-ben-zothiophene 60), but suggest fairly tight SAR. The lactoneoxygen and the preference for H-bond acceptor in the appendedring appear to be a requirement for activity in the class. It isnot entirely clear whether it is necessary to have a thioamideor an amide in the A-subunit, with variable activity in both setsof examples. Although the 2-linked B-subunit is required foractivity in the monocycles, the point of connection is moreflexible for the bicyclic series. Substitution on the B-subunitcan also result in improved activity in some cases.

The inhibitory action of the dihydrofuro[3,4-c]pyridinonesis quite specific for perforin, with no effect observed on abacterial pore-forming protein with a similar mechanism ofaction, or on death-receptor pathways mediated through FasLand TNFR. Lead compounds 1 and 60 show inhibition ofKHYG-1 NK cell-mediated target cell killing while stillretaining NK cell viability. Compound 1 also shows excellentinhibitory activity against intact primary human NK cells. Thisinitial study should therefore provide some substantial clues forthe further development of this class of compounds.

Experimental Section

Medicinal Chemistry. Analyses were performed by the Micro-chemical Laboratory, University of Otago, Dunedin, NZ. Meltingpoints were determined using an electrothermal model 9200 andare as read. NMR spectra were measured on a Bruker Advance400 spectrometer and referenced to Me4Si. Mass spectra wererecorded either on a Varian VG 7070 spectrometer at nominal 5000resolution or a Finnigan MAT 900Q spectrometer. All finalcompound purities were determined to be >95% by HPLC.

Ethoxyacetylacetone (3). NaH (60% in mineral oil, 6.5 g, 162mmol) was rinsed twice with hexane and then suspended in Et2O(100 mL). The mixture was placed under an inert atmosphere andcooled at -5 °C. Ethyl 2-ethoxyacetate (2) (20 mL, 147 mmol)and a solution of acetone (12 mL, 162 mmol) in Et2O (50 mL)were added. The mixture was stirred 3 h at -5 °C then 1 h at RT.An ice cold solution of concd HCl (50 mL) in water (100 mL) wasadded carefully. The two layers were separated, and the aqueousphase was extracted with Et2O (100 mL). The combined organiclayers were dried over MgSO4, and the solvent was evaporated afterfiltration. The crude product was purified by silica gel columnchromatography (hexane, EtOAc, 95:5, v/v) to afford 3 (15.08 g,71%) as a pale-yellow oil. 1H NMR (400 MHz, CDCl3, 298 K):The 1H NMR spectra shows two sets of signals corresponding tothe two tautomeric forms of the molecule. Form 1: δ 1.23 (t, J )7.0 Hz, 3 H), 2.26 (s, 3 H), 3.54 (q, J ) 7.0 Hz, 2 H), 3.63 (s, 2H), 4.01 (s, 2 H); Form 2: δ 1.25 (t, J ) 7.0 Hz, 3 H), 2.09 (s, 3H), 3.58 (q, J ) 7.0 Hz, 2 H), 4.01 (s, 2 H), 5.81 (s, 1 H), 15.23(s, 1 H). LCMS (APCI+) calcd for C6H11O3, 131 (MH+), found131.

4-(Ethoxymethyl)-6-methyl-2-oxo-1,2-dihydropyridine-3-car-bonitrile (4). 2-Cyanoacetamide (5.75 g, 68.4 mmol) was suspendedin EtOH (60 mL) and the mixture was heated to 75 °C, at whichpoint 3 (8.9 g, 62.2 mmol) and piperidine (710 µL, 8.1 mmol) wereadded and the mixture was stirred vigorously at 75 °C for 2 h. Themixture, which contained a precipitate, was allowed to cool to RTand stored 4 h at 4 °C. The resulting solid was collected by filtration

and rinsed with EtOH (2 × 40 mL). Drying under vacuum afforded4 (6.8 g, 56%) as a white powder. 1H NMR (400 MHz, DMSO-d6,298 K) δ 1.17 (t, J ) 7.0 Hz, 3 H), 2.28 (s, 3 H), 3.53 (q, J ) 7.0Hz, 2 H), 4.46 (s, 2 H), 6.28 (s, 1 H), 12.45 (s, 1 H). LCMS(APCI+) calcd for C10H13N2O2, 193 (MH+), found 193.

6-Methylfuro[3,4-c]pyridine-3,4(1H,5H)-dione (5). Compound4 (6.4 g, 33 mmol) was dissolved in concd HCl (58 mL). Themixture was stirred at 120 °C for 10 h then allowed to cool to RT.A mixture of ice and water (∼100 mL) was added to the crudemixture, and the resulting precipitate collected by filtration, washedwith water (3 × 20 mL), and dried to yield 5 (4.31 g, 79%) as awhite powder. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.29 (s,3 H), 5.13 (s, 2 H), 6.30 (s, 1 H), 12.08 (s, 1 H). LCMS (APCI+)calcd for C8H8NO3, 166 (MH+), found 166.

4-Chloro-6-methylfuro[3,4-c]pyridin-3(1H)-one (6). Com-pound 5 (4.0 g, 24.2 mmol) was mixed with POCl3 (25 mL) andPCl5 (7.3 g, 35.1 mmol) was added portionwise. The mixture wasstirred at 110 °C for 30 min then allowed to cool to RT. POCl3

was removed by evaporation, the residue was dissolved in CH2Cl2

(40 mL), and washed with water. The aqueous layer was extractedwith CH2Cl2 and the combined organic layers were dried overMgSO4, which was then removed by filtration. After evaporationof the solvent, the crude mixture was purified by silica gel columnchromatography (hexane, EtOAc, 65:35, v/v). The title compound6 (2.99 g, 66%) was isolated as a white solid. 1H NMR (400 MHz,DMSO-d6, 298 K) δ 2.59 (s, 3 H), 5.38 (s, 2 H), 7.60 (s, 1 H).LCMS (APCI+) calcd for C8H7ClNO2, 184.5 (MH+), found 184.5.

6-Methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one(7). Compound 6 (2.3 g, 12.6 mmol) was dissolved in butan-1-ol(105 mL). Thiourea (958 mg, 12.6 mmol) was added, and themixture was stirred at 120 °C for 10 h. The reaction was allowedto cool to RT and then stored at 4 °C for 16 h. The resulting solidwas collected by filtration and rinsed with EtOH (3 × 40 mL).Drying under vacuum afforded 7 (2.01 g, 88%) as a beige solid.1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.41 (s, 3 H), 5.14 (s, 2H), 6.78 (s, 1 H), 13.48 (s, 1 H). LCMS (APCI+) calcd forC8H8NO2S, 182 (MH+), found 182.

6-Methylfuro[3,4-c]pyridin-3(1H)-one (8). Compound 6 (730mg, 3.97 mmol) was dissolved in EtOH (100 mL) and Et3N (6mL), and 10% Pd/C (150 mg) were added. The reaction was carriedout in a pressure vessel under H2 (30 psi) at RT for 2.5 h. ThePd/C was removed by filtration over celite, and the filtrate wasevaporated. The resulting residue was diluted with CH2Cl2 (20 mL)and washed with water (20 mL). The organic layer was dried overMgSO4, which was removed by filtration. Evaporation yielded 8(440 mg, 74%) as a white foam. 1H NMR (400 MHz, DMSO-d6,298 K) δ 2.62 (s, 3 H), 5.43 (s, 2 H), 7.58 (s, 1 H), 8.95 (s, 1 H).LCMS (APCI+) calcd for C8H8NO2, 150 (MH+), found 150.

6-Methyl-1H-pyrrolo[3,4-c]pyridine-3,4(2H,5H)-dione (9). Amixture of 4 (813 mg, 4.23 mmol) dissolved in concd H2SO4 (8mL) was stirred at RT for 3 days. The flask was cooled to 0 °Cwith ice/water and EtOH (32 mL) was added to the mixture whichproduced a precipitate. This solid was collected by filtration anddried. Dissolution in water was followed by neutralization byaddition of a saturated aqueous solution of NaHCO3. A solidprecipitated and was collected by filtration. Drying afforded thetitle compound 9 (564 mg, 81%) as a white solid. 1H NMR (400MHz, DMSO-d6, 298 K) δ 2.28 (s, 3 H), 5.13 (s, 2 H), 6.28 (s, 1H), 7.67 (s, 1 H), 12.18 (s, 1 H). LCMS (APCI+) calcd forC8H9N2O2, 165 (MH+), found 165.

