JP2000511191A - Analogs of CC-1065 and duocarmycin - Google Patents

Abstract

  (57) [Summary] Systematic and scalable modification of the DNA binding subunit bound to the 1,2,9,9a-tetra-hydro-cyclo-propa [c] benz [e] indol-4-one (CBI) alkylating subunit An analog of the antitumor antibiotics CC-1065 and duocarmycin is synthesized. The analog has potent cytotoxic activity and is an effective antitumor compound.

Description

DETAILED DESCRIPTION OF THE INVENTION               Analogs of CC-1065 and duocarmycinField of the Invention:   The present invention relates to antitumor antibiotics. More particularly, the invention relates to antitumor antibiotics. The present invention relates to analogs of CC-1065 and duocarmycin having substance activity.background:   (+)-CC-1065 (1) and duocarmycin are very potent antitumor It represents the earliest of antibiotics. This antitumor antibiotics substance is a double DN The reversible, stereoselectively controlled sequence-selective alkylation of A Bring out physical effects. (H. Sugiyama, et al., Tetrahedron Lett. 1990, 31, 7197; C.H. Lin, et al. J. Am. Chem. Soc. 1992, 114, 10658; Sugiyama, et al., Tetrahedron Le tt. 1993, 34, 2179; Yamamoto, et al., Biochemistry 1993, 32, 1059; A sai, et al. Am. Chem. Soc. 1994, 116, 4171; and D.L. Boger, et al., T etrahedron 1991, 47, 2661.) (+)-CC-1065 (1) was published in 1981 by L.J. Hanka, et al. By For the first time (J. Am. Chem. Soc. 1981, 103, 7629.). Zuocarmycin Was first disclosed in 1988 and 1990 (Takahashi, et al. J. Antibiot. 19 88, 41, 1915; Yasuzawa, et al., Chem. Pharm. Bull. 1988, 36, 3728; I chimura, et al. Antibiot. 1988, 41, 1285; Ichimura, et al., J. Ant ibiot. 1990, 43, 1037; M.H. Ichimura, et al., J. Antibiot. 1991, 44, 1045 ; K. Ohba, et al. Antibiot. 1988, 41, 1515; and S.M. Ishii, J .; Antibiot. 1989, 42, 1713.)   Following their disclosure, a great deal of effort has been made to determine the selectivity and And its structural origin was established. (D.L. Boger, Acc. Chem. Res. 1995, 28, 20; D.L. Boger, Proc. Natl. Sci. U.S.A. in press; D.L. Boger, Chemtracts: Org. Chem. 1991, 4, 329; D.L. Boge r, In Proceed. R. A. Welch Found. Conf. on Chem. Res., XXXV. Chem. at the Frontiers of Medicine 1991, 35, 137; D.L. Boger, In Advances in He terocyclic Natural Products Synthesis, Vol. 2, Pearson, W .; H.Ed.; JAI Pre ss: Greenwich, CT, 1992, 1-188; D.L. Boger, Pure Appl. Chem. 1993, 65, 112 3; D.L. Boger, Pure Appl. Chem. 1994, 66, 837; R.S. Coleman, In Studies in  Nat. Prod. Chem., Vol 3, Rahman, A.-u.-, Ed .; Elsevier: Amsterdam, 1989, 3 01; and D.L. Boger, In Heterocycles in Bioorganic Chemistry; Bergman, H .C. van der Plas, and M. Simonyl, Eds; Royal Society of Chemistry: Cambrid ge, 1991, 103.) It should also characterize the relationship between DNA alkylation and its attendant biological properties. And progress has been made with respect to (D.L. Boger, et al., Bioorg. Med. Chem. Lett. (1994, 4, 631) In addition, much effort has been made to determine structural, chemical reactivity, and biological characteristics. The basic principles underlying the relationship between genders have been defined. (W. Wierenga, et al., Adv. Enzyme Regul. 1986, 25, 141; M.A. Warpehoski , Et al., J. Mol. Med. Chem. 1988, 31, 590; D.L. Boger, et al. Am. Chem. S oc. 1993, 115, 9025; D.L. Boger, et al. Am. Chem. Soc. 1992,114,100 56; Muratake, et al.,. Tetrahedron Lett. 1994, 35, 2573; Fukuda, et  al., Tetrahedron 1994, 50, 2793; Fukuda, et al., Tetrahedron 1994, 50 , 2809; Fukuda, et al., Bioorg. Med. Chem. Lett. 1992, 2, 755; Fukud a, et al., Tetrahedron Lett. 1990, 31, 6699; W. Wierenga, J .; Am. Chem. So c. 1981, 103, 5621; Magnus, et al.,. J. Am. Chem. Soc. 1987, 109, 270 6; G.A. Kraus, et al., J. Mol. Org. Chem. 1985, 50, 283; D.L. Boger, et al.,   J. Am. Chem. Soc. 1988, 110, 1321, 4796; R.E. Bolton, et al., J.S. Chem. Soc., Perkin Trans. 1 1988, 2491; R.J. Sundberg, et al. Org. Chem. 19 88, 53, 5097; R.J. Sundberg, et al. Org. Chem. 1991, 56, 3048; P. M Artin, Helv. Chim. Acta 1989, 72, 1554; Toyota, et al., J. Chem. Soc., P erkin Trans. 1 1992, 547; and L.F. Tietze, et al., J.A. Org. Chem. 1994, 59 , 192.) It also discusses the relationship between the structure, chemical reactivity, and biological properties of CI-based analogs. Even though it was characterized. (D.L. Boger, et al., Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1431; D.L. Boger, et al. Am. Chem. Soc. 1991, 113, 3980; D.L. Boger, et al. Org. Chem. 1989, 54, 1238; D.L. Boger, et al. Am. Chem. Soc. 1990, 11 2, 5230; K.J. Drost, et al. Org. Chem. 1989, 54, 5985; J.H. Tidwell, e t al., J.S. Org. Chem. 1992, 57, 6380; .J. Sundberg, et al., Tetrahedron Le tt. 1986, 27, 2687; Wang, et al., Heterocycles 1993, 36, 1399; Wang , Et al. Med. Chem. 1993, 36, 4172; L.F. Tietze, et al., Chem. Ber. 1 993, 126, 2733; and T.C. Sakamoto, et al. Chem. Soc., Perkin Trans. 1 1 993, 1941.) Also, C_TwoRelationship between structure, chemical reactivity, and biological properties of BI-based analogs Was also characterized. (D.L. Boger, et al., J. Am. Chem. Soc. 1992, 114. And 9318; and D.L. Boger, et al., Bioorg. Med. Chem. 1993, 1, 27.) The relationship between the structure, chemical reactivity, and biological properties of Q-based analogs is also notable. Was imposed. (D.L. Boger, et al., J. Am. Chem. Soc. 1994, 116, 6461; and D.L. Boger, et al., J. Am. Chem. Soc. 1994, 116, 11335.) F. Mohamadi et al. On the relationship between structure, chemical reactivity, and biological properties of CFI-based analogs confirmed. (J. Med. Chem. 1994, 37, 232.) p-quinone methide analogs are described in D.L. . Characterized by Boger et al. (J. Org. Chem. 1994, 59, 4943.)   Along with the structure / function studies described above, we will use the structure of natural products to increase in vivo efficacy. Significant efforts have also been made in the development of potential clinical objects. (D.L. Boger, et al., J. Org. Chem. 1984, 49, 2240; M.A. Warephoski, M.A. . Tetrahedron Lett. 1986, 27, 4103; Li, L .; H .; Invest. New Drugs 1991, 9, 137; B.K. Bhuyan, et al., Cancer Res. 1992, 52, 5687; B.K. Bhuyan, et al. , Cancer Res. 1993, 53, 1354; L.H. Li, et al., Cancer Res. 1992, 52, 4904 ; M.A. Mitchell, et al., J. Mol. Am. Chem. Soc. 1991, 113, 8994. Lee, C.-S .; Gi bson, N .; W. Cancer Res. 1991, 51, 6586. Lee, C.-S .; Gibson, N.W. Biochemi stry 1993, 32, 9108; Wierenga, W .; Drugs Fut. 1991, 16, 741; Gomi, et al ., Jpn. J. Cancer Res. 1992, 83, 113. Okamoto, A .; Okabe, M .; Gomi, K. Jpn . J. Cancer Res. 1993, 84, 93; Kobayashi, et al., Ca ncer Res. 1994, 54, 2404; and H. Ogasawara, Jpn. J. Cancer Res. 1994, 85, 418. ) For a Phase I clinical trial of this class of drug subjects, see G. F. Fleming et al. (J. Natl. . Cancer Inst. 1994, 86, 368). Also, (+)-CC The development of analogues with reduced delayed toxicity compared to the native form of Effort has been made. (J.P. McGovren, et al., Cancer Res. 1993, 53, 5690)   This unique property is due to the fact that ent-(-)-CC- 1065, and is not found in natural products, as is a simplified analog of a natural product. It is important that neither is found in carmycin.   1,2,9,9a-tetrahydrocyclopropa [c] benz [e] indole For the preparation and testing of drugs containing -4-one (CBI) alkylated subunits CC-1065 and deep sheet structure change in alkylated subunit And duocarmycin. (D.L. Boger , Et al., J. Mol. Am. Chem. Soc. 1989, 111, 6461; and D.L. Boger, et al., J. Org . Chem. 1990,55,5823.)   These drugs have structural features associated with sequence-selective alkylation of double DNA. Identify and define the fundamentals between structural, chemical or functional reactivity and biological properties It was used as a tool to define a simple relationship.   Prior to the present invention, the specificity of the CPI subunit of naturally occurring CC-1065 Alkylation activity is hypothesized to decrease as this part of the molecule changes structurally. Was. (L.H. Hurley, et al., Science 1984, 226, 843; V.L. Reynolds, et al., Bio chemistry 1985, 24, 6228. L.H. Hurley, et al., Biochemistry 1988, 27, 3886; L.H. Hurley, et al., J . Am. Chem. Soc. 1990, 112, 4633; M.A. Warpehoski, et al., J. Mol. Biochemistr y 1992, 31, 2502; D.L. Boger, et al., Bioorg. Med. Chem. 1994, 2, 115; D.L . Boger, et al. Am. Chem. Soc. 1990, 112, 4623; M.A. Warpehoski, et a l., In Advances in DNA Sequence Specific Agents; Hurley, L.H., Ed .; JAI Pr ess: Greenwich, CT, 1992, Voll, 217; M.A. Warpehoski, Drugs Fut. 1991, 16 , 131; M.A. Warpehoski, et al., In Molecu1ar Basis of Specificity in Nucleic Ac id-Drug Interactions; B. Pullman and J. Jortner, Eds .; Kluwer: Netherlands; 1990, 531; M.A. Warpeh oski, et al., Chem. Res. Toxicol. 1988, 1, 315; Hurley, L .; H .; InMolecul ar Aspects of Anticancer Drug-DNA Interactions; Neidle, S., Waring, M., E ds .; CRC Press: Ann Arbor, MI 1993, Vol, 89; and L.H. Hurley, et al., Acc. Ch em. Res. 1986, 19, 230. The present invention discloses that the above hypothesis is incorrect. In addition, CBI based The natural enantiomer of the (+)-CC-1065 analog is the natural enantiomer of CC-1065. Approximately 4-fold higher than the corresponding drug incorporating the CPI alkylated subunit It is biologically stable, indicating that it is about 4-fold more biologically effective. (D.L.Bog er, et al., Tetrahedron Lett. 1990, 31, 793; D.L. Boger, et al. Org. Ch em. 1992, 57, 2873; and D.L. Boger, et al. Org. Chem. 1995,60,0000.)   Also, CBI analogs are much more synthetic than naturally occurring CPI compounds. Easy to use. (+)-CBI-indole_TwoCytotoxic titer is (+)-CC-1 065, a potential clinical object (+)-CPI-A introduced by Upjohn Ndore_TwoIt shows a value (4x) larger (4x) than (U71, 184). Also, (+) -CBI-indole_TwoShows strong and effective in vivo antitumor activity. (D.L.B. oger, et al., Bioorg. Med. Chem. Lett. 1991, 1, 115) (+)-CBI-in Doll_Two(27) is a structurally altered and simplified DNA alkylation subunit. The most important anti-tumor activity was due to the CC-1065 analog having the same. further The drug lacked the delayed lethal toxicity of (+)-CC-1065.   The natural enantiomer of a CBI-ase analog can be compared to the corresponding CPI analog. Have been shown to alkylate DNA without altering sequence selectivity. (D.L.Bo ger, et al. Am. Chem. Soc. 1994, 116, 7996; and P.A. Aristoff, et al., J. Med. Chem. 1993, 36, 1956.) Additionally, DNA alkyls of CBI-based analogs The conversion is performed using the corresponding CPI analog (D.L. Boger, J. Am. Chem. Soc. 1991, 113, 2779). And occurs more efficiently than the corresponding CPI analog. (D.L.B. oger, et al. Am. Chem. Soc. 1992, 114, 5487)   Reversible and offset AT-rich enantiomeric drugs and their structural analogs Zuocarmycin DN adapted to DNA alkylation selectivity of adenine N3 Exact models of the A-alkylation reaction have been developed. (D.L. Boger, et al., J. Org. Chem. 1990, 55, 4499; D.L. Boger, et al.,   J. Am. Chem. Soc. 1990, 112, 8961; D.L. Boger, et al. Am. Chem. Soc . 1991, 113, 6645; D.L. Boger, et al., .J. Am. Chem. Soc. 1993, 115, 9872 ; D.L. Boger, et al., Bioorg. Med. Chem. Lett. 1992, 2, 759; and D.L. Boge r, et al. Am. Chem. Soc. 1994, 116, 1635.) Reversible and offset AT-enriched enantiomers and their structural analogs D of CC-1065 adapted to the selectivity of the DNA alkylation reaction of Denin N3 A similar model of the NA alkylation reaction has been developed. (D.L. Boger, et al., Bioorg. Med. Chem. 1994, 2, 115; and D.L. Boger, et al. Am. Chem. Soc. 1990, 112, 4623.) From these motels, dimers derived from unnatural enantiomers The astereomeric adduct is the C7 center of CPI (CH_Three) Or C8 center of CBI; Base adjacent to the alkylated adenine that is not present in the natural enantiomeric adduct It has a drawback that the steric interaction of becomes extremely unstable. further , As the inherent three-dimensional bulk around this center is reduced or removed, The salient features of the unnatural enantiomer are reduced or disappear. Unnatural Enantiomers have steric interactions around the C7 center of CPI or C8 center of CBI. Non-natural enantiomers of CBI-based analogues Omers have the corresponding Cs, which similarly increase the kinetics and efficiency of DNA alkylation. It is especially more effective than PI analogs.   What is needed is incorporation into analogs of CC-1065 and duocarmycin Is an alternative alkylating agent with a different reactivity compared to CBI.Summary of the Invention   A first aspect of the present invention relates to a DNA alkylating agent represented by the following structure. In the above structure, R₁Is an alkyl (C1-C6) and a radical represented by the following structure: Selected from the group consisting of:   In a first embodiment of the above structure, R_TwoAnd R_ThreeIs the indicated vinylene group Together with the carbon atom of the group W, ie R_FourAnd R_FiveA vinylene group-containing N in which is hydrogen Forming a substituted pyrrolidine ring. More specifically, the N-substituted pyrrolidine ring has the following It can be shown by the structure of Where R_TwoBinds to C and R_ThreeIs bonded to N to form the indicated pyrrolidine ring;₆ Is NH_TwoAnd compounds selected from the group consisting of:   In a second embodiment, R_TwoIs hydrogen, R_ThreeIs N represented by the following compound Is a substituted subgroup.Where R_FourAnd R_FiveIs hydrogen.   In a third embodiment, R_TwoIs hydrogen, R_ThreeIs hydrogen and OCH_ThreeGroup consisting of Selected from R_FourIs hydrogen and OCH_ThreeAnd R is selected from the group consisting of_FiveIs water Elementary and OCH_ThreeSelected from the group consisting of:   A second aspect of the present invention relates to a DNA alkylating agent represented by the following structure. Here, R is selected from the group consisting of H and -OMe.   A third aspect of the present invention relates to a DNA alkylating agent represented by the following structure. Where R is -SMe, -S (O) Me, and-⁺SMe_TwoSelected from the group consisting of You.   A fourth aspect of the present invention relates to a DNA alkylating agent represented by the following structure.Where R is -SMe and-⁺Sme_TwoSelected from the group consisting of:   A fifth aspect of the present invention relates to a DNA alkylating agent represented by the following structure. Where R is -SMe and-⁺Sme_TwoSelected from the group consisting of:   A sixth aspect of the present invention relates to a DNA alkylating agent represented by the following structure.   A seventh aspect of the present invention provides a DNA for each of the above DNA alkylating agents. It relates to the process of alkylation.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the structures of (+)-CC-1065 (1) and duocarmycin 2-3. .   FIG. 2 shows the synthesis of Intermediate 6 (top) and Intermediate 12 (bottom).   FIG. 3 shows the synthesis of improved N-BOC CCBI intermediate 25 and CCBI intermediate 26. Is shown.   FIG. 4 shows the synthesis of improved intermediate 21.   FIG. 5 shows CCBI duocarmycin analogs CCBI-TMI35, CCBI- Indole_Two37 and CCBI-CDPI₁3 shows the synthesis of 39.   FIG. 6 shows CBI and MC compared to CCBI-based drugs for cytotoxic activity. The BI drugs are listed side by side in the table.   FIG. 7 shows the chemical stability (−log k) and in vitro cytotoxicity titer (L1210). , Log1 / IC₅₀) And the linear relationship between Shown.   FIG. 8 has in vitro cytotoxic activity and CCBI giving the most potent drug Hammett σ of C7 substituent_pShows a linear relationship with constants.   FIG. 9 compares the three currently available drugs following the trend of 44-49. Show.   FIG. 10 shows three drugs currently available following the trend of all relevant structural analogs. 3 shows a comparison.   FIG. 11 shows that the drug with DNA was incubated for 72 hours at 37 ° C. Duplex after removing unbound drug and incubating at 100 ° C. for 30 minutes Heat-induced strand cleavage of DNA (SV40 DNA fragment, 144 bp, nucleotide number 5 238-138, clone w794), 8% denatured PAGE, and autoradiograph Shows Raffy. Lane 1, (+)-N-BOC-MCBI (1 × 10^-2M); 2, ent-(-)-N-BOC-MCBI (1 × 10^-2M); Lane 3, control D NA; Lane 4, (+)-CC-1065 (1 × 10^-2M); Lanes 5-8, Sanger G, C, A and T reactions; lanes 9-11, (+)-N-BOC-CCBI (1 × 1 0^-2~ 1 × 10^-FourM); Lanes 12-14, ent-(-)-N-BOC-CCB I (1 × 10^-2~ 1 × 10^-FourM).   FIG. 12 shows that the drug-DNA was incubated for 24 hours at 25 ° C. and unbound. Double-stranded DNA after drug removal and incubation at 100 ° C. for 30 minutes Heat-induced strand cleavage (SV40 DNA fragment, 144 bp, nucleotide number 523) 8-138, clone w794), 8% denatured PAGE, and autoradiograph Indicates Lane 1, control DNA; Lanes 2-4, (+)-Duocarmycin S A (2, 1 × 10^-Five~ 1 × 10^-7M); Lanes 5-7, (+)-CCBI-TMI (1 × 10^-Five~ 1 × 10^-7M); Lanes 8-11, Sanger G, C, A and T reactions; 12 to 14, (+)-CC-1065 (1, 1 × 10^-Five~ 1 × 10^-7M); 15-17, (+)-CCBI-indole_Two(1 × 10^-Five~ 1 × 10^-7M) ； レ 18-18, (+)-CCBI-CDPI₁(1 × 10^-Five~ 1 × 10^-7M).   FIG. 13 shows a 72 hour incubation at 25 ° C. to remove unbound drug, Heat-induced strand breaks of the replicated DNA after incubation at 100 ° C. for 30 minutes ( SV40 DNA fragment, 144 bp, nucleotide numbers 5238-138, claw 794), 8% denatured PAGE, and autoradiography. lane 1, control DNA; lane 2, (+)-CC-1065 (1 × 10^-6M); Lanes 3 to 6, Sanger G, C, A and T sequencing reactions; lane 7, ent-(-)-zoocal Mycin SA (1 × 10^-6); Lanes 8-9, ent-(-)-CCBI-TMI ( 1 × 10^-6And 10^-7M); Lanes 10-11, ent-(-)-CCBI-in. Doll_Two(1 × 10^-6And 10^-7M); Lanes 12-13, ent-(-)-CCB I-CDPI₁(1 × 10^-6And 10^-7M).   FIG.³²Obtained by autoradiography of P-terminal labeled DNA Shown is a plot of the percent integrated optical density (IOD%) plotted against time. 5, 37, (+)-CBI-TMI, (+)-MCBI-TMI, (+)-CBI-A Ndore_Two, And (+)-MCBI-indole_Two5'-AATTA high affinity site of Used to monitor the relative rate of w794 alkylation at   FIG. 15 is a family of clinically effective glycopeptide antitumor antibiotics. Bleomycin A_TwoThis shows bleomycin containing (4) as a main component.   FIG. 16 shows a series of CC-1065 / zuocarmycin and bleomycin. Shows the hybrid agents 105 to 111. These are bleomycin A_TwoC-terminal of Of the former natural product linked to a di- and tripeptide S DNA binding region of Incorporates a CBI analog of the NA alkylation subunit.   FIG. 17 shows the synthesis of Intermediate 16.   FIG. 18 shows the synthesis of analogs 6 and 7.   FIG. 19 shows the synthesis of analog 9.   FIG. 20 shows the synthesis of analog 11.   FIG. 21 shows a comparative test of drugs 105 to 111 and CBI acylated only with a linking group. Representative range of additional comparative samples, including ingredients 114-116, and natural drugs 1-3. The L1210 cytotoxic activity was summarized below.   