Page  98 General Papers ARKIVOC 2006 (xii) 98-110 Synthesis and biological evaluation of new 5-methyl-N-(3-oxo-1-thia4- azaspiro[4.5]-dec-4-yl)-3-phenyl-1H-indole-2-carboxamide derivatives Özlen Güzel*, Nalan Terzioglu, Gültaze Çapan, and Aydin Salman Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry 34116 Beyazit, Istanbul, Turkey E-mail: [email protected] Abstract A series of new 5-methyl-N-(3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)-3-phenyl-1H-indole-2carboxamide derivatives (2 and 3) was synthesized and characterized by IR, 1H-NMR, HSQC, HMBC, 13C-NMR (APT), APCI mass spectral data and elemental analysis. All synthesized compounds were evaluated for in vitro antituberculosis activity against M.tuberculosis H37Rv. Compound 3c was found to provide the highest (90%) inhibition of mycobacterial growth in the primary screen conducted at 6.25 µg/mL. Compounds 2a, 2b, 3a, 3b and 3c chosen as prototypes were evaluated against the full panel of 55 human tumour cell lines in the National Cancer Institute’s in vitro primary cytotoxicity assay. 2b showed the most favourable cytotoxic effects on ovarium cancer cell line IGROV1 (log10 GI50 value –7.31) and renal cancer cell line UO-31 (log10 GI50 value –7.56), whereas 3a showed favorable cytotoxicity on renal cancer cell line RXF-393 (log10 GI50 value – 6.38). Keywords: Indole, spirothiazolidinones, antituberculosis activity, anticancer activity Introduction Tuberculosis (TB), an infection of Mycobacterium tuberculosis, still remains the leading cause of worldwide death among infectious diseases. One-third of the population is infected with M. Tuberculosis and the World Health Organization (WHO) estimates that within the next 20 years about 30 million people will be infected with the bacillus1. Active disease following new infection, as well as reactivation of latent tuberculosis, is particularly prevalent in individuals with compromised immune systems, such as those that are HIV positive. Duration of the treatment of cases is very prolonged especially when caused by resistant bacteria. ISSN 1424-6376 Page 98 ©ARKAT

Page  99 General Papers ARKIVOC 2006 (xii) 98-110 Although advances in cancer detection and treatment over the past 10 years have led an increase in the expected survival period, the majority of patients diagnosed with the disease relapse after cytoreductive surgery and standard adjuvant chemotherapy. While much research is being done to establish active salvage regimens, successful treatment depends upon effective adjuvant therapy. Intrinsic or acquired resistance of cancer cells to chemotherapeutics in several malignant tumors decrease the response rate and new therapeutic agents are needed. Indole derivatives have been shown to possess antibacterial, antifungal and anti HIV activities2-7. There have also been several reports on the anticancer properties of compounds bearing this ring system8,9. A very recent report deals with the apoptosis inducing effect of 5methyl- 3-phenyl-indole-2-carboxylic acid benzylidene hydrazides9. On the other hand, 4-thiazolidinones and their spiroheterocyclic analogs have been reported to exhibit antibacterial, antifungal and antimycobacterial activity10-17. Furthermore 2aryl- 4-oxothiazolidin-3-yl amides have been reported to demonstrate antiproliferative activity in prostate cancer18. As a consequence of this research and with the aim of obtaining new and more potent antituberculosis and antitumor compounds which can improve the current chemotherapeutic antituberculosis and antitumor treatments, we have synthesized and evaluated a series of new 5methyl- N-(3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)-3-phenyl-1H-indole-2-carboxamide derivatives bearing various substituents on the spiroheterocyclic residue. Results and Discussion 