4-(Ethoxymethyl)-6-methyl-2-thioxo-1,2-dihydropyridine-3-carbonitrile (10). In a flask wrapped with aluminum foil, 2-cyan-othioacetamide (1.53 g, 15.3 mmol) was mixed with EtOH (20 mL).The mixture was heated at 75 °C, and then 3 (2.0 g, 13.8 mmol)and piperidine (157 µL, 1.8 mmol) were added. The reaction wasstirred at 75 °C for 2 h and then allowed to cool to RT. After 2 hat 4 °C, the precipitate was collected by filtration and rinsed withEtOH (2 × 10 mL). Drying gave 10 (1.17 g, 41%) as a brownsolid. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 1.18 (t, J ) 7.0Hz, 3 H), 2.41 (s, 3 H), 3.56 (q, J ) 7.0 Hz, 2 H), 4.50 (s, 2 H),


6.78 (s, 1 H), 13.42 (s, 1 H). LCMS (APCI+) calcd for C10H13N2OS,209 (MH+), found 209.

6-Methyl-4-thioxo-4,5-dihydro-1H-pyrrolo[3,4-c]pyridin-3(2H)-one (11). Compound 10 (362 mg, 1.74 mmol) was mixed withconcentrated H2SO4 (3.5 mL), and the reaction was performed inexactly the same manner as for 9. The title compound 11 (172 mg,55%) was obtained as a dark-green solid. 1H NMR (400 MHz,DMSO-d6, 298 K) δ 2.43 (s, 3 H), 5.16 (s, 2 H), 6.84 (s, 1 H),9.74 (s, 1 H), 13.68 (s, 1 H). LCMS (APCI+) calcd for C8H9N2OS,181 (MH+), found 181.

General Procedure for the Aldolization-Crotonization Step.A suspension of the appropriate bicyclic starting material (5, 7, 8,9, or 11) in EtOH (0.07 mmol/mL) was heated at 60 °C. Piperidine(0.1 equiv) and a solution of aldehyde (1.1 equiv) in EtOH (0.3mmol/mL) were added. The mixture was heated to 75 °C. Thereaction was monitored by MS and stopped when the peakcorresponding to the starting material had disappeared (reactiontime varied from 40 min. to 20 h.). The mixture was allowed tocool to RT and stored 2 h at 4 °C. The resulting solid was collectedby filtration, rinsed with EtOH (2 × 5 mL), Et2O (2 × 5 mL), anddried to give the desired product.

(Z)-6-Methyl-1-((5-methylfuran-2-yl)methylene)-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (1). Yield 59%, bright-orange solid, mp 292-296 °C. 1H NMR (400 MHz, DMSO-d6,298 K) δ 2.38 (s, 3 H), 2.40 (s, 3 H), 6.37 (d, J ) 3.3 Hz, 1 H),6.91 (d, J ) 3.3 Hz, 1 H), 6.99 (s, 1 H), 7.13 (s, 1 H), 13.40 (s, 1H). Anal. (C14H11NO3S) C, H, N.

(Z)-1-(Furan-2-ylmethylene)-6-methyl-4-thioxo-4,5-dihydro-furo[3,4-c]pyridin-3(1H)-one (12). Yield 49%, brown solid, mp> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.42 (s, 3 H),6.72 (dd, J ) 1.7, 3.4 Hz, 1 H), 7.01 (d, J ) 3.4 Hz, 1 H), 7.08 (s,1 H), 7.19 (s, 1 H), 8.04 (d, J ) 1.3 Hz, 1 H), 13.49 (s, 1 H). 13CNMR (100 MHz, DMSO-d6, 298 K) δ 19.13, 101.00, 102.98,113.25, 115.91, 117.33, 139.40, 145.68, 148.43, 150.22, 154.95,163.64, 175.97. HRMS (EI+) calcd for C14H12NO3S, 274.05379(MH+), found 274.05404.

(Z)-6-Methyl-1-((5-phenylfuran-2-yl)methylene)-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (13). Yield 96%, red solid,mp 290-294 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.42(s, 3 H), 7.14 (m, 2 H), 7.38 (tt, J ) 1.1, 1.1, 1.7, 7.4 Hz, 1 H),7.50 (t, J ) 7.7 Hz, 2 H), 7.81 (dd, J ) 1.1, 8.5 Hz, 2 H), 13.47(s, 1 H). Anal. (C19H13NO3S) C, H, N.

(Z)-6-Methyl-1-(thiophen-2-ylmethylene)-4-thioxo-4,5-dihy-drofuro[3,4-c]pyridin-3(1H)-one (14). Yield 81%, brown solid,mp 280-284 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.42(s, 3 H), 7.12 (s, 1 H), 7.21 (dd, J ) 3.7, 5.1 Hz, 1 H), 7.40 (s, 1H), 7.49 (d, J ) 3.5 Hz, 1 H), 7.87 (d, J ) 5.0 Hz, 1 H) 12.8 (s,1 H). Anal. (C13H9NO2S2 ·1.25H2O) C, H, N.

(Z)-6-Methyl-1-((5-methylthiophen-2-yl)methylene)-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (15). Yield 61%, brownsolid, mp 286-290 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ2.41 (s, 3 H), 2.51 (s, 3 H), 6.92 (dd, J ) 1.1, 3.7 1 H), 7.10 (s, 1H), 7.31 (m, 2 H), 13.36 (s, 1 H). Anal. (C14H11NO2S2) H, N. C:calcd, 58.1; found 57.6.

(Z)-6-Methyl-1-((5-phenylthiophen-2-yl)methylene)-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (16). Yield 77%, orangesolid, mp > 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ2.43 (s, 3 H), 7.13 (s, 1 H), 7.38 (t, J ) 7.3 Hz, 1 H), 7.41 (s, 1 H),7.46 (t, J ) 7.2 Hz, 2 H), 7.50 (d, J ) 4.2 Hz, 1 H), 7.63 (d, J )3.9 Hz, 1 H), 7.77 (m, 2 H), 13.48 (s, 1 H). Anal. (C19H13NO2S2)H, N. C: calcd, 64.9; found 64.4.

(Z)-1-((1H-Pyrrol-2-yl)methylene)-6-methyl-4-thioxo-4,5-di-hydrofuro[3,4-c]pyridin-3(1H)-one (17). Yield 94%, red solid, mp> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.40 (s, 3 H),6.28 (m, 1 H), 6.72 (m, 1 H), 6.89 (s, 1 H), 7.02 (s, 1 H), 7.10 (m,1 H), 11.26 (s, 1 H), 13.23 (s, 1 H, NH). Anal. (C13H10N2O2S ·0.5H2O) C, H, N.

(Z)-1-((1H-Imidazol-2-yl)methylene)-6-methyl-4-thioxo-4,5-di-hydrofuro[3,4-c]pyridin-3(1H)-one (18). Yield 64%, orange solid,mp > 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.42 (s,3 H), 7.00 (s, 1 H), 7.19 (br s, 1 H), 7.28 (s, 1 H), 7.33 (br s, 1 H),

12.17 (s, 1 H), 13.49 (s, 1 H). HRMS (EI+) calcd for C12H9N3O2S,259.04155 (M+), found 259.04162.

(Z)-6-Methyl-1-((1-methyl-1H-pyrrol-2-yl)methylene)-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (19). Yield 60%, dark-red solid, mp > 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K)δ 2.41 (s, 3 H), 3.77 (s, 3 H) 6.25 (dd, J ) 2.6, 3.8 Hz), 6.91 (dd,J ) 1.5, 4 Hz, 1 H), 7.0 (s, 1 H), 7.08 (t, J ) 2.0 Hz, 1 H), 7.22(s, 1 H), 13.06 (s, 1 H). Anal. (C14H12N2O2S ·0.5H2O) C, H, N.

(Z)-1-Benzylidene-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]py-ridin-3(1H)-one (20). Yield 53%, yellow solid, mp 299-303 °C.1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.45 (s, 3 H), 7.01 (s, 1H), 7.19 (s, 1 H), 7.41 (t, J ) 7.3 Hz, 1 H), 7.48 (t, J ) 7.3 Hz, 2H), 7.81 (d, J ) 7.3 Hz, 2 H), 13.52 (s, 1 H). HRMS (EI+) calcdfor C15H12NO2S, 270.05888 (MH+), found 270.05923.

(Z)-1-(4-Fluorobenzylidene)-6-methyl-4-thioxo-4,5-dihydro-furo[3,4-c]pyridin-3(1H)-one (21). Yield 11%, yellow solid, mp> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2. 43 (s, 3H), 7.00 (s, 1 H), 7.14 (s, 1 H), 7.34 (t, J ) 8.8 Hz, 2 H), 7.86 (dd,J ) 5.7, 8.8 Hz, 2 H), 13.61 (s, 1 H). HRMS (EI+) calcd forC15H11FNO2S, 288.04945 (MH+), found 288.04941.