FIG. 22 shows low nM cytotoxic activity (5-6 nM IC₅₀, L1210), now Of 115 and 116 natural, counted as the most effective and simplest derivatives disclosed Shows the enantiomers (see 123-127).   FIG. 23 shows that the drug with DNA was incubated at 37 ° C. for 72 hours, Duplex after removing unbound drug and incubating at 100 ° C. for 30 minutes Heat-induced strand cleavage of DNA (SV40 DNA fragment, 144 bp, nucleotide number 5 238-138, clone w794), 8% denatured PAGE, and autoradiograph Shows Raffy. Lane 1, control DNA; Lane 2, (+)-CC-1065 (1 , 1 × 10^-6M); Lane 3, (+)-Duocarmycin SA (2, 1 × 10^-6M) Lanes 4-7, Sanger G, C, A and T sequencing reactions; lanes 8 and 9, (+) −N-BOC-CBI ((+)-123, 1 × 10^-1And 1 × 10^-2M); Lane 1 0-12, (1S) -114 (1 × 10^-1~ 1 × 10^-3M); lanes 13-15, (+) −116 (1 × 10^-1And 1 × 10^-3M; lanes 16 and 17, (-)-116 (1 × 10^-1And 1 × 10^-2M).   FIG. 24 shows that the drug with DNA was incubated for 48 hours at 37 ° C. Duplex after removing unbound drug and incubating at 100 ° C. for 30 minutes Heat-induced strand cleavage of DNA (SV40 DNA fragment, 144 bp, nucleotide number 5 238-138, clone w794), 8% denatured PAGE, and autoradiograph Shows Raffy. Lane 1, control DNA; Lane 2, (+)-CC-1065 (1 , 1 × 10^-6M); Lane 3, (+)-Duocarmycin SA (2, 1 × 10^-6M) Lanes 4-7, Sanger G, C, A and T sequencing reactions; lanes 8 and 9, (+) −N-BOC-CBI ((+)-123, 1 × 10^-1And 1 × 10^-2M); Lane 1 0 and 11, (−)-N-BOC-CBI ((−)-123, 1 × 10^-1And 1 × 1 0^-2M); Lanes 12 and 13, 108 (1 × 10^-2And 1 × 10^-3M); Lane 14, 109 (1 × 10^-3M); lanes 15, 111 (1 × 10^-3M).   FIG. 25 shows a table of drugs outlining calf thymus DNA alkylation and recovery.   FIG. 26 shows the initial analog of such as involving substitution or functionalization of the reaction center in natural products. Difluoro-substituted cyclopropyl of 1 to 3 alkylated subunits 9,9-Difluoro-1,2,9,9a-tetrahydrocyclo, a pan analog Prop [c] benzo [e] indole-4-one (F_TwoCBI).   FIG. 27 shows that carbeno was added to 1,1-difluoroalkene using quinonediazide. With key intramolecular metal to insert_Two1 shows the retrosynthesis of CBI.   FIG. 28 shows the synthesis of improved intermediate 219.   FIG. 29 shows the synthesis of improved intermediates 216 and 221.   FIG. 30 shows the synthesis of analog 227.   FIG. 31 shows an aqueous buffer solution of 50% methanol (pH 3.0, 4: 1: 20 (v / v / v)). 0.1M citric acid, 0.2M Na_TwoHPO_Four, And) H_TwoN-BOC-F in O_TwoC BI (219, upper figure) and F_TwoSolvolysis studies on CBI (218, below) (UV spectrum). If you record the spectrum on a regular basis, only a few It appeared clearly. Upper figure: 1, 0 minutes; 2, 2 minutes; 3, 5 minutes; 4, 11 minutes; 5, 17 minutes; 6, 25 minutes; 7, 33 minutes; 8, 48 minutes. Lower figure: 1, 0 hours; 2, 0.5 hours; 3 4, 1.5 hours; 5, 3.5 hours; 6, 4.5 hours; 7, 5 hours 8, 5.5 hours.   FIG. 32 shows the solvolysis reactivity of selected drugs.   FIG. 33 shows a nucleophilic addition producing 218 under basic conditions.   FIG. 34 shows the in vitro cytotoxic activity of selected drugs.   FIG. 35 shows the relative drug relative to the corresponding CBI drug of FIG. In vitro cytotoxic activity based on specific reactivity. This is, qualitatively, exceptionally Drugs with stronger activity appear to be more stable (500-fold) And closely follow the trends established in previous studies. FIG. 36 shows heat-induced (100 ° C., 30 minutes) strand breaks in w794 DNA after drug treatment. 8 shows 8% denatured PAGE and autoradiography. Lanes 1-2, ent -(-)-CBI-TMI (25 ° C, 10^-FiveAnd 10^-6M); Lanes 3-5, (+)- CBI-TMI (25 ° C, 10^-FiveAnd 10^-7M); Lanes 6-9, Sanger G, C, A and T sequencing reactions; lane 10, DNA reference; lanes 11-13, F_TwoCB I-TMI (25 ° C., 10^-3-10^-FiveM). Lanes 14-16, F_TwoCBI-T MI (4 ° C) 10^-3-10^-FiveM).   FIG. 37 shows the synthesis of the SA methoxy analog.   FIG. 38 shows that all five drugs show nearly identical DNA alkylation selectivity, The differences observed are found in the rate and overall efficiency of DNA alkylation. It is a table which shows that.   FIG. 39 shows that the presence of the methoxy group affects the analog and its hard subunit Through the sub-groove itself.Detailed description The present invention relates to analogs of the antitumor antibiotics CC-1065 and duocarmycin. You. Analogs were synthesized and 1,2,9,9a-tetra-hydrocyclo-propa [c ] Linked to the alkylated subunit of benz [e] indole-4-one (CBI) It has systematic and wide variations of the DNA binding subunits that are used. Similar The body has potent cytotoxic activity and is an effective antitumor compound. One embodiment of the present invention An embodiment is a substituted CCBI derivative having a C7 cyano substituent at the para position of the C4 carbonyl. Form: 7-cyano-1,2,9,9a-tetra-hydrocyclo-propa [c] ben [E] Indol-4-one (CCBI). Second Reality of the Invention An embodiment is a DNA alkylation subunit of CC-1065 / zuocarmycin. Containing the C-terminal DNA binding region of bleomycin linked to an analog of Including the synthesis of bridging agents. A third embodiment of the present invention relates to 9,9-difluoro- 1,2,9,9a-tetrahydrocyclopropa [c] benz [e] indole- 4-one (F_TwoCBI) of duocarmycin incorporating an alkylated subunit It relates to the synthesis of fluorocyclopropane analogs. A fourth embodiment of the present invention provides: Includes the synthesis of duocarmycin SA methoxy substituted analogs.Example 1 CCBI derivative, 7-cyano-1,2,9,9a-tetra-hydroxy Synthesis of Clo-propa [c] benz [e] indol-4-one (CCBI)   In this example, a substituted CBI derivative having a C7 cyano group, 7-cyano -1,2,9,9a-tetrahydrocyclopropa [c] benz [e] indole The synthesis of -4-one (CCBI) is described. CCBI alkylation subunit Precisely functionalized naphthas with improved Stobbe condensation / Friedel-Crafts acylation To form a ren precursor and by the 5-exo-trig allyl radical-alkene cyclization , 1,2-dihydro-3H-benz [e] indole skeleton and finally Ar Prepared by introducing active cyclopropane by -3 'alkylation. most According to a concise approach, the CCBI subunit and its immediate precursors are 1 In 4-15 steps, it was produced with excellent overall conversion (15-20%). Resolution of the direct CCBI precursor and its conversion to CC-1065 and duocarmycin The incorporation into both enantiomers of the analogs, 34-39, is described below. Shown in detail. Solvolysis reactivity and regioselectivity of N-BOC-CCBI (25) Studies show that the introduction of C7 nitrile, albeit surprisingly small, It has been found that the speed of volatilization is reduced. Classic Hammett's quantification of its effect According to the remarkably small p (-0.3), the electron of the C7 substituent for functional reactivity The effect is exceptionally small. Further thermodynamic studies show that acid-catalyst Nuclear addition is inconsistent with protonation of C4 carbonyl as a slow and rate limiting step However, cyclotrons are rapidly reversible and require both the presence and assistance of nucleophiles. A mechanism by which a slow rate-limiting nucleophilic addition to propane continues (S_N2 mechanism) I found out. This affects the selectivity of this class of drugs for DNA alkylation. Undoubtedly give, not protonation of C4 carbonyl, but acceptable Positioning of a Novel Nucleophile (N3 of Adenine) Is Sequence Selection for DNA Alkylation It suggests that it is the rate-limiting step in controlling selectivity. Affect the speed of solvolysis This small electronic effect is due to the regioselectivity of the solvolysis and the activated cyclopropane. For the stereoselectively controlled nucleophilic addition to the carbon with the fewest substituents It turned out to have no effect. CBI system gives the most potent analogues For CCBI-inducing drugs, stability and specificity of solvolysis were consistent with previous studies. A linear relationship to vesicle injury titers was found, and these observations indicate a predictable Hammett's Related to the transposition effect. For natural enantiomers, the effect on functional reactivity of Unusually small electronic effects have an effect on the selectivity of DNA alkylation I couldn't. Similar effects are observed for the C7 cyano substituent of the unnatural enantiomer. Which is 4-10 times higher than the corresponding CBI-based unnatural enantiomer It is effective and is 4- to 70-fold less potent than the natural enantiomer of CCBI. won. Synthesis of CCBI (26) and N-BOC-CCBI (25)   T-B of 3-bromobenzaldehyde (4) with diethyl succinate (1.5 equivalents) Stobbe in the presence of uOK (1.1 eq, t-BuOH, reflux, 2 hours, 75-100%) The condensation gave a 3.2: 1 mixture 5 of the half-ester with very good conversion (FIG. 2). This mixture 5 was subjected to Friedl-Crafts acylation (1 to 1.1 equivalents of NaOAc, Ac_Two O, reflux, 6 hours), 6, its ortho-acylated product 7, and the major isomer A mixture of products 8 and 9 was obtained. Friedl-Crafts acylation moderately diluted (0.1M vs. 0.5M). Next, The resulting mixture was subjected to o-acetation by ethanolysis (3M HCl-EtOH). The hydrolysis of 7 and 9 yielded a 7: 1 mixture of 6 and its isomer 8. 6 and 8 are Easily, or even more conveniently, from the mixture by chromatography By recrystallization. In this method, the total of the three stages is as high as 53%. Produced 6 in yield, but is usually produced at a lower 20-30% conversion. This The decrease in the conversion as in the above indicates that E is incorporated into the Friedl-Crafts acylation reaction. And mixtures of Z-5 and the aggressiveness required for the isomerization of Z to E for cyclization Due to poor reaction conditions and time.   This can be considerably improved by performing Stobbe condensation in a more controlled manner. It was good. 4 and Wadsworth-Horner-Emmons substance 10 (1.03 equivalents, 1.1 equivalents of NaH , THF, 0-25 ° C, 12 hours, 95-100%) to give 11 Obtained. In this case, the required E-isomer takes precedence as shown in the second diagram of FIG. (> 11: 1 E: Z). 11 was deprotected with an acid catalyst (100%), 5 was Ac_TwoO- Treatment with KOAc (reflux, 1 hour, 30 minutes), Friedl-Crafts acylation, Recrystallization provided 7 in 65% yield without any 9 present. In part, The improved transformation to give 7 shows that the correct E isomer of 5 is easily ring closed and used. May be due to milder reaction conditions and shorter reaction times . Under the reaction conditions, isomerization of Z to E is no longer required . ο-Acetate 7_TwoCO_Three-EtOH (reflux, 0.5-1 hour, 81- 100%) hydrolysis gave 6 neatly. This approach is reliable The total conversion rate in 4 stages is 45 to 50% to give 6, and the necessary E-5 is further removed. Advantage that the intermediate 6 of the pathway can be purified and characterized showed that. Free phenol 6 was protected to give its benzyl ether 12 ( 100%) (FIG. 2).   At this stage, 12 is easily treated with CuCN in refluxing DMF In a very clean and efficient reaction, C6 nitrile was introduced to give 13 (96%) (FIG. 3). After hydrolysis of the ethyl ester, 14 was converted to Shioiri-Yamada reagent (1.2 equivalents D PPA, 1.2 equivalent ET_ThreeAcetic acid with N, t-BuOH, reflux, 14 hours, 87%) To give 15 by Curtius rearrangement. Optimizing this latter reaction Using strictly dried t-BuOH and dilute (0.005M) or moderately dilute ( 0.025M), and adding 4Å molecular sieve to reduce the anhydrous reaction conditions. It can be seen that maintaining it can significantly reduce the production of symmetric urea. won. Preliminarily reversing the order of the 12 to 15 conversion stage, The ethyl ester is converted to the corresponding t-butyl carbamate and then the C6 The experiment of substituting bromide to give 15 failed. 15 with low temperature acid catalyst Bromination of C4 (1.2 equivalents NBS, catalyst H_TwoSO_Four, THF, -60 ° C, 4 hours, 87 %) To give 16 and the sodium salt of 16 (1.3 eq NaH, DMF, 25 ° C, 30 min) with 1-bromo-3-methyl-2-butene (3 equivalents, DMF, 0-25 ° C, (12 hours, 99-100%) to give 17. Careful reaction conditions 17 low-temperature ozonolysis, immediately reduce the crude ozonide, Me_TwoS), aldehyde 18 was obtained (91%). The reaction time becomes longer or excessive O_Three Failure to immediately dissipate resulted in faster production of more oxidized products. Bi Nyl ether 19 was introduced (74%) in THF at low temperature in Ph._ThreeP = CHOTHHP And then reacting with 18 in THF-HMPA to maintain the reaction time Was found to be the most effective. 19 to Bu_ThreeProcessing with SnH (2 Equivalent, 0.2 equivalent AIBN, C₆H₆, 80 ° C, 2 hours, 98-100%), clean Cyclization of 5-exo-trig allyl radical-alkene gives excellent yields 20 was obtained. The THF was then deprotected (100%) and the salt was removed from primary alcohol 21. (100%) and deprotection of the phenol (100%) to give 23. 23 was treated with NaH (3 eq, 0 ° C., 30 min, 95%) and deprotected by acid catalysis (4M HCl-EtOAC, 25 ° C., 30 minutes) and spirocyclization to give N-BO. C-CCBI (25) was obtained and then the crude indoline hydrochloride 24 was 5% NaHCO_Three-Treatment with aqueous THF solution (1: 1, 25 ° C, 1.5 hours, 10 hours) 0%) and CCBI (26) was obtained cleanly. Especially because of the high acidity of phenol , 23 with 5% NaHCO_Three-Simple treatment with an aqueous THF solution (1: 1, 25 ° C., 9 hours, 100%) without the ensuing hydrolysis of active BOC occurring Spirocyclization gives 25. This approach to 25-26 uses an inert, non-functionalized acceptor. 5-exo-trig allyl radical-alkene of substrate 28 containing an alkene Performing the cyclization Tempo trap further shortened and improved it. N at low temperature By treatment with IS, 15 C4s can be selectively iodinated with an acid catalyst to give 2 7 (1.5 equiv. NaH, DMF, 25 ° C., 2 hours) with allyl bromide (5 equivalents, DMF, 25 ° C., 2 hours, 92%) to give 28. 2 8 in benzene in the presence of Tempo (5 equivalents)_ThreeSnH (5 equivalents, 60 ° C, 1.5 ~ (2 hours) to give a clean 29. Zn (80 equivalents, 3: 1: 3 THF-H OAc-H_TwoO, 70 ° C, 7 hours, 73%) to reductively open 29. It was split to give 21 (FIG. 4).   Resolution of the final synthetic intermediate was performed on a Chiralcel-OD semi-preparative HPLC column (2 × 25%) using 7% i-PrOH-hexane eluate (7 mL / min), α = 1.38 Was carried out by chromatographic separation of the 22 enantiomers. to this Thus, 22 enantiomers are always obtained at 97% recovery over 99.9% ee Obtained. Intermediate 22 is the only one that can be effectively resolved on a Chiralcel-OD column. It was found to be a final intermediate, 21-fold with N-BOC-CCBI itself. Did the same for 23 but was unsuccessful. 22 enantiomers The benzyl ether was removed under the conditions of catalytic hydrogenation to give an optically active agent 25-26. And 34-39. The absolute configuration is the final drug 25, 26, 33, Provisionally assigned based on the rotations of 37 and 39. A strong positive rotation is all dense Similar to those clearly established in the relevant and subsequent biological studies Assigned to the natural configuration. Most notably, conventional stereochemical assignment Distinct DNA libraries of natural or unnatural enantiomers that have been clearly established The killing selectivity characteristics were found to be consistent with the initial assignment. See Table 1 below Shine Table 1 Chromatographic resolution^a Drug solvent α   21 3-7% i-PrOH / hexane 1.09             Slope   225 5% i-PrOH / hexane 1.33   22.7% i-PrOH / hexane 1.38   23 2 or 3% i-PrOH / hexane 1   25 15, 25 or 30% 1i-PrOH / hexane ^aAnalysis 4.6 × 250 mm, 10 μm     Chiralcel-OD column, 1mL / min   CCBI-TMI (35), CCBI-indole_Two(37) and CCBI- CDPI (39) As shown in FIG. 5, CC-1065 and zuocarmycin analogs 34-39 A CBI alkylated subunit was introduced. 23 is deprotected with an acid catalyst (4 M HCl-EtOAc, 25 ° C., 30 min, quantitative), 5,6,7-trimethoxy Indole-2-acetic acid (30, 3 equivalents EDCI, DMF, 25 ° C, 14 hours, 8 5%), 31 (3 equivalents EDCI, DMF, 25 ° C., 16 hours, 87%), and CD PI₁(32, 3 equivalents EDCI, DMFN 25 ° C, 16 hours, 81%) with base Without adding, coupling 24 indoline hydrochlorides, 34, 36 and 38 were obtained respectively. Interestingly, drug 24 is the previous drug It is found that coupling is harder than that of the strain (eg, CDPI_TwoIn) The slow coupling reaction was not successful. These slow coupling reactions are Physically, acetic acid is difficult to dissolve even in DMF. Has the disadvantage of a competitive ring closure to CCBI (26). Partly, about 24 This difference is attributed to the deprotection and increased acidity of the phenol that competes with the coupling. This may be due to spiro cyclization becoming easier. Then the coupled drug The agent is treated with NaH (3 eq, 20% DMF-THF, 0 ° C., 30 min) In conversion, CCBI-TMI (35, 99%) and CCBI-indole_Two(37 , 66%). More remarkably, 36 was replaced by 3% NaHCO_Three-TH F (1: 1, 25 ° C., 3 hours, 68%) or 38 to DMF-H_TwoIn O ( 5: 2, 25 ° C., 9-10 hours, 69%) KHCO_ThreeSimply exposure to 37 and 39 were obtained without hydrolysis of the active amide.   In vitro cytotoxic activity. Comparison of cytotoxic properties of CCBI-based drugs. The results of the study are shown in FIG. 6 alongside the corresponding CBI and MCBI agents. Preliminary research The natural enantiomers of CCBI-based drugs are among the strongest in the CBI system It was found to show a high cytotoxic activity (FIG. 6).   The drug, consistent with previous observations, has a narrow range of reactivity tested in the system. Indicates the chemical stability (-logk) and in vitro cytotoxicity titer (L1210, log 1 / IC₅₀) Follow a linear relationship. This is shown in FIG. The body was shown. This is probably due to the more efficient provision of more stable drugs. Due to feeding, the intracellular target and the rate of solvolysis are dependent on the relative function of the drug. Would accurately represent the typical reactivity / stability.   Less obvious, but more fundamentally, from its observations, the in vitro cytotoxic activity And Hammett σ of C7 substituent having CCBI which is the most effective drug_pRelation to constant Was found to follow a predictable linear relationship (FIG. 8). This basic relationship is It is shown to be useful for designing new analogs with further enhanced properties.   The latter two correlations are limited to comparisons among the three currently available drugs, They correspond to 44-49 (FIG. 9) and all structural analogs related to them (FIG. 10). ), Following the trends established in the experiments on Carry. In particular, 25 is the most stable and easiest to synthesize alkyl disclosed so far. One of the subunits.   