5-Methyl-3-phenyl-1H-indole-2-carbohydrazide (1), obtained via a multistep synthesis19, an appropriate cyclic ketone and mercaptoacetic acid or a-mercaptopropionic acid were refluxed in dry benzene using a Dean-Stark water separator to produce 5-methyl-N-(3-oxo-1-thia-4azaspiro[ 4.5]dec-4-yl)-3-phenyl-1H-indole-2-carboxamide derivatives (2 and 3) in a one-pot reaction (Scheme 1). The structures of the new compounds were established by IR, 1H-NMR, HMBC, HSQC, 13C-NMR (APT), atmospheric pressure chemical ionization [APCI(+)] mass spectrometry and elemental analysis (Table 1). The IR spectra exhibited N-H and C=O bands in the 3241-3370 cm-1 and 1651-1678 cm-1 regions attributed to the common CONH functions of 2 and 3 20. Observation of new endocyclic C=O bands (1692-1713 cm-1) characteristic for such structures besides C=O amide bands (16511678 cm-1) in the IR spectra of 2 and 3 supported the aimed cyclization12,13. 1H-NMR spectra of 2 showed the spirodecane C2-H2 at d 3.52-3.63 ppm and C2-H of 3 at d 3.74-3.92 ppm 13,21,22. The remaining spirodecane protons resonated at about d 0.81-2.10 ppm together with the alkyl substituents on the spiroheterocycle. Peaks associated with the indole subunit were observed in the expected regions20. ISSN 1424-6376 Page 99 ©ARKAT

Page  100 General Papers ARKIVOC 2006 (xii) 98-110 CH3 CH3 CH3 NH2 CH3 N2Cl CH3 O C6H5 i N N COOC2H5 CH3 N H C6H5 N H C6H5 O R CH3 N C6H5 HN O N S O R1 R HN NH2 O OC2H5 O + ii iii iv v+ OC2H5 O + - C6H5 H H 1 2, 3 Scheme 1. Reagents and conditions: i) NaNO2, HCl, 0°C; ii) KOH, 0°C; iii) 37% HCl, reflux, 4h; iv) H2NNH2.H2O, EtOH, reflux, 6h; v) HSCH2COOH/HSCH(CH3)COOH, dry C6H6, reflux, 6h. 13C-APT run on 2c and 2D NMR experiments HSQC and HMBC run on 3c allowed explicit assignments for the proton and carbon chemical shifts. The spectra substantiated the expected conversion and revealed the typical spirodecane C2, C3 (lactam C=O) and C5 resonances at d 28.57-37.45, 168.05-170.71 and 71.40-73.00 ppm, respectively13,22. Cross peaks observed between 2-CH3 and C2-H protons and spirodecane C3 (lactam C=O) in the HMBC spectrum of 3c enabled definite assignment of the lactam C=O (d 170.71 ppm) and CONH (d 162.57 ppm) carbons. Abundant (M+H)+ ions observed in the APCI mass spectra of 2d and 3c (2d: m/z 496; 3c: m/z 448) confirmed their molecular weights. The molecules fragmented via loss of m/z 74 or m/z 88 fragments or rupture of the exocyclic CO-NH bond which afforded the base peaks (2d: m/z 422; 3c: m/z 234)13,20. A proposed mass fragmentation route of 2d and 3c recorded employing APCI in the positive ionization mode is depicted in Scheme 2. Further spectral details are presented in the experimental section. The in vitro antimycobacterial activity evaluation of 2 and 3 against M. Tuberculosis H37Rv (ATCC 27294) was initially carried out in BACTEC 12B medium using the microplate alamar blue assay (MABA) at a concentration of 6.25 µg/mL at the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF)23 (Table 1). ISSN 1424-6376 Page 100 ©ARKAT

Page  101 General Papers ARKIVOC 2006 (xii) 98-110 Table 1. Formulas, physical constants, elemental analysis and primary in vitro antimycobacterial activity evaluation of 2 and 3 against M. Tuberculosis H37Rv R C6H5CH3 H N NS N O H R1 O 2, 3 Comp. R R1 Formula Yield M.p. Analysis (%) GI (M.W.) (%) (°C) (calc./found) (%)a C H N 2a 6-CH3 H C25H27N3O2S 47 176-8 69.26 6.28 9.69 72 (433.57) 68.73 6.32 9.54 2b 7-CH3 H C25H27N3O2S.H2O 25 156-8 66.49 6.49 9.31 63 (451.58) 66.03 6.25 9.16 2c 8-C3H7 H C27H31N3O2S 71 196-8 70.25 6.77 9.10 85 (461.62) 70.12 6.81 8.98 2d 8-C6H5 H C30H29N3O2S 57 108-10 72.70 5.90 8.48 56 (495.64) 72.33 5.89 8.16 3a 6-CH3 CH3 C26H29N3O2S 43 156-8 69.77 6.53 9.39 63 (447.60) 70.30 6.69 9.40 3b 7-CH3 CH3 C26H29N3O2S 14 197-8 69.77 6.53 9.39 71 (447.60) 69.45 6.49 9.84 3c 8-CH3 CH3 C26H29N3O2S 46 172-4 69.77 6.53 9.39 90 (447.60) 70.04 6.69 9.37 3d 8-C3H7 CH3 C28H33N3O2S 48 170-3 70.70 6.99 8.83 86 (475.65) 70.18 7.22 9.14 3e 8-C6H5 CH3 C31H31N3O2S.1.5H2O 79 132-5 69.37 6.38 7.82 69 (536.66) 69.69 6.29 7.32 aGrowth Inhibition of virulent H37Rv strain of M. Tuberculosis at 6.25µg/ml. MIC of Rifampin: 0.125 µg/mL versus M. Tuberculosis H37Rv (97% inhibition). ISSN 1424-6376 Page 101 ©ARKAT

