Efficient synthesis of 3,3-diheteroaromatic oxindole analogues and their in vitro evaluation for spermicidal potential

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Efficient synthesis of 3,3-diheteroaromatic oxindole analogues and their in vitro evaluation for spermicidal potential
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  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyright  Author's personal copy Efficient synthesis of 3,3-diheteroaromatic oxindole analogues and their in vitro evaluation for spermicidal potential Priyankar Paira, Abhijit Hazra, Shrabanti Kumar, Rupankar Paira, Krishnendu B. Sahu, Subhendu Naskar,Pritam saha, Shyamal Mondal, Arindam Maity, Sukdeb Banerjee, Nirup B. Mondal * Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032, India a r t i c l e i n f o  Article history: Received 26 March 2009Revised 11 June 2009Accepted 12 June 2009Available online 17 June 2009 Keywords: Isatin3,3-diindolyl and dipyrrolyl oxindolesIodineSpermicidal potentialTransmission electron microscopy a b s t r a c t Syntheses of 3,3-diheteroaromatic oxindole derivatives has been achieved by coupling indole-2,3-dione(isatin) with differently substituted indoles and pyrrole in presence of I 2  in  i -PrOH. The in vitro spermi-cidal potentials and the mode of spermicidal action of the synthesized analogues were evaluated and thederivative, 3,3-bis (5-methoxy-1 H  -indol-3-yl) indolin-2-one ( 3d ) exhibited most significant activity.   2009 Elsevier Ltd. All rights reserved. The vaginal contraceptive products available for many years aremostly based on Nonoxynol-9 (N-9), 1 which is a mixture of oligo-mers. 2 Asthehealthcareindustrymovestowardsusingeitherpurecompounds or mixtures whose individual components meet safetystandards, 3 the use of N-9 may or may not meet future safety reg-ulations. Several European nations have banned or restricted theuse of N-9 and related compounds on the basis of health risksand potential environmental toxicity. 4 These limitations of usingN-9 in protecting sexually transmitted diseases (STDs) 5 haveencouraged researchers for the development of better alternativesof N-9 that would have dual function of contraception and STDprotection for women.Oxindoles are known to possess antibacterial, antiprotozoal,and anti-inflammatory activities and are also patented as PR (pro-gesterone receptors) agonists. 6 The naturally occurring oxindolederivative convolutamydine has been found to exhibit potentactivityinthedifferentiationofHL-60humanplomyelocyticleuke-mic cells. 7 The varied biological activities of oxindole derivativeshave attracted the synthetic chemists to a number of syntheticstrategies. 8 Most of the strategies are based on acid catalyzed con-densation of arenes with isatins and though claimed to be efficient,have some shortcomings regarding cost, reaction time, operationalparameters,% of yield, and selectivity. 9 Therefore,the developmentof methodology using newer reagents with greater efficiency, sim-pler operational procedure, milder reaction condition, higher% of yield of products coupled with potential bioactivity is important.We have already reported the anti-spermatogenic potential of anovel indole derivative. 10 This finding intrigued us to go in pursuitofneweroxindolederivativesfortheexplorationoftheirimpactonsperm morphology.Recently, molecular iodine has received considerable attentionas an inexpensive, nontoxic, and readily available catalyst for var-iousorganictransformations,affordingthecorrespondingproductswith high selectivity and in excellent yields. 11 It is well docu-mented that the mild Lewis acidity associated with iodineenhances its usage in organic synthesis to perform several organictransformations using stoichiometric levels to catalytic amounts.In continuation of our interest on the catalytic applications of molecular iodine, 12 we herein report the first metallic catalyst freedirect synthesis of 3,3-diindolyl and 3,3-dipyrrolyl oxindoles bythe condensation of isatin with substituted indoles or pyrrole inpresence of iodine under neutral conditions.At the outset, we reacted isatin ( 1 ) with indole ( 2 ) in the pres-ence of molecular iodine in isopropanol for 5 min, which yieldedthe di-indolyl oxindole  3a  in 98% yield (Scheme 1, Table 1, entry 1). Only 5 mol % of iodine is sufficient to catalyze the reaction;no significant change in the yield of the products was observedusing higher mol % of iodine. But in absence of iodine the reactiondid not yield any product even after 4 h.Encouraged by this result, we turned our attention towards arange of indole derivatives. Interestingly, 1- or 2-substitutedindoles underwent smooth coupling with isatin to give the corre-sponding bisindolyl oxindoles in high yields (Table 1, entry 3, 5, 0960-894X/$ - see front matter    2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.bmcl.2009.06.049 *  Corresponding author. Tel.: +91 33 2473 3491; fax: +91 33 2473 5197. E-mail address:  nirup@iicb.res.in (N.B. Mondal).Bioorganic & Medicinal Chemistry Letters 19 (2009) 4786–4789 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl  Author's personal copy and 6). The characterizations of the products were accomplishedby spectroscopic analysis (NMR and Mass) and also by comparisonwith the data reported in the literature. 