As with conventional observations, the corresponding seco precursors 23, 34, 36, and 38 are drug-containing It showed cytotoxic activity indistinguishable from cyclopropane.   Selectivity and Efficiency of DNA Alkylation The DNA alkylation properties of drugs are w79 4 tested within double DNA, ie a 144 base pair segment of double DNA, The comparison results can also be used for related drugs. Identification and reaction of alkylation sites Evaluation of reaction selectivity in possible sites is based on a single 5 'end labeled double D This was done by thermally induced strand scission after irradiation with NA. Then, the end-labeled double DNA is Treatment was performed over a range of agent concentrations to remove unbound by EtOH precipitation of the DNA. So DNA was re-dissolved in a buffered aqueous solution, and the DNA Strand breaks at the site of killing, according to Sanger dideoxynucleotide sequencing standards Denaturing high resolution polyacrylamide gel electrophoresis (PAGE) and autoradiography Raffy identified DNA cleavage and alkylation sites. As described herein DNA alkylation reaction observed under incubation conditions for selected drugs The selectivity is shorter at longer or longer reaction times or at different temperatures (37 or Same as the alkylation selectivity observed when performed at 4 ° C, 0.5-7 d). I knew there was. As described below, the rate of DNA alkylation and the final relative The efficiency, which was not the target efficiency, was changed by changing the reaction temperature.   DNA alkylation properties of (+)-and ent-(-)-N-BOC-CCBI   DNA alkylation properties of both enantiomers of N-BOC-CCBI (25) A tabular comparison was made between both enantiomers of N-BOC-MCBI (40) in w794 DNA. This is shown in FIG. N-BOC-CCBI (25) and N-BOC-MC No essential difference was detected with BI (40). For both natural enantiomers , 10^-3M (37 ° C., 72 hours)^-2Manifest in M Significantly, the rates of DNA alkylation can be compared, with both being the corresponding unnatural enantiomers. Just a little (1-2 times) higher. This means that the DNA alkylation rate is ( +)-N-BOC-CBI (41) but similar to N-BOC-CBI non-natural It was shown to be distinguishable from the enantiomer: (+)-N-BOC-CBI / e nt-(-)-N-BOC-CCBI (5 to 10 times) vs. (+)-N-BOC-CCBI / Ent-(-)-N-BOC-CCBI and (+)-N-BOC-MCBI / en t-(-)-N-BOC-MCBI (1-2 times). Found in 41 enantiomers, This difference, not seen in 40 or 25, is due to the relative cytotoxicity titers of the drugs. But it was accurately reflected. That is, both N-BOC-CCBI (4 times) and NB The unnatural enantiomer of OC-MCBI (2 ×) is N-BOC-CBI (11 ×) Is closer to the titer of the corresponding natural enantiomer than to the cytotoxic titer of . As examined with the BOC derivative described above, 25 two enantiomers are DNA , 10^-2-10^-3M (37 ° C., 24-27 hours) 9 (10^FourTimes less efficient than two base pairs AT-rich 34 to 39 showing killing selectivity (5'-AA> 5'-TA) show no selectivity And for the same site alkylation. Alkylate the same site The unusual behavior of these two enantiomers is similar to previous observations. It Is the reversible bond orientation of the two enantiomers and the alkylated adenine Diastere of the two adducts that will cover exactly the same binding site around the It is a natural consequence of an omnipresent relationship.   CCBI-TMI (35) and CCBI-indole_Two(37), and CCBI- CDPI₁Natural enantiomeric DNA alkylation properties of (39) 35, 37; And 39 for DNA alkylation by natural enantiomers, w7 (+)-Duocarmycin SA (2) and (+)-CC-1065 in 94 DNA Shown in FIG. 12, side by side with (1), is a typical It is a comparison. (+)-Duocarmycin SA (2) and (+)-CCBI-TMI ( 35) are indistinguishable and the two agents have the same selection for DNA alkylation. And efficiency. This is better illustrated in FIG. That is, these two The drug is 10^-6-10^-75′-AATTA at M (25 ° C., 24 hours) Of the same high affinity site can be detected. This means that Our studies on carmycin SA (2) and CBI-TMI or MCBI-TMI Similar to the observations made in previous comparisons.   Each alkylation site detected is located at three base pair sites according to the following preferences: Adenine following two 5'A or T bases: 5'-AAA> 5'-TTA > 5'-TAA> 5'-ATA. Shorter drugs CCBI-TMI and CCBI -CDPI₁Has a higher priority, but the fourth 5 'base is A or Does not absolutely require T vs G or C, and this preference is due to the high affinity site pair Distinguish many of the low affinity sites (eg, 5'-AAAA). Longer drug, CCB I-indole_TwoRegarding (1), not only has a higher priority, but also The ancestry is extended and also includes the fifth 5'A or T base (e.g., 5'-AAAAAA) . Thus, similar to the above drug, the CCBI-based drug is a 3 'adenine N At the 3 alkylation site, 3 'covering 3.5 or 5 base pairs in the minor groove → AT-rich adenine N3 alkylation, which starts by binding to the drug in the 5 'direction Showed selectivity.   CCBI-TMI (35), CCBI-indole_Two(37), and CCBI-C DPI₁NA-alkylation properties of the unnatural enantiomer of (39). CCBI A typical comparison of DNA alkylation by unnatural enantiomers of base drugs Unnatural enantiomer of carmycin SA (1) and CC- in w794 of DNA It is shown in FIG. 13 along with the natural enantiomer of 1065 (1). CBI based drugs Some important findings similar to those found in our previous studies on This was also done for I-based drugs. First, a non-natural enantiomeric DNA alkyl The conversion is fairly slow and from the results shown in FIG. 13 for the unnatural enantiomer, A 25 ° C. incubation (24 hours, FIG. 12) for the natural enantiomer In contrast, it was obtained only by incubation at 25 ° C. (72 hours). More intense anti Even at conditioned conditions (37 ° C.) or longer reaction times, unnatural enantiomers The degree of alkylation does not require detectable high concentrations of drug. This D A striking difference in the rate and efficiency of NA alkylation is due to the small drug CCBI-T Most prominent and easily recognized for MI (35) and duocarmycin SA (2) But medium-sized drug CCBI-CDPI₁(39) and CCBI-India Rule_Two(37) is inconspicuous. This trend is apparent for enantiomeric pairs. As observed in the relative cytotoxicity titers.   ent-(-)-CCBI-TMI (35) and ent-(-)-zuocarmycin The selectivity and efficiency of DNA alkylation observed for SA (2) is due to the latter drug. The agent is a little efficient and almost indistinguishable to some extent. This observation is based on (-)-MCB Similar to our previous comparison for I-TMI, except that (−)-CBI-TMI Unlike that for, the difference is much greater (10-fold). Larger drugs I.e., (-)-CCBI-indole_Two> (-)-CCBI-CDPI₁> (−) − Like CCBI-TMI, it is efficient in alkylating DNA, Even at 72 hours of incubation at room temperature, more effective agents are The effect observed for the enantiomer was not met. This is (+)-CC-1 About 065 (1)^-6CCBI unnatural of unreacted DNA observed in M This is better illustrated in FIG. 13, which shows a comparison for the entire set of enantiomers. Ma Also, selectivity of DNA alkylation of CCBI-based drugs with unnatural enantiomers No differences were observed for. Each alkylation site is almost always A Or adenine adjacent to both sides of the T base. The preference for the h-site is 5'-AAA> 5'-TAA> 5'-AAT> 5'- TAIt was T. For shorter drugs, the second 3 'base is A or T (eg, For example, 5'-AAAA) with a strong preference, and for larger drugs, 3 The same applies to the third 3 'base (e.g., 5'-AAAAA). Therefore, non-natural 2. Each alkylation site of the enantiomer surrounds the alkylation site within the minor groove. Crosses the 5 or 5 base pair AT-rich site with the drug in the opposite 5 '→ 3' direction. It was found to be consistent with the binding adenine N3 alkylation. This is a choice Is extended in the opposite 5 ′ → 3 ′ direction in the minor groove, and the diastereomer of the adduct is Are offset by one base pair against the natural enantiomer Other than that, it is similar to the alkylation selectivity of the natural enantiomer.   Kinetics of DNA alkylation CCBI-TMI (35) and CCBI-indigo Le_TwoRelative rates for DNA alkylation of the natural enantiomer of (37) versus The relative velocities of CBI and MCBI drugs were determined using w794, 5'-AATT.AOnly in Measured at high affinity sites. MCBI-TMI, CBI-TMI, and Zuo In a previous study of the relative rates for carmycin SA DNA alkylation, 4 ° C. Was found to be almost the same as 1.8: 1.0: 0.9, respectively. Main research institute In a similar study, similar relative velocities were observed including CCBI-TMI (35), The latter drug has the highest relative velocity (10^-6, 25 ° C) CCBI-TMI (2.5 times)> MCBI-TMI (1.9 times)> C BI-TMI (1.0)> Duocarmycin SA (0.9-fold) (FIG. 14). CCBI -Indole_Two(2.5x), MCBI-indole_Two(1.4 times) and CBI Ndore_TwoThe relative speed of (1.0) at 25 ° C. was measured and showed almost the same tendency. Although difficult to distinguish because the study is limited to a very narrow range of reactivity, The rate of alkylation is related to the relative reactivity of the drug to acid-catalyzed solvolysis. I could not attach. Is this due to other or additional factors due to speed? Or, it suggests that it depends on the catalyst of the DNA alkylation reaction. In previous studies, Are strongly aware of similar observations for drugs that cover a wider range of relative reactivities. I've adjusted it.   More interestingly, the final DNA alkylation observed under this reaction condition The relative efficiencies closely followed the relative reactivity trends of the drug. The more chemically stable CCBI-based drugs 35 and 37 are essentially distinguishable. Archiving DNA 1.5 to 2 times more efficiently than MCBI or CBI based drugs Has been converted to Almost the same tendency, but the stability is very close to 1.06 to 1.2 times, Relative of drugs with CCBI 1.6-2.1 times more stable than BI or MCBI Observed in stability. These observations are consistent with our previous studies, It is not the rate of DNA alkylation that is related to the cytotoxicity titer of The ultimate efficiency of A-alkylation will be more related to the development of its biological properties Suggest that Just as important, the other factor is the DNA alkylation reaction. And other or additional features include the protonation of C4 carbonyl or It also suggests that it may depend on the catalyst, beyond complexation of the isocyanic acid. this The most common of these possibilities is that DNA binds and changes conformation And destabilization of the ground state resulting from activation of the drug for nucleophilic addition Activation. Research addressing these issues is ongoing, An interim report will be made.   Conclusions A detailed description of the short and efficient synthesis of CCBI and its intermediate precursors I do. Considering that, the effect of substituents on the chemical and functional reactivity and collision of the drug About the electronic effects and the effects this would have on the biological properties of the drug. Was evaluated correctly. did. This effect is due to the N-BOC-CCBI Research on the comparison of the reactivity with its related substances has revealed a strong electron-withdrawing property. The C7 cyano group slows down solvolysis, but its effect is very small. Was shown. According to the classic Hammett's quantification of the effect, p (-0.3) is significantly Exceptionally small electronic effect of C7 substituent on functional reactivity Is represented. Further thermodynamic studies show that the protonation of C4 carbonyl is It is not the rate-limiting step of rubolysis or acid-catalyzed nucleophilic addition, but rather it is fast and reversible , Then slow-limiting, cyclopropane requiring the presence and help of nucleophiles It was found that nucleophilic addition to_N2). This is the alkyl of DNA Rather than protonating the C4 carbonyl or forming a Lewis acid complex, The positioning of the available nucleophile (adenine N3) is dependent on the sequence of DNA alkylation. This suggests that it is the rate-limiting step in controlling decision selectivity. Of this solvolysis The exceptionally small electronic effect on velocity has no effect on the regioselectivity of solvolysis No effect, stereoselective control of activated cyclopropane to the least substituted carbon The observed nucleophilic addition was seen exclusively. Consistent with previous studies, stable solvolysis Shows a linear relationship between sex and cytotoxicity titer, predictable relationship with Hammett's substituent effect Was attached. For the natural enantiomer, very little effect on functional reactivity The electronic effect had no effect on the selectivity of DNA alkylation. The range of reactivity measured for drugs is very narrow and difficult to distinguish, The efficiency, not the rate of NA alkylation, is related to the relative reactivity of the drug, Of the most stable drugs are the most efficient at alkylating DNA and the most potent It was found to confer cytotoxic activity. In unnatural enantiomers, C7 A similar effect of the ano group was detected, indicating that the corresponding CBI-based non-natural 4 to 10 times more effective than the enantiomer and the corresponding CCBI natural enantiomer -4- to 70-fold lower.Example 2: CC-1065 /'s Okaru mycin and bleomycin A ₂ hybridized Agent: DNA alkylation promotion by attachment to non-complementary DNA binding subunit Lack of progress   CBI of CC-1065 and duocarmycin DNA alkylation subunit Bleomycin A linked to an analog_TwoContaining a C-terminal DNA binding region of Brid agents 105 to 111 were prepared and evaluated. The drug is DNA alkylated With little or no efficiency, and in some cases, when combined, The properties are reduced compared to the single alkylated subunit itself. Further The DNA drug alkylation selectivity of the obtained drug (5'-AA> 5'-TA) Is C Selectivity of a single derivative of the BI alkylated subunit, such as N-BOC-CBI And was found to be identical. Therefore, bleomycin A_TwoTo the DNA binding domain Ligation does not alter this inherent DNA alkylation selectivity, and bleomycin A_Twoof It is not reflected in the selectivity of DNA binding or cleavage, and it does not reflect CC-1065 or Is the larger 5- or 3.5 base pair A observed respectively with duocarmycin. It was not reflected in T-rich selectivity. Similar to these observations, 105-111 Cytotoxic properties of the CC-1065 / zuocarmycin alkylating subunit , Such as N-BOC-CBI.   Bleomycin is a family of clinically effective glycopeptide antitumor antibiotics -Bleomycin A_TwoIs the main component (FIG. 15). They are metal ions and Mediates oxidative cleavage of double DNA or RNA by oxygen-contributed processes It is generally believed that the ability elicits a therapeutic effect. Sulfonium mosquito Bleomycin A containing thione and bithiazole_TwoC-terminal trinucleotide The tide S subunit provides most of the DNA binding affinity (K_app= 0.26 to 1 . 0x10^FiveM^-1), While the adjacent erythro-hydroxy-L-histidineamino The linking amino-terminal pyrimidbraminic acid subunit is a metal chelation / Oxygen activates and recognizes polynucleotides. N-BOC di-, tri-, te DNA binding properties of S and related structures of tiger and pentapeptides (K_app= 0.1, 0.26 and 0.23 × 10^FiveM^-1) Determines the size of those distinct binding sites (2.2, 3.6, 3.7, and 3.8 base pairs of 4.2 to 104) The agent is a conformation that is curvedly bound to tripeptide S completely bound to DNA. The tripeptide S-tetrapeptide S bond to the peptide backbone It suggests that it has taken inversion and swivel points. Oligonucleo Bleomycin A bound to a cleavage site in the tide_TwoThe structure was determined by NMR. The basic tenant of the unstructured model was specifically found (FIG. 15).   In this example, we used CC-1065 / zuocarmycin and bleomycin. Preparation and evaluation of hybrid agent systems 105-111 for . The hybrid agent is a CB of the former natural product DNA alkylation subunit. Incorporating I analog, bleomycin A_TwoC-terminal di- and tripeptide S DN of It is linked to the A-binding region (FIG. 16). Drugs 105-111 are CBI alkylated Subunits are prepared in an equivalent manner for preformed cyclopropane-containing drugs. Incorporates precursors known to be used. Very interested in the obtained drug By examining 105-111 in addition to the deep biological properties, CC-1065 / Alkylated subunits of duocarmycin and / or bleomycin A_Twoof Gain deeper insight into the unique recognition of C-terminal unique polynucleotides Was expected. (FIG. 16).   Preparation of 114-116: unlinked CBI-based drug di- and tripeptide S For direct comparison with the linked drug, CBI drugs 114-116 were prepared and all acyl Group to convert the CBI precursor into bleomycin A_TwoBinds to the DNA binding region of Was. 112 was acid-catalyzed deprotected (3.6 N HCl-ETOAc, 25 ° C., 30 minutes) And then 113 with ethyl oxalyl chloride (2 equivalents, 3 equivalents NaHCO_Three, (25 ° C, THF, 2 hours, 95-100%) for direct coupling and clean 11 4 was obtained (FIG. 17). Similarly, freshly formed 113 is converted to ethyl succinyl chloride. (1 equivalent, 2.5 equivalents NaHCO_Three, THF, 25 ° C, 1 hour, 95-100%) 115 directly with 5% NaHCO_Three-THF aqueous solution (1: 1, 25 (9 ° C, 9 hours, 79%) to give 116. This is illustrated in FIG. Although only the enantiomer system is shown, both enantiomers of drugs 114-116 are Prepared for comparative testing. (FIG. 17).   Preparation of 105-107: dibeptide S using a rigid dicarbonyl linking group The first system of CBI-prepared drugs linked to the CBI alkylation subunit The precursor was linked to dipeptide S by a rigid dicarbonyl linking group. 5 to 107 (FIG. 18). 117 is converted to ethyl oxalyl chloride (2 equivalents, D (MF, 25 ° C., 20 hours, 72%), and 118 was added with ethyl ester. Hydrolyze (5 eq, LiOH) 3: 1: 1 THF-CH_ThreeOH-H_TwoO, 25 ° C, 2 Time, 100%), making sure that no added base is present, 113 was directly coupled with 113 (1.5 equivalents EDC, DMF, 25 ° C, 1 7 hours) 105 was obtained neatly. When exposed to air for a long time during the purification process of 105 Indicates that the corresponding sulphoxide is separated in considerable amounts (20-25%) and confirmed solely Was done. This interesting variant of 105 and 107 is bleomycin A₁DNA of The binding region, a naturally occurring minor component of the bleomycin family, is specifically illustrated. You. S-methylation of 105 (100 equivalents CH_ThreeI, DMF, 25 ° C, 67 hours , 100%) to give a clean sulfonium salt 107 (FIG. 18).   