Solid-Phase Synthesis and Combinatorial Technologies

OMe

SKIP = no monomer

Figure 7.45 Monomer sets used for the synthesis of the natural products-biased libraryL11.

Finally, the large encoded library L12 was prepared using three spacers on the resin (Gly, aminocaproic acid and a skip spacer), (+) and (–) 7.71 and the three nitrones 7.72a–c, to give 18 iodobenzyl tetracyclic scaffolds [7.74a–f, 7.75a–f, 7.84a–f from

(+) 7.71 (a–c) and from (–) 7.71 (d–f)] (Fig. 7.46). These scaffolds, using mix-and- split synthesis, were coupled to the previously rehearsed monomers: 31 alkynes (30 + the skip codon, 558 compounds, 7.76, 7.77, 7.85), 63 primary amines (62 + skip, 34,596 compounds, 7.78, 7.79, 7.86, + 558 from the previous skip lactone), and 63 carboxylic acids (62 + skip, 2,179,548 compounds, L12, Fig. 7.46). The binary encoding technique (189) required two tags for the spacers, for the epoxycyclohexenol isomers, and for the nitrones; five tags for the alkyne couplings; and six tags each for the amine and the carboxylic acid couplings; the reported encoding procedure (189) was significantly optimized to afford cleaner and more reliable decoding results. A total of 23 tags, inserted after each monomer coupling onto SP, was sufficient to encode the 3 × 2 × 3 × 31 = 558 butyrolactones and the 3 × 2 × 3 × 31 × 62 × 63 = 2,179,548 final library individuals, producing an ≈2,180,000-member library. Details of the analytical characterization of single beads were not given, but the decoding of single beads from each library pool produced satisfactory results (vide infra).

The extreme flexibility of these scaffolds would definitely allow the preparation of other large, primary libraries using chemistry-friendly synthetic schemes. The epoxide could be touched, as could the N–O function, which could be reductively cleaved and eventually used to obtain two new handles on constrained, stereo-determined novel scaffolds. The potential of such an integrated approach, where all the steps toward a large bead-based library are carefully assessed, is clearly enormous, especially if any library prepared would contain embedded biological information (starting from natu-

326 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE POOL LIBRARIES

S

O

O

O

L12

2,179,548-member library

63 pools, 34,596 compounds/pool

Figure 7.46 SP synthesis of the large, natural products–biased library L12.

ral and/or active scaffolds) as in the example (240). Possibly the library would be prepared in a >50 library equivalents-quantity to be tested on more than one specific assay. The efforts required to prepare a >2,000,000 library should be considered acceptable especially considering the developed miniaturized HTS formats (see next section); such chemistry, which should also be feasible using alternative chemical starting points and/or reaction schemes, should allow the generation of invaluable proprietary chemical diversity.

7.5 NEW TRENDS IN SOLID-PHASE POOL LIBRARIES 327

7.5.2 Bead-Based Libraries: Miniaturized High Throughput Screening

The library described in the previous section was tested on a miniaturized nanodroplet assay to spot ligand-protein interactions (242), and the authors decided to use 2.5 grams of photolinker-resin construct (around 2.7 million beads/gram, around 6,600,000 beads) to ensure the successful preparation of three library equivalents, corresponding to a >95% probability of representation for each library individual. The compounds were partitioned into polydimethylsiloxane plates (6500 assays in a 10 cm-plastic dish) by using a wetting- (pipetting a beads-cells-agar suspension into the wells) dewetting technique (pipetting off the excess liquid and leaving in the well uniform nanodroplets of 50–150 nL due to surface tension). Using a 10 mg suspension of beads in 0.006% aqueous agar resulted in a significant amount of wells (>60%) containing one to three beads in a nanodroplet, thus allowing the testing, and then the decoding, of such beads in an automated manner. As soon as the nanodroplets containing the beads, the cells and the media were deposited, the photolinker was UV-cleaved at 365 nM, releasing the library individuals from the beads. Active beads showed an effect on cells and were finally decoded (189). After the whole screening, a family of compounds from the library (7.87, Fig. 7.47), was identified as active for the specific biological target.