(Z)-1-(4-Chlorobenzylidene)-6-methyl-4-thioxo-4,5-dihydro-furo[3,4-c]pyridin-3(1H)-one (22). Yield 72%, yellow solid, mp> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.44 (s, 3 H),7.02 (s, 1 H), 7.16 (s, 1 H), 7.57 (d, J ) 8.6 Hz, 2 H), 7.82 (d, J) 8.6 Hz, 2 H), 13.55 (s, 1 H). Anal. (C15H10ClNO2S) H, N. C:calcd, 59.3; found 58.8.

(Z)-1-(4-Bromobenzylidene)-6-methyl-4-thioxo-4,5-dihydro-furo[3,4-c]pyridin-3(1H)-one (23). Yield 44%, yellow solid, mp> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.44 (s, 3 H),7.00 (s, 1 H), 7.16 (s, 1 H), 7.74 (m, 4 H), 13.62 (s, 1 H). HRMS(EI+) calcd for C15H11

79BrNO2S, 347.96939 (MH+), found347.97048; calcd for C15H11

81BrNO2S, 349.96734 (MH+), found349.96858.

(Z)-6-Methyl-1-(4-methylbenzylidene)-4-thioxo-4,5-dihydro-furo[3,4-c]pyridin-3(1H)-one (24). Yield 73%, yellow solid, mp> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.36 (s, 3 H),2.44 (s, 3 H), 6.98 (s, 1 H), 7.17 (s, 1 H), 7.31 (d, J ) 8.1 Hz, 2H), 7.72 (d, J ) 8.2 Hz, 2 H), 13.52 (s, 1 H). Anal. (C16H13NO2S)C, H, N.

(Z)-6-Methyl-4-thioxo-1-(4-(trifluoromethyl)benzylidene)-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (25). Yield 17%, yellowsolid, mp 290-294 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ2.45 (s, 3 H), 3.82 (s, 3 H), 7.09 (s, 1 H), 7.19 (s, 1 H), 7.85 (d, J) 8.4 Hz, 2 H), 7.99 (d, J ) 8.4 Hz, 2 H), 13.64 (s, 1 H). Anal.(C16H10F3NO2S ·0.25H2O) C, H, N.

(Z)-1-(4-Methoxybenzylidene)-6-methyl-4-thioxo-4,5-dihydro-furo[3,4-c]pyridin-3(1H)-one (26). Yield 84%, yellow solid, mp> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.41 (s, 3 H),3.82 (s, 3 H), 6.97 (s, 1 H), 7.08 (d, J ) 8.9 Hz, 2 H), 7.13 (s, 1H), 7.78 (d, J ) 8.9 Hz, 2 H), 13.44 (s, 1 H). Anal. (C16H13NO3S)C, H, N.

(Z)-6-Methyl-4-thioxo-1-(4-(trifluoromethoxy)benzylidene)-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (27). Yield 8%, orangesolid, mp 279-282 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ2.44 (s, 3 H), 7.04 (s, 1 H), 7.16 (s, 1 H), 7.49 (d, J ) 8.3 Hz, 2H), 7.92 (d, J ) 8.8 Hz, 2 H), 13.62 (s, 1 H). HRMS (EI+) calcdfor C16H11F3NO3S, 354.04117 (MH+), found 354.04143.

(Z)-6-Methyl-1-(4-(methylthio)benzylidene)-4-thioxo-4,5-dihy-drofuro[3,4-c]pyridin-3(1H)-one (28). Yield 43%, yellow solid,mp 308-311 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.44(s, 3 H), 2.49 (s, 3 H), 6.99 (s, 1 H), 7.16 (s, 1 H), 7.37 (d, J ) 8.6Hz, 2 H), 7.75 (d, J ) 8.6 Hz, 2 H), 13.48 (s, 1 H). Anal.(C16H13NO2S2) C, H, N.

(Z)-1-(4-(Dimethylamino)benzylidene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (29). Yield 25%, dark-redsolid, mp 281-284 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ2.40 (s, 3 H), 3.02 (s, 6 H), 6.81 (d, J ) 9.1 Hz, 2 H), 6.89 (s, 1H), 7.07 (s, 1 H), 7.68 (d, J ) 9.0 Hz, 2 H), 13.28 (s, 1 H). Anal.(C17H16N2O2S ·0.8CH2Cl2) C, H, N.


(Z)-N-(4-((6-Methyl-3-oxo-4-thioxo-4,5-dihydrofuro[3,4-c]py-ridin-1(3H)-ylidene)methyl)phenyl)acetamide (30). Yield 33%,yellow solid, mp > 300 °C. 1H NMR (400 MHz, DMSO-d6, 298K) δ 2.07 (s, 3 H), 2.43 (s, 3 H), 6.95 (s, 1 H), 7.16 (s, 1 H), 7.71(d, J ) 8.9 Hz, 2 H), 7.76 (d, J ) 8.9 Hz, 2 H), 13.47 (s, 1 H).Anal. (C17H14N2O3S) C, H, N.

(Z)-1-(Biphenyl-4-ylmethylene)-6-methyl-4-thioxo-4,5-dihy-drofuro[3,4-c]pyridin-3(1H)-one (31). Yield 70%, yellow solid,mp > 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.46 (s,3 H), 7.07 (s, 1 H), 7.21 (s, 1 H), 7.41 (t, J ) 7.4 Hz, 1 H), 7.50(t, J ) 7.3 Hz, 2 H), 7.75 (d, J ) 8.5 Hz, 2 H), 7.83 (d, J ) 8.5Hz, 2 H), 7.91 (d, J ) 8.5 Hz, 2 H), 13.62 (s, 1 H). Anal.(C21H15NO2S ·0.75H2O) C, H, N.

(Z)-6-Methyl-1-((5-methylfuran-2-yl)methylene)furo[3,4-c]py-ridine-3,4(1H,5H)-dione (32). Yield 64%, yellow solid, mp >290°C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.30 (s, 3 H), 2.37(s, 3 H), 6.35 (d, J ) 3.3 Hz, 1 H), 6.70 (s, 1 H), 6.87 (d, J ) 3.3Hz, 1 H), 6.89 (s, 1 H), 12.15 (s, 1 H). Anal. (C14H11NO4 ·0.25H2O)C, H, N.

(Z)-6-Methyl-1-((5-phenylthiophen-2-yl)methylene)furo[3,4-c]pyridine-3,4(1H,5H)-dione (33). Yield 89%, yellow solid, mp> 295 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.33 (s, 3 H),6.69 (s, 1 H), 7.31 (s, 1 H), 7.36 (t, J ) 7.3 Hz, 1 H), 7.46 (m, 3H), 7.60 (d, J ) 3.9 Hz, 1 H), 7.57 (d, J ) 7.2 Hz, 2 H), 12.11 (s,1 H). Anal. (C19H13NO3S) C, H, N.

(Z)-1-((1H-Pyrrol-2-yl)methylene)-6-methylfuro[3,4-c]pyridine-3,4(1H,5H)-dione (34). Yield 68%, brown solid, mp > 300 °C.1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.30 (s, 3 H), 6.26 (t, J) 2.8 Hz, 1 H), 6.58 (d, J ) 0.5 Hz, 1 H), 6.69 (d, J ) 3.3 Hz, 1H), 6.80 (s, 1 H), 7.07 (s, 1 H), 11.24 (s, 1 H), 11.97 (s, 1 H).Anal. (C13H10N2O3) C, H, N.

(Z)-6-Methyl-1-((5-methylfuran-2-yl)methylene)-4-thioxo-4,5-dihydro-1H-pyrrolo[3,4-c]pyridin-3(2H)-one (35). Yield 31%,brown solid, mp > 290 °C. 1H NMR (400 MHz, DMSO-d6, 298K) δ 2.36 (s, 3 H), 2.42 (s, 3 H), 6.35 (d, J ) 3.3 Hz, 1 H), 6.84(s, 1 H), 6.87 (d, J ) 3.3 Hz, 1 H), 7.19 (s, 1 H), 9.80 (s, 1 H),13.66 (s, 1 H). HRMS (EI+) calcd for C14H13N2O2S, 273.06977(MH+), found 273.07023.