Page  102 General Papers ARKIVOC 2006 (xii) 98-110 R R + C6H5 C6H5 SS HN N N + H+ CH3 H R1 CH3 HN R1 NOO NOO HH 2d R=C6H5 R1=H m/z 496 (M+H)+ OS 3c R=CH3 R1=CH3 m/z 448 (M+H)+ R1 -m/z 74 R1=H -m/z 88 R1=CH3 R C6H5 CH3 C6H5 + CH3 HNN + O HN H NOH m/z 234 2d R=C6H5 R1=H m/z 422 3c R=CH3 R1=CH3 m/z 360 Scheme 2. Proposed mass fragmentation pattern of 2d and 3c under APCI (+). As can be seen in Table 1, 2 and 3 produced growth inhibitions ranging from 56% to 90%. The correlation between structure and antituberculosis activity in this series was not straightforward, however, it may be speculated that introduction of a methyl group at 2- position of the spirodecane system increased activity (except 3a) and thus compounds 3 were more active than 2 and 3c, the 8-C6H5 and 2-CH3 substituted entry showed the highest inhibition (90%). Compounds 2a, 2b, 3a, 3b, 3c chosen by the National Cancer Institute were screened for antitumor activity. Primary anticancer assay was performed in accordance with the protocol of the Drug Evaluation Branch of the National Cancer Institute (Bethesda)24-26. For the compounds, the 50% growth inhibition (GI50) and total growth inhibition (TGI) were obtained for each cell line. The log10 GI50 and log10 TGI defined as the mean of the log10’s of the invidual GI50 and TGI values were then determined. Negative values indicated the most sensitive cell lines. Compounds having values –4 and <-4 were declared to be active. As shown in Table 2. log10 GI50 values of compounds 2a, 2b, 3a, 3b and 3c were smaller than –4 for all tested tumour cell lines. Among the compounds tested, compound 2b demonstrated the most marked effects in the National Cancer Institute’s 55 human tumour cell line in vitro screen on ovarium cancer cell line IGROV1 (log10 GI50 value –7.31), renal cancer cell line UO-31 (log10 GI50 value –7.56), and CNS cancer cell line SNB-19 (log10 GI50 value –4.73). On the same cancer cell line, the log10 GI50 ISSN 1424-6376 Page 102 ©ARKAT

Page  103 General Papers ARKIVOC 2006 (xii) 98-110 values of thioguanine used as an anticancer agent were –5.32, -5.98 and –4.10, respectively. When these data are examined, it is observed that 2b is more active than thioguanine on ovarium cancer cell line IGROV1, renal cancer cell line UO-31 and CNS cancer cell line SNB-19. On the other hand compound 3a exhibited high cytotoxicity on renal cancer cell line RXF 393 and CNS cancer cell line SNB-19. The log10 GI50 values of compound 3a and thioguanine were –6.38, -4.57 and -5.83, -4.10 on the same cancer cell lines, respectively. Table 2. Invitro tumor cell growth inhibition of 2a, 2b, 3a, 3b and 3c Panel/cell line 2a 2b 3a 3b 3c Thioguanine log10GI50log10TGI log10GI50 log10TGI log10GI50log10TGI log10GI50 log10TGI log10GI50 log10TGI log10GI50log10TGI Leukemia CCRF-CEM -4.77 -4.41 -4.97 -4.50 -4.66 -4.14 -4.13 >-4.00 -4.69 -4.10 -6.81 -4.59K-562 -4.97 -4.38 -4.90 -4.41 -4.73 -4.22 -4.46 >-4.00 -4.79 -4.21 -5.96 -4.75MOLT-4 -5.03 -4.55 -5.06 -4.50 -4.91 -4.39 -4.71 >-4.00 -5.10 -4.49 -6.39 -4.15RPMI-8226 -4.86 -4.45 -4.86 -4.50 -4.67 -4.19 -4.27 >-4.00 -4.94 -4.28 -6.57 -4.81 Non-small cell lung cancer A549/ATCC -4.88 -4.55 -4.82 -4.52 -4.80 -4.48 -4.51 >-4.00 -4.85 -4.50 -5.45 -3.98EKVX -4.77 -4.47 -4.62 -4.23 -4.61 -4.21>-4.00 >-4.00 -4.71 -4.25 -5.72 -3.91HOP-62 -4.75 -4.47 -4.72 -4.45 -4.70 -4.42>-4.00 >-4.00 -4.74 -4.42 -6.13 -4.54HOP-92 -4.90 -4.56 -4.88 -4.55 -4.71 -4.41>-4.00 >-4.00 -4.78 -4.44 -5.39 -4.01NCI-H226 -4.76 -4.47 -4.74 -4.45 -4.76 -4.47>-4.00 >-4.00 -4.76 -4.45 -5.97 -4.64NCI-H23 -4.77 -4.48 -4.70 -4.44 -4.61 -4.27>-4.00 >-4.00 -4.82 -4.50 -5.13 -3.65NCI-H322M -4.77 -4.45 -4.75 -4.42 -4.65 -4.29>-4.00 >-4.00 -4.73 -4.37 -6.17 -4.70NCI-H460 -4.74 -4.36 -4.74 -4.42 -4.66 -4.23 -4.08 >-4.00 -4.81 -4.36 -6.04 -5.20 NCI-H522 -4.88 -4.55 -4.92 -4.59-5.62 -4.78 -4.11 >-4.00 -4.89 -4.51Colon cancer COLO 205 -4.69 -4.31 -4.76 -4.45 -4.74 -4.41 -4.03 >-4.00 -4.75 -4.39 -5.77 -5.05HCC-2998 -4.79 -4.50 -4.84 -4.54 -4.71 -4.45 -4.12 >-4.00 -4.78 -4.50 -6.00 -4.74HCT-116 -4.84 -4.47 -4.84 -4.51 -4.76 -4.45 -4.12 >-4.00 -4.82 -4.46 -6.27 -4.79HCT-15 -4.75 -4.41 -4.74 -4.43 -4.74 -4.41 -4.06 >-4.00 -4.71 -4.27 -5.96 -4.29HT29 -4.84 -4.43 -4.86 -4.53 -4.81 -4.41 -4.47 >-4.00 -4.88 -4.53 -5.94 -3.98KM12 -4.83 -4.46 -4.79 -4.46 -4.73 -4.40 -4.18 >-4.00 -4.81 -4.45 -5.76 -4.50SW-620 -4.62 -4.22 -4.59 -4.17 -4.70 -4.27>-4.00 >-4.00 -4.50 -4.08 -5.81 -3.86 CNS cancer SF-268 -4.77 -4.43 -4.81 -4.49 -4.73 -4.36 -4.19 >-4.00 -4.83 -4.45 -5.94 -3.80SF-295 -4.80 -4.50 -4.76 -4.48 -4.75 -4.45 -4.16 >-4.00 -4.85 -4.48 -5.99 -3.80SF-539 -4.71 -4.42 -4.68 -4.40 -4.65 -4.35>-4.00 >-4.00 -4.68 -4.34 -5.99 -4.16SNB-19 -4.72 -4.37 -4.73 -4.44 -4.57 -4.11>-4.00 >-4.00 -4.64 -4.20 -4.10 -3.61SNB-75 -4.71 -4.42 -4.77 -4.46 -4.67 -4.38 -4.22 >-4.00 -4.61 -4.29 -5.84 -4.38 ISSN 1424-6376 Page 103 ©ARKAT

Page  104 General Papers ARKIVOC 2006 (xii) 98-110 U251 -4.78 -4.49 -4.76 -4.47 -4.79 -4.48 -4.31 >-4.00 -4.80 -4.47 -5.31 -3.62 Melanoma LOX IMVI -4.72 -4.45 -4.70 -4.43 -4.72 -4.45>-4.00 >-4.00 -4.75 -4.46 -6.68 -5.04MALME-3M -4.76 -4.45 -4.74 -4.46 4.69 -4.42 -4.16 >-4.00 -4.77 -4.48 -5.87 -4.54M14 -4.71 -4.42 -4.73 -4.45 4.70 -4.41>-4.00 >-4.00 -4.74 -4.41 -6.23 -4.84SK-MEL-2 -4.68 -4.41 -5.04 -4.64 4.92 -4.58>-4.00 >-4.00 -4.75 -4.44 -6.03 -4.97SK-MEL-28 -4.70 -4.42 -4.71 -4.42 -4.71 -4.41>-4.00 >-4.00 -4.75 -4.44 -5.05 -3.81SK-MEL-5 -4.82 -4.50 -4.76 -4.46 -4.80 -4.47>-4.00 >-4.00 -4.74 -4.44 -5.45 -4.78UACC-257 -4.78 -4.50 -4.72 -4.46 -4.69 -4.42 -4.18 >-4.00 -4.72 -4.35 -5.70 -4.01UACC-62 -4.89 -4.57 -4.86 -4.53 -4.87 -4.55 -4.11 >-4.00 -4.93 -4.59 -6.30 -5.34 Ovarian cancer IGROV1 -7.31 -4.92 -4.23 >-4.00 -4.87 -4.44 -5.32 -3.84OVCAR-3 -4.86 -4.52 -4.86 -4.52 -4.84 -4.50 -4.01 >-4.00 -4.93 -4.56 -6.15 -5.17OVCAR-4 -4.78 -4.47 -4.73 -4.40 -4.80 -4.44 -4.30 >-4.00 -4.83 -4.46 -5.82 -4.00OVCAR-5 -4.71 -4.44 -4.74 -4.46 -4.69 -4.40>-4.00 >-4.00 -4.77 -4.46 -6.18 -4.32OVCAR-8 -4.76 -4.45 -4.81 -4.48 -4.68 -4.36 -4.46 >-4.00 -4.67 -4.27 -6.16 -4.22SK-OV-3 -4.67 -4.33 -4.67 -4.32 -4.42 >-4.00 >-4.00 >-4.00 -4.48 >-4.00 -6.26 -4.78 Renal cancer 786-O -4.76 -4.42 -4.71 -4.41 -4.65 -4.29 -4.23 >-4.00 -4.72 -4.28 -6.08 -3.97A498 -4.76 -4.46 -4.76 -4.44 -4.82 -4.49>-4.00 >-4.00 -4.76 -4.46 -5.17 -4.02ACHN -4.76 -4.48 -4.74 -4.44 -4.76 -4.44>-4.00 >-4.00 -4.70 -4.35 -5.71 -3.95CAKI-1 -4.79 -4.49 -4.81 -4.47 -4.77 -4.39 -4.02 >-4.00 -4.81 -4.42 -6.27 -4.80SN12C -4.75 -4.47 -4.78 -4.49 -4.80 -4.50 -4.63 >-4.00 -4.86 -4.52 -6.12 -5.15TK-10 -4.76 -4.47 -4.76 -4.46 -4.72 -4.45 -4.05 >-4.00 -4.89 -4.55 -5.90 -3.76UO-31 -4.81 -4.51-7.56 -4.98 -4.77 -4.49 >-4.00 >-4.00 -4.85 -4.53 -5.98 -4.10RXF 393 -5.23 -4.69-6.38 -4.68 -4.77 >-4.00 -4.85 -4.53 -5.83 -4.49 Prostate cancer PC-3 -4.90 -4.52 -4.85 -4.53 -4.81 -4.42 -4.26 >-4.00 -4.86 -4.34 -5.75 -3.76DU-145 -4.74 -4.36 -4.73 -4.37 -4.66 -4.29>-4.00 >-4.00 -4.66 -4.27 -6.10 -3.87 Breast cancer MCF7 -4.83 -4.41 -4.81 -4.41 -4.64 >-4.00 -4.49 >-4.00 -4.76 >-4.00 -6.15 -4.37MDA-MB 231/ATCC -4.77 -4.46 -4.76 -4.47 -4.76 -4.46>-4.00 >-4.00 -4.81 -4.50 -6.23 -4.44HS 578T -4.56 -4.02 -4.55 -4.17 -4.84 -4.36 -4.06 >-4.00 -4.38 >-4.00 -5.71 -3.78MDA-MB 435 -4.75 -4.46 -4.75 -4.46 -4.79 -4.48>-4.00 >-4.00 -4.78 -4.48 -5.13 -3.74BT-549 -4.70 -4.43 -4.71 -4.44 -4.73 -4.46 -4.12 >-4.00 -4.71 -4.44 -6.07 -4.49T-47D -4.79 -4.40 -4.69 -4.26 -4.61 -4.10>-4.00 >-4.00 -4.71 -4.07 -5.44 -3.79 MG MID -4.78 -4.44 -4.88 -4.47 -4.77 -4.40 -4.15 -4.00 -4.77 -4.38 -5.90 -4.33 Delta 0.25 0.13 2.67 0.51 1.60 0.65 0.62 0.06 0.32 0.21 Range 0.47 0.55 3.01 0.81 1.96 1.06 0.77 0.07 0.72 0.59 ISSN 1424-6376 Page 104 ©ARKAT