MacBeath et al. recently reported a miniaturized screening format, inspired by the DNA microarray technique and called Small Molecule Printing (SMP), to maximize the synthetic efforts to produce a bead-based large library (244). In a validation experiment, SP beads were delivered to polypropylene plates (one per well, using bead pickers) and cleaved in a small volume of solvent to afford a concentrated solution (high M). The authors used a high precision robotic instrument (252) to deliver 1 nL-aliquots from each well to the surface of many chemically derivatized microscope slides. The grafted functionality on the slides reacted with the library individuals, immobilizing each of them on its surface at a very high density (>1000 spots per cm2); each slide carried thousands of compounds, representing a subset of the bead-based library, and could be tested with an on-slide screen format using soluble targets (a

OMe

MeO

R1

NH

O N

O

O

O

O

O

7.87

Figure 7.47 Structure of a family of protein ligands (7.87) from the miniaturized screening of the large, natural products-biased library L12.

328 SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE POOL LIBRARIES

fluorescent-tagged isolated receptor in the example). A throughput of 150 printed slides per print run of the robot could easily be obtained; up to 3,000 slide spots (thus up to 3,000 screens) could derive from the releasate of a single 425 m diameter PS bead. The slides were then processed through a slide scanner to detect the positives (a UV scanner in ref. 244). The constraints imposed by hooking appropriate chemical groups of the library individuals on the slides (see Section 7.2.3) are more than compensated by the extremely high throughput of the miniaturized assay which requires extremely low quantities of chemicals and biological reagents often expensive or difficult to obtain. HTS campaigns for all the orphan receptors and for many recombinant proteins deriving from the expression of genetic libraries may assess their relevance via the discovery of chemicals interacting with them (high throughput chemical genetics, see also Section 9.1.4); this may become at least in part possible by using such miniaturized approaches, and may unravel novel relevant mechanisms to cure various important diseases.

Other interesting reports related to the synthesis and screening of large bead-based SP pool libraries can be found in the literature (204, 253, 254), reinforcing the concepts that were presented for the specific example reported here. In a nutshell, the saga of large, SP pool libraries of small organic molecules should gain new strength in the future, rather than being completely overpowered by other library formats and synthesis techniques.

REFERENCES

1.Furka, A., Sebestyen, F., Asgedom, M. and Dibo, G.,Int. J. Pept. Protein Res. 37, 487–493 (1991).

2.Lam, K. S., Salmon, S. E., Hersh, E. M., Hruby, V. J., Kazmierski, W. M. and Knapp, R. J., Nature 354, 82–84 (1991).

3.Houghten, R., Pinilla, C., Blondelle, S. E., Appel, J. R., Dooley, C. T. and Cuervo, J. H., Nature 354, 84–86 (1991).

4.Baldwin, J. J., Burbaum, J. J., Henderson, I. and Ohlmeyer, M. H. J., J. Am. Chem. Soc. 117, 5588–5589 (1995).

5.Dankwardt, S. M., Newman, S. R. and Krstenansky, J. L., Tetrahedron Lett. 36, 4923– 4926 (1995).

6.Burbaum, J. J., Ohlmeyer, M. H. J., Reader, J. C., Henderson, I., Dillard, L. W., Li, G., Randle, T. L., Sigal, N. H., Chelsky, D. and Baldwin, J. J.,Proc. Natl. Acad. Sci. USA 92, 6027–6031 (1995).

7.Storer, R., Drug Discovery Today 1, 248–254 (1996).

8.Gravert, D. J. and Janda, K. D., Curr. Opin. Chem. Biol. 1, 107–113 (1997).

9.Bailey, N., Cooper, A. W. J., Deal, M. J., Dean, A. W., Gore, A. L., Hawes, M. C., Judd, D. B., Merritt, A. T., Storer, R., Travers, S. and Watson, S. P.,Chimia 51, 832–837 (1997).

10.Carell, T., Wintner, E. A. and Rebek, J. Jr., Angew. Chem. Int. Ed. Engl. 33, 2061–2064 (1994).

11.An, H., Haly, B. D. and Cook, P. D., J. Med. Chem. 41, 706–716 (1998).

REFERENCES 329

12.Boger, D. L., Jiang, W. and Goldberg, J., J. Org. Chem. 64, 7094–7100 (1999).

13.Lam, K. S., Lebl, M. and Krchnak, V., Chem. Rev. 97, 411–448 (1997).

14.Hardin, J. H. and Smietana, F. R., Mol. Diversity 1, 270–274 (1995).