(Z)-6-Methyl-1-((5-methylfuran-2-yl)methylene)-1H-pyrrolo[3,4-c]pyridine-3,4(2H,5H)-dione (36). Yield 22%, green solid, mp200-204 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.30 (s, 3H), 2.33 (s, 3 H), 6.33 (d, J ) 0.3 Hz, 1 H), 6.66 (s, 1 H), 6.74 (s,1 H), 6.85 (d, J ) 2.9 Hz, 1 H), 8.59 (s, 1 H), 12.14 (s, 1 H).HRMS (EI+) calcd for C14H13N2O3, 257.09262 (MH+), found257.09273.

(Z)-6-Methyl-1-((5-phenylthiophen-2-yl)methylene)-4-thioxo-4,5-dihydro-1H-pyrrolo[3,4-c]pyridin-3(2H)-one (37). Yield 9%,dark-brown solid, mp > 290 °C. 1H NMR (400 MHz, DMSO-d6,298 K) (E and Z isomers coexist in this spectra but are notdistinguished) δ 2.46 (s, 3 H), 2.49 (s, 3 H), 6.85 (s, 1 H), 7.13 (s,1 H), 7.25 (m, 3 H), 7.37 (m, 3 H), 7.45 (m, 4 H), 7.60 (d, J ) 3.8Hz, 1 H), 7.64 (d, J ) 7.6 Hz, 2 H), 7.74 (d, J ) 7.5 Hz, 2 H),8.92 (s, 1 H), 9.89 (s, 1 H), 10.38 (s, 1 H), 13.81 (s, 2 H). Anal.(C19H14N2OS2 ·1.5H2O) C, H, N.

(Z)-6-Methyl-1-((5-phenylthiophen-2-yl)methylene)-1H-pyr-rolo[3,4-c]pyridine-3,4(2H,5H)-dione (38). Yield 24%, brownsolid, mp > 295 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ2.35 (s, 3 H), 6.35 (s, 1 H), 7.21 (m, 2 H), 7.38 (m, 3 H), 7.61 (d,J ) 7.3 Hz, 2 H), 8.23 (m, 2 H), 12.50 (s, 1 H). HRMS (EI+)calcd for C19H15N2O2S, 335.08542 (MH+), found 335.08558.

(Z)-1-(Furan-3-ylmethylene)-6-methyl-4-thioxo-4,5-dihydro-furo[3,4-c]pyridin-3(1H)-one (39). Yield 60%, yellow solid, mp290-294 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.42 (s, 3H), 6.89 (d, J ) 1.8 Hz, 1 H), 6.97 (s, 1 H), 7.11 (s, 1 H), 7.81 (t,J ) 1.5 Hz, 1 H), 8.13 (d, J ) 0.7 Hz, 1 H), 13.48 (s, 1 H). Anal.(C13H9NO3S) C, H, N.

(Z)-6-Methyl-1-(thiophen-3-ylmethylene)-4-thioxo-4,5-dihy-drofuro[3,4-c]pyridin-3(1H)-one (40). Yield 46%, brown solid,mp > 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.42 (s,3 H), 7.10 (s, 1 H), 7.13 (s, 1 H), 7.53 (dd, J ) 1.1, 5.1 Hz, 1 H),

7.69 (dd, J ) 2.9, 5.0 Hz, 1 H), 7.99 (dd, J ) 0.8, 2.9 Hz, 1 H)13.48 (s, 1 H). Anal. (C13H9NO2S2) C, H. N: calcd, 5.1; found 4.4.

(Z)-6-Methyl-1-((5-phenylthiophen-3-yl)methylene)-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (41). Yield 73%, yellowsolid, mp > 295 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ2.43 (s, 3 H), 7.05 (s, 1 H), 7.12 (s, 1 H), 7.37 (t, J ) 7.3 Hz, 1 H),7.47 (t, J ) 7.4 Hz, 2 H), 7.68 (d, J ) 7.2 Hz, 2 H), 7.80 (d, J )1.1 Hz, 1 H), 7.98 (s, 1 H), 13.51 (s, 1 H). Anal.(C19H13NO2S2 ·1.2H2O) C, H, N.

(Z)-1-(Benzofuran-2-ylmethylene)-6-methyl-4-thioxo-4,5-di-hydrofuro[3,4-c]pyridin-3(1H)-one (42). Yield 70%, orange solid,mp 291-295 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.44(s, 3 H), 7.02 (s, 1 H), 7.25 (s, 1 H), 7.31 (t, J ) 7.1 Hz, 1 H), 7.41(t, J ) 7.2 Hz, 1 H), 7.43 (s, 1 H), 7.65 (dd, J ) 0.7, 8.3 Hz, 1 H),7.76 (d J ) 7.5 Hz, 1 H), 13.60 (s, 1 H). Anal. (C17H11NO3S) C,H, N.

(Z)-1-((4-Methoxybenzofuran-2-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (43). Yield 27%,orange solid, mp 290-294 °C. 1H NMR (400 MHz, DMSO-d6,298 K) δ 2.44 (s, 3 H), 3.94 (s, 3 H), 6.84 (d, J ) 7.8 Hz, 1 H),7.15 (s, 1 H), 7.21 (s, 1 H), 7.24 (d, J ) 8.3 Hz, 1 H), 7.33 (s, 1H), 7.36 (t, J ) 7.9 Hz, 1 H), 13.57 (s, 1 H). Anal. (C18H13NO4S)C, H, N.

(Z)-1-((5-Methoxybenzofuran-2-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (44). Yield 38%,orange solid, mp > 295 °C. 1H NMR (400 MHz, DMSO-d6, 298K) δ 2.44 (s, 3 H), 3.80 (s, 3 H), 7.02 (dd, J ) 2.6, 9 Hz, 1 H),7.17 (s, 1 H), 7.24 (s, 1 H), 7.25 (d, J ) 2.5 Hz, 1 H), 7.36 (s, 1H), 7.55 (d, J ) 9 Hz, 1 H), 13.56 (s, 1 H). Anal.(C18H13NO4S ·0.75H2O) C, H, N.

(Z)-1-((6-Methoxybenzofuran-2-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (45). Yield 21%,red solid, mp > 295 °C. 1H NMR (400 MHz, DMSO-d6, 298 K)δ 2.43 (s, 3 H), 3.85 (s, 3 H), 6.94 (dd, J ) 2.2, 8.6 Hz, 1 H), 7.16(s, 1 H), 7.23 (s, 1 H), 7.29 (d, J ) 1.2 Hz, 1 H), 7.36 (s, 1 H),7.62 (d, J ) 8.6 Hz, 1 H), 13.52 (s, 1 H). Anal. (C18H13NO4S ·0.5H2O) C, H, N.

(Z)-1-((7-Methoxybenzofuran-2-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (46). Yield 10%,yellow solid, mp 286-290 °C. 1H NMR (400 MHz, DMSO-d6,298 K) δ 2.44 (s, 3 H), 3.97 (s, 3 H), 7.01 (d, J ) 7.8 Hz, 1 H),7.22 (m, 3 H), 7.31 (d, J ) 7.7 Hz, 1 H), 7.40 (s, 1 H), 13.59 (s,1 H). HRMS (EI+) calcd for C18H14NO4S, 340.06435 (MH+), found340.06469.

(Z)-1-((6-(Benzyloxy)-5-methoxybenzofuran-2-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (47).Yield 73%, red solid, mp 278-282 °C. 1H NMR (400 MHz,DMSO-d6, 298 K) δ 2.43 (s, 3 H), 3.82 (s, 3 H), 5.19 (s, 2 H),7.13 (s, 1 H), 7.20 (s, 1 H), 7.26 (s, 1 H), 7.31 (s, 1 H), 7.34 (t, J) 7.2 Hz, 1 H), 7.71 (t, J ) 7.5 Hz, 2 H), 7.43 (s, 1 H), 7.48 (d,J ) 7.0 Hz, 1 H), 13.48 (s, 1 H). Anal. (C25H19NO5S) C, H, N.

(Z)-1-(Benzo[b]thiophen-2-ylmethylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (48). Yield 86%, yellowsolid, mp > 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ2.41 (s, 3 H), 7.19 (s, 1 H), 7.44 (m, 2 H), 7.49 (s, 1 H), 7.80 (s,1 H), 7.95 (m, 1 H), 8.03 (m, 1 H), 13.48 (s, 1 H). Anal.(C17H11NO2S2) C, H, N.

(Z)-1-((5-Methoxybenzo[b]thiophen-2-yl)methylene)-6-meth-yl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (49). Yield47%, red solid, mp > 290 °C. 1H NMR (400 MHz, DMSO-d6,298 K) δ 2.44 (s, 3 H), 3.86 (s, 3 H), 7.04 (dd, J ) 2.2, 8.7 Hz, 1H), 7.16 (s, 1 H), 7.43 (s, 1 H), 7.61 (d, J ) 1.7 Hz, 1 H), 7.71 (s,1 H), 7.85 (d, J ) 8.6 Hz, 1 H), 13.49 (s, 1 H). Anal.(C18H13NO3S2 ·0.75H2O) C, H, N.