Preparation of 105-107: Dipeptide S with flexible 4-carbon linking group Rigid and very unstable dicarbonyl linkages of linked CBIs 105-107 Since the linking group was found not to specifically affect the properties of the drug, the CBI The precursor of the alkylated subunit is separated by a flexible four-carbon linking group 108 and 109 were prepared by coupling to peptide S (FIG. 19). Raw without optimization The freshly formed 113 is replaced with t-butyl, taking care that no added base is present. Direct coupling with hemisuccinate (3 equivalents EDCI, DMF) at 25 ° C, 2 1 hour), 120 was obtained. 120 was deprotected with an acid catalyst (HCO_TwoH, 25 ° C, 3 H), the crude carboxylic acid 21 (121?) Is re-added with no base present. Carefully couple with 17 (117?) (3 equivalents EDCI, DM F, 25 ° C, 17 hours, 40%), 108. Then 108 is S-methyl (100 equivalent CH_ThreeI, DMF, 25 ° C, 144 hours, 100%), clean To give 109.   The first 117 couplings were alternatively performed with succinic anhydride (2.5 equivalents, Medium CoCl_Two, 2 equivalents i-Pr_TwoNEt, CH_Three(CN, 25 ° C, 58 hours, 70%) Produced the resulting carboxylic acid, taking care that the added base is not present Coupling with all 113 to produce 108 is a competitive intermolecular imino Failure due to lactone formation.   Preparation of 110 and 111: Tripeptide using flexible 4-carbon linking group The final drug system prepared for the CBI test in conjunction with S The precursor of the alkylated subunit is converted to a 110 and 111 coupled to the node S. A detailed description of 108 and 109 As with the approach, 120 was deprotected with an acid catalyst (HCO_TwoH, 25 ° C, 1.5 hours ), 121 coupled directly with 122 (2.5 eq EDCI, 1.1 eq H (OBt, 25 ° C., 47 hours, 65%), and 110 was obtained neatly (FIG. 20). Then , 110 are S-methylated (100 equivalents CH_ThreeI, DMF, 25 ° C., 88 hours, 100%) and 111 was obtained neatly.   In vitro cytotoxic activity FIG. 21 shows drugs 105-111, simply linked Samples 114-116 for CBI acylated with a group, and natural products 1- The L1210 cytotoxic activity for representative additional comparative samples including Shown. Comparative drugs 114-116 are consistent with previous observations and are simple N-acyl CBI derivatives are in the range of 200-5 nM over natural or modified CBI-based analogs. Around 10^Three-10^FourShow twice the potency and the natural enantiomer is the corresponding non-natural enantiomer It was found to be 2-50 times more potent than the enantiomer. As with previous observations, The difference between the cyclic precursor 115 and the corresponding drug 116 containing cyclopropane is I couldn't see it. Interestingly, the 115 and 116 natural enantiomers Shows low nM cytotoxic activity (5-6 nM IC₅₀L1210), they are now The most potent simple derivatives disclosed up to, for example, 123-127 (FIG. 22) It is counted in.   In sharp contrast, bleomycin A linked to drugs 114-116^TwoNo -Or agents 105-111 incorporating the DNA binding region of tripeptide S , 114-116 more than itself^Two-10^ThreeLower cytotoxic activity than natural products To 10^Five-10⁶It showed a low titer. Only 105 two enantiomers Is close to the cytotoxic titer of 114, and its corresponding CBI building block and its structure Bleomycin A_TwoIncorporates the least essential components of the DNA binding domain Represents the structure of the system. The remaining drugs had essentially reduced properties.   DNA Alkylation Properties The DNA alkylation properties of drugs 114-116 are It turned out to be similar to the properties of -BOC-CBI (Figure 23). Within w794 DNA And the drug is 10^-1-10^-3M concentration, 10 from 1-3^Three-10^FourWith twice the efficiency , Alkylating DNA and irrespective of its absolute configuration the same site (5 ' -AA> 5'-TA) Was alkylated. As well as their relative cytotoxicity titers , Natural enantiomers are about 10 times more efficient than the corresponding non-natural enantiomers there were. Further consistent with its cytotoxic properties, the 116 natural enantiomers Alkylation efficiency of DNA is 10-100 times higher than N-BOC-CBI (123) It was efficient, and 114 was also found to be more efficient. Therefore, combined The linking group did not reduce, but did enhance, DNA alkylation.   In contrast, the first set of hybrids tested, including 105-107, lost Lost, high drug concentration 10^-2Even under vigorous reaction conditions at 37 ° C (72 hours) No detectable thermosensitive DNA alkylation could be found. medicine Similar to the relative cytotoxic activity of the drug, the CBI alkyl When the ligated subunit is linked to the thiazole C-terminus of bleomycin, As a result, the adenine N3 alkylation properties of 1-3 were reduced. Similarly, flexible Methyl sulfide 108 incorporating a novel four-carbon linking group, and non-methylated di- And the methyl sulfide 110 incorporating the C-terminus of tripeptide S^-3M The following concentrations did not alkylate the DNA and did not produce thermally active adducts. This is better illustrated in FIG. 24 with respect to 108, where both N-BOC-CBI The enantiomer is 10^-2Alkylate the DNA at M, but Does not respond at all. Flexible 4-carbon linking group and fully functional Only drugs 109 and 111 incorporating the C-terminus of the di- and tripeptides S Showed a thermally active DNA alkylation reaction, but only slightly more effective than 116 It was just. Furthermore, in order to detect alkylation, vigorous reaction conditions (37 C, 48-72 hours), the reaction time needs to be extended, and N-BOC-CBI and 116 (5'-A).A, 5'-TAProceed with) did. Thus, the drug has better properties than 105-107 or 108 and 110 But only similar to 115 and 116. Therefore, their DNA The killing efficiency is bleomycin A_TwoDepending on the ligation to the C-terminus, Not enhanced, the selectivity of the intrinsic DNA alkylation did not change.   Drugs with calf thymus DNA were treated under similar conditions (37 ° C., 48-72 hours). , 1:51 drug: base pair ratio) Incubate and extract (105,106, 108 and 110) or DNA precipitation (109 and 111) As a result of collection and observation, DNA alkylation ability was It was confirmed that no thermally active adduct could be produced (FIG. 2). 5). 105 and 106 enantiomers and 108 and 110 natural enantiomers Under long and vigorous reaction conditions (37 ° C., 72 hours) in the presence of excess DNA , Was almost quantitatively recovered from the DNA reaction mixture. 10 Only 9 and 111 showed a recognizable covalent bond with calf thymus DNA and were sequenced. This was consistent with the moderate DNA alkylating power observed in the study of the above.   Thus, bleomycin A specific to CC-1065 and duocarmycin_Two Ligation of the CBI alkylated subunit to the C-terminal DNA binding region of The DNA alkylation or cytotoxic properties of the selected hybrid agents do not increase and decrease In some cases, it was reduced. This indicates that the conventional D of CC-1065 and related analogs The effect of the NA binding domain has a DNA alkylation and cytotoxicity titer of 10^Three-10^FourDouble This is in stark contrast to turning. In natural products or related analogs Important complementary properties of the two functions of DNA binding and subsequent DNA alkylation As shown, the results indicate that bleomycin A_TwoAnd CC-1065 / s It shows that both behaviors of ocarmycin have significant significance. Most obvious What is Bleomycin A_TwoC-terminus is CC-1065 and Does not act as an AT-rich minor groove binding region similar to the right subunit It is. Minor groove binding is bleomycin A_TwoProductive DNA binding mode for And has even been suggested to be sequence-selective polynucleotide recognition, The results are further in line with the expectations obtained from the bithiozol insertion bond. This result In the combined mode, selectively supplying the alkylating subunit to the minor groove of DNA Unexpected, in fact, would block such supplies. Further, 109 and 11 The alkylation selectivity of 1 is bleomycin A_TwoN-BOC without DNA binding region -Equal to the selectivity of CBI (123) and 116, all much more selective than 1-3 Is small. Thus, bleomycin A_TwoLigation of the DNA binding region of This component of the mycin will result in a CBI protein that will reflect somewhat in sequence-selective binding. It does not alter the selectivity of the DNA alkylation of the killed subunit, and Selectivity approaching that of the five base pair AT-rich alkylation for There was no enhancement.Example 3: 9,9-difluoro-1,2,9,9a-tetrahydrocyclopropa [C] Benzo [e] indole-4-one (F ₂ CBI) alkylated subunit And characterization of fluorocyclopropane analogs of duocarmycin incorporating thiol   9,9-difluoro-1,2,9,9a-tetrahydrocyclopropa [c] Nzo [e] indole-4-one (F_TwoCBI), that is, the reaction center in natural products CC-1065 and Zocal, which represent the most important of such agents, including substitutions For the synthesis of difluorocyclopropane analogs of the alkylated subunit of mycin I will talk about it. F_TwoThe core structure of CBI is represented by the following formula using p-quinonediazide. Difluoroalkene (74%) prepared by intramolecular metal carbenoid insertion reaction F_TwoImported into CBI-TMI (233), similar to the key of duocarmycin I got a body. N-BOC-F_TwoThe CBI (219) study of solvolysis showed that Introduction of the fluorocyclopropane substitution results in a difluoro substituted C9 cyclopropyl. Reaction without changing the inherent regioselectivity caused by nucleophilic addition to ropane carbon It has been shown that sex is increased 500 times. 217 single crystal X-ray structure analysis and C B Comparison of the X-ray structures of I and related analogs shows the cleavage reaction of cyclopropane. The structural origin of the difluoro substitution effect on the reactivity and regioselectivity of Call C-CF of cyclopropane_TwoThe -C bond angle is increased and the difluoro substituted The opposite carbon-carbon bond is longer, favoring the compressed exocyclic FCF bond angle Adaptively, producing strain energy. This enhances responsiveness and has been According to established trends, drugs should be compatible with no substitution of difluorocyclopropane. It was found to be 500-1000 times less cytotoxic than the CBI derivatives. As well In addition, diemdifluoro substitution does not affect drug DNA alkylation selectivity. , The drug undergoes the addition of specific adenine N3 to the C9 cyclopropane carbon However, it reduces efficacy according to the correlation between cytotoxicity and stability ( 675-725 times).   In this example, 9,9-difluoro-1,2,9,9a-tetrahydro Cyclopropa [c] benzo [e] indol-4-one (F_TwoCBI), ie Representative of the most important analogs containing substitution or functionalization of the reaction center in natural products Of difluoro-substituted cyclopropanes of alkylation subunits of 1 to 3 And the test will be described in detail. Typically, the anharmonic induced electron withdrawing property of the fluorine substituent and Its resonance stabilizing properties are similar to those of substrates with hydrogen substituents. With the decrease in reactivity, the regioselectivity of the resonance stabilization reaction is enhanced. These characteristics In order to complement, the size of the fluorine substituent is exactly the same as hydrogen, Although it is sterically large, it can be regarded as not hindering hydrogen substitution. But However, the fluorine substitution of cyclopropane is unique due to the effect of other halogens. Has been shown to have a significant effect. Experimentally, the effect was 4. It has been shown to increase ring strain at 5-5.0 kcal / mol, which is Reduce the bond on the opposite side of the substituted carbon by 9 to 10 kcal / mol, but It has been experimentally determined that it has a weakening effect of 0 to 2 kcal / mol without any significant effect. Follow Thus, the gemdifluoro substitution of CBI cyclopropane provides a stand for nucleophilic ring opening. It is not clear whether regioselectivity of body-selectively controlled reactions changes or increases Furthermore, it is clear that this substitution enhances or reduces the intrinsic nucleophilic functional reactivity. There is no. So we work hard and F_TwoPreparing a CBI alkylated subunit, Difluoro The effect of substitution on the structure, reactivity, and regioselectivity of the drug The effects of the 1-3 analogs on the biological properties were investigated.   N-acetyl-F_TwoCBI, N-BOC-F_TwoCBI and F_TwoSynthesis of CBI F_TwoThe synthesis of the CBI nucleus is shown in FIG. Here, quinonediazide and 1,1-diph CC-1065 Using Insertion of Key Intramolecular Metal Carbenoids into Fluoroalkenes The retrosynthesis of the alkylated subunit of   204 (1.3 eq NaH, DMF, 0-25 ° C.) 14 h, 97%; Boger, D.L. Yun, W .; Teegarden, B.R.J.Org. Chem. 1992, 57, 2873) Alkylation with lomo-3-methyl-2-butene gave 205 neatly (FIG. 28). While carefully monitoring the reaction conditions, the low-temperature ozonolysis of 205 followed by By reducing the crude ozonide, (Me_TwoS), when oxidation progresses Aldehyde 206 was obtained in spite of the reaction time being slightly exceeded. The Fluoroalkenes follow a three-step procedure developed by Sabol and McCarthy. Introduced. Α-lithiophenyldifluoromethylsulfone (in the presence of 206 LHMDS, THF-HMPA, -78 ° C) to produce β-hydroxy Sulfone 207 was obtained (51%, Stahly, G.P.J.Fluorine Chem. 1989, 43, 53. ler, T.G .; Thanassi, J.W.J. Org. Chem. 1960, 25, 2009), this reaction is difficult to optimize Met. Produce α-lithiosulfone and then add aldehyde 206 However, competitive conversion occurred due to reagent instability, and 207 was not obtained. . Finally, in situ production of α-lithiosulfone in the presence of aldehyde 206 It was found to be most convenient to carry out the reaction with Finally, the substrate was recovered (20-40%). Chromatography of 206 and 207 Due to the properties, the crude reaction mixture or following according to the rapid plug chromatography Carefully chromatograph the conversion of 207 to mesylate 208. And recovering unreacted 206 and reusing it to bring the overall conversion rate closer to 75% I did. This improved what was the only problematic stage of the synthesis. 207 to 208 (2 equivalents MsCl) and 10 equivalents Et_ThreeN, 3.5 hours, 87%), 5% Na ( Hg) (6 equivalents, 4 equivalents Na_TwoHPO_Four, CH_Three(OH, 0 ° C, 1 hour, 77%) By reductive elimination, a key difluoroalkene 209 was obtained. This latter Reaction is performed using the substrate: Na_TwoHPO_Four: Na (Hg) is optimal when the ratio is 1: 4: 6 If the equivalent number of Na (Hg) exceeds 10 equivalents, a large amount of desulfonylation The rate (20-40%) separated. 207 tosylate (Stahly, G.P.J.Fluorine  Chem. 1989, 43, 53. Miller, T.G .; Thanassi, J.W.J. Org. Chem. 1960, 25, 2009) When the treatment is carried out with Na (Hg) under the optimal conditions, 209 can be obtained at a good conversion rate. Obtained. SmI to promote reductive elimination of_TwoI tried this, but this substrate Does not work, and promotes reductive elimination by using 207 itself with Na (Hg). Nor was it productive (FIG. 28).   Diethyl (difluoromethyl) phosphonate, diphenyldifluoromethylphosphonate For the introduction of terminal difluoroalkenes, including fin oxides and related wittig reagents An alternative olefination process for this would form a competitive route to 109 (209?). I couldn't do that. Similarly, reported reactions with stronger nucleophiles From 104 (204?), Its own sodium or lithium salt With 3,3,3-trifluoropropene as S_NBy the 2 'reaction, 109 (2 09? Attempts to produce) only recovered the starting material. (References on page 44, line 28 to page 45, line 14)   To introduce quinonediazide, deprotect the benzyl ether and preferentially The acid-active protecting group had to be reintroduced. After removing it, the acid catalyst Forms an azonium salt, but still reduces the arylnitro group to an arylamine. Stable to conditions and try to prevent oxidation to p-quinone monoimine That's why. Depending on this indicator, the protecting group of N-BOC can be introduced at the correct position. Did not. Therefore, the N-BOC group is replaced with an N-acetyl group, and 211 is replaced with CF._Three CO_TwoNO_Two(2.5 to 3.5 equivalent Bu_FourHNO3, 0.002 equivalent TFAA, CH_Two Cl_Two, 25 ° C., 16-32 hours, 70%) to give a clean 212. . It is derived by C-4 nitration and the isomeric C-2 nitration product is Only small amounts (about 10%) were produced. Decompose the benzyl ether (2. 0 equivalent BBr_Three, CH_TwoCl_Two, -78 ° C, 30 minutes, 92%), 213. The enol was again protected to ethyl carbonate 214 (88%). Difluoro NaBH in the presence of alkene_Four / 5% Pd-C (H_TwoO-CH_ThreeOH) 0 ° C, 15 minutes ) To reduce the nitro. Without purifying or storing the amine 215, An acid catalyzed cleavage of the carbonate such that the intermediate diazonium salt is directly converted to 216. Under the i-C_FiveH₁₁ONO (4M HCl-CH as catalyst_ThreeOH, -30 ° C, 1 6 hours) to give the key quinonediazide 216 directly. This way Thus, the overall conversion yield from 214 to 216 is 80%, with a typical conversion of 60 %Met. The only significant by-product of this series of processes is With zimidazole 220a, its formation shortens the reduction time (15 minutes) and free Amine 215 can be minimized by not storing or purifying it before use. did it. The use of large amounts of strong acids in the diazotization reaction also reduces their formation. A little. In a further optimization of this series of routes, iC_FiveH₁₁The amount of ONO and In both cases the conversion steadily increased and eventually used as solvent, reducing the reaction time by 1 To 3 hours, and finally to 17 hours. The acid is iC_FiveH₁₁It was found to be optimal to add after ONO. Alternative (Zn, CaCl_Two, 95% EtOH). No. 4 failed, resulting in recovery of starting material (FIG. 29). ,   The use of tert-butyl carbonate 221 (FIG. 29) was also studied and Produced 222 or 216 with some conversion, but using ethylene carbonate 214 It turned out that there was no difference or advantage.   Metal carbenoid formation and insertion into difluoroalkenes should be protected from light. 216 to Rh_Two(OAc)_Four(0.1-0.2 equivalent, toluene, reflux, 0.5 hour , 74%) to give N-acetyl-F_TwoCBI (217) was obtained (FIG. 28). . Similarly, at a lower rate, Cu (acac)_TwoConverted even with catalyst (58%), both low At the reaction temperature, the conversion decreases, and the conversion decreases as the amount of the catalyst exceeds 0.2 equivalent. It seemed to be. 217 is converted to LiOH (1.2 equivalents, CH_ThreeOH, -10 ° C, 15 minutes, 92%)_TwoObtained CBI (218) . Deprotection with NaH and BOC_TwoBy the substitution reaction with O, N- BOC-F_TwoConverted to CBI (219).   