15.DeWitt, S. H., Ann. Rep. Combi. Chem. Mol. Div. 1, 69–77 (1997).

16.Sugarman, J. H., Rava, R. P., Kedar, H., Dower, W. J., Barrett, R. W., Gallop, M. A. and Needels, M. C., WO Patent 9512608 A1, May 5th, 1995.

17.Zuckermann, R. N., Kerr, J. M., Siani, M. A. and Banville, S. C.,Int. J. Pept. Protein Res. 40, 497–506 (1992).

18.Lashkari, D. A., Hunicke-Smith, S. P., Norgren, R. M., Davis, R. W. and Brennan, T.,

Proc. Natl. Acad. Sci. USA 92, 7912–7915 (1995).

19.Boutin, J. A. and Fauchere, J.-L., Am. Biotechnol. Lab. 14, 36–40 (1996).

20.Stankova, M. and Lebl, M., Mol. Diversity 2, 75–80 (1996).

21.Needels, M. and Sugarman, J., in Combinatorial Chemistry and Molecular Diversity in Drug Discovery, E. M. Gordon and J. Kerwin (Eds.). Wiley-Liss, New York, 1998, pp. 339–348.

22.Stanchfield, J., Wright, D., Hsu, S., Lamsa, M. and Robbins, A., BioTechniques 20, 292–296 (1996).

23.Fitch, W. L., Ann. Rep. Combi. Chem. Mol. Div. 1, 59–68 (1997).

24.Fitch, W. L., Look, G. C. and Detre, G., in Combinatorial Chemistry and Molecular Diversity in Drug Discovery, E. M. Gordon and J. F. Kerwin, Jr. (Eds.). John Wiley and Sons, New York, 1998, pp. 349–368.

25.Sepetov, N. and Issakova, O., in Combinatorial Chemistry and Combinatorial Technolo - gies: Methods and Applications , S. Miertus and G. Fassina (Eds.). Marcel Dekker, New York, 1999, pp. 169–203.

26.Fathi, R., Patel, R. and Cook, A. F., Mol. Diversity 2, 125–134 (1997).

27.Whitehouse, D. L., Nelson, K. H., Jr., Savinov, S. N., Lowe, R. S. and Austin, D. J.,Bioorg. Med. Chem. 6, 1273–1282 (1998).

28.Richter, H. and Jung, G., Mol. Diversity 3, 191–194 (1998).

29.Kurth, M. J., Randall, L. A. A. and Takenouchi, K.,J. Org. Chem. 61, 8755–8761 (1996).

30.Phillips, G. B. and Wei, G. P., Tetrahedron Lett. 37, 4887–4890 (1996).

31.Haap, W. J., Kaiser, D., Walk, T. B. and Jung, G., Tetrahedron 54, 3705–3724 (1998).

32.Amparo Lago, M., Nguyen, T. T. and Bhatnagar, P., Tetrahedron Lett. 39, 3885–3888 (1998).

33.Zhu, T. and Boons, G.-J., Angew. Chem. Int. Ed. Engl. 37, 1898–1900 (1998).

34.Chen, C., Ahlberg Randall, L. A., Miller, R. B., Jones, A. D. and Kurth, M. J.,Tetrahedron 53, 6595–6609 (1997).

35.Whitehouse, D. L., Nelson, K. H., Jr., Savinov, S. N. and Austin, D. J., Tetrahedron Lett. 38, 7139–7142 (1997).

36.Dunayevskiy, Y., Vouros, P., Carell, T., Wintner, E. A. and Rebek, J. Jr., Anal. Chem. 67, 2906–2915 (1995).

37.Metzger, J. W., Wiesmuller, K.-H., Kienle, S., Brunjes, J. and Jung, G., in Peptide and Non-Peptide Libraries: A Handbook for the Search of Lead Structures , G. Jung (Ed.). VCH, Weinheim, Germany, 1997, pp. 247–285.

330SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE POOL LIBRARIES

38.Heerding, D. A., Takata, D. T., Kwon, C., Huffman, W. F. and Samanen, J., Tetrahedron Lett. 39, 6815–6818 (1998).

39.Thompson, L. A. and Ellman, J. A., Chem. Rev. 96, 555–600 (1996).

40.Balkenhohl, F., von dem Bussche-Huennefeld, C., Lansky, A. and Zechel, C., Angew. Chem. Int. Ed. Engl. 35, 2288–2337 (1996).