(Z)-1-((6-Methoxybenzo[b]thiophen-2-yl)methylene)-6-meth-yl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (50). Yield46%, orange solid, mp > 300 °C. 1H NMR (400 MHz, DMSO-d6,298 K) δ 2.44 (s, 3 H), 3.83 (s, 3 H), 7.06 (dd, J ) 2.5, 8.8 Hz, 1H), 7.19 (s, 1 H), 7.47 (s, 1 H), 7.48 (d, J ) 2.4 Hz, 1 H), 7.72 (s,1 H), 7.89 (d, J ) 8.8 Hz, 1 H), 13.43 (s, 1 H). Anal.(C18H13NO3S2 ·H2O) C, H, N.


(Z)-1-(Benzo[b]thiophen-2-ylmethylene)-6-methylfuro[3,4-c]pyridine-3,4(1H,5H)-dione (51). Yield 74%, yellow solid, mp> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.35 (s, 3 H),6.77 (s, 1 H), 7.42 (m, 3 H), 7.44 (s, 1 H), 7.94 (m, 1 H), 8.02 (m,1 H), 12.20 (s, 1 H). Anal. (C17H11NO3S ·0.7H2O) C, H, N.

(Z)-1-((1H-Indol-2-yl)methylene)-6-methyl-4-thioxo-4,5-dihy-drofuro[3,4-c]pyridin-3(1H)-one (52). Yield 16%, dark-brownsolid, mp > 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ2.44 (s, 3 H), 7.03 (m, 3 H), 7.17 (m, 3 H), 7.55 (d, J ) 8.3, 1 H),7.31 (d, J ) 8.0, 1 H), 11.23 (s, 1 H), 13.02 (s, 1 H). Anal.(C17H12N2O2S ·0.25H2O) C, H, N.

(Z)-1-((5-Methoxy-1H-indol-2-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (53). Yield 41%, darkbrown solid, mp > 295 °C. 1H NMR (400 MHz, DMSO-d6, 298K) δ 2.43 (s, 3 H), 3.76 (s, 3 H), 6.85 (dd, J ) 2.5, 8.9 Hz, 1 H),6.91 (d, J ) 1.5 Hz, 1 H), 7.04 (s, 1 H), 7.08 (d, J ) 2.5 Hz, 1 H),7.14 (s, 1 H), 7.45 (d, J ) 8.9 Hz, 1 H), 11.10 (s, 1 H), 13.11 (s,1 H). Anal. (C18H14N2O3S.0.25H2O) C, H, N.

(Z)-1-((6-Methoxy-1H-indol-2-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (54). Yield 97%, dark-red solid, mp 250-254 °C. 1H NMR (400 MHz, DMSO-d6, 298K) δ 2.42 (s, 3 H), 3.82 (s, 3 H), 6.71 (dd, J ) 2.3, 8.7 Hz, 1 H),6.92 (d, J ) 1.6 Hz, 1 H), 7.04 (s, 1 H), 7.08 (d, J ) 2.1 Hz, 1 H),7.12 (s, 1 H), 7.49 (d, J ) 8.7 Hz, 1 H), 11.10 (s, 1 H), 13.27 (s,1 H). Anal. (C18H14N2O3S ·0.25H2O) C, H, N.

(Z)-1-((6-(Benzyloxy)-5-methoxy-1H-indol-2-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (55).Yield 83%, brown solid, mp 240-244 °C. 1H NMR (400 MHz,DMSO-d6, 298 K) δ 2.44 (s, 3 H), 3.79 (s, 3 H), 5.13 (s, 2 H),6.88 (s, 1 H), 7.01 (s, 1 H), 7.10 (d, J ) 7.3 Hz, 2 H), 7.18 (s, 1H), 7.34 (t, J ) 7.3 Hz, 1 H), 7.41 (t, J ) 7.1 Hz, 2 H), 7.49 (d,J ) 7.0 Hz, 1 H), 10.95 (s, 1 H) 13.27 (s, 1 H). Anal. (C25H20N2O4S)C, H, N.

(Z)-1-((5-Fluoro-6-methoxy-1H-indol-3-yl)methylene)-6-meth-yl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (56). Yield28%, red solid, mp > 280 °C. 1H NMR (400 MHz, DMSO-d6,298 K) δ 2.43 (s, 3 H), 3.88 (s, 3 H), 6.92 (d, J ) 1.7 Hz, 1 H),7.05 (s, 1 H), 7.12 (s, 1 H), 7.24 (d, J ) 7.6 Hz, 1 H), 7.40 (d, J) 11.7 Hz, 1 H), 11.15 (s, 1 H), 13.39 (s, 1 H). Anal.(C18H13FN2O3S ·0.75H2O) C, H, N.

(Z)-1-((5-(Benzyloxy)-1H-indol-2-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (57). Yield 70%,brown solid, mp 247-251 °C. 1H NMR (400 MHz, DMSO-d6,298 K) δ 2.43 (s, 3 H), 5.10 (s, 2 H), 6.90 (d, J ) 1.6 Hz, 1 H),6.92 (dd, J ) 2.4, 8.9 Hz, 1 H), 7.03 (s, 1 H), 7.14 (s, 1 H), 7.18(d, J ) 2.4 Hz, 1 H), 7.33 (t, J ) 7.25 Hz, 2 H), 7.39 (d, J ) 7.1Hz, 2 H), 7.46 (m, 2 H) 11.11 (s, 1 H) 13.28 (s, 1 H). Anal.(C24H18N2O3S) C, H, N.

(Z)-6-Methyl-1-((1-methyl-1H-indol-2-yl)methylene)-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (58). Yield 78%, redsolid, mp > 295 °C.1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.45(s, 3 H), 3.99 (s, 3 H), 7.08 (t, J ) 7.7 Hz, 1 H), 7.25 (m, 2 H),7.29 (s, 1 H), 7.38 (s, 1 H), 7.50 (d, J ) 8 Hz, 1 H), 7.65 (d, J )8.0, 1 H), 13.52 (s, 1 H). Anal. (C18H14N2O2S ·0.5H2O) C, H, N.

(Z)-1-(Benzofuran-3-ylmethylene)-6-methyl-4-thioxo-4,5-di-hydrofuro[3,4-c]pyridin-3(1H)-one (59). Yield 76%, yellow solid,mp > 290 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.46 (s,3 H), 7.27 (s, 1 H), 7.32 (s, 1 H), 7.43 (m, 1 H), 7.68 (m, 2 H),8.07, (t, J ) 4.1 Hz, 1 H) 8.49 (s, 1 H), 13.55 (s, 1 H). HRMS(EI+) calcd for C17H12NO3S, 310.05379 (MH+), found 310.05463.

(Z)-1-(Benzo[b]thiophen-3-ylmethylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (60). Yield 23%, yellowsolid, mp > 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ2.47 (s, 3 H), 7.48 (m, 3 H), 7.55 (td, J ) 1.1, 7.1 Hz, 1 H), 8.08(d, J ) 7.9 Hz, 1 H), 8.34 (d, J ) 8 Hz, 1 H), 8.43 (s, 1 H), 13.51(s, 1 H). Anal. (C17H11NO2S2 ·0.75H2O) C, H, N.

(Z)-1-[(4-Methoxy-1-benzothien-3-yl)methylene]-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridine-3(1H)-one (61). Yield 42%,yellow solid. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.50 (s, 3H), 4.06 (s, 3 H), 7.05 (d, J ) 7.9 Hz, 1 H), 7.15 (s, 1 H), 7.41 (t,J ) 8.0 Hz, 1 H), 7.62 (d, J ) 8.0 Hz, 1 H), 7.72 (s, 1 H), 8.39 (s,

1 H), 8.39 (s, 1 H), 13.48 (br s, 1 H). HRMS (FAB+) calcd forC18H14NO3S2, 356.04151(MH+), found 356.04162.

(Z)-1-((5-Methoxybenzo[b]thiophen-3-yl)methylene)-6-meth-yl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (62). Yield28%, yellow solid, mp 237-241 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.48 (s, 3 H), 3.92 (s, 3 H), 7.12 (dd, J ) 2.3, 8.8 Hz,1 H), 7.39 (s, 1 H), 7.42 (s, 1 H), 7.83 (d, J ) 2.4 Hz, 1 H), 7.94(d, J ) 8.8 Hz, 1 H), 8.42 (s, 1 H), 13.26 (s, 1 H). HRMS (EI+)calcd for C18H14NO3S2, 356.04151 (MH+), found 356.04112.