F_TwoPreparation of CBI-TMI (233)   An improved analog 224 of duocarmycin deprotects 218 (1.1 equivalents Na H, DMF, 25 ° C) and reacted with 223 (DMF, 1 hour, 25 ° C, 45%; Muratake, H .; Abe, I .; Natsume, M. Teterahedron Lett. 1994, 35, 2573; recovered 64% based on the measured 218) (FIG. 30).   Solvolysis: Reactivity Previous studies have shown that two alkylating subunits The basic properties of have been found to be important. One is the cyclopropane stereo Selectively controlled acid-catalyzed ring opening, the least substituted of cyclopropane That is, the nucleophile is preferentially added to carbon. Modified alkylated sub For the CBI system of units, the stereoselective alignment of this C8b-C9 bond Are optimized and exclusive (≧ 20) with cleavage of the C8b-C9 bond. 1) Addition to the C9 center is observed. The second characteristic is the relative speed of solvolysis. Degree, which accurately describes the functional responsiveness of a drug and Stability and in vitro cytotoxicity titers follow a very clear linear relationship I know that. Thus, the difluorocyclopropane substitution is not sufficient for the reactivity of CBI. And the effect of the reaction on the regioselectivity.   217-219, pH 3 (50% CH_ThreeOH-buffer solution, buffer solution = 4: 1: 20 (v / v / v) 0.1 M citric acid, 0.2 M Na_TwoHPO_Four, H_TwoO) and And pH 7 (1: 1H_TwoO-CH_ThreeOH) UV solvolysis spectrophotometry According to F_TwoShort-wave due to disappearance of long-wavelength absorption band of CBI chromophore and ring-opening product The appearance of length was measured and evaluated (FIG. 31).   N-BOC-F_TwoCBI (219) is a significant reaction to acid-catalyzed solvolysis Showed sex. It has a half life of 0.26 hours at pH 3 (k = 7.05 × 10 5^-Fours^{- 1} ), Indicating that N-BOC-CBI (t_1/2= 133 Interval, k = 1.45 × 10^-6s^-1) Was about 500 times higher in reactivity. Sa Furthermore, 219 is the most modified alkylation subunit studied so far. It is counted as having high reactivity. Even at pH 7 where N-BOC-CBI is stable, Luvolysis (t_1/2= 2.33 hours, k = 8.27 × 10^-Fives^-1). Also, N -Acetyl-F_TwoCBI (217) and F_TwoAlso check CBI (218) 32 are summarized in FIG. For the reactivity of 217 and 219, an essential phase No differences were observed, but 218 was found to be significantly more stable than CBI (At pH 3 t_1/2= 4.2 hours, k = 4.54 × 10^-Fives^-1). this thing Is similar to previous observations, indicating that 218 is about 220 times more reactive than CBI. won.   Solvolysis: Regioselectivity 217 and 219 to CH_ThreeIn OH (0 ° C) Medium CF_ThreeSO_ThreeSingle feature quickly and cleanly when treated with H (0.1 equivalent) The resulting product 225 (30 min, 90%) or 226 (10 min, 79%) was obtained ( (FIG. 33). This is spectroscopically related to N-BOC-CBI and its related drugs. Similar to the normal solvolysis seen, difluoro substituted C9 cycloprop CH to carbon_ThreeIt can be shown that it was induced by the addition of OH. 22 5 and 226¹⁹The F NMR spectrum shows the resonance of one fluorine and both fluorines This means that the elements are magnetically equivalent. Ring expansion position isomers are magnetically non- It had an equivalent diastereotopic fluorine. In addition, 226¹H-¹³C The HMB CNMR spectrum shows that C1 (δ51.0) and CF_TwoCarbon (δ50.3, t) and C9b (δ125.0). Ring expansion position Key CF required for sex_TwoCarbon (δ50.3, t) and C9b (δ12 5.0) was not detected. This means that THF-H_TwoIn O, CF_Three SO_ThreeBy performing solvolysis with H catalyst to obtain 227 neatly (18 hours, 85%) and became clear (FIG. 33). 218 (Fig. Nucleophilic addition under basic conditions, as demonstrated by obtaining Occurs preferentially to the substituent.   Under solvolysis conditions, small amounts of unusual solvolysis products are lost. It may have been decomposed or decomposed, but from the studies described above, It has been determined that the addition to the oro-substituted C9 carbon occurs with a preference of 9: 1 or higher. . This is because CBI-based drugs add exclusively (> 20: 1) to the C9 carbon. It is similar to the observation. Thus, gemdifluorocyclopropane substitution is active Regioselection of Stereoselectively Controlled Acid-Catalyzed Nucleophilic Addition to Reactive Cyclopropane Did not change sex. Conventionally, drugs incorporating the CBI nucleus show the most regioselectivity, C C-1065 (about 4: 1), duocarmycin SA (6.5-4: 1), duocarmy For a CPI derivative or CBQ derivative (3: 2) containing syn A (4-1: 1), , A milder selectivity has been observed. A comparison made in a CBQ structural study. Thus, the observed decrease or loss of regioselectivity for a drug is two possible scenarios. May be due to the relative degree of stereoselective alignment of clopropane binding unknown. If structural information is available, the degree of selectivity is two possible Reflecting the relative degree of stereoselective alignment of cyclopropane linkages, Alone, despite the fact that 217 and 219 are very reactive, Explain that the reaction site selectivity is observed. Furthermore, it contributes to this position selectivity What is done is fluorine that can accommodate the partial positive charge that develops on C9 This is the effect of resonance stabilization of the substituent.   In vitro cytotoxic activity In preliminary studies, 217-219 and 224 (F_TwoCB The in vitro cytotoxic activity of I-TMI) was determined using racemic samples. Drug Consistent with predictions based on the relative reactivity of Was also found to be less effective 500- to 1000-fold (FIG. 34). This means that quantitative In general, the relative reactivity of drugs that show more effective activity for exceptionally more stable drugs ( 500 times), and was established in a previous study. This tendency is well followed (FIG. 35).   DNA alkylation properties F_TwoAbout the DNA alkylation properties of CBI drugs A study was conducted which showed that it behaved similarly to the corresponding CBI-based drug. won. As comparison results for past drugs are available, within w794 DNA Was tested for the DNA alkylation reaction. Alkylation site identification refers to drug After bombardment, it was obtained by thermal strand cleavage of a single 5 'end labeled double DNA. Then the drug The unbound drug is treated by EtOH precipitation of the DNA Removed. The DNA was redissolved in an aqueous buffer solution and pyrolyzed at 100 ° C (30 minutes). , Depurination and chain scission at the adenine N3 or guanine N3 minor groove alkylation sites. Prompting and denaturing high resolution PAGE and autoradiography accompanying Sanger sequencing standards. The fee provided DNA cleavage and alkylation sites.   Racemic F_TwoDNA alkylation by CBI-TMI (224) and ent- Representative comparisons with (-)-and (+)-CCBI-TMI (35) are shown in FIG. There are three important conclusions that can be drawn from the comparison in FIG. First, F_Two CBI-TMI alkylates DNA in a manner similar to CBI-TMI, And with the same sequence selectivity. No new site of alkylation detected, limiting drug Adenine N3 alkylation is detected under the condition that DNA is excessive Was done. In particular, in such sequencing studies, the highest affinity alkylation sites Only minor sites with comparable position and comparable affinity (1-0.01 fold) are detected. Many Lower affinity because it requires a much higher density of agents to promote the alkylation of No alkylation sites are detected and cleavage of the DNA to produce short DNA fragments Not observed on sequencing gel. 224 is also like duocarmycin A itself Guanine without access to or available adenine N3 sites N3 alkylation appears to be possible, but it is not a significant primary or secondary reaction. As demonstrated by a comparison of ent-(-)-and (+)-CBI-TMI, T The natural enantiomer of the CBI-based drug in the MI system is greater than the unnatural enantiomer Also found that the efficiency of DNA alkylation was 50 to 100 times higher, indicating that the CBI system (CB I, MCBI, CCBI). Racemic F_TwoCBI- By testing for TMI, we found that w794D, as shown in FIG. Governing to the extent that it contributes to the properties detected in the NA is the natural enantiomer Can be confidently suggested.   Second, F_TwoDNA alkyl of CBI-TMI and CBI-TMI itself Although there is no significant difference in the selectivity of the alkylation, the relative efficiency of DNA alkylation Differences. Consistent with both its relative stability and its relative cytotoxicity titer, F_Two CBI-TMI is 100 to 1000 times less efficient than CBI-TMI. To this, and this and F_TwoCorrelation of CBI-TMI with other biological properties , Turned out to be very accurate. This difference in efficiency is determined by densitometry. Quantification and averaging of several comparison results to give racemic F_TwoCBI-TMI is , An average rating of 675-725 times less efficient than (+)-CBI-TMI Obtained. Stable alkylated subunits (CPI, DSA, CBI, MCBI, CBI (CBI), but similar to the most reactive (CI, DA, CBQ) analogs To In contrast to CBI-TMI and other 1-3 analogs, F_TwoCBI-TMI The efficiency of the alkylation increases as the temperature decreases from 37 ° C. to 25 ° C. and 4 ° C. It turned out to be steadily higher. This is due to the non-production of drugs that antagonize alkylation. It may be due to productive competitive solvolysis. This phenomenon has been studied Only seen for the most reactive drugs given.   Finally, in these studies, the exclusive adenine N3 to C9 cyclopropane carbon was Observation of addition indicates that gemdifluoro substitution does not affect intrinsic regioselectivity It turns out that there is no contradiction with the prediction that it will be. This is about 217 and 219 Shows that the acid-catalyzed nucleophilic addition of OH occurs at C9 without ring expansion solvolysis CPI-based drugs that can be predicted based on chemical solvolysis studies And CC-1065 (4: 1 position selectivity), duocarmycin A (4-1: 1 position). Selectivity), duocarmycin SA (6-4: 1 regioselectivity), and CBQ-based drugs Drugs with lower regioselectivity, including drugs (3: 2 regioselectivity) From the previous work on the drug, the most unsubstituted cyclopropane Only adducts induced by adenine N3 addition were detected. Also this this Each study was quantified for adduct formation, and duocarmycin A (86-92%) , Zuocarmycin SA (95-100%), and CBQ-based drugs (> 75%) Thus, the regioselectivity of DNA alkylation is greater than simple solvolysis Led the observation that. Some explanations for these observations can be offered, The best of all is that the orientation of the bond (close proximity) facilitates normal adenine N3 addition. Effect) and torsional strain and steric interaction that significantly destabilize with abnormal addition Is to adopt the service first. A diagram showing these effects is shown in our earlier It has been disclosed in research and suggests that this subtle effect is of paramount importance. Obedience What, F_TwoAs for CBI-TMI, its regioselectivity for cyclopropane addition is higher. Expected to detect abnormal adenine N3 addition, albeit milder I didn't.   Conclusion F_TwoCBI, ie, the alkaloid of CC-1065 and duocarmycin Completed efficient synthesis of difluorocyclopropane analogs of the killed subunit , It represents the first of such drugs that include functionalized reaction centers in natural products I ing. The core structure is converted to the difluoroalkene using the key p-quinonediazide.  Assembled employing the Sundberg intramolecular insertion of situ generated metal carbenoids.   At the beginning of our study, fluorocyclopropane substitution is a typical To change or increase the reactivity, increase or decrease the electrophilic functional reactivity It was not clear about. When two fluorine substituents are introduced, Most unsubstituted cyclopropane carbon is substituted, and the electron To remove electrons from the electron density and stabilize the resonance. Nevertheless, the positive charge developed in the reaction center can be stabilized. Bigger Substituents reduce nucleophilic addition to the C9 carbon, but for relatively small fluorines It was difficult to assess in advance its effect on nucleophilic addition. C9 electron Reduce the potential resonance stabilization of the positive charge developing on the density and C9, The complementary electron-withdrawing property of the fluorine substituent depends on the position of the ring-opening reaction of the nucleophilic cyclopropane. It will increase selectivity. In contrast, the opposite side of the two fluorine substituents The significant effect on the bond is essentially to lengthen and weaken the C8b-C9a bond, Tentatively, the nucleophilic addition to C9a is directed again, and an unusual solute with ring expansion occurs. It could be expected to cause levolysis.   N-BOC-F_TwoCBI (219) and N-acetyl-F_TwoAcid to CBI (217) Studies of catalytic nucleophilic addition indicate that substitution of difluorocyclopropane can Difluoro-substituted C9 cyclopropyl, even though the substituents are inductively electron withdrawing Without altering the inherent regioselectivity resulting from the nucleophilic addition to the ropane carbon, It was found that the response was increased by 500 times. Maintaining positional selectivity is twofold. Stabilization of potential partial positive charge by fluorine substituent and cleavage of C8 Due to stereoselective control of the reaction such that only the b-C9 bond is arranged for the reaction will do. This is not an unaligned C8b-C9a bond, but something else. Happens despite preferentially weakening. Next, the orientation of this cyclopropane is This can be explained by the geometrical constraints imposed by the joining five loss rings.   Ground-state effects were found to be the reason for increasing reactivity. Of cyclopropane C-CF2-C bond angle increases, and cyclopropane on the opposite side of difluoro substitution The bond length becomes essentially longer, and the FCF bond angle of the preferentially compressed exo ring is reduced. And the strain energy is added,_TwoThe reactivity of CBI increases. This basal The increase in strain and reactivity due to instability of the state is similar to that seen in simple related systems. Exactly the same, not unique or F_TwoEven upset by incorporation into CBI It will not be done. In addition, it is due to the electron-attracting effect of fluorine substitution. The potential stability of can be overcome.   Consistent with this increase in reactivity (500-fold) and in accordance with previously established relationships, Quantitatively, the drug is 500-1000 times less cytotoxic than the corresponding CBI derivative. I understood. Similarly, difluorocyclopropane substitution can be applied to the drug's DNA Has no detectable effect on the selectivity for Makes an intrinsic adenine N3 addition to the lopropane carbon, which is cytotoxic / stable It was found to follow the gender correlation well, with a decrease in efficiency (675-725 times).Example 4: Synthesis of methoxy substituent of duocarmycin SA and its role Research   From the preparation and testing of 304-307, (+)-305 and (+)- SA was indistinguishable. In contrast, 306 and 307 are 304 Wherein the C6 and C7 methoxy substituents of duocarmycin SA are And little or no contribution to its properties. Therefore, The C5 methoxy substituent on the 5,6,7 trimethoxyindole subunit of SA It is necessary and sufficient to observe the total titer of natural products.   One important structural component of natural products is the rate, efficiency, and choice of DNA alkylation. And increase biological potency by 10^Three-10^FourN that has been shown to increase^Two Right subunit linked to alkylated subunit through amide . In this example, we used the trimethoxyindole-2 of duocarmycin SA. -Examine the carboxylate subunit in detail and determine its three methoxy substituents. Test with the intention of defining the significance and potential role of each. The results show that DNA is deeply buried in the minor groove during alkylation. The particular importance of the C5 methoxy substituent is clearly emphasized, and Advocate an unaware role. In comparing 1 and 304, the individual contributions for one of the three methoxy groups are Since the determination could be made between 305 and 307, 304 to 307 drugs were targeted for evaluation. Optically active duocarmycin SA alkylated subunit, N-BOC-DSA (308) to Boger et al. Am. Chem. Soc. 1992, 114, 10056; Boger et al., J. Am. Chem. Soc. Prepared by the method previously disclosed in 1993, 115, 9025. The division of 308 , A semi-preparative chiral cell OD HPLC column (10 μm 2 × 25 cm, 30% 2-pro Directly on panol / hexane, 7 mL / min, α = 1.24, ≧ 99.9% ee) Achieved by chromatographic separation. This is because of the intermediate precursor bis- (R)- Derivation of o-Acetyl Mandelate and Chromatography of Its Corresponding Diastereomer Reproduction of the pure enantiomer by hydrolysis of the mandelate ester following luffy separation (Boger et al., J. Am. Chem. Soc. 1992, 114, 10056; Boger et al., J. Am. Chem. Soc. 1993, 115, 9025), which is more efficient and convenient than the method we have previously reported. I found out. Clean addition of HCl to activated cyclopropane at 308 (4 M HCl-ETOAc, 25 ° C., 30 min, 95-100 %) To give the seco HCl salt 309 (FIG. 37). Immediately carefully remove the added base. And coupled with indole-2-carboxylic acid 310-313 (3 equivalent ED CI, DMF, 25 ° C, 4-15 hours), with excellent conversion (70-82%), pen ultimate precursors 314-317 were obtained. NaH (3 equivalents, THF-D MF 4-2: 1, 0 ° C., 30 min) spirocyclization, with excellent conversion (87-96). %), Both enantiomers of drugs 304-307. 309 couplings NaHCO_ThreeWhen performed in the presence of a weak base containing The spirocyclization reaction mixture is linked by the presence of externally added moisture. N^TwoHydrolysis of the amide will follow.   In vitro cytotoxic activity In vitro cells of both enantiomers 304-307 The cytotoxic activity is summarized in FIG. 38 together with one cytotoxic activity. To natural enantiomers In turn, removal of the three methoxy groups reduced the titer by 6.5-fold. 5-me The toxic derivative (+)-305 is indistinguishable from duocarmycin SA, and C6 and C The methoxy group of 7 shows little contribution to its properties. Like this In addition, (+)-307 has the same strength as (+)-304, and the C7 methoxy group has (+) It does not contribute to the characteristic of -1. On the other hand, (+)-306 Showing activity and enhancing the C5 methoxy substituent effect. Similar but A more pronounced effect was observed with the unnatural enantiomer. 304, 306, and 3 The unnatural enantiomer of 07 is essentially equal in strength, 1 to more than ent-(-)-1 Ent-(-)-305 is 3 to 18 times weaker, while ent-(-)-1 It was closer.   