41.Nefzi, A. and Houghten, R. A., Chem. Rev. 97, 449–472 (1997).

42.Houghten, R. A., Pinilla, C., Appel, J. R., Blondelle, S. E., Dooley, C. T., Eichler, J., Nefzi,

A.and Ostresh, J. M., J. Med. Chem. 42, 3743–3778 (1999).

43.Pei, Y., Houghten, R. A. and Kiely, J. S., Tetrahedron Lett. 38, 3349–3352 (1997).

44.Krchnak, V. and Weichsel, A. S., Tetrahedron Lett. 38, 7299–7302 (1997).

45.Cao, J., Cuny, G. D. and Hauske, J. R., Mol. Diversity 3, 173–179 (1998).

46.Nefzi, A., Bioorg. Med. Chem. Lett. 8, 2273–2278 (1998).

47.Neustadt, B. R., Smith, E. M., Lindo, N., Nechuta, T., Bronnenkant, A., Wu, A., Armstrong,

L.and Kumar, C., Bioorg. Med. Chem. Lett. 8, 2395–2398 (1998).

48.Gennari, C., Longari, C., Ressel, S., Salom, B., Piarulli, U., Ceccarelli, S. and Mielgo, A.,

Eur. J. Org. Chem., 2437–2449 (1998).

49.Kelly, M. A., Liang, H., Sytwu, I.-I., Vlattas, I., Lyons, N. L., Bowen, B. R. and Wennogle,

L.P., Biochemistry 35, 11747–11755 (1996).

50.Blom, K. F., Larsen, B. S. and McEwen, C. N., J. Comb. Chem. 1, 82–90 (1999).

51.Davis, R. G., Anderegg, R. J. and Blanchard, S. G.,Tetrahedron 55, 11653–11667 (1999).

52.Dunayevskiy, Y. M., Lai, J.-J., Quinn, C., Talley, F. and Vouros, P.,Rapid Commun. Mass Spectrom. 11, 1178–1184 (1997).

53.Chu, Y.-H., Kirby, D. P. and Karger, B. L., J. Am. Chem. Soc. 117, 5419–5420 (1995).

54.Chu, Y.-H., Dunayevskiy, Y. M., Kirby, D. P., Vouros, P. and Karger, B. L.,J. Am. Chem. Soc. 118, 7827–7835 (1996).

55.Cheng, C. C. and Chu, Y.-H., Am. Lab. 30, 79–81 (1998).

56.Dunayevskiy, Y. M., Lyubarskaya, Y. V., Chu, Y.-H., Vouros, P. and Karger, B. L.,J. Med. Chem. 41, 1201–1204 (1998).

57.Chu, Y.-H. and Cheng, C. C., Cell. Mol. Life Sci. 54, 663–683 (1998).

58.Sun, S., Headrick, J., Staller, T. and Sepaniak, M., J. Microcolumn Separations 10, 653–660 (1998).

59.Lyubarskaya, Y. V., Carr, S. A., Dunnington, D., Prichett, W. P., Fisher, S. M., Appelbaum,

E.R., Jones, C. S. and Karger, B. L., Anal. Chem. 70, 4761–4770 (1998).

60.Wieboldt, R., Zweigenbaum, J. and Henion, J., Anal. Chem. 69, 1683–1691 (1997).

61.Nedved, M. L., Habibi-Goudarzi, S., Ganem, B. and Henion, J. D., Anal. Chem. 68, 4228–4236 (1996).

62.Wu, J., Takayama, S., Wong, C.-H. and Siuzdak, G., Chem. Biol. 4, 653–657 (1997).

63.Berlin, K., Jain, R. K., Tetzlaff, C., Steinbeck, C. and Richert, C., Chem. Biol. 4, 63–77 (1997).

64.Hsieh, F., Keshishian, H. and Muir, C., J. Biomol. Screening 3, 189–198 (1998).

65.Siegel, M. M., Tabei, K., Bebernitz, G. A. and Baum, E. Z., J. Mass Spectrom. 33, 264–273 (1998).

66.Holtzapple, C. K. and Stanker, L. H., Anal. Chem. 70, 4817–4821 (1998).