Page  105 General Papers ARKIVOC 2006 (xii) 98-110 In conclusion, these preliminary results are promising and some of these compounds especially 2b, 3a and 3c may be potential candidates for new antituberculosis and anticancer lead molecules. Experimental Section General Procedures. Cyclic ketones, mercaptoacetic acid and 2-mercaptopropionic acid were commercially available. 5-methyl-3-phenyl-1H-indole-2-carbohyrazide (1) was synthesized as depicted in Scheme 1 according to ref. 19. The reactions were monitored by TLC on silica gel HF254 coated plates (E.Merck, Darmstadt, Germany). Melting points were measured in open capillary tubes with a Buchi 530 melting point apparatus and are uncorrected. IR (KBr) spectra were recorded using a Perkin-Elmer 1600 FTIR spectrophotometer and all values are expressed as .max cm-1. 1H-NMR, HSQC, HMBC and 13C-NMR (APT) spectra were recorded on Bruker AC 200 MHz and VarianUNITYINOVA 500 MHz spectrometers using DMSO-d6. Mass spectra (LC/MS-APCI) were recorded on a Finnigan TM LCQ TM Mass Spectrometer in the positive ionization mode. Elemental analyses were performed on a Carlo Erba Model 1106 elemental analyzer. Synthesis of 5-methyl-N-(3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)-3-phenyl-1H-indole-2carboxamide derivatives (2 and 3). A mixture of 1 (0.005 mol), an appropriate cyclic ketone (0.005 mol) and mercaptoacetic acid or a-mercaptopropionic acid (0.02 mol) was refluxed in 20 ml dry benzene for 5-6 h using a Dean-Stark water separator. Excess benzene was evaporated in vacuo. The resulting residue was triturated with saturated NaHCO3 solution until CO2 evolution ceased and was allowed to stand overnight or in some cases refrigerated until solification. The solid thus obtained was washed with water, dried, and recrstallized from C2H5OH. 5-Methyl-N-(6-methyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)-3-phenyl-1H-indole-2carboxamide (2a). IR(KBr): . 3279 (NH/OH), 1704, 1667 (C=O); 1H-NMR (200 MHz): d 0.81 (s, 3H, 6-CH3 sp.1), 1.03-1.10 (m, 4H, sp.), 1.34-1.81 (m, 5H, sp.), 2.37 (s, 3H, 5-CH3 ind.2), 3.52 (s, 2H, C2-H2), 7.12 (d, J=8.35 Hz, 1H, C6-H ind.), 7.32-7.56 (m, 7H, C4,7-H ind. and Ar-H), 9.80 (s, 1H, CONH), 11.81 (s, 1H, NH ind.). 5-Methyl-N-(7-methyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)-3-phenyl-1H-indole-2carboxamide (2b). IR(KBr): . 3241 (NH/OH), 1711,1651 (C=O); 1H-NMR (500 MHz): d 0.89 (s, 3H, 7-CH3 sp.), 1.30-1.80 (m, 9H, sp.), 2.38 (s, 3H, 5-CH3 ind.), 3.58 (s, 2H, C2-H2), 7.12 (dd, J=8.3, 1.5 Hz, 1H, C6-H ind.), 7.32 (s, 1H, C4-H ind.), 7.36 (t, J=7.3 Hz, 1H, Ar-H), 7.41 (d, J=8.3 Hz, 1H, C7-H ind.), 7.46 (t, J=7.6 Hz, 2H, Ar-H), 7.53 (dd, J=8.3, 1.4 Hz, 2H, Ar-H), 10.02 (s, 1H, CONH), 11.92 (s, 1H, NH ind.). 5-Methyl-N-(3-oxo-8-propyl-1-thia-4-azaspiro[4.5]dec-4-yl)-3-phenyl-1H-indole-2carboxamide (2c). IR (KBr): . 3329, 3255 (NH/OH), 1692, 1674 (C=O); 1H-NMR (200 MHz): 1 sp: spirodecane 2 ind: indole ISSN 1424-6376 Page 105 ©ARKAT