Selectivity, efficiency, and rate of DNA alkylation   Selectivity of DNA Alkylation for the Natural Enantiomers 304-307 And efficiency were compared to 1 in w794 DNA. All 5 drugs are almost equal D Demonstrates the selectivity of NA alkylation and demonstrates the rate and overall efficiency of DNA alkylation. Differences were observed. Incubation with w794 DNA at 25 ° C for 24 hours 305 is essentially indistinguishable from 1 itself, and 306 and 307 are Is 5 to 10 times less efficient than 305 (306> 307), and 304 is the most effective drug The rate was low, 20 times lower than 1 or 305 (FIG. 38). This DNA alkylation The trend for overall efficiency parallels the relative trend in cytotoxicity titers. Similarly, the relative rates of DNA alkylation for 1, 305, and 304 are also w 794 (10-5M) at 25 ° C, 1-72 hours) with a single high affinity site, 5'-d ( AATTA). (+)-Duocarmycin SA (1) and 305 1 is slightly faster (k_rel= 1.3-2.3), almost indistinguishable, Essentially 304 (k_rel= 18-33).   The C7 and C6 methoxy groups are on the outer surface of the DNA-drug conjugate, but separately It hardly contributes to the properties of ocarmycin SA (C6> C7). In contrast, minor grooves The C5 methoxy group deeply embedded in the DNA significantly affects the properties of duocarmycin SA. Give. Drugs containing one C5 methoxy substituent are duocarmycin SA and It is indistinguishable and it turns out that it is sufficient to observe the full titer of natural products. won. This means that the C5 methoxy group is located deep in the minor groove, Role to stabilize the essentially reversible DNA alkylation reaction more non-covalently Consistent with the above, the lack of such an effect for the C6 / C7 methoxy substituent indicates that Consistent with quantitative modeling studies. Furthermore, the C5 methokishi of duocarmycin SA The groups extend the rigid length of the subunit that binds to the DNA. It exists As a result, in the helical conformation of the DNA binding agent,^TwoAt the site The inherent twist increases with the helical increase in the modulated drug. This N^TwoAmi The conformational torsion in the alkylation subunits Drug-specific reactions that disrupt mid conjugates and contribute to the catalysis of DNA alkylation reactions Enhance the nature. Removal of the C5 methoxy group shortens the right subunit, Linking N in NA-binding conformation^TwoThe inherent twist of the amide is reduced Second, the drug does not work effectively on DNA alkylation.                                 Synthesis method   Ethyl 7-bromo-4-hydroxy-2-naphthalenecarboxylate (6) Method A : M-bromobenz at 45 ° C in a solution of t-BuOK (7.19 g, 64.1 mmol) in t-BuOH (100 mL) Aldehyde (4, 10.78 g, 58.3 mmol) and diethyl succinate (15.23 g, 87.4 mmol) were added dropwise. I dropped it. The reaction mixture was heated at reflux for 2 hours. After cooling to 25 ° C, the mixture % HCl aqueous solution (pH = 1) for neutralization, and t-BuOH was removed from the organic layer under reduced pressure. . The residue was extracted with EtOAc (3 × 30 mL). 5% NaHCO_ThreeAqueous solution (5 × 30mL) from organic layer The half ester was extracted. The amphoteric aqueous layer was re-acidified with 3 M aqueous HCl, and EtOAc (3 × 30 mL). Both organic layers were washed with a saturated aqueous solution of NaCl and dried (MgSO_Four). Dissolution The medium was removed to give the two isomeric half-esters 5 (13.7 g, theoretically) as an amber oil. Yielded a mixture of 18.2 g (75%).   The half-ester 5 (13.66 g) was added to 300 mL of Ac._TwoDissolved in O. NaOAc anhydride (3.93 g, 4 8.0 mmol) was added and the mixture was heated at reflux for 6 hours. Under reduced pressure, Ac_TwoRemove O, 10% Na residue_TwoCO_ThreeSuspended in aqueous solution (100 mL) and extracted with EtOAc (3 × 30 mL). Mixed Dry the combined organic layer (Na_TwoSO_Four) And concentrated. The residue was dissolved in 150 mL of 3M HCl-EtOH at 0 ° C. Dissolved and the solution was heated to 25 ° C. and stirred for 16 hours. The solvent was removed under reduced pressure. Black Matography (SiO_Two, 5 × 20 cm, 20% EtOAc-hexane) and then toluene Recrystallization afforded 6 as a pure white solid without impurity 8.   Method B: NaH (2.99 g, 74.6 mmol, 1.05 eq, 60%, dispersion in mineral oil) with Et_TwoO (3 × 20 mL) and suspended in anhydrous THF (100 mL) under Ar. Cool the suspension to 0 ° C After cooling, 10 (24.77 g, 72.3 mmol, 1.03 equivalent) was added dropwise under Ar, and the reaction mixture was added to 0%. Stirred at C for 2 hours. The solution was then added to m-bromobenz in 80 mL of THF at 0 ° C. Transferred via cannula to the aldehyde solution (4, 13.15 g, 71.1 mmol, 1 eq). Obtained The reaction mixture was stirred at 0 ° C. for 1 hour, heated to 25 ° C. and stirred overnight. Melting under reduced pressure Remove the medium and remove the residue with H_TwoO and CH_TwoCl_TwoDivided into The aqueous phase is CH_TwoCl_Two(3 × 200mL) And the combined organic layers were dried (MgSO_Four) Concentrated. The diester is SiO_Two(10% EtOAc- (Hexane) as a clean oil  90% CF_ThreeCO_TwoA solution of 11 (16.6 g, 46.5 mmol) in aqueous H (75 mL) was stirred at 25 ° C. for 30 minutes. Stirred. The solvent was removed under reduced pressure and the residue was azeotroped twice with benzene. Half-S Tell 5 NaHCO_ThreePurified by dissolving in an aqueous solution (50 mL). The aqueous phase is combined with a 10% aqueous HCl solution (pH = 1), extract with EtOAc (3 × 50 mL), then dry the combined organic phases (Na_TwoSO_Four) Concentrate to give pure 5 as a pale yellow oil, which is recrystallized under reduced pressure   Half-ester 5 (14.56 g, 46.5 mmol) was treated with Ac under Ar_TwoIn O (310mL, 0.15M) Was dissolved. KOAc (5.93 g, 60.5 mmol, 1.3 eq) was added and the reaction mixture was allowed to stand for 30 minutes. Heated to reflux. H the hot solution_TwoPoured into O (400 mL), stirred and cooled to 25 ° C. Raw Fractionate the product, CH_ThreePure 7 (collected by recrystallization from OH and free of impurities 9) 10.06 g, theoretically 15.68 g, 65%). About 7: melting point 113  The acetate 7 (15.7 g, 46.6 mmol) was dissolved in EtOH (200 mL)._TwoCO_Three(32.2g, 2 33 mmol, 5 eq.). The reaction mixture was heated at reflux for 1 hour, cooled to 25 ° C. H_TwoPoured into O (200 mL). The mixture was acidified with 10% aqueous HCl (pH = 1) and the required The product was extracted into EtOAc (3 × 250 mL). Wash the combined organic layers with a saturated aqueous solution of NaC1 And dried (MgSO_Four) And the solvent was removed under reduced pressure. Recrystallize the obtained solid with toluene To give 6. (14.62 g, theoretically 14.62 g, 10     C₁₃H₁₁BrO_ThreeAnalysis calculation: C, 52.91; H, 3.76. Found: C, 53.17; H, 3.46.   Ethyl 4-benzyloxy-7-bromo-2-naphthalenecarboxylate (12) under Ar A solution of 6 (8.04 g, 2.2 mmol) in anhydrous DMF (150 mL) with K_TwoCO_Three(5.65 g, 40.9 mmol), bromide Benzyl (5.59 g, 32.7 mmol, 1.2 equiv) and Bu_FourNI (402mg, 1.1mmol, 0.04eq) Processed. After stirring for 11 hours at 25 ° C., the reaction mixture was_TwoPour into O (200 mL), Extracted with EtOAc (3 × 200 mL). The combined organic layer was washed with a saturated aqueous solution of NaCl and dried ( MgSO_Four) And concentrated. The resulting solid was recrystallized from 5% EtOAc-hexane to give a white 12 were obtained as needles (8.36 g, theoretically 10.49 g, 80%). In addition, 2.13 g (20%) Of the recrystallized mother liquor (SiO 2_Two, 4 × 20cm, 0%    C_TwoOH₁₇BrO_ThreeAnalysis calculation: C, 62.35; H, 4.45. Found: C, 62.23; H, 4.61.   Ethyl 4-benzyloxy-7-cyano-2-naphthalenecarboxylate (13). Under Ar, a solution of 12 (9.46 g, 27.2 mmol) in anhydrous DMF (12.6 mL) was added to CuCN (2.92 g, 32.6 mm). ol, 1.2 equivalents) and the mixture was heated at reflux for 20 hours. The reaction mixture is Cool to 250 ° C and 250 mL H_TwoPour into O and swirl FeCl_Three(5.29 g, 32.6 mmol, 1.2 equivalent Volume) was added. The solution was extracted with EtOAc (3 × 200 mL) and the combined organic layers were Wash with liquid and dry (MgSO_Four) And concentrated. By recrystallization from 20% EtOAc-hexane 13 was obtained as a white solid (6.70 g, theoretically 9.00 g, 74%). Recrystallization mother liquor Matography (SiO_Two, 4 × 20 cm, 0% EtOAc-hexane), as a white solid , And 13 (2.00g, 22%, combined)     C_{twenty one}H₁₇NO_ThreeAnalysis: C, 76.11; H, 5.17; N, 4.23. Found: C, 75.96; H, 5. 42; N, 4.31.   4-benzyloxy-7-cyano-2-naphthalenecarboxylic acid (14). 260ml THF -CH_ThreeOH-H_TwoA solution of 13 (8.67 g, 26.2 mmol) in O (3: 1: 1) was LiO-H_TwoO (5.49 g, 131 mmol , 5 eq.) And the mixture was stirred at 25 ° C. for 25 h. 10% HCl aqueous solution The solution was acidified (pH <1) and the product was partially precipitated. The product is filtered And the residual aqueous phase was extracted with EtOAc (3 × 200 mL). Dry the combined organic layers (Mg SO_Four) And concentrated in vacuo to give 14 (7.94 g, theoretical)   N- (tert-butyloxycarbonyl) -4-benzyloxy-7-cyano-2-naphthylami (15). A solution of 14 (500 mg, 1.65 mmol) in freshly distilled t-BuOH (165 mL) was added to Et._Three Treated with N (0.276 mL, 1.98 mmol, 1.2 eq) and 5 g of activated 4Å molecular sieve did. Diphenylphosphoryl azide (0.426 mL, 1.98 mmol, 1.2 equivalents) was added and The reaction mixture was heated to reflux for 14 hours. The mixture was cooled to 25 ° C and the solvent was removed under reduced pressure. I left. The residue was dissolved in EtOAc and the organic layer was washed with 10% aqueous HCl, dried (Na_TwoSO_Four ) And concentrated in vacuo. Chromatography (SiO_Two, 3 × 20cm, 20% EtOAc-hexa To give 15 as a white crystalline solid (534 mg, 618 mg theoretically, 87%). : Melting point 145 ° C (white needles, 10% EtOAc-hex    C_{twenty three}H_{twenty two}N_TwoO_ThreeAnalysis: C, 73.78; 5.92; N, 7.48. Found: C, 73.20; H, 5.84 ; N, 7.23   N- (tert-butyloxycarbonyl) -4-benzyloxy-1-bromo-7-cyano-2- Naphthylamine (16). 15 (137 mg, 0.366 mmol) in freshly distilled THF (7.3 mL) The solution of was cooled to -78 ° C under Ar, and 1 μL / mL H_TwoSO_FourTreat with 10 μL of solution, After stirring for 20 minutes, NBS (78 mg, 439 mmol, 1.2 eq) was added. The reaction mixture is -60 Heated to ° C. and stirred for 4 h when the reaction was complete by TLC. Et_TwoO (7.3 mL) was added. 5% NaHCO_ThreeAqueous solution (1 × 10 mL), washed with saturated aqueous NaCl solution (1 × 10 mL), dried Dry (MgSO 4_Four) And concentrated under reduced pressure. Chromatography (SiO_Two, 2 × 20cm, 0% EtOAc-F Xan) afforded 16 as a white crystalline solid (145 mg, theoretically 166 mg, 87%). : 179 ° C dec (white needles, 10% EtOAc-hex     C_{twenty three}H_{twenty one}BrN_TwoO_ThreeAnalysis for: C, 60.94; H, 4.67; N, 6.18. Found: C, 61.12; H , 4.75; N, 6.04.   N- (tert-butyloxycarbonyl) -N- (3-methyl-2-buten-1-yl) -4-benzyl Ruoxy-1-bromo-7-cyano-2-naphthylamine (17). Under Ar, in anhydrous DMF (20 mL) A solution of 16 (1.77 g, 3.90 mmol) in NaH (206 mg, 5.1 mmol, 1.3 eq, 60% oil dispersion) And the reaction mixture was stirred for 30 minutes. The mixture was cooled to 0 ° C. Chill-2-butene (1.35 mL, 11.7 mmol, 3 eq) was added dropwise via cannula. The solution After stirring at 0 ° C. for 1 hour, the mixture was heated to 25 ° C. and stirred overnight. Add water (20 mL) and add Was extracted with EtOAc (3 × 15 mL). Wash the combined organic phases with a saturated aqueous solution of NaCl (1 × 30 mL), Dry (Na_TwoSO_Four) And the solvent was removed under reduced pressure. Chromatography (SiO_Two, 4 × 20cm, 1 0% EtOAc-hexane) provided 17 as an amber oil (2.05 g,     C₂₈H₂₉BrN_TwoO_ThreeAnalysis calculation: C, 64.60; H, 5.62; N, 5.38. Found: C, 64.51; H , 5.74; N, 5.18.   N- (tert-butyloxycarbonyl) -N- (formylmethyl) -4-benzyloxy-1- Bromo-7-cyano-2-naphthylamine (18). 22mL (0.016M) 20% CH_ThreeOH-H_TwoCl_TwoDuring ~ The solution of 17 (180 mg, 0.345 mmol) was cooled to -78 ° C. 3% O in the solution for 72 seconds_Three/ O_Two(1 (60 L / min) and aerated. Immediately add 0.81 mL of dimethyl sulfide The reaction was stopped, and the mixture was stirred at -78 ° C for 5 minutes, then heated to 25 ° C and stirred for 5 hours . The solvent was removed under reduced pressure. Chromatography (SiO_Two, 2 × 20cm, 30% EtOAc-hex Sun) to give 18 as a white foamed solid (155 mg,    C_{twenty five}H_{twenty three}BrN_TwoO_FourAnalysis: C, 60.62; H, 4.68; N, 5.65. Found: C, 60.39; H , 4.61; N, 5.69.   2- [N- (tert-butyloxycarbonyl) -N- (3-tetrahydropyranyloxy-2- Propen-1-yl)] amino-4-benzyloxy-1-bromo-7-cyanonaphthalene (1 9). Triphenyl [(2-tetrahydropyranyloxy) methyl] phos in THF (5 mL) Honium chloride⁵⁴(635 mg, 1.51 mmol, 3 equivalents) in n-BuLi at −78 ° C. (1.44 mmol, 0.58 mL, 2.86 eq in 2.5 M hexane) was added dropwise. -78 reaction mixture Stirred at 0 ° C for 10 minutes and warmed to 0 ° C in 20 minutes. The mixture was cooled to -78 ° C and HMPA (2.11m L, 12.1 mmol, 24 equiv.) Immediately after addition of 18 (250 mg, 0.505 mmol) in 2.5 mL of THF. Was added. After stirring at -78 ° C for 1.5 hours and at 25 ° C for 24 hours, 20 mL of phosphate buffer solution (pH 7.0) was added to stop the reaction. The mixture was extracted with EtOAc (3 × 50 mL) and mixed. Dry the combined organic phases (Na_TwoSO_Four) And concentrated under reduced pressure. Chromatography (SiO_Two, 2 × 30cm , 2% Et_ThreeN-containing 10% EtOAc-hexane), as a mixture of E- and Z-isomers, oil 1 9 (221 mg, theoretically 300 mg,   5-benzyloxy-3- (tert-butyloxycarbonyl) -8-cyano-1-[(tetrahi [Dropranyloxy) methyl] -1,2-dihydro-3H-benz [e] indole (20). A solution of 19 (41 mg, 69 μmol) in freshly distilled benzene (3.5 mL) was added under argon to AIBN (2 mg, 0.2 equivalent), then Bu_ThreeTreated with SnH (40 mg, 0.014 mmol, 2 eq). Reaction mixture The mixture was heated at reflux for 2 hours, cooled to 25 ° C._TwoThe solvent was removed under a stream of air. Residue T Azeotroped with HF (1 × 2 mL). Chromatography (Si0_Two, 1 × 20cm, 0% EtOAc- Hexane) gave 20 as a clean oil (35 mg, theoretically 35.5   5-benzyloxy-3- (tert-butyloxycarbonyl) -8-cyano-1- (hydroxy (Scimethyl) -1,2-dihydro-3H-benz [e] indole (21). From 20: new steam Retained CH_ThreeA solution of 20 (35 mg, 69 mol) in OH (1 mL) was added to 0.5 mg of Amberlyst-15 Treated with on-exchange resin and the reaction mixture was stirred at 45 ° C. for 5 hours. Filter the resin Remove with excess CH_ThreeWashed with OH (1 × 2 mL). Combine the filtrates and N_TwoRemove solvent under air flow did. Chromatography (SiO_Two, 1 × 20cm, 20% EtOAc-hexane) 21 was obtained as a solid (29.7 mg, theoretically 29.7 mg,   From 29: THF-OAc-H_TwoA solution of 29 (341 mg, 0.599 mmol) in O (3: 1: 1, 20 mL) was treated with Zn Flour (3.13 g, 80 equiv) and the mixture was heated at 70 ° C. for 6 hours. Celite Zn powder The mixture was removed by filtration with e, and the mixture was concentrated under reduced pressure. Chromatography (SiO_Two, 1.3 x 13 cm, 0-25% EtOAc-hexane), as an off-white solid on all surfaces Gave the same 21 as above (188 mg, theoretically 258 mg, 73%).   5-benzyloxy-3- (tert-butyloxycarbonyl) -1- (chloromethyl) -8-cy Ano-1,2-dihydro-3H-benz [e] indole (22). Freshly distilled anhydrous CH_TwoCl_Two A solution of 21 (47 mg, 0.10 mmol) in (0.35 mL) under Ar under Ph_ThreeP (82mg, 0.31mmol, 3 equivalents Amount) and CCl_Four(91 μL, 0.94 mmol, 9 equivalents). The reaction mixture at 25 ° C Stir for 2 hours. N_TwoThe solvent was evaporated under a stream of air. Chromatography (SiO_Two, 0.8 × 10 cm0% EtOAc-hexane) to give 22 as a white solid.   Resonance of 22 A sample of racemate 22 was analyzed by preparative HPLC chromatography on Diacel.  On a Chiralcel-OD column (10 μm, 2 × 25 cm), eluate of 7% i-PrOH-hexane (7 mL / m in). The enantiomer has a retention time of 20.70 minutes (unnatural enantiomer Thiomer) and 28.54 minutes (natural enantiomer), eluting with α = 1.38.     (1S) -22: [α]^Five _D-9.5 (c0.5, CHCl_Three)     ent- (1R) -22: [α]^Five _D+9.5 (c0.3, CHCl_Three)   3- (tert-butyloxycarbonyl) -1- (chloromethyl) -8-cyano-5-hydroxy -1,2-dihydro-3H-benz [e] indole (23). 22 in anhydrous EtOAc (5 mL) (71.5 mg , 0.159 mmol) and a solution of 10% Pd-C (40 mg)_TwoDegassed for 30 minutes with airflow. Got Mixture with H_TwoIt was placed under an atmosphere and stirred at 25 ° C. for 2.5 hours. Dilute the mixture with THF (1 mL) And filtered through Celite (EtOAc wash). The solvent was removed in vacuo. Chroma Tomography (SiO_Two, 1.5 × 6 cm, 20% EtOAc-hexane)     (1S) -23: [α]^{twenty three} _D-15 (c0.08, CHCl_Three)     ent- (1R) -23: [α]^{twenty three} _D+16 (c0.09, CHCl_Three).   N- (tert-butyloxycarbonyl) -7-cyano-1,2,9,9a-tetrahydrocycloprop Lopa [c] benz [e] indole-4-one (25, N-BOC-CCBI). Method A: 2: 1 DMF-HF (1 A solution of 23 (1.4 mg, 3.91 μmol) in 12 μL) was added at 0 ° C. to NaH (1.6 mg, 39 μmol). , 60% oil dispersion) and the mixture was stirred for 30 minutes. N_TwoUnder airflow and reduced pressure And the solvent was removed. PTLC (SiO_Two, 0.25mm x 10 x 15cm, 30% EtOAc-hexane) 25 was obtained as a colored solid (1.20 mg, theoretically 1.26 mg, 95%). :     (+)-N-BOC-CCBI (25): [α]^Three _D+124 (c0.04, THF).     ent-(-)-N-BOC-CCBI (25): [α]^Three _D-121 (c0.03, THF).   Method B: A solution of 23 (8.6 mg, 24.0 μmol) was added to 1: 1 THF-5% NaHCO 3_ThreeIn aqueous solution (2 ml) And the mixture was stirred at 25 ° C. for 9 hours. The THF is removed by evaporation and the product E Extracted with tOAc (4 × 1 mL). Chromatography (SiO_Two, 0.8 × 5cm, 0-25% EtOAc- Hexane gradient) gave 25 as above as a white solid (7.7 mg, theoretically). Is 7.7 mg, 100%).   7-cyano-1,2,9,9a-tetrahydrocyclopropa [c] benz [e] indole-4-o (26, CCBI). A solution of 23 (2.5 mg, 7.0 μmol) in 4M HCl-EtOAc (400 μL) Under Ar at 25 ° C. for 30 minutes. N_TwoThe solvent was removed under a stream of air. After drying under reduced pressure , Residue 30¹⁴Was dissolved in THF (200 μL), and 200 μL of 5% NaHCO 3_ThreeTreated with aqueous solution . After stirring the reaction mixture at 25 ° C. for 5 hours, the solvent was removed under reduced pressure. PTLC (SiO_Two, 0.25mm x 20 x 20cm, 70% THF-hexane)     (+)-CCBI (26): [α]^Three _D+64 (c0.05, THF)     ent-(-)-CCBI (26): [α]^Three _D-67 (c0.05, THF).   N- (tert-butyloxycarbonyl) -4-benzyloxy-7-cyano-1-indole 2-naphthylamine (27). 