REFERENCES 331

67.van Breemen, R. B., Huang, C.-R., Nikolic, D., Woodbury, C. P., Zhao, Y.-Z. and Venton,

D.L., Anal. Chem. 69, 2159–2164 (1997).

68.Zhao, Y.-Z., van Breemen, R. B., Nikolic, D., Huang, C.-R., Woodbury, C. P., Schilling,

A.and Venton, D. L., J. Med. Chem. 40, 4006–4012 (1997).

69.Nikolic, D. and van Breemen, R. B., Combi. Chem. High. Throughput Screen. 1, 47–55 (1998).

70.Woodbury, C. P. and Venton, D. L., Am. Lab. 30, 16–19 (1998).

71.Bruce, J. E., Anderson, G. A., Chen, R., Cheng, X., Gale, D. C., Hofstadler, S. A., Schwartz, B. L. and Smith, R. D., Rapid Commun. Mass Spectrom. 9, 644–650 (1995).

72.Cheng, X., Chen, R., Bruce, J. E., Schwartz, B. L., Anderson, G. A., Hofstadler, S. A., Gale, D. C., Smith, R. D., Gao, J. and Sigal, G. B., J. Am. Chem. Soc. 117, 8859–8860 (1995).

73.Gao, J., Cheng, X., Chen, R., Sigal, G. B., Bruce, J. E., Schwartz, B. L., Hofstadler, S. A., Anderson, G. A., Smith, R. D. and Whitesides, G M., J. Med. Chem. 39, 1949–1955 (1996).

74.Hofstadler, S. A., Sannes-Lowery, K. A., Crooke, S. T., Ecker, D. J., Sasmor, H., Manalili,

S.and Griffey, R. H., Anal. Chem. 71, 3436–3440 (1999).

75.Shuker, S. B., Hajduk, P. J., Meadows, R. P. and Fesik, S. W., Science 274, 1531–1534 (1996).

76.Hajduk, P. J., Meadows, R. P. and Fesik, S. W., Science 278, 498–499 (1997).

77.Marek, D., poster presented at Thirty ninth Experimental Nuclear Magnetic Resonance Conference, P227, Asilomar, CA (1998).

78.Hajduk, P. J., Dinges, J., Miknis, G. F., Merlock, M., Middleton, T., Kempf, D. J., Egan,

D.A., Walter, K. A., Robins, T. S., Shuker, S. B., Holzman, T. F. and Fesik, S. W.,J. Med. Chem. 40, 3144–3150 (1999).

79.Hajduk, P. J., Sheppard, G., Nettesheim, D. G., Olejniczak, E. T., Shuker, S. B., Meadows,

R.P., Steinman, D. H., Carrera, G. M., Marcotte, P. A., Severin, J., Walter, K., Smith, H., Gubbins, E., Simmer, R., Holzman, T. F., Morgan, D. W., Davidsen, S. K. and Fesik, S. W., J. Am. Chem. Soc. 119, 5818–5827 (1997).

80.Hajduk, P. J., Gerfin, T., Boehlen, J.-M., Haeberli, M., Marek, D. and Fesik, S. W.,J. Med. Chem. 42, 2315–2317 (1999).

81.Hajduk, P. J., Dinges, J., Schkeryantz, J. M., Janowick, D., Kaminski, M., Tufano, M., Augeri, D. J., Petros, A., Nienaber, V., Zhong, P., Hammond, R., Coen, M., Beutel, B., Katz, L. and Fesik, S. W., J. Med Chem. 42, 3852–3859 (1999).

82.Lin, M., Shapiro, M. J. and Wareing, J. R., J. Am. Chem. Soc. 119, 5249–5250 (1997).

83.Lin, M., Shapiro, M. J. and Wareing, J. R., J. Org. Chem. 62, 8930–8931 (1997).

84.Shapiro, M. J., Chin, J., Chen, A., Wareing, J. R., Tang, Q., Tommasi, R. A., Marepalli,

H.R., Tetrahedron Lett. 40, 6141–6143 (1999).

85.Bleicher, K., Lin, M., Shapiro, M. J. and Wareing, J. R., J. Org. Chem. 63, 8486–8490 (1998).

86.Anderson, R. C., Lin, M., and Shapiro, M. J., J. Comb. Chem. 1, 69–72 (1999).

87.Hajduk, P. J., Olejniczak, E. T. and Fesik, S. W., J. Am. Chem. Soc. 119, 12257–12261 (1997).