Page  106 General Papers ARKIVOC 2006 (xii) 98-110 d 0.88 (t, J=7.03 Hz, 3H, 8-CH2CH2CH3 sp.), 1.14 (s, 4H, 8-CH2CH2CH3 sp.), 1.29 (s, 4H, sp.), 1.71 (br.s, 5H, sp.), 2.35 (s, 3H, 5-CH3 ind.), 3.56 (s, 2H, C2-H2), 7.11 (d, J=8.39 Hz, 1H, C6-H ind.), 7.31-7.54 (m, 7H, C4,7-H ind. and Ar-H), 9.94 (s, 1H, CONH), 11.75 (s, 1H, NH ind.). 13C- NMR (APT)(125 MHz): d 14.81 (8-CH2CH2CH3 sp.), 20.24 (8-CH2CH2CH3 sp.), 21.94 (5-CH3 ind.), 28.57 (C2 sp.), 30.02 (C7,9 sp.), 35.52 (C8 sp.), 39.04 (8-CH2CH2CH3 sp.), 37.12 (C6,10 sp.), 73.00 (C5 sp.), 112.85 (C7 ind.), 119.07 (C3 ind.), 119.92 (C4 ind.), 126.80 (C2 ind.), 127.21 (C6 ind.), 127.45 (C3a ind.), 128.40 (C4 3-C6H5 ind.), 128.89 (C3,5 3-C6H5 ind.), 130.90 (C2,6 3-C6H5 ind.), 134.60 (C1 3-C6H5 ind.), 129.77 (C5 ind.), 134.88 (C7a ind.), 162.62 (amide C=O), 168.05 (lactam C=O). 5-Methyl-N-(3-oxo-8-phenyl-1-thia-4-azaspiro[4.5]dec-4-yl)-3-phenyl-1H-indole-2carboxamide (2d). IR(KBr): . 3284 (NH/OH), 1709, 1659 (C=O); 1H-NMR (200 MHz): d 1.67 2.10 (m, 9H, sp.), 2.38 (s, 3H, 5-CH3 ind.), 3.63 (br.s, 2H, C2-H2), 7.11-7.60 (m, 13H, Ar-H), 10.11 (s, 1H, CONH), 11.89 (s, 1H, NH ind.); MS (APCI): m/z 496 (M+H)+ (41%), 498 (6%), 497 (20%), 422 (100%), 234 (96%) . 5-Methyl-N-(2,6-dimethyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)-3-phenyl-1H-indole-2carboxamide (3a). IR(KBr): . 3370,3288 (NH/OH), 1702, 1678 (C=O); 1H-NMR (200 MHz): d 0.79 (br.s, 3H, 6-CH3 sp.), 1.09-1.13 (m, 4H, sp.), 1.39 (d, J=6.89 Hz, 3H, 2-CH3 sp.), 1.51-1.64 (m, 5H, sp.), 2.37 (s, 3H, 5-CH3 ind.), 3.79-3.87 (2q, J=6.97 Hz, 1H, C2-H), 7.12 (d, J=8.45 Hz, 1H, C6-H ind.), 7.31-7.55 (m, 7H, C4,7-H ind. and Ar-H), 9.82 (s, 1H, CONH), 11.84 (s, 1H, NH ind.). 5-Methyl-N-(2,7-dimethyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)-3-phenyl-1H-indole-2carboxamide (3b). IR(KBr): . 3250 (NH/OH), 1693, 1665 (C=O); 1H-NMR (500 MHz): d 0.89 (d, J=5.9 Hz, 3H, 7-CH3 sp.), 1.30-1.80 (m, 12H, sp. and 2-CH3 sp.), 2.38 (s, 3H, 5-CH3 ind.), 3.87 (br.s, 1H, C2-H), 7.13 (dd, J=8.5, 1.4 Hz, 1H, C6-H ind.), 7.32 (s, 1H, C4-H ind.), 7.36 (t, J=7.3 Hz, 1H, Ar-H), 7.41 (d, J=8.3 Hz, 1H, C7-H ind.), 7.46 (t, J=7.6 Hz, 2H, Ar-H), 7.53-7.54 (m, 2H, Ar-H), 9.91 (s, 1H, CONH), 11.75 (s, 1H, NH ind.). 5-Methyl-N-(2,8-dimethyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)-3-phenyl-1H-indole-2carboxamide (3c). IR(KBr): . 3290 (NH/OH), 1712, 1654 (C=O); 1H-NMR (500 MHz): d 0.97 (d, J=6.83 Hz, 3H, 8-CH3 sp.), 1.05-1.20 (m, 2H, C7/9-H sp.), 1.31-1.35 (m, 1H, C8-H sp.), 1.67 (d, 2H, J=13.18 Hz, C7/9-H sp.), 1.41 (d, J=6.83 Hz, 3H, 2-CH3 sp.), 1.73-1.85 (m, 2H, C6/10-H sp.), 1.87-1.97 (m, 2H, C6/10-H sp.), 2.37 (s, 3H, 5-CH3 ind.), 3.85-3.88 (m, 1H, C2-H), 7.12 (d, J=8.54 Hz, 1H, C6-H ind.), 7.33-7.36 (m, 2H, C4-H ind. and C4-H 3-C6H5 ind.), 7.41 (m, 1H, C7- H ind.), 7.45 (t, J=7.56 Hz, 2H, C3,5-H 3-C6H5 ind.), 7.54 (d, J=7.56 Hz, 2H, C2,6-H 3-C6H5 ind.), 9.89 (s, 1H, CONH), 11.73 (s, 1H, NH ind.). 13C-NMR (125 MHz): d 20.49 (2-CH3 sp.), 21.93 (5-CH3 ind.), 22.60 (8-CH3 sp.), 31.07 (C8 sp.), 31.97, 32.39 (C7/9 sp.), 37.28, 38.15 (C6/10 sp.), 37.45 (C2 sp.), 71.40 (C5 sp.), 112.81 (C7 ind.), 119.12 (C3 ind.), 120.00 (C4 ind.), 126.65 (C6 ind.), 126.80 (C3a ind.), 127.27 (C4 3-C6H5 ind.), 127.47 (C2 ind.), 128.94 (C3,5 3-C6H5 ind.), 129.82 (C5 ind.), 130.91 (C2,6 3-C6H5 ind.), 134.49 (C1 3-C6H5 ind.), 134.55 (C7a ind.), 162.57 (amide C=O), 170.71 (lactam C=O); MS (APCI): m/z 448 (M+H)+ (20%), 449 (6%), 360 (72%), 234 (100%). ISSN 1424-6376 Page 106 ©ARKAT

Page  107 General Papers ARKIVOC 2006 (xii) 98-110 5-Methyl-N-(2-methyl-3-oxo-8-propyl-1-thia-4-azaspiro[4.5]dec-4-yl)-3-phenyl-1H-indole2- carboxamide (3d). IR(KBr): . 3291 (NH/OH), 1694, 1658 (C=O); 1H-NMR (200 MHz): d 0.75 (br.s, 3H, 8-CH2CH2CH3 sp.), 1.02-1.28 (m, 8H, sp. and 8-CH2CH2CH3 sp.), 1.59 (br.s, 3H, 2-CH3 sp.), 2.23 (br.s, 5H, sp.), 2.36 (s, 3H, 5-CH3 ind.), 3.74 (br.s, 1H, C2-H), 6.99-7.38 (m, 8H, Ar-H), 10.09 (s, 1H, CONH), 11.88 (s, 1H, NH ind.). 5-Methyl-N-(2-methyl-3-oxo-8-phenyl-1-thia-4-azaspiro[4.5]dec-4-yl)-3-phenyl-1H-indole2- carboxamide (3e). IR(KBr): . 3262 (NH/OH), 1713, 1672 (C=O); 1H-NMR (200 MHz): d 1.42 (d, J=6.89 Hz, 3H, 2-CH3 sp.), 1.50-2.10 (m, 9H, sp.), 2.36 (s, 3H, 5-CH3 ind.), 3.92 (q, J=6.89 Hz, 1H, C2-H), 7.09-7.53 (m, 13H, Ar-H), 10.45 (s, 1H, CONH), 12.22 (s, 1H, NH ind.). In vitro evaluation of antituberculosis activity [microplate alamar blue assay (MABA)]. Antimicrobial susceptibility testing was performed in black, clear-bottomed, 96-well microplates (black view plates; Packard Instrument, Meriden, Conn.) in order to minimize background fluorescence. Outer perimeter wells were filled with sterile water to prevent dehydration in experimental wells. Initial drug dilutions were prepared in either dimethyl sulfoxide or distilled deionized water, and subsequent twofold dilutions were performed in 0.1 ml of 7H9GC (no Tween 80) in the microplates. BACTEC 12B-passaged inocula were initially diluted 1:2 in 7H9GC, and 0.1 ml was added to wells. Subsequent determination of bacterial titer yielded 1x106 CFU/ml in plate wells for H37Rv. Frozen inocula were initially diluted 1:20 in BACTEC 12B medium followed by a 1:50 dilution in 7H9GC. Addition of 1/10 ml to wells resulted in a final bacterial titer of 2.0x105 CFU/ml for H37Rv. Wells containing drug only were used to detect autofluorescence of compounds. Addition control wells consisted of bacteria only (B) and medium only (M). Plates were incubated at 37 °C. Starting at day 4 of incubation, 20 µl of 10x Alamar Blue solution (Alamar Biosciences/Accumed, Westlake, OH) and 12.5 µl of 20% Tween 80 were added to one B well and one M well, and plates were reincubated at 37 °C. Wells were observed at 12 and 24 h for a color change from blue to pink and for a reading of =50,000 fluorescence units (FU). Fluorescence was measured in a Cytofluor II microplate fluorometer (PerSeptive Biosystems, Framingham, MA.) in bottom-reading mode with excitation at 530 nm and emission at 590 nm. If the B wells became pink by 24 h, reagent was added to the entire plate. If the well remained blue or =50,000 FU was measured, additional M and B wells were tested daily until a color change occurred, at which time reagents were added to all remaining wells. Plates were then incubated at 37 °C, and results were recorded at 24 h post-reagent addition. Visual MICs were defined as the lowest concentration of drug that had prevented a color change. For fluorometric MICs, a background subtraction was performed on all wells with a mean of triplicate M wells. Percent inhibition was defined as 1-(test well FU/mean FU of triplicate B wells)x100. The lowest drug concentration effecting an inhibition of =90% was considered as the MIC. Methodology of the in vitro cancer screen. The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM Lglutamine. For a typical screening experiment, cells were inoculated into 96 well microtiter plates in 100 µL at plating densities ranging from 5,000 to 40,000 cells/well depending on the ISSN 1424-6376 Page 107 ©ARKAT

Page  108 General Papers ARKIVOC 2006 (xii) 98-110 doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37° C, 5 % CO2, 95 % air and 100 % relative humidity for 24 h prior to addition of experimental drugs. After 24 h, two plates of each cell line were fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental drugs were solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 µg/ml gentamicin. Additional four, 10-fold or ½ log serial dilutions were made to provide a total of five drug concentrations plus control. Aliquots of 100 µl of these different drug dilutions were added to the appropriate microtiter wells already containing 100 µl of medium, resulting in the required final drug concentrations. Following drug addition, the plates were incubated for an additional 48 h at 37°C, 5 % CO2, 95 % air, and 100 % relative humidity. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 µl of cold 50 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded, and the plates were washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 µl) at 0.4 % (w/v) in 1 % acetic acid was added to each well, and plates were incubated for 10 minutes at room temperature. After staining, unbound dye was removed by washing five times with 1 % acetic acid and the plates were air dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology was the same except that the assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 µl of 80 % TCA (final concentration, 16 % TCA). Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth was calculated at each of the drug concentrations levels. Percentage growth inhibition was calculated as: [(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz [(Ti-Tz)/Tz] x 100 for concentrations for which Ti<Tz. Three dose response parameters were calculated for each experimental agent. Growth inhibition of 50 % (GI50) was calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which was the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment was calculated from [(Ti- Tz)/Tz] x 100 = -50. Values were calculated for each of these three parameters if the level of activity was reached; however, if the effect was not reached or was exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested. ISSN 1424-6376 Page 108 ©ARKAT

Page  109 General Papers ARKIVOC 2006 (xii) 98-110 Acknowledgements Antimycobacterial activity was determined by the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) through a research and development contract with the U.S. National Institute of Allergy and Infectious Diseases. Thanks are addressed to Dr.Joseph A. Maddry (TAACF) and his team for their collaboration. We thank the Division of Cancer Research, National Cancer Institute, Bethesda, Maryland, for the anticancer activity screening. This work was supported in part by The Research Fund of Istanbul University (Project Number 1267/050599). References 1. Savini, L.; Chiasserini, L.; Gaeta, A.; Pellerano, C. Bioorg.Med.Chem. 2002, 10, 2193. 2. Mahboobi, S.; Eichhorn, E.; Popp, A.; Sellmer, A.; Elz, S.; Möllmann, U. Eur. J. Med. Chem. 2006, 41, 176. 3. Hoemann, M. Z.; Kumaravel, G.; Xie, R. L.; Rossi, R. F.; Meyer, S.; Sidhu, A.; Cuny, G. D.; Hauske, J. R. Bioorg. Med. Chem. Lett. 2000, 10, 2675. 4. Kutschy, P.; Such., M.; Andreani, A.; Dzurilla, M.; Kovácik, V.; Alföldi, J.; Rossi, M.; Gramatová, M. Tetrahedron 2002, 58, 9029. 5. Levy, L. M.; Cabrera, G. M.; Wright, J. E.; Seldes, A. M.; Phytochemistry 2000, 54, 941. 6. Sugiyama, H.; Yokokawa, F.; Aoyama, T.; Shioiri, T. Tetrahedron Lett. 2001, 42, 7277. 7. Font, M.; Monge, A.; Cuartero, A.; Elorriaga, A.; Martínez-Irujo, J. J.; Alberdi, E.; Santiago, E.; Prieto, I.; Lasarte, J. J.; Sarobe, P.; Borrás, F. Eur. J. Med. Chem. 1995, 30, 963. 8. Adreani, A.; Granaiola, M.; Leoni, A.; Locatelli, A.; Morigi, R.; Rambaldi, M.; Garaliene, V.; Farruggia, G.; Masotti, L. Bioorg. Med. Chem. 2004, 12, 1121. 9. Zhang, H.-Z.; Drewe, J.; Tseng, B.; Kasibhatla, S.; Cai, S. X. Bioorg. Med. Chem. 2004, 12, 3649. 10. Andres, C. J.; Bronson, J. J.; D’Andrea, S. V.; Deshpande, M. S.; Falk, P. J.; Grant-Young, K. A.; Harte, W. E.; Ho, H. T.; Misco, P. F.; Robertson, J. G.; Stock, D.; Sun, Y.; Walsh, A.W. Bioorg. Med. Chem.Lett. 2000, 10, 715. 11. Bonde, C. G.; Gaikwad, N. J. Bioorg. Med. Chem. 2004, 12, 2151. 12. Çapan, G.; Ulusoy, N.; Ergenç, N.; Kiraz, M. Monatsh. Chem. 1999, 130, 1399. 13. Ulusoy, N. Arzneim-Forsch /Drug Res. 2002, 52(7), 565. 14. Babaoglu, K.; Page, M. A.; Jones, V. C.; McNeil, M. R.; Dong, C.; Naismith, J. H.; Lee, R. E. Bioorg. Med. Chem.Lett. 2003, 13, 3227. 15. Srivastava, T.; Gaikwad, A. K.; Haq, W.; Sinha, S.; Kati, S. B. Arkivoc 2005, ii, 120. 16. Sharma, R.; Nagda, D. P.; Talesara, G. L. Arkivoc 2006, i, 1. ISSN 1424-6376 Page 109 ©ARKAT

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