10 mL THF-CH_Three23 (250 mg, 0.67 mmol) in OH (1: 1) At -40 ° C. in 2 mL of a catalytic amount of TsOH-H_TwoO (20 mg) and NIS (180 mg, 0.80 mmol , 1.2 equivalents). The reaction mixture was stirred at -40 ° C for 1 hour under Ar and then to 0 ° C. Warmed up. In addition, TsOH-H_TwoO (10 mg) and NIS (75 mg, 0.5 eq) were added. Reaction mixing After stirring the material for 1 hour at 25 ° C., 5 mL of NaHCO_ThreeAdd saturated aqueous solution and cool, Et_TwoO (4 × 1 5 mL). The combined organic layer was washed with a saturated aqueous solution of NaCl and dried (Na_TwoSO_Four). PC TLC (2mm SiO_Two, 0-10% EtOAc-hexane gradient) to give 27 as a solid (230 mg, Theoretically 338 mg, 68%). : Melting point     C_{twenty three}H_{twenty one}IN_TwoO_ThreeAnalysis for: C, 55.21; H, 4.23; N, 5.60. Observed: C, 55.28; H, 4.03; N, 5.40.   N- (tert-butyloxycarbonyl) -N- (2-propenyl) -4-benzyloxy-7-cy Ano-1-indole-2-naphthylamine (28). (160 mg, 27) in anhydrous DMF (5 mL). A solution of 0.31 mmol) was treated with NaH (19 mg, 0.47 mmol, 1.5 eq, 60% oil dispersion) under A. The reaction mixture was stirred at 0 ° C. for 30 minutes. Allyl bromide (194mg, 139μL, 1.55m mol, 5 equivalents) dropwise over 5 minutes, warm the solution to 25 ° C. and stir for 2 hours did. NaHCO_ThreeA saturated aqueous solution (10 mL) was added, and the aqueous phase was extracted with EtOAc (4 × 10 mL), Dry (Na_TwoSO_Four) And concentrated. Chromatography (SiO_Two, 2 × 15cm, 10% EtOAc-hex Sun) to give a clean oil which was recrystallized under reduced pressure to give 28 (155 mg,     C₂₆H_{twenty five}IN_TwoO_ThreeAnalysis: C, 57.99; H, 4.66; N, 5.18. Measurement: C, 57.6 0; H, 4.41; N, 5.27.   5-benzyloxy-3- (tert-butyloxycarbonyl) -8-cyano-1-[(2 ', 2', 6 ' , 6'-Tetramethylpiperidino) oxy] methyl-1,2-dihydro-3H-benz [e] india (29). A solution of 28 (200 mg, 0.37 mmol) in freshly distilled benzene (12 mL) , TEMPO (3.0 equivalents), then Bu_ThreeTreated with SnH (1.0 eq). Bring the reaction mixture to 60 ° C Heated. 20 minutes later, another equivalent of Bu_ThreeSnH was added. Half an hour Later, TEMPO (2 equivalents) and Bu_ThreeSnH (1.0 equivalent) was added. 20 minutes later, two more And equivalent TEMPO and Bu_ThreeSnH was added in two portions at 15 minute intervals. 45 After a minute, at 60 ° C., the solvent was removed by evaporation. PCTLC (4mm SiO_Two, 0-25% EtOAc-hex (Sun gradient) gave 29 as a semi-solid (144 mg, 212 mg theoretical, 68%). :   seco-CCBI-TMI (34). 23 in 150 μL of 4M HCl-EtOAc (1.5 mg, 4.2 μmmol) was stirred at 25 ° C. for 20 minutes. N_TwoThe solvent was removed under a stream of air. Decompression After drying under reduced pressure, residue 30 (1.0 mg, 4.2 μmmol, 1 equivalent; Boger et al., J. Am Chem. Soc. 199) 3 115, 9025) and EDCI (2.9 mg, 12.6 μmmol, 3 equivalents) were dissolved in anhydrous DMF. The reaction mixture was stirred at 25 ° C. for 16 hours. Remove the solvent under reduced pressure and dissolve the residue in THF And directly packed on a silica gel column. Chromatography (SiO_Two, 0.5 × 6cm , 50% EtOAc-hexane) to give 34 as a mustard-colored solid (1.7 mg, m.p.     (1S) -34: [α]^Five _D-19 (c0.12, CHCl_Three).     ent- (1R) -34: [α]^Five _D+19 (c0.12, CHCl_Three). CCBI-TMI (35). 34 in 20% DMF-THF (0.25 mL, 0.018 M) (2.2 mg, 4. (4 μmol) was cooled to 0 ° C. under Ar and NaH (0.5 mg, 3 eq) was added. Reaction mixing After stirring the material at 0 ° C. for 30 minutes, care was taken to keep the temperature at 0 ° C._TwoDown the air And the solvent was removed. PTLC (SiO_Two, 0.25mm x 10cm x 15cm, 50% EtOAc-hexane) 35 was obtained as a pale yellow solid (2.1 mg, theoretically 2.1 mg, 99%). :     (+)-CCBI-TMI (35): [α]^Three _D+144 (c0.05, acetone).     ent-(-)-CCBI-TMI (35): [α]^{twenty three} _D-135 (c0.04, acetone)   seco-CCBI-indole_Two(36). 23 in 250 μL of 4M HCl-EtOAc (2.8 mg, 7. (8 mol) was stirred at 25 ° C. for 20 minutes. N_TwoUnder a stream of air, the solvent was removed and the crude salt The acid salt was dried under reduced pressure. 24,31 (2.5 mg, 7.8 μmol, 1.0 equivalent) and EDCI (4.5 mg, 23.5 μmol, 3 equiv.) and the mixture was mixed with 142 μL (0.55 M) of anhydrous D Slurried in MF. After stirring the reaction mixture at 25 ° C. for 16 hours, the solvent was removed under reduced pressure. I left. PTLC (SiO_Two, 0.25mm x 15 x 20cm, 30% DMF-toluene)     (1S) -36: [α]^Five _D+49 (c0.17, DMF).     ent- (1R) -36: [α]^Five _D-44 (c0.21, DMF).   CCBI-indole_Two(37). Method A: 36 in 20% DMF-THF (0.34 mL) (3.4 mg, 6. Solution was cooled to 0 ° C. under Ar, and NaH (0.8 mg, 18.3 μmol, 60% l in oil, 3 Equivalent). After stirring the reaction mixture for 30 minutes at 0 ° C., the temperature is maintained at 0 ° C. While N_TwoThe solvent was removed under a stream of air. PTLC (SiO_Two, 0.25mm × 15 × 20cm, 5% DMF-to Ruen) to give 37 as a tan solid (2.1 mg, theoretically     (+)-CCBI-Indole_Two(37): [α]^Five _D+80 (c0.04, THF).     ent-(-)-CCBI-indole 2 (37): [α]^{twenty five} _D-81 (c0.09, THF).   Method B: 1: 1 THF-3% NaHCO_ThreeDissolution of 36 (2.7 mg, 4.8 μmol) in aqueous solution (700 μL) The liquid was stirred at 25 ° C. for 10 hours. N_TwoThe solvent was removed under a stream of air. PTLC (SiO_Two, 0. 25 mm × 20 × 20 cm, 10% DMF-toluene) gave 37 as a tan solid (1.7 mg, theoretically 2.5 mg, 68%). seco-CCBI-CDPI₁(38). 23 in 500 μL of 4M HCl-EtOAc (4.0 mg, 11.1 μmol) was stirred at 0 ° C. for 30 minutes. N_TwoUnder a stream of air, the solvent is removed and the crude The hydrochloride was dried under reduced pressure. The salt and 32 (3.0 mg, 12.3 μmol, 1.1 eq.) It was dissolved in 300 μL of anhydrous DMF and treated with EDCI (6.4 mg, 33.3 μmol, 3 equivalents). The reaction mixture was stirred at 25 C for 14 hours. The crude reaction mixture is concentrated and prep TL Loaded directly on C plate. Chromatography (SiO_Two, 0.25mm × 20 × 15cm, 20 % DMF-toluene) to give 38 as a yellow solid (4.4 mg, theoretical)     (1S) -38: [α]^Five _D+37 (c0.15, DMF).     ent- (1R) -38: [α]^Five _D-36 (c0.22, DMF).   CCBI-CDPI₁(39). DMF-H_Two38 in O (5: 2, 600 μL + 240 μL) (3.6 mg, 7 .4μmol) solution in KHCO_Three(20 equivalents). The reaction mixture at 25 ° C. for 9 hours Stirred. After removing the solvent, PTLC (SiO_Two, 0.25mm × 20 × 20cm, 20% DMF-to Ruen) to give 39 as a tan solid (2.3 mg, 3.3 mg theoretically, 69%). :     (+)-CCBI-CDPI₁(39): [α]^{twenty three} _D+124 (c0.10, DMF).     ent-(-)-CCBI-CDPI₁(39): α]^{twenty three} _D-117 (c0.07, DMF).   Reactivity of aqueous solvolysis. pH3: N-BOC-CCBI (25, 100 μg)_Three0H (1.5mL) Dissolved and mixed with pH 3 aqueous buffer solution (1.5 mL). The buffer solution is 4: 1: 20 (v: v : v) respectively, 0.1M citric acid, 0.2M Na_TwoHPO_Four, And H_TwoIncluding O. That sorboli The cis solution was sealed and kept at 25 ° C protected from light. The UV spectrum of the first Regularly every 2 hours a day, then every 12 hours for a week, and every 24 hours for the next week Was measured. Reduction of long wavelength absorption at 322 nm and increase of short wavelength absorption at 268 nm Was monitored (FIG. 1). Solvolysis rate constant (k = 9.90 × 10^-7s^-1) And half-life (t_{1 / 2} = 213 hours) from the data recorded at the short wavelength by the least squares method (r = 0.999) , Ln [(A_f-A_i) / (A_f-A)] from the slope of the plot.   pH 2: 25 (100 μg) and 26 (50 μg) samples were CH_ThreeDissolve in OH (1.5 mL) The mixture was mixed with a buffer solution (pH 2.05, 1.5 mL). The buffer solution was 4: 1: 20 (v: v: v), respectively. 0.1M citric acid, 0.2M Na_TwoHPO_Four, And H_TwoIncluding O. Immediately after mixing, CH_ThreeOH (1.5 mL) The UV spectrum of the solution against a control solution containing 1.5 mL of aqueous buffer solution. And the reading was used as the initial absorption value. Stop solution, protect from light, 25 ° C Kept. Constant values for long wavelength and short wavelength UV spectra were recorded at regular intervals until The solvolysis rate constant is , 25 using the short wavelength measurement and 26 using the long wavelength measurement. 1n [(A_f-A_i) / (A_f−A)] plot of the straight line obtained from the least squares method Determined by the gradient. The first-order rate constant determined under these conditions is N-BOC-C 7.94 x 10 for CBI (25)^-6s^-1(T_1/2= 24.2 hours, r = 0.999), CCB For I (26), 2.1 × 10^-6s^-1(T_1/2= 91.5 hours, r = 0.99).   THF-CH_ThreeSolvolysis of N-BOC-CCBI in OH N-BOC -Solvolysis of CCBI (25) is 0.1 to 0.25 equivalent CF_ThreeSO_ThreeIn the presence of H, 20 ~ 500 equivalents of CH_ThreePerformed in THF containing OH. The following is CF_ThreeSO_ThreeH (0.1 equivalent ) In the presence of 20 equivalents of CH_ThreeFor solvolysis of 25 in THF with OH Is the way. The storage solution is CF_ThreeSO_ThreeH (5.6 μL) and anhydrous CH_ThreeOH (502μL) Prepared by addition to anhydrous THF (99.49 mL). 25 samples (100 μg) in a UV cell were THF (1950 μL), CF_ThreeSO_ThreeH (0.1 eq), CH_ThreeOH (20 equivalents) And dissolved in THF storage solution (50 μL). Seal the solvolysis solution and Cycle program allows UV spectra to be collected at regular intervals (10 min / cycle) It was measured. Monitor the decrease in long wavelength absorption at 313 nm and after 80 hours, Has completed. Solvolysis speed (k = 0.2 × 10^-Fours^-1And half-life (t_1/2= 9.2 hours), 3 From data recorded at 13 nm, time vs. ln [(A_f-A_i) / (A_f−A)] plot Calculated by the least squares method (r = 0.984) for the gradient of   Solvolysis regioselectivity: 3- (tert-butyloxycarbonyl) -8-cyano-5-hydride Roxy-1-methoxymethyl-1,2-dihydro-3H-benz [e] indole (42). 0.12 Equivalent CF_ThreeSO_ThreeH-containing CH_Three25 (2.5 mg, 7.8 μmol) in OH (1 mL) Was stirred at 0 ° C. for 24 hours. NaHCO_Three(2.5 mg) and the reaction mixture was added. Stir at 0 ° C., heat to 25 ° C., filter through a plug of Celite and concentrate. PTLC (SiO_Two, 0.25 mm × 20 × 20 cm, 20% EtOAc-hexane) to give 42 as a semi-solid (2 .3mg, theoretically 2.7mg, 85%, 95% based on conversion), 2   DNA alkylation studies: selectivity and efficiency³²P5 'end Preparation of labeled double-stranded DNA, study of drug binding, gel electrophoresis, and autoradiography Ography was performed according to standard methods. In TE buffer solution (10 mM Tris, 1 mM Eppendorf tubes containing 9 μL of 5 ′ end labeled DNA (EDTA, pH 7.5) were placed in DMSO Medium (at 1 μL unique concentration) was treated with drug. Vortex the solution briefly Mixing by separation, followed by 24 hours at 4 ° C. or 25 ° C. (natural enantiomers) Incubated at 25 ° C. or 37 ° C. for 72 hours (unnatural enantiomer). share Bound-modified DNA is separated from unbound drug by EtOH precipitation and TE buffered. The suspension was resuspended in the solution (10 μL). DNA solution in Eppendorf tubes sealed with parafilm Heat at 100 ° C for 30 minutes to cut at the alkylation site, cool to 25 ° C and centrifuge Was. Add the formamide dye (0.03% xylene cyanol FF, 0.03% buffer) to the supernatant. Lomophenol blue, 8.7% Na_TwoEDTA (250 mM) was added (5 μL). Electrophoresis Prior to heating, the sample was denatured by heating at 100 ° C for 5 minutes, placed in an ice bath, and centrifuged. On release, the supernatant was loaded directly onto the gel. Sanger dideoxynucleotide sequence The determination reaction was performed according to criteria close to the reaction sample. Polyacrylamide gel electric Electrophoresis (PAGE) was performed on an 8% sequencing gel under denaturing conditions (8 M urea), TBE buffer solution. In the liquid (100 mM Tris, 100 mM boric acid, 0.2 mM Na_TwoEDTA), auto radio Obtained a graphy.   (+)-CCBI-TMI (35), (+)-CBI-TMIN and (+)-MCBI -DNA alkylation reaction rate of TMI According to the method described above, TE buffer Eppendorf tube containing 5 'end labeled w794 DNA (9 [mu] L) in solution (pH 7.5) With (+)-CCBI-TMI (35), (+)-CBI-TMI, or (+)-MCB I-TMI (1 μL, 10 in DMSO^-6M). Mix the solution and at 25 ° C, Incubation was for 2, 4, 6, 9, 12, 15, and 24 hours, respectively. Next Separation of alkylated DNA by EtOH precipitation, resuspension in TE buffer solution (10 μL, pH 7.5), heat treatment (30 minutes, 100 ° C), PAGE, and autoradiography Was performed as described above. High affinity 5'-AATT of w794AAl at the site The relative rate of killing is determined by the percentage integrating optics of the high affinity alkylated cleavage zone over time. Derived from the slope of the density (IDO) plot.   (+)-CCBI-indole_Two(37), (+)-CBI-indole_Two, And (+) -MCBI-indole_TwoDNA alkylation relative rate of the method described above Therefore, Epp containing 5 'end labeled w794 DNA (9 [mu] L) in TE buffer solution (pH 7.5) Endorf tubes were replaced with (+)-CCBI-indole_Two(37), (+)-CBI-indole_Two , And (+)-MCBI-indole_Two(1 μL, 10 in DMSO^-6M). Mix the solution and incubate at 25 ° C for 2, 4, 6, 9, 12, 15, and 24 hours, respectively. It was cubified. Then, separation of the alkylated DNA by EtOH precipitation, T Resuspension in E buffer solution (10 μL, pH 7.5), heat treatment (30 minutes, 100 ° C), PAGE, And autoradiography were performed as described above. High affinity 5'- of w794 AATTAThe relative rate of alkylation at the site is high affinity alkylation versus time Derived from the slope of the plot of the percentage integrated optical density (IDO) of the cut zone. 1-chloromethyl-3- (2-ethoxy-1,2-dioxoethyl) -5-hydroxy-1,2-dihydride B-3H-benz [e] indole (114). Treated with 3.6N HCl-EtOAc (25 ° C, 30 minutes) 112 (3.6 mg. 0.01 mmol; Boger, DL; Colletti, SL; Honda, T .; Menezes, R . F. J. Am. Chem. Soc. 1994, 116, 5607. Boger, D.L .; Col1etti, S.L. L.; Tera lnoto, S .; Ramnsey, T .; R.; Zhou, J. Bioorg. Med. Chem. 1995, 3, 1281) The newly generated 113 samples were_Three(2.7 mg, 0.03 mmol, 3.0 equivalents) In the presence of ethyl oxalyl chloride (3.0 mg, 0.022 mmol, 2.0 equivalents) in THF (0.5 mL). And the reaction mixture was stirred at 25 ° C. for 2 hours. Solvent to N_Twoair flow Removed below. Chromatography (SiO_Two, 0.8 × 5 cm, 50% EtOAc-hexane)    Natural (1S) -114: [a]^{twenty five} _D-81 (c0.2, CHCl_Three).     Ent- (1R) -114: [a]^{twenty five} _D+90 (c0.25, CHCl_Three).   1-chloromethyl-3- (4-ethoxy-1,4-dioxobutyl) -5-hydroxy-1,2-dihi Doro-3H-benz [e] indole (115). Treated with 3.6N HCl-EtOAc (25 ° C., 30 Min) 12¹⁵(5.3 mg, 0.016 mmol) were collected from a sample of NaHCO_Three (0.5 mg) in THF in the presence of (3.3 mg, 0.40 mmol, 2.5 equiv.) Treated with chloride (2.6 mg, 0.016 mmol, 1.0 eq) and the reaction mixture was allowed to stand at 25 ° C. for 1 hour Stirred. Solvent to N_TwoIt was removed under airflow. PTLC (SiO_Two, 0.25mm × 20 × 20cm, 50% EtOAc -Hexane) to give 115 as a solid (5.8 mg,     Natural (1S) -115: []^{twenty five} _D-32 (c0.3, THF).     Ent- (1R) -115: []^{twenty five} _D+39 (c 0.15, THF).   N^Two-(4-ethoxy-1,4-dioxobutyl) -1,2,9,9a-tetrahydrocyclopropa [c ] Benz [e] indole-4-one (116). 115 samples (3.5mg, 9.7μmol) , 5% NaHCO_Three-THF aqueous solution (1: 1, 500 μL), and the mixture is stirred at 25 ° C. for 9 hours , N_TwoThe solvent was removed under a stream of air. PTLC (SiO_Two, 0.25mm x 20 x 20cm, 50% THF-hexane) Yielded 116 as a white solid (2.5 mg, theoretically 3.2 mg,     Natural (+)-116: []^{twenty five} _D+133 (c0.13, THF).     Ent-(-)-116: []^{twenty five} _D+150 (c0.12, THF).   3- [2 '-(2- (2-ethoxy-1,2-dioxoethyl) aminoethyl) -2,4'-bithiazo 4-Carboxamido] propylmethyl sulfide (118). 117 (13.7mg , 0.04 mmol; Boger, D .; L .; Colletti, S .; L .; Honda, T .; Menezes, R .; F. J . Am. Chem. Soc. 1994, 116, 5607. Boger, D .; L .; Colletti, S .; L.; Teramoto, S .; Ramsey, T .; R.; Zhou, J. Bioorg. Med. Chem. 1995, 3, 1281) with DM Treatment with ethyl oxalyl chloride (9.1 μL, 0.08 mmol, 2.0 equiv) in F (0.04 mL) After stirring at 25 ° C. under Ar for 20 hours, the solvent was removed under reduced pressure. Chromatography (SiO_Two , 0.8 × 12 cm, 70% EtOAc-hexane) to give 118 as an off-white solid (12.7 mg, theoretically 17.7 mg, 72%). : R_f0.62   3- [2 '-(2- (2-hydroxy-1,2-dioxoethyl) aminoethyl) -2,4'-bitia Sol-4-carboxamido] propylmethyl sulfide (119). THF-H_TwoO-CH_Three A solution of 118 (12.7 mg, 0.029 mmol) in OH (3: 1: 1, 0.45 mL) was added to LiOH (6.0 mg, 0.14 mL). (5.0 mmol), and the mixture was stirred at 25 ° C for 2 hours, and then the solvent was removed under reduced pressure. I left. The crude product is H_TwoO, acidified by adding 10% HCl aqueous solution H0.5. 30% isopropanol-CHCl_Three(7 × 1.2 mL) and mix The combined extract is concentrated to an off-white solid that is suitable for direct use in the next reaction. Pure 119 was obtained (11.9 mg, theoretically   3- [2 '-(2- (2- (1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] i Ndol-3-yl) -1,2-dioxoethyl) aminoethyl) -2,4'-bithiazol-4- Carboxamido] propylmethyl sulfide (105). Treat with 4N HCl-EtOAc (25 ° C, 30 minutes), 12¹⁵(5.3mg, 0.016mmol, 1.5eq) newly generated 11 A sample of 3 was prepared by mixing 119 (4.4 mg, 0.011 mmol, 1.0 equiv) and EDCI in DMF (0.2 mL) under Ar. (3.0 mg, 0.016 mmol, 1.5 eq.) And the mixture was stirred at 25 ° C. for 17 hours. DM F is removed and the crude product is_TwoO (0.2 mL). CHCl aqueous phase_Three(3 × 0.3mL) and 50 Extracted with% hexanes EtOAc (2 × 0.3 mL). The combined organic extracts were concentrated under reduced pressure. PCTLC (SiO_Two, 0.25mm × 20 × 20cm, 5% CH_ThreeOH-CH_Two Cl_Two) Gave 105 as a tan solid (2.9 mg, 6.7 mg theoretically,     Natural (1S) -105: []^{twenty five} _D−22 (c0.05, CHCl_Three).     Ent- (1R) -105: []^{twenty five} _D+22 (c0.05, CHCl_Three).   3- [2 '-(2- (2- (1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] i Ndol-3-yl) -1,2-dioxoethyl) aminoethyl) -2,4'-bithiazol-4- Carboxamido] propylmethylsulfoxide (106). 106 samples     Natural (1S) -106: []^{twenty five} _D-10 (c0.07, CHCl_Three).     Ent- (1R) -106: []^{twenty five} _D+10 (c0.050, CHCl_Three).   3- [2 '-(2- (2- (1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] i Ndol-3-yl) -1,2-dioxoethyl) aminoethyl) -2,4'-bithiazol-4- Carboxamido] propyldimethylsulfonium iodide (107). DMF (0.2 mL) of 105 (2.9 mg, 0.0046 mmol) in CH_ThreeI (29 μL, 0.46 mmol, 100 equivalents Volume) and the mixture was stirred under Ar at 25 ° C. for 67 hours. Evaporation of solvent and CHC l_ThreeTrituration with (5 x 0.1 mL) gave pure 108 as a yellow solid (3.6     Natural (1S) -107: []^{twenty five} _D-8.3 (c0.08, CH_ThreeOH).     Ent- (1R) -107: []^{twenty five} _D+8.5 (c0.06, CH_ThreeOH).   1-chloromethyl-5-hydroxy-1,2-dihydro-3H-3- [N- (4-tert-butyloxy- 1,4-dioxobutyl)]-benz [e] indole (120). 112 (10 mg, 0.03 mmol; Boger, D .; L .; McKie, J .; A. J. Org. Chem. 1995, 60, 1271) in DMF (0.75 mL) And 113 samples freshly prepared by treatment with 4N HCl-EtOAc (25 ° C., 30 minutes) Butyl hemisuccinate (7.8 mg, 0.045 mmol, 1.5 equivalents) and EDCI (17.3 mg, 0.09 mmol) 1,3.0 equiv.) and the mixture was stirred at 25 ° C. under Ar for 21 h. Solvent under reduced pressure Was removed. Chromatography (SiO_Two, 8 × 10cm, 7% Et_TwoO-CH_TwoCl_Two)     Natural (1S) -120: []^{twenty five} _D-58 (c0.1, CHCl_Three).     Ent- (1R) -120: []^{twenty five} _D+60 (c0.4, CHCl_Three).   