(Z)-1-((6-Methoxybenzo[b]thiophen-3-yl)methylene)-6-meth-yl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (63). Yield84%, yellow solid, mp > 300 °C. 1H NMR (400 MHz, DMSO-d6,298 K) δ 2.46 (s, 3 H), 3.86 (s, 3 H), 7.15 (dd, J ) 2.4, 8.9 Hz, 1H), 7.38 (s, 1 H), 7.42 (s, 1 H), 7.65 (d, J ) 2.3 Hz, 1 H), 8.20 (d,J ) 8.9 Hz, 1 H), 8.22 (s, 1 H), 13.51 (s, 1 H). Anal.(C18H13NO3S2 ·0.5H2O) C, H, N.

(Z)-1-((5-Bromobenzo[b]thiophen-3-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (64). Yield32%, orange solid, mp > 300 °C. 1H NMR (400 MHz, DMSO-d6,298 K) δ 2.47 (s, 3 H), 7.43 (s, 1 H), 7.44 (s, 1 H), 7.61 (dd, J )1.8, 8.6 Hz, 1 H), 8.06 (d, J ) 8.6 Hz, 1 H), 8.48 (s, 1 H), 8.62 (d,J ) 1.8 Hz, 1 H), 13.49 (s, 1 H). Anal. (C17H10BrNO2S2 ·0.7H2O)C, H, N: calcd, 3.4, found 4.4.

(Z)-1-(Benzo[b]thiophen-3-ylmethylene)-6-methylfuro[3,4-c]pyridine-3,4(1H,5H)-dione (65). Yield 75%, yellow solid, mp> 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.37 (s, 3 H),7.03 (s, 1 H), 7.35 (s, 1 H), 7.46 (t, J ) 7.9 Hz, 1 H), 7.54 (t, J )7.9 Hz, 1 H), 8.08, (d, J ) 7.9 Hz, 1 H), 8.34 (d, J ) 7.9 Hz, 1 H),8.39 (s, 1 H), 12.17 (s, 1 H). Anal. (C17H11NO3S) C, H, N.

(Z)-1-((1H-Indol-3-yl)methylene)-6-methyl-4-thioxo-4,5-dihy-drofuro[3,4-c]pyridin-3(1H)-one (66). Yield 53%, orange solid,mp > 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.43 (s,3 H), 7.21 (m, 2 H), 7.24 (s, 1 H), 7.44 (s, 1 H), 7.49 (dd, J ) 1.7,6 Hz, 1 H), 8.01 (dd, J ) 1.9, 7 Hz, 1 H), 8.04 (s, 1 H), 11.97 (s,1 H), 13.23 (s, 1 H). Anal. (C17H12N2O2S ·0.15H2O) C, H, N.

(Z)-6-Methyl-1-((1-methyl-1H-indol-3-yl)methylene)-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (67). Yield 92%, orangesolid, mp > 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ2.47 (s, 3 H), 3.93 (s, 3 H), 7.23 (s, 1 H), 7.25 (m, 1 H), 7.30 (t,J ) 7.9 Hz, 1 H), 7.42 (s, 1 H), 7.54 (d, J ) 7.9 Hz, 1 H), 8.02 (d,J ) 7.4 Hz, 1 H), 8.07 (s, 1 H), 13.08 (s, 1 H). Anal.(C18H14N2O2S ·0.5H2O) C, H, N.

(Z)-6-Methyl-1-((1-methyl-1H-indol-3-yl)methylene)furo[3,4-c]pyridine-3,4(1H,5H)-dione (68). Yield 66%, yellow solid, mp> 290 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.32 (s, 3 H),3.90 (s, 3 H), 6.81 (s, 1 H), 7.24 (t, J ) 7 Hz, 1 H), 7.29 (t, J )7 Hz, 1 H), 7.32 (s, 1 H), 7.53 (d, J ) 7.7 Hz, 1 H), 8.02 (m, 2 H),11.88 (s, 1 H). HRMS (EI+) calcd for C18H14N2O3 306.10044 (M+),found 306.10025.

(Z)-1-(Benzo[b]thiophen-3-ylmethylene)-6-methyl-4-thioxo-4,5-dihydro-1H-pyrrolo[3,4-c]pyridin-3(2H)-one (69). Yield 26%,brown solid, mp 272-275 °C. 1H NMR (400 MHz, DMSO-d6,298 K) δ 2.48 (s, 3 H), 7.30 (s, 1 H), 7.46 (s, 1 H), 7.48 (m, 1 H),7.53 (t, J ) 7.1 Hz, 1 H), 8.07, (d, J ) 7.9 Hz, 1 H), 8.31 (d, J )7.9 Hz, 1 H), 8.39 (s, 1 H), 9.31 (s, 1 H) 13.74 (s, 1 H). Anal.(C17H12N2OS2 ·0.75H2O) C, H, N.

(Z)-1-((1H-Indol-7-yl)methylene)-6-methyl-4-thioxo-4,5-dihy-drofuro[3,4-c]pyridin-3(1H)-one (70). Yield 79%, orange solid,mp > 300 °C. 1H NMR (400 MHz, DMSO-d6, 298 K) δ 2.46 (s,3 H), 7.01 (s, 1 H), 7.23 (t, J ) 7.8 Hz, 1 H), 7.35 (s, 1 H), 7.47(m, 2 H), 7.51 (t, J ) 2.8 Hz, 1 H), 7.85 (d, J ) 7.5 Hz, 1 H),11.37 (s, 1 H), 13.30 (s, 1 H). Anal. (C17H12N2O2S ·0.5H2O) C, H,N.

Inhibition of Perforin-Mediated Lysis of Sheep Red BloodCells (SRBC). A commercial library of ∼100000 compounds wasscreened for the ability to reproducibly inhibit perforin-mediatedlysis of SRBC at a compound concentration of 100 µM.23

Compounds were assayed as single points using 384-well platemethodology measuring change in turbidity. Lysis of the SRBCresults in a change of the turbidity of the reaction mixture, whereasinhibition of cell lysis results in reduction or abolition of the change


in turbidity reading. A solution of the compound in DMSO wasadded to 10 µL of 0.5 µg/mL perforin in buffer or controls,respectively, with at least 30 min preincubation with compoundroutinely. 0.04 mL of SRBC was then added in RBC buffer.Samples were initially read (t ) 0 min) at an absorbance of 650nM (in Envision; using an Envision reader, automation ABS@650nm), incubated for 15 min. at 37 °C, then read at an absorbance of650 nm (in Envision) to assess the change in turbidity of the reactionmixture. As the inhibitor compounds were routinely dissolved inDMSO, the same concentration of DMSO was used as a negativecontrol for the inhibition of perforin. Secondary screening wascarried out using the same methodology, employing a series ofconcentrations; 100, 20, 4, 0.8, and 0.16 µM to determine an IC50.

Inhibition of PLO-Mediated Lysis of SRBC. To determinewhether the active inhibitors identified in the above screen (Perforin/SRBC) were also able to block the lytic function of PLO, theinhibitors were then tested at a concentration of 20 µM in thepresence PLO instead of perforin.

Inhibition of Perforin-Mediated Lysis of Jurkat Cells. Theability of the compounds to inhibit the lysis of nucleated (Jurkat Tlymphoma) cells in the presence of 0.1% BSA, as measured byrelease of 51Cr was measured. Jurkat target cells were labeled byincubation in medium with 100 µCi 51Cr for one hour. The cellswere then washed three times to remove unincorporated isotopeand resuspended at 1 × 105 cells per mL in RPMI buffersupplemented with 0.1% BSA. Each test compound was preincu-bated to concentrations of 20, 10, 5, 2.5, and 1.25 µM withrecombinant perforin for 30 min with DMSO as a negative control.51Cr labeled Jurkat cells were then added and cells were incubatedat 37 °C for 4 h. The supernatant was collected and assessed forits radioactive content on a γ counter (Wallac Wizard 1470automatic γ counter). Each data point was performed in triplicate,and an IC50 was calculated from the range of concentrationsdescribed to above. Compounds with an IC50 < 1 µM were titrateddown to lower concentrations in the same manner as above todetermine an accurate IC50.