88.Meyer, B., Weimar, T. and Peters, T., Eur. J. Biochem. 246, 705–709 (1997).

332SYNTHETIC ORGANIC LIBRARIES: SOLID-PHASE POOL LIBRARIES

89.Henrichsen, D., Ernst, B., Magnani, J. L., Wang, W.-T., Meyer, B. and Peters, T.,Angew. Chem. Int. Ed. 38, 98–102 (1999).

90.Fejzo, J., Lepre, C. A., Peng, J. W., Bemis, G. W., Ajay, Murcko, M. A. and Moore, J. M., Chem. Biol. 6, 755–769 (1999).

91.Comprehensive Medicinal Chemistry (CDC) database, version CDC3D98.1, MDL Information Systems, San Leandro, CA, US.

92.Bemis, G. W. and Murcko, M. A., J. Med. Chem. 39, 2887–2893 (1996).

93.Available Chemical Directory (ADC) database, version ACD98.2, MDL Information Systems, San Leandro, CA, US.

94.Chen, A. and Shapiro, M. J., J. Am. Chem. Soc. 120, 10258–10259 (1998).

95.Mayer, M. and Meyer, B., Angew. Chem. Int. Ed. 38, 1784–1788 (1999).

96.Klein, J., Meinecke, R., Mayer, M. and Meyer, B., J. Am. Chem. Soc. 121, 5336–5337 (1999).

97.Youngquist, R. S., Fuentes, G. R., Miller, C. M., Ridder, G. M., Lacey, M. P. and Keough, T., Adv. Mass Spectrometry 14, 423–448 (1998).

98.Sussmuth, R. D. and Jung, G., J. Chromatogr., B: Biomed. Sci. Applic. 725, 49–65 (1999).

99.Keifer, P. A., Curr. Opin. Biotechnol. 10, 34–41 (1999).

100.Moore, J. M., Curr. Opin. Biotechnol. 10, 54–58 (1999).

101.Gounarides, J. S., Chen, A. and Shapiro, M. J., J. Chromatogr., B: Biomed. Sci. Applic. 725, 79–90 (1999).

102.Chen, A. and Shapiro, M. J., Anal. Chem. 71, 669A–675A (1999).

103.Woodbury, C. P., Jr. and Venton, D. L., J. Chromatogr., B: Biomed. Sci. Applic. 725, 113–137 (1999).

104.Eliseev, A. V., Curr. Opin. Drug Discovery Dev. 1, 106–115 (1998).

105.Lam, K. S. and Lebl, M., Methods 6, 372–380 (1994).

106.Lam, K. S., Wade, S., Abdul-Latif, F. and Lebl, M., J. Immunol. Methods 180, 219–223 (1995).

107.Lam, K. S. and Lebl, M., Peptide and Non-Peptide Libraries: A Handbook for the Search of Lead Structures, G. Jung (Ed.). VCH, Weinheim, Germany, 1996, pp. 173–201.

108.Lam, K. S., Lake, D., Salmon, S. E., Smith, J., Chen, M.-L., Wade, S., Abdul-Latif, F., Knapp, R. J., Leblova, Z. et al., Methods 9, 482–493 (1996).

109.Lam, K. S., Methods Mol. Biol. 87, 7–12 (1998).

110.Lebl, M., Krchnak, V., Sepetov, N. F., Seligmann, B., Strop, P., Felder, S. and Lam, K. S., Biopolymers 37, 177–198 (1995).

111.Ohlmeyer, M. H. J., Swanson, R. N., Dillard, L., Reader, J. C., Asouline, G., Kobayashi, R., Wigler, M. and Still, W. Clark., Proc. Natl. Acad. Sci. USA 90, 10922–10926 (1993).

112.Boyce, R., Li, G., Nestler, H. P., Suenaga, T. and Still, W. Clark., J. Am. Chem. Soc. 116, 7955–7956 (1994).

113.Nestler, H. P., Mol. Diversity 2, 35–40 (1996).

114.Yu, Z. and Chu, Y.-H., Bioorg. Med. Chem. Lett. 7, 95–98 (1997).

115.Hwang, S., Tamilarasu, N., Ryan, K., Huq, I., Richter, S., Still, W. Clark and Rana, T. M.,

Proc. Natl. Acad. Sci. USA 96, 12997–13002 (1999).