9 In case of 5-methoxyindole ( 1d ), the reaction occurred at slower rate (Table 1, entry4). However, an increment in catalyst loading from 5 mol % to10 mol % reduced the reaction time. Reactions with 3-substitutedindoles, viz. 3-methyl indole ( 1g  ) and indole-3-acetic acid ( 1h )took (Scheme 2) longer time (1–2 h) for completion with respectto the earlier ones (Table 1, entry 7 and 8). The spectral analysisof the compounds revealed the formation of 3,3-di-indolyl-2-oxin-doles.It is noteworthythatnoreports onthereaction of isatin with3-substituted indoles have so far appeared in the literature, theonly exception being the condensation of 3-hydroxy-3-indolylin-dolin-2-ones with skatole. 9b Furthermore, pyrrole ( 4 ) also reactedefficiently with isatin under similar condition, to afford 3,3-dipyrr-olyl-2-oxindole  5  (Scheme 3, Table 1, entry 9). A series of in vitro experiments were performed to determinewhether the synthesized analogues ( 3a – h  and  5 ) had any effectonspermmotility.Theresultsof theSander–Cramer’stestrevealedthat the oxindole analogues exerted dose-dependent sperm immo-bilizing effects. The minimum effective concentrations (MECs)were calculated for the analogues that caused 100% sperm immo-bilization within 20 s with no subsequent revival of motility inBaker’s buffer after 60 min incubation at 37   C. The data summa-rized in Table 1 reveal that compound  3d  showed maximum effi-cacy with MEC 0.34 mg/ml, which is much lower than that of thestandard spermicide N-9. 13 The RBC hemolysis assay, known tobe advantageous in the screening of new topical preparations for R 3 NR 1 R 2 NHOOI 2 , (CH 3 ) 2 CHOHNHONR 1 R 3 R 1 NR 2 R 2 R 3 rt+ 1(a-f)23(a-f)a ; R 1  = R 2  = R 3  = H d ; R 1 = R 2  = H, R 3  = OMe b : R 1  = R 2 = H, R 3 = Br e ; R 1  = Me, R 2  = R 3 = H c ; R 1  = R 3  = H, R 2 = Me f  ; R 1  = R 3  = H, R 2  = COOH Scheme 1.  The reactions of 3-unsbstituted indoles and isatin leading to 3,3-di(3-indolyl)-2-oxindoles.  Table 1 I 2  catalyzed synthesis of oxindole derivatives  3a – h ,  5  from isatin ( 2 ), and minimumeffective concentration (MEC) of the products for immobilization of rat spermatozoa Entry Substrate Product a Time (min) Yield (%) MEC b (mg/ml)1.  1a 3a 9a 15 98 2.40 ± 0.0582.  1b 3b 9c 15 95 3.18 ± 0.0413.  1c 3c 9a 30 92 2.82 ± .0.0574.  1d 3d 9c 180 80 0.34 ± 0.0185.  1e 3e 9b 30 90 2.36 ± 0.0456.  1f 3f   45 90 1.11 ± 0.0617.  1g 3g   60 85 1.00 ± 0.0588.  1h 3h  120 85 0.53 ± 0.0339.  4 5 9c 15 90 3.15 ± 0.087 a All the reaction was performed in isopropanol in presence of 5 mol % iodine. b The standard sample N-9 has been reported 14 to have the MEC of    0.500. NHNHOOI 2 , (CH 3 ) 2 CHOHrt+ 1(g-h)2 R g ; R = CH 3 h  ; R = CH 2 COOH 3(g-h) NHOHNRNHR Scheme 2.  The reactions of 3-substituted indoles and isatin leading to 3,3-di(2-indolyl)-2-oxindoles. NHNHOOI 2 , (CH 3 ) 2 CHOHrt+NHONHNH 425 Scheme 3.  The reaction of pyrrole and isatin leading to 3,3-di(2-pyrrolyl)-2-oxindole. P. Paira et al./Bioorg. Med. Chem. Lett. 19 (2009) 4786–4789  4787  Author's personal copy their local irritation potential, 14 was carried out on  3d  using rabbiterythrocytes. The hemolytic index (concentration required for 50%hemolysis of red blood cells) of the compound was 3.3 ± 0.24 mg/ml, which is much higher (  10-fold) than its spermicidal MEC.The large window between the spermicidal MEC and hemolyticindex indicates that the compound has less possibility of generalmembrane disruption if used as a spermicide (see Fig. 1).In the process of sperm migration and fertilization the plasmamembrane plays a pivotal role and a number of spermicidal agentsare known to exert their effect by structural and functional modu-lation of the plasma membrane. 15 Thus we examined whether thespermicidal effect of   3d  was due to some adverse modulation of sperm membrane integrity. Two most commonly employed tech-niques to assess sperm membrane integrity are hypo-osmoticswelling test and fluorescent staining with SYBR14 and PI. Thehypo-osmotic swelling test is generally employed to check thefunctional integrity of the sperm membrane. When exposed tohypo-osmotic environment, an intact membrane permits free pas-sage of the fluid to reach the osmotic equilibrium. As a result thesperm volume increases and the plasma membrane bulges. More-over, The plasma membrane around the sperm tail fiber is moreloosely attached than that around the other parts. As a result thesperm tail is particularly susceptible and responds by coiling. Thisfeature of functionally active sperm membrane was observed inmore than 90% of the control sperm population while almost noneof the  3d  exposed sperm (at MEC) showed such functional pertur-bation in hypo-osmotic condition indicating that the functionalintegrity of the sperm membrane was lost following exposure tothe oxindole  3d . This observation was further supported by the dif-ferential staining of normal and  3d  exposed sperm with SYBR14/PI.The functionally intact membrane in the live sperm offers selectivepermeability and retards the entry of large sized dyes like propidi-um iodide. Thus when control and  3d  treated spermatozoa weresubjected to this stain combination and excited at 488 nm, morethan 95% of the control spermatozoa took only SYBR 14 andappeared green while sperm nuclei treated with  3d  at MEC exhib-ited red fluorescence (Fig. 2) indicating sperm death. TransmissionElectron Microscopic observation on  3d  treated sperm populationat MEC revealed that the spermicidal action was due not only toloss of functional integrity but also to the adverse effect of   3d  onsperm membrane architecture. The electron micrographs of con-trol spermatozoa showed a smooth oval shaped head with a welldefined acrosome covering about 70% of the head while in caseof the treated sperm there is a dissolution of acrosomal cap whichmade the sperm nuclei almost uncovered (Fig. 3).The structural features of the tested compounds revealed thatthe presence of functional groups in the analogues ( O –CH 3  in  3d and –CH 2 –COOH in  3h ) might have played a vital role regardingtheirefficacy,althoughat thisstagetheroleofthefunctionalgroupis obscure. Detailed study regarding the mechanism of action is inprogress.In summary, we have disclosed an iodine-catalyzed reaction of isatin and different derivatives of indole and pyrrole to give thecorresponding 3,3-disubstituted oxindole derivatives in highyields. To the best of our knowledge this is the first report of iodine-catalyzed coupling reaction of isatin with indoles andpyrrole. The spermicidal potential of 3,3-bis (5-methoxy-1 H  -in-dol-3-yl) indolin-2-one ( 3d ) and 3,3-bis (3-carboxymethyl-1H-in-dol-2-yl) indolin-2-one ( 3h ) are higher than or comparable tothat of the standard spermicide N-9, signifying that both thecompounds have a possibility to be used as a non-detergent typespermicidal agent as an alternative to N-9.  Acknowledgements The authors express their gratitude to the CSIR and DBT forproviding the fund as well as fellowship to P. Paira, A. Hazra, S.Kumar, R. Paira, K. B. Sahu, S. Naskar, P. Saha, S. Mondal, A. Maity,and to Dr. N. P. Sahu, Emeritus Scientist, CSIR, for his valuablesuggestions. 0 100 200 300 400 500 6000255075100 concentration (   g/ml)    %   o   f   H   O   S  +  v  e  c  e   l   l  s Figure 1.  Dose dependent HOS reactivity of spermatozoa exposed to differentconcentrationsofcompound 3d .ThenumberofHOSpositive cellsexhibitingtypicaltail coiling was counted under a phase contrast microscope (40  ). Each pointrepresents mean ± SEM of at least six observations. Figure 3.  Transmission electron microscopic observation of sperm sample treatedwith compound  3d  at MEC. (A) Control spermatozoa with intact acrosome coveringthe sperm head; (B)  3d  treated spermatozoa with disintegrated sperm acrosomalcap indicating membrane damage; (C) (9+1) axoneme doublet of control spermshowed intactness of the sperm membrane in tail portion; (D) (9+1) axonemestructure of the  3d  treated sperm showed loss of membrane coverage. Figure 2.  Sperm viability assessment by SYBR-14/PI staining. (A) Control spermappeared green due to uptake of SYBR14 only; (B) compound  3d  treated spermappeared red due to uptake of PI when observed under a fluorescence microscope.4788  P. Paira et al./Bioorg. Med. Chem. Lett. 19 (2009) 4786–4789  Author's personal copy Supplementary data Supplementary data associated with this articlecan be found, inthe online version, at doi:10.1016/j.bmcl.2009.06.049. 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