Inhibition of Fas Ligand- or TRAIL-Mediated Lysis ofJurkat Cells. Fifty thousand 51Cr-labeled Jurkat cells (0.05 mL)were preincubated with inhibitor to a final concentration of 20 µMfor 30 min at 37 °C in the wells of a round-bottom, sterile 96-wellplate in a humidified CO2 incubator. Two negative controls wereused: (i) the diluent (DMSO) used to dissolve the inhibitors wasadded at the same concentration as used in the other wells, and (ii)a compound known not to inhibit perforin (designated B3) wasalso used at the same concentration as the perforin inhibitor. Eachdata point was performed in triplicate. Each plate was set up induplicate so that the capacity to block both Fas- and TRAIL-mediated cell death could be evaluated independently. To each wellof the duplicate plate was added either anti-Fas agonistic mono-clonal antibody CH11 or recombinant (rec)-TRAIL to initiate celldeath. Each agent was used at a final concentration of 250 ng/mL,and the reaction was carried out in a final volume of 200 µL RPMImedium containing 1% BSA. As further controls, three wellscontained Jurkat cells in which the detergent SDS was added, andthese wells were used to estimate the maximum release of 51Cr.After 2 h, the cells were pelleted by centrifugation and 50 µL ofthe assay supernatant was collected and assessed for its radioactivecontent on a γ counter. The results in Figure 5 (SupportingInformation) show the mean 51Cr release ((standard error mean).

KHYG-1 Cytotoxicity Assay. KHYG-1 cells were washed andresuspended in RPMI + 0.1% BSA at 4 × 105 cells/ml and 50 µLof KHYG-1 cells were dispensed to each well of a 96-wellV-bottom plate. Test compounds were added to KHYG-1 cells atvarious concentrations up to 20 µM and incubated at RT for 20min. One × 106 K562 target cells were labeled with 75 µCi 51Crin 200 µL RPMI for 90 min at 37 °C, cells were washed asdescribed above, and resuspended in 5 mL RPMI + 0.1% BSA.50 µL of 51Cr labeled K562 leukemia target cells were added toeach well of the KHYG-1 plate (effector:target 2:1) and incubatedat 37 °C for 4 h. 51Cr release was assayed using a Skatron harvestingpress and radioactivity estimated on a Wallac Wizard 1470

automatic γ counter (Turku, Finland). The percentage of specificcytotoxicity was calculated by the formula:

% specific lysis)(experimental release- spontaneous release)

(maximum release- spontaneous release)× 100 (1)

and expressed as the mean of triplicate assays ( standard error ofthe mean.

Toxicity to KHYG-1 NK Cells. The toxicity assay was carriedout in exactly the same manner as the killing assay above, butinstead of adding the labeled K562 target cells, 100 µL of RPMI0.1% BSA was added. Cells were incubated for 4 h at 37 °C thenwashed ×3 in RPMI + 0.1% BSA. Cells were then resuspendedin 200 µL of complete medium and incubated for 18 to 24 h at 37°C. Trypan blue was added to each well and viable (clear) cellscounted as a percentage of total (clear + blue) cell number (%viability).

Inhibition of Perforin Secreted by Primary Human NK Cells.Human CD3-CD56+ NK cells were isolated from the buffy coatsof healthy blood donors (Australian Red Cross Blood Bank). Themononuclear cell fraction was initially isolated by centrifugationon a Hypaque-Ficoll cushion. NK cells were then isolated using aMACS human NK isolation kit (Miltenyi Biotec). Non-NK cellswere labeled, and NK cells were purified by negative selection usingan AutoMACS separator. The cells were washed three times inRPMI buffer containing 0.1% BSA, then used on the same day orcultured in media containing 100 U/mL IL-2 for up to 3 days. K562leukemia cells were washed and labeled with 51Cr as describedabove and used as target cells in the NK killing assay. NK cells(up to 2 × 105) were placed in triplicate wells of a V-bottom 96well plate, together with each of the inhibitors indicated, or withDMSO diluent or with medium alone, which were used as thenegative controls. The reaction volume was 200 µL, in RPMI buffercontaining 0.1% BSA. The plate was incubated at RT for 30 min,then labeled K562 cells (1 × 104 in 50 µL of the same solution)were then added to each well, and the plate was incubated for 4 hat 37 °C as before. 51Cr released into the supernatant was harvestedusing a Skatron harvesting press and radioactivity estimated on aWallac Wizard 1470 automatic γ counter and % specific lysiscalculated as previously.

Acknowledgment. J.A.T. is supported by a Program Grantand a Senior Principal Research Fellowship from the NationalHealth and Medical Research Council of Australia. This workwas supported by Incitive Limited (Australia) and the AucklandDivision of the Cancer Society of New Zealand. We also thankPamela Murray for conducting the Ames testing of selectedcompounds, Sisira Kumara for the HPLC studies, and MarutaBoyd for NMR studies.

Supporting Information Available: Experimental for thepreparation of selected intermediates, elemental analyses, NMR,HRMS, HPLC data on final compounds and selected figures asindicated in the text. This material is available free of charge viathe Internet at http://pubs.acs.org.

References(1) Voskoboinik, I.; Smyth, M. J.; Trapani, J. A. Perforin-mediated target-

cell death and immune homeostasis. Nat. ReV. Immunol. 2006, 6, 940–952.

(2) Lieberman, J. Mechanisms of granule-mediated cytotoxicity. Curr.Opin. Immunol. 2003, 15, 513–515.

(3) Ashton-Rickardt, P. G. The granule pathway of programmed cell death.Crit. ReV. Immunol. 2005, 25, 161–182.

(4) Russell, J. H.; Ley, T. J. Lymphocyte-mediated cytotoxicity. Annu.ReV. Immunol. 2002, 20, 323–370.

(5) Lichtenheld, M. G.; Olsen, K. J.; Lu, P.; Lowrey, D. M.; Hameed,A.; Hengartner, H.; Podack, E. R. Structure and function of humanperforin. Nature 1988, 335, 448–451.

(6) Podack, E. R. Perforin: Structure, function and regulation. Curr. Top.Microbio. Immun. 1992, 178, 175–185.


(7) Trapani, J. A.; Smyth, M. J. Functional significance of the perforin/granzyme cell death pathway. Nat. ReV. Immunol. 2002, 2, 735–747.

(8) Voskoboinik, I.; Trapani, J. A. Addressing the mysteries of perforinfunction. Immunol. Cell Biol. 2006, 84, 66–71.

(9) Rosado, C. J.; Buckle, A. M.; Law, R. H. P.; Butcher, R. E.; Kan,W.-T.; Bird, C. H.; Ung, K.; Browne, K. A.; Baran, K.; Bashtannyk-Puhalovich, T. A.; Faux, N. G.; Wong, W.; Porter, C. J.; Pike, R. N.;Ellisdon, A. M.; Pearce, M. C.; Bottomley, S. P.; Emsley, J.; Smith,A. I.; Rossjohn, J.; Hartland, E. L.; Voskoboinik, I.; Trapani, J. A.;Bird, P. I.; Dunstone, M. A.; Whisstock, J. C. A common fold mediatesvertebrate defence and bacterial attack. Science 2007, 317, 1548–1551.

(10) Kagi, D.; Ledermann, B.; Burki, K.; Seiler, P.; Odermatt, B.; Olsen,K. J.; Podack, E. J.; Zinkernagel, R. M.; Hengartner, H. Cytotoxicitymediated by T cells and natural killer cells is greatly impaired inperforin-defficient mice. Nature 1994, 369, 31–37.

(11) Kagi, D.; Odermatt, B.; Seiler, P.; Zinkernagel, R. M.; Mak, T. W.;Hengartner, H. Reduced incidence and delayed onset of diabetes inperforin-deficient nonobese diabetic mice. J. Exp. Med. 1997, 186,989–997.

(12) Katano, H.; Cohen, J. I. Perforin and lymphohistiocytic proliferativedisorders. Br. J. Haematol. 2005, 128, 739–750.

(13) Henter, J.-I.; Arico, M.; Elinder, G.; Imashuku, S.; Janka, G. Familialhemophagocytic lymphohistiocytosis. Primary hemophagocytic lym-phohistiocytosis. Hematol. Oncol. Clin. North Am. 1998, 12, 417–433.

(14) Voskoboinik, I.; Thia, M.-C.; De Bono, A.; Browne, K.; Cretney, E.;Jackson, J. T.; Darcy, P. K.; Jane, S. M.; Smyth, M. J.; Trapani, J. A.The functional basis for hemophagocytic lymphohistiocytosis in apatient with co-inherited missense mutations in the perforin (PFN1)gene. J. Exp. Med. 2004, 200, 811–816.

(15) Catalfamo, M.; Henkart, P. A. Perforin and the granule exocytosiscytotoxicity pathway. Curr. Opin. Immunol. 2003, 15, 522–527.

(16) Pearl-Yafe, M.; Kaminitz, A.; Yolcu, E. S.; Yaniv, I.; Stein, J.;Askenasy, N. Pancreatic islets under attack: cellular and moleculareffectors. Curr. Pharm. Des. 2007, 13, 749–760.

(17) Veale, J. L.; Liang, L. W.; Zhang, Q.; Gjertson, D. W.; Du, Z.;Bloomquist, E. W.; Jia, J.; Qian, L.; Wilkinson, A. H.; Danovitch,G. M.; Pham, P.-T. T.; Rosenthal, J. T.; Lassman, C. R.; Braun, J.;Reed, E. F.; Gritsch, H. A. Noninvasive diagnosis of cellular andantibody-mediated rejection by perforin and granzyme B in renalallografts. Hum. Immunol. 2006, 67, 777–786.

(18) Choy, J. C.; Kerjner, A.; Wong, B. W.; McManus, B. M.; Granville,D. J. Perforin mediates endothelial cell death and resultant transplantvascular disease in cardiac allografts. Amer. J. Pathol. 2004, 165, 127–133.

(19) Barry, M.; Bleackley, R. C. Cytotoxic T lymphocytes: All roads leadto death. Nature ReV. Immunol. 2002, 2, 401–409.

(20) Togashi, K.; Kataoka, T.; Nagai, K. Characterization of a series ofvacuolar type H+-ATPase inhibitors on CTL-mediated cytotoxicity.Immunol. Lett. 1997, 55, 139–144.

(21) Kataoka, T.; Shinohara, N.; Takayama, H.; Takaku, K.; Kondo, S.;Yonehara, S.; Nagai, K. Concanamycin A, a powerful tool forcharacterization and estimation of contribution of perforin- and Fas-based lytic pathways in cell-mediated cytotoxicity. J. Immunol. 1996,156, 3678–3686.

(22) Kataoka, T.; Taniguchi, M.; Yamada, A.; Suzuki, H.; Hamada, S.;Magae, J.; Nagai, K. Identification of low molecular weight probesof perforin- and Fas-based killing mediated by cytotoxic T-lympho-cytes. Biosci. Biotechnol. Biochem. 1996, 60, 1726–1728.

(23) Holloway, G. A.; Baell, J. B.; Fairlamb, A. H.; Novello, P. M.; Parisot,J. P.; Richardson, J.; Watson, K. G.; Street, I. P. Discovery of2-iminobenzimidazoles as a new class of trypanothione reductaseinhibitor by high-throughput screening. Bioorg. Med. Chem. Lett. 2007,17, 1422–1427.

(24) Trapani, J. A. and Smyth, M. J. Retroviral vectors encoding recom-binant mouse perforin, its expression and therapeutic uses thereof.Patent WO 2005083098 A1, March, 1, 2005.

(25) Kaigodorova, E. A.; Kvak, S. N.; Ugrak, B. I.; Zaplishnyi, V. N.;Kul’nevich, V. G. Studies of furopyridines. VI. Synthesis and steric

structure of hetarylmethylene-4-thiofuro[3,4-c]pyridin-3-ones. Russ.J. Org. Chem. 1995, 31, 1650–1652.

(26) Kaigorodova, E. A.; Mikhailichenko, S. N.; Vasilin, V. K.; Terekhov,V. I.; Kul’nevich, V G. Investigation of furopyridines. Part 7. Synthesisand antibacterial activity of 1-arylidene-3-oxo-4-thioxofuro[3,4-c]-pyridines. Pharm. Chem. J. 1998, 32, 194–196.

(27) Arustamova, I. S.; Kaigorodova, E. A.; Kul’nevich, V. G.; Ugrak,B. I. Research in the field of furopyridines. 2. Synthesis and stericstructure of 6-methyl-1-hetarylidene-3,4-dioxo[3,4-c]pyridines. Chem.Heterocycl. Compds. 1993, 29, 539–544.

(28) Bruce, W. F.; Coover, H. W. Pyridine Derivatives. I. 3-Cyano-4-ethoxymethyl-6-methyl-2-pyridone and some related transformationproducts. J. Am. Chem. Soc. 1944, 66, 2092–2094.

(29) Harris, S. A.; Stiller, E. T.; Folkers, K. Structure of Vitamin B6. II.J. Am. Chem. Soc. 1939, 61, 1242–1244.

(30) Prostenik, M.; Butula, I. Selective reduction of some halogen-substituted pyridines of the vitamin B6 series. Chem. Ber. 1977, 110,2106–2113.

(31) Shul’ga, N. A.; Balyakina, M. V.; Gunar, V. I. Peculiarities of thenitration of 2-methyl-4-methoxymethyl-5-cyano-6-pyridone in a mix-ture of HNO3 and H2SO4. Pharm. Chem. J. 1983, 17, 665–669.

(32) (a) Preparation of 71: Karminski-Zamola, G.; Dogan, J.; Bajic, M.;Blazevic, J.; Malesevic, M. Synthesis of some furyl- and thienylacry-lates or diacrylates and acrylic acids by the palladium catalysedvinylation of substituted bromofurans and bromothiophenes. Hetero-cycles 1994, 38, 759–767. (b) Hassan, J.; Sevignon, M.; Gozzi, C.;Schulz, E.; Lemaire, M. Aryl-aryl bond formation one century afterthe discovery of the Ullmann reaction. Chem. ReV. 2002, 102, 1359–1469.

(33) Preparation of benzofurans: Chapleo, C. B.; Myers, P. L.; Butler,R. C. M.; Davis, J, A.; Doxey, J. C.; Higgins, S. D.; Myers, M.; Roach,A. G.; Smith, C. F. C.; Stillings, M. R.; Welbourn, A. P. R-Adreno-receptor reagents. 2. Effects of modifications of the 1,4-benzodioxanring system on R-adrenoreceptor activity. J. Med. Chem. 1984, 27,570–576.

(34) (a) Preparation of benzothiophenes: Bridges, A. J.; Lee, A.; Schwartz,C. E.; Towle, M. J.; Littlefield, B. A. The synthesis of three4-substituted benzo[b]thiophene-2-carboxamidines as potent and selec-tive inhibitors of urokinase. Bioorg. Med. Chem. 1993, 1, 403–410.(b) Titus, R. L.; Titus, C. F. 2-Amino-3-(6-methoxybenzo[b]thien-3-yl)propanoic acid. J. Heterocycl. Chem. 1973, 10, 679–681. (c)Campaigne, E.; Rogers, R. B. Benzo[b]thiophene derivatives. XXI.On the proof of structure of the sulfur isostere of Psilocin. J. Het.Chem. 1973, 10, 963–966.

(35) Preparation of indoles Marco, J. L. Improved preparation of N-pro-pargyl-2-(5-benzyloxy-indolyl)methylamine. J. Heterocycl. Chem.1998, 35, 475–476.

(36) (a) Reduction-oxidation reactions Mitsumori, S.; Tsuri, T.; Honma,T.; Hiramatsu, Y.; Okada, T.; Hashizume, H.; Inagaki, M.; Arimura,A.; Yasui, K.; Asanuma, F.; Kishino, J.; Ohtani, M. Synthesis andbiological activity of various derivatives of a novel class of potent,selective, and orally active prostaglandin D2 receptor antagonists. 1.Bicyclo[2.2.1]heptane derivatives. J. Med. Chem. 2003, 46, 2436–2445. (b) Chapman, N. B.; Clarke, K.; Saraf, S. D. Pharmacologicallyactive benzo[b]thiophen derivatives. Part III. 2-Substituted compounds.J. Chem. Soc. C 1967, 731–735. (c) Ogiso, A.; Kurabayashi, M.;Takahashi, S.; Mishima, H.; Woods, M. C. The structure and totalsynthesis of Futoenone, a constituent of Piper futokadzura. Chem.Pharm. Bull. 1970, 18, 105–114. (d) Zaidlewicz, M.; Chechlowska,A.; Prewysz-Kwinto, A.; Wojtczak, A. Enantioselective synthesis of2- and 3-benzofuryl �-amino alcohols. Heterocyles 2000, 55, 569–577.

(37) Boyd, P. D. W.; Lena, G.; Spicer, J. A. Ethyl 4-hydroxymethyl-2-methylpyridine-5-carboxylate. Acta Crystallogr., Sect. E 2008, 64,o883.

(38) Maron, D. M.; Ames, B. N. Revised methods for the Salmonellamutagenicity test. Mutat. Res. 1983, 113, 173–215.

(39) Trapani, J. A. Dual mechanisms of apoptosis induction by cytotoxiclymphocytes. Int. ReV. Cytol. 1998, 182, 111–192.

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