3- [2 '-(2- (2- (1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] i Ndol-3-yl) -1,4-dioxobutyl) aminoethyl) -2,4'-bithiazol-4- Carboxamido] propyl methyl sulfide (108). 120 samples (3.7 mg, 0 .009 mmol) was treated with formic acid (2 mL) at 25 ° C. for 3 hours. Formic acid to N_TwoEvaporate under air flow Removed. The crude acid 121 was dissolved in 117 (3.9 mg, 0.011 mmol, 1. 2 equivalents; Boger, D .; L .; Colletti, S .; L .; Honda, T .; Menezes, R .; F. J. Am. Chem . Soc. 1994, 116, 5607. Boger, D .; L .; Colletti, S .; L.; Teramoto 、 S.; Ramsey , T .; R.; Zhou, J. Bioorg. Med. Chem. 1995, 3, 1281), EDCI (5.5 mg, 0.029 mmo l, 3.0 equiv.), the mixture was stirred at 25 ° C under Ar for 38 h, and the solvent was evaporated under reduced pressure. Was removed. PCTLC (SiO_Two, 0.25mm × 20cm × 20cm, 3% CH_ThreeOH-CH_TwoCl_Two), Bright yellow 108 was obtained as a colored solid (2.3 mg, theoretically 6.2 mg, 37%):     Natural (1S) -108: []^{twenty five} _D+10 (c0.13, CHCl_Three).   3- [2 '-(2- (4- (1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] i Ndol-3-yl) -1,4-dioxobutyl) aminoethyl) -2,4'-bithiazol-4- Carboxamido] propyldimethylsulfonium iodide (109). A solution of 108 (1.9 mg, 0.003 mmol) in DMF (0.19 mL) was added to CH_ThreeI (41 mg, 0.29 mmol , 100 eq.) And the mixture was stirred under Ar at 25 ° C. for 120 h. Further CH_ThreeI ( 41 mg, 0.29 mmol, 100 equivalents), and 22 hours later, DMF was added under reduced pressure. Removed. The residue was washed with CHCl_Three(7 × 0.3 mL) and purified to obtain 109 (2. Three    Natural (1S) -109: []^{twenty five} _D-12 (c0.18, DMSO).   3- [2 '-(2-((4- (1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] i Ndol-3-yl) -1,4-dioxobutyl) -L-threonyl) aminoethyl) -2,4'-bi Thiazole-4-carboxamido] propylmethyl sulfide (110). 120 (3 .6 mg, 0.009 mmol) was treated with formic acid (1 mL) for 1.5 hours at 25 ° C._TwoMelting under air current The medium was removed. The crude 121 was prepared by adding 122 (4.0 mg, 0.009 mmol, 1) in DMF (0.3 mL). .0 eq; Boger, D.E. L .; Colletti, S .; L .; Honda, T .; Menezes, R .; F. J. Am. Che m. Soc. 1994, 116, 5607. Boger, D .; L .; Colletti, S .; L.; Teramoto 、 S.; Rajns ey, T .; R.; Zhou, J. Bioorg. Med. Chem. 1995, 3, 1281), EDCI (4.4mg, 0.023m mol, 2.5 eq) and HOBt (1.4 mg, 0.01 mmol, 1.1 eq) and the mixture was treated with 2 Stirred at 5 ° C. for 47 hours. The solvent was removed under reduced pressure. PCTLC (SiO_Two, 0.25mm × 20 × 20cm, 5%, CH_ThreeOH-CH_TwoCl_Two), Orange solid     Natural (1S) -110: []^{twenty five} _D-218 (c0.2, CHCl_Three).   3- [2 '-(2-((4- (1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz [e] i Ndol-3-yl) -1,4-dioxobutyl) -L-threonyl) aminoethyl) -2,4'-bi Thiazole-4-carboxamido] propyldimethylsulfonium iodide (11 1). A solution of 110 (2.2 mg, 0.003 mmol) in DMF (0.17 mL) was added in CH_ThreeI (41.2mg, 0.29m mol, 100 equiv.) and the mixture was stirred at 25 ° C. under Ar for 88 h. By evaporation To remove the solvent. CHCl_Three(8 x 0.5 mL) to give pure 110, yellow solid 111 (2.6 mg, 2.6 mg theoretically, 100%). :   Natural (1S) -111: []^{twenty five} _D-9.2 (c0.1, DMSO).   w794 DNA in a TE buffer solution (10 mM Tris, 1 mM EDTA, pH 7.2) Eppendorf tubes containing 5 'end labeled DNA (9 [mu] L) were placed in DMSO (at 1 μL) and treated with drug. Vortex the solution and mix by simple centrifugation And then at 37 ° C. for 76 hours (105, 106, 107, 114, 116 and 123 Both enantiomers) and 48 hours (108, 109, 110 and 111) incubate Was an option. The covalently modified DNA is removed by EtOH precipitation. Separated from unbound drug and resuspended in TE buffer solution (10 μL). With Teflon tape Heat the DNA solution in the sealed Eppendorf tube at 100 ° C for 30 minutes at the alkylation site. Cut, cooled to 25 ° C and centrifuged. In the DNA solution, formamide dye (0.03% Xylene cyanol FF, 0.03% bromophenol blue, 8.7% Na_TwoEDTA 250mM) (5 μL). Prior to electrophoresis, the sample was denatured by heating at 100 ° C for 5 minutes, The solution was placed in an ice bath, centrifuged, and the solution (4 μL) was loaded on the gel. Reaction trial Sanger dideoxynucleotide sequencing reactions were performed according to near standard standards. Po Liacrylamide electrophoresis (PAGE) was performed on an 8% sequencing gel under denaturing conditions (8M urine Base) in a TBE buffer solution (10 mM Tris, 100 mM boric acid, 0.2 mM Na_TwoEDTA) and auto Radiography was obtained.   DNA alkylation of calf thymus One aliquot of drug (5 μL, 0.01 M in DMSO) , Calf thymus DNA solution (0.45 mL, 3.79 mg / mL, 10 mM sodium phosphate, pH 7.0, Base pair: drug = 51: 1). The DNA-drug mixture was added to 105, 10 For both enantiomers 6 and 107, 108, 10 For 9, 110 and 111, incubation was performed at 37 ° C. for 48 hours. For both enantiomers 105, 106, 108 and 110, Was extracted with EtOAc (0.5 mL × 4). Dry the combined extracts, dissolve in EtOAc (0.9 mL), The amounts of 105, 106 and 108 and 110 were measured by UV. Further Et Extraction with OAc (0.5 mL × 4) was performed and the amounts of both substances were measured by UV. Additional things No quality was recovered. For ionic drugs 107, 109 and 111, Unreacted substances were recovered from the supernatant of the EtOH precipitation of the DNA. EtOH in the supernatant was_Two Removed by airflow. Transfer the supernatant to H_TwoDilute to 0.9 mL with O, 107, 109 and The recovered amount of 111 was measured by UV.   These studies were performed in tandem with a control reaction in which no DNA was present. 1 aliquot Drug (5 μL, 0.01 M in DMSO) with sodium phosphate buffer solution (0.45 mL, 10 mM, pH 7.0) Was added. The drug-buffer solution mixture was added to both 105, 106 and 107 Enantiomers for 108, 109, 110 and 111 at 37 ° C for 72 hours. Was incubated at 37 ° C. for 48 hours. 105, 106, 108 and For and 110, the material was extracted with EtOAc (0.5 mL x 4). Dry the combined extract Dry, dissolve in EtOAc (0.9 mL) and measure the amount of drug by UV. Ionic drugs For agents 107, 109 and 111, the drug-buffer solution mixture was_TwoO for 0. Diluted to 9 mL and measured the amount by UV.   3- [N- (tert-butyloxycarbonyl) -N- (3-methyl-2-buten-1-yl)] amino 1-benzyloxynaphthalene (205). NaH (0.53 g, 22.0 mm) in anhydrous DMF (10 mL) ol) The suspension was treated with 204 (5.98 g, 17.0 mmol; Boger, DL) in DMF (50 mL) at 25 ° C. under Ar. .; Yun, W .; Teegarden, B .; R. J. Org. Chem. 1992, 57, 2873). Was stirred at 25 ° C. for 0.5 h. The mixture was cooled to 0 ° C and 4-bromo- 2-Methyl-2-butene (5.9 mL, 51.0 mmol) was added slowly. Bring the mixture to 25 ° C After heating and stirring for 14 hours, H_TwoPoured into O (60 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (3 × 50 mL). Mix the organic solution with H_TwoWash with O (70 mL) and saturate NaCl aqueous solution (100 mL). And dried (MgSO 4_Four) And concentrated under reduced pressure. Chromatography (SiO_Two, 15% EtOAc- Hexane) to give 205 as a white solid (6.91 g,     C₂₇H₃₁NO_ThreeAnalysis: C, 77.67; H, 7.48; N, 3.35. Found: C, 77.30; H, 7. 60; N, 3.30.   3- [N- (tert-butyloxycarbonyl) -N- (formylmethyl)] amino-1-benzyl Luoxynaphthalene (206). 5: 1 CH_TwoCl_Two-CH_ThreeSolution of 205 in 0H (350mL) (3.31 g, 7.94 mmol) in 3% O at -78 ° C_Three/ O_TwoTreated by airflow (160 L / h, 4 minutes). Immediately add 14 mL of Me to the reaction mixture._TwoAdd S and cool, cool the resulting mixture to 25 ° C After stirring (12 hours), the solvent was removed under reduced pressure. Chromatography (SiO_Two, 10-20% Et OAc-hexane gradient elution) gave 206 as a white solid (2.55     C_{twenty four}H_{twenty five}NO_FourAnalysis calculated for: C, 73.64; H, 6.44; N, 3.58. Found: C, 73.50; H, 6.4 1; N, 3.67.   3- [N- (tert-butyloxycarbonyl) -N- (3,3-difluoro-2-hydroxy-3-phenyl Enylsulfonyl-1-propyl)] amino-1-benzyloxynaphthalene (207). 206 (33.1 mg, 0.085 mmol) and PhSO in anhydrous THF (3.5 mL)_TwoCF_TwoH (31.0mg, 0.16mmol) And HMPA (0.5 mL) were cooled to −78 ° C. under Ar. In THF (200 μL, 0.20 mmol) A solution of 1.14M LiHMDS is added dropwise and the resulting orange solution is heated to 25 ° C. And stirred for 4 hours. The reaction mixture was poured into saturated aqueous NaCl (10 mL) and Et._TwoO (3 × 5m L). The organic layers were combined, dried (MgSO_Four), Filtered and concentrated under reduced pressure . Chromatography (SiO_Two, 10% EtOAc-hexane) to recover 206 (10.0 mg, 30%) to give 211 as a white foam (25.2 mg, 49.4 mg theoretically, 51%; recovery 206). 73% based on mp): 45-46.5 ° C;     C₃₁H₃₁F_TwoNO₆Analysis of S Calc: C, 63.80; H, 5.35; N, 2.40. Found: C, 63.48; H , 5.11; N, 2.41.   3- [N- (tert-butyloxycarbonyl) -N- (3,3-difluoro-2-methanesulfonyl Ruxy-3-phenylsulfonyl-1-propyl)] amino-1-benzyloxynaphtha Len (208). CH_TwoCl_TwoA solution of 207 (205 mg, 0.35 wol) in (5 mL) was cooled to -30 ° C under Ar Reject, Et_ThreeTreated with N (0.30 mL, 3.5 mmol). After stirring for 5 minutes, MsCl (98 μL, 0.70 mm ol) was added and the reaction mixture was stirred for a further 5 hours. NH_FourCl saturated aqueous solution (10 mL) Was added to cool the reaction mixture. The organic layer was removed and the aqueous layer was extracted with EtOAc (3 × 15 mL). Issued. Combine the organic solutions and dry (MgSO_Four), Filtered and concentrated under reduced pressure. Chromat Graphy (SiO_Two, 10% EtOAc-hexane) as a beige foam     C₃₂H₃₃F_TwoNO₈S_{Two of}Analysis Calculations: C, 58.08; H, 5.03; N, 2.12. Observed: C, 58.45; H , 5.16; N, 2.02.   3- [N- (tert-butyloxycarbonyl) -N- (3,3-difluoro-2-propen-1-yl )] Amino-1-benzyloxynaphthalene (209). CH_ThreeOH in (4 mL) 208 (116 mg, 0 .17 mmol) was cooled to 0 ° C. and_TwoHPO_Four(99mg, 0.70mmol) and 5% Na (Hg) (500 mg, 1.1 mmol). After stirring vigorously at 0 ° C. for 1 hour, the reaction mixture Heated to 25 ° C., Et_TwoO (20 mL) was added. Filtration of solid Hg residue with a fluff plug And the ether solution was concentrated under reduced pressure. Chromatography (SiO_Two, 3% EtOA c-hexane) to give 209 as a colorless oil (57 mg, 74 mg theoretically, 77% ) And recrystallized in a refrigerator to give a white solid. : Melting point 57.0-     C_{twenty five}H_{twenty five}F_TwoNO_ThreeAnalysis for: C, 70.57; H, 5.92; N, 3.29. Observed: C, 70.82; H, 5.88; N, 3.05.   3- [N- (3,3-difluoro-2-propen-1-yl)] amino-1-benzyloxynaphtha Len (210). A solution of 209 (700 mg, 1.64 mmol) under Ar in EtSH (4.0 mL) was treated with BF_Three ・ Et_TwoO (305 μL, 2.45 mmol), and the resulting solution was stirred at 25 ° C. for 1 hour. After stirring, H_TwoO (5 mL) was added and the mixture was cooled. Et water layer_TwoExtract with O (3 x 5 mL) and mix organic solution Was washed with a saturated aqueous solution of NaCl (15 mL) and dried (MgSO_Four), Filtered and concentrated under reduced pressure. Black Matography (SiO_Two, 5% EtOAc-hexane) as a red-rust viscous oil.   3- [N- (3,3-difluoro-2-propen-1-yl) acetamido] -1-benzyloxy Naphthalene (211). A solution of (5 mL) 210 (149 mg, 0.46 mmol) in dioxane was added to Ar Below, DMAP (50 mg, 0.40 mmol), pyridine (0.37 mL, 4.6 mmol) and Ac_TwoO (0.2mL, 2.3mm ol) and stirred at 25 ° C. for 19 hours. To the reaction solution, add 10% HCl aqueous solution (10 mL) and And EtOAc (10 mL) were added and cooled. The aqueous layer was removed and extracted with EtOAc (3 × 5 mL). Combine the organic solutions, wash with saturated aqueous NaCl (15 mL), dry (MgSO 4_Four), Concentrated under reduced pressure did. Chromatography (SiO_Two, 15% EtOAc-hexane)   2- [N- (3,3-difluoro-2-propen-1-yl) acetamido] -4-benzyloxy- 1-nitronaphthalene (212). CH_TwoCl_Two(20 mL) and 211 (581 mg, 1.58 mmol) and Bu_FourNN O_Three(1.20 g, 3.90 mmol) was treated with TFAA (0.25 mL) under Ar. 16 at 25 ° C After stirring for an additional hour, additional TFAA (10 μL) was added and the reaction mixture was stirred at 25 ° C. for an additional 4 hours. Stirred. NaHCO_ThreeSaturated aqueous solution (20 mL) and CHCl_Three(10 mL) was added to cool the solution. The organic layer was removed and the aqueous layer was washed with CHCl_Three(3 × 15 mL). Dry the combined organic solution (Mg._Four), Filtered and concentrated under reduced pressure. Chromatography (SiO_Two, 10% EtOAc- Xan) afforded 212 as a yellow oil (457 mg, 652 mg theoretically, Thus, the regiochemistry of nitration was confirmed. HMBC studies show that carbon-carbon connectivity is , NO with carbon (δ157.5) attached to C2 (δ132.6) and C8a carbon (δ125.8)_TwoResult Showed compatibility. This means¹H NMR was predicted by NOE experiments. That is, by irradiation of C3-H resonance (δ6.55), OCH at δ5.30_TwoPh resonance increased by 5% Plus CH at δ1.83_ThreeCO resonance increases by 4%. Similarly, by the 2D-NOE experiment, OCH_TwoPh and CH_ThreeA characteristic cross peak of C3-H due to CO was shown.   Often, the isomeric nitro product, 3- [N- (3,3-difluoro-2-propen-2-yl ) Acetamide] -1-benzyloxy-2-nitronaphthalene (about 10%)   3- [N- (tert-butyloxycarbonyl) -N- (3,3-difluoro-2-methanesulfonyl   3- [N-tert-butyloxycarbonyl) -N- (3,3-difluoro-3-phenylsulfoni Ru-2-tosyloxy-1-propyl)] amino-1-benzyloxynaphthalene   3- [N- (tert) -butyloxycarbonyl) -N- (3,3-difluoro-2-hydroxy-1-   3- (5-difluoromethyl-oxazolidinone-3-yl) -1-benzyloxynaphtha   1-amino-4-[(tert-butyloxycarbonyl) oxy] -2- [N- (3,3-difluoro-    DNA alkylation studies: selectivity and efficiency in TE buffer solution (10 mM Tris, 1mM EDTA, pH7.5), single³²P5 'end labeled w794 DNA⁵⁸Eppen containing (9μL) The dorf tubes were treated with the drug in DMSO (1 μL at a unique concentration). Vortex the solution Mix by brief centrifugation, then incubate at 25 ° C or 4 ° C for 72 hours. Privation. The modified DNA is treated with unbound drug by EtOH precipitation of the DNA. Separated. EtOH precipitation was performed using t-RNA as carrier, 3M NaOAc (0.1 volume) and And EtOH at -20 ° C (2.5 vol) was added. Mix the solution and wait at least 1 hour for REVCO Cooled to -78 ° C in a riser. Centrifuge DNA at 4 ° C for 15 minutes to pellet The cells were reduced and washed with 70% EtOH at −20 ° C. in a TE buffer solution containing 0.2 M NaCl. The pellet Was dried on a Savant Speed Vac concentrator and resuspended in TE buffer solution (10 μL). The solution of the alkylated DNA was heated at 100 ° C. for 30 minutes to give an adenine N3 alkyl. At the cleavage site. After a brief centrifugation, add formamide dye solution (5 μL). Added. Prior to electrophoresis, the sample was denatured by heating at 100 ° C for 5 minutes. The mixture was placed in a bath, centrifuged briefly, and the supernatant (2.8 μL) was loaded on the gel. D According to the standard close to the drug used in the NA reaction sample, Sanger A dideoxynucleotide sequencing reaction was performed. Polyacrylamide electrophoresis (P AGE) was run on an 8% sequencing gel under denaturing conditions (19: 1 acetylamide: N, N'- Tylene bisacrylamide, 8 M urea) in TBE buffer solution (100 mM Tris, 100 mM ho Acid, 0.2mM Na_TwoEDTA). PAGE should be performed for 30 minutes before loading the sample. Pre-run with de-dye solution. Autoradiography of the dried gel at -78 ° C Picker Spectra to enhance Kodak X-Omat AR film and screen with^TMTo It was performed using. Synthesis of Compound 309 308 in 2.5 M HCl-EtOAc (400 μL) (2.5 mg, 7.0 μmol; Boger et al., J. Am. Chem. Soc. 1992 114, 10056) was stirred at 25 ° C. under Ar for 30 minutes. N_TwoUnder the airflow, The solvent was removed. After drying under reduced pressure, the residue was dissolved in THF (200 μL), and 200 μL of 5% NaH CO_ThreeTreated with aqueous solution. After stirring the reaction mixture at 25 ° C for 5 hours, the solvent was removed under reduced pressure. I left. PTLC (SiO_Two, 0.25mm x 20 x 20cm, 70% THF-hexane) 309 was obtained as body (1.6 mg, theoretically 1.6 mg, 100%). Synthesis of compounds 314, 315, 316, or 317 After drying under reduced pressure, the residue, 39 (1.0 mg, 4.2 μmol, 1 equivalent; see above), EDCI (2.9 mg , 12.6 μmol, 3 equivalents; Aldrich) and 310, 311, 312 or 313 (1.1 (Ed .; Aldrich) in anhydrous DMF and the reaction mixture is allowed to stand at 25 ° C. for 16 hours. Stirred. The solvent was removed under reduced pressure, and the residue was dissolved in THF. Contact filling. Chromatography (SiO_Two, 0.5 × 6cm, 50% EtOAc-hexane) 314, 315, 316 or 317 were obtained as a rust solid (1.7 mg, theory 2.0mg, 85%). Synthesis of compounds 304, 305, 306 or 307 314, 315, 316 or 317 (1.4 mg, 3.91 in 2: 1 DMF-THF (112 μL) μmol) at 0 ° C. with NaH (1.6 mg, 39 μmol, 60% oil dispersion), The mixture was stirred for 30 minutes. N_TwoThe solvent was removed under a stream of air and reduced pressure. PTLC (SiO_Two, 0.25 mm x 10 x 15 cm, 30% EtOAc-hexane), 304, 305, 306 Or 307 was obtained.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 31/427 A61K 31/425 602 C07D 403/06 C07D 403/06 417/14 417/14 487/04 137 487/04 137 (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF) , BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, SD, SZ, UG), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, C Z, DE, DK, EE, ES, FI, GB, GE, GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU

Claims (1)

[Claims] 1. A compound represented by the following structure: Where R₁Is composed of alkyl (C1 to C6) and a radical represented by the following structure: Selected from group. Where R_TwoAnd R_ThreeTogether with the carbon atoms of the vinylene group shown, the group W, ie the vinyl Forming an N-substituted pyrrolidine ring containing a nylene group (provided that R_FourAnd R_FiveIs hydrogen You. ), Or R_TwoIs hydrogen and R_ThreeIs an N-substituted subgroup represented by the following compound, (However, R_FourAnd R_FiveIs hydrogen. ) Or R_TwoIs hydrogen and R_ThreeIs hydrogen and OCH_ThreeR is selected from the group consisting of_FourIs hydrogen And OCH_ThreeAnd R is selected from the group consisting of_FiveIs hydrogen and OCH_ThreeGroup consisting of More choice. 2. The first claim, wherein the N-substituted pyrrolidine ring has the following structure: The compound according to item.Where R_TwoBinds to C and R_ThreeIs bonded to N to form a pyrrolidine ring as shown,₆ Is NH_TwoAnd compounds selected from the group consisting of: 3. A compound represented by the following structure: Wherein R is selected from the group consisting of H and -OMe. 4. A compound represented by the following structure: Wherein R is -SMe, -S (O) Me, and-⁺SMe_TwoSelected from the group consisting of You. 5. A compound represented by the following structure:Wherein R is -SMe and-⁺Sme_TwoSelected from the group consisting of: 6. A compound represented by the following structure: Wherein R is -SMe and-⁺Sme_TwoSelected from the group consisting of: 7. A compound represented by the following structure: