JPH06500115A - Dynemycin-like compounds: synthesis, formulation and usage - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Synthesis, Prescription and Method of Use of Dynemycin Similar Compounds This application was filed in March 1991. The date 21 is related to the Continuation-in-Part (CIP) application of Patent Application No. 673.199. The patent application is also filed under Patent Application No. 562.269 dated August 1, 1990. Concerning the CIP application of No. The disclosures of these two applications are incorporated into the present invention.

Technical field The present invention relates to novel NA cleavage, cytotoxic and anti-tumor compounds, and More specifically, enediyne macrocyclic rings and epoxy Concerning fused ring systems containing side rings as well as chimeric compounds containing such fused ring systems It is.

Background technology Dynemicin A (compound shown below): (where , Me is a methyl group) has recently been developed by Micromonospora Chelsina (Micro .

Engineering (Golik et al., J, Am, Chem, S ac,, 1987.109. I) Ri.

3461-3462; 1 bid, 1987. 109. pp, 3462-3 464; Lee et al., 1bid, 1987.　109K 1ll), 3464-3466; x Ellestad et al., 1 bid, 1987. 109. pp, 3466-34U8) Anthracycline ne AntibiotiCS) J, H, S, El Khadem (Khade m) I! , Academic Press, NY (1982) and “Anthracyclino Recent Aspects in Anthracy Clinone Chemistry) J, Tetrahedron Symposia I Tetrahedron Symposia-in-Print ) No, 17. Edited by T. R. Kelly, Tetrahedron (Tet) rahedron).

This provided an opportunity for canned goods to be sold.

calicheamicin and nisperamycin (esperamicin) derivatives are perhaps the best known enediyne compounds. It must be something. Regarding the literature disclosing the first synthesis of the key calicheamycinone, References include: (a) Cabel et al., J. Am., Chea. Soc,, 1990.112. See p. 3253. kalicheama Other selected studies of model systems in the field of icins-esperamycins Regarding this, the following references: (b) =: Nicolaou et al., J. A. m, CheIn, Soc.

1988, 110. pp, 4866-4868; (c) Nie 7 Lau et al. J, Aa Chea Sac, 1988.1 Bile.

pp, 7247-7248; (d) Schoenen et al. Tetrahedron Lett, 1989.30゜pp, 3765- 3768; (e) 7 Magnus et al., J. ALCheILS ac,, 198g, 110. I) h).

6921-6923; (f) Kende et al., Tetrahedro n Lett, 1988.29. pp, 4217-S220 can be referred to.

Brief summary of the invention The present invention includes an epoxide ring and an enediyne macrocyclic ring, and therefore It involves a novel fused ring system with structural features similar to SynA. These compounds The substance has DNA cleavage, antibiotic and anti-tumor activity. Groups containing these compounds compositions and methods of making and using the same. The clamping ring compound of the present invention is as follows: It has a structure corresponding to the formula.

Here, A represents a double bond or a single bond, and R1 is H, Cr-Cs alkyl, Noxycarbonyl, benzyloxycarbonyl, C, -C, alkoxycarbonyl substituted C5-Cm alkoxycarbonyl (especially substituted ethoxycarbonyl), and 9-fluorenylmethyloxycarbonyl group. and R2 is H, carboxyl, hydroxymethyl and carbonyloxy C+- Cm represents a group selected from the group consisting of alkyl; R3 is H and C, -C, alkyl; Represents a group selected from the group consisting of koxy groups, R4 is H, hydroxyl, Cr-C 1 alkoxy, oxyacetic acid, oxyacetic acid C, -C, hydrocarbyl or benzy ester, oxyacetamide, oxyimidazylthiocarbonyl and C,- C, acyloxy group, and R6 and 7 each represent H or an intervening group. 1.2 or 3 fused aromatic 6-membered aromatic atoms together with the unsaturated carbon atoms of the vinylene group present Represents a ring ring system; W is 1.2 or 3 carbon atoms of the intervening vinylene group as indicated The fused ring compound contains a substituted aromatic hydrocarbyl ring system containing a 6-membered ring of 3.4 or containing five fused rings, two of which are aromatic rings, or In the hydrocarbyl ring system, W is attached to the structure shown as [a, b] (i.e., bonded to the nitrogen atom-containing ring in the structure shown as [a, b]); and P is a hydrogen atom. or methyl group, but W together with the carbon atom of the intervening vinylene group represents a 9.10-di When representing an oxoanthra group, it must represent a hydrogen atom.

In a preferred embodiment of the invention, W forms a benzo ring with an intervening vinylene group. , therefore the compound of the present invention has the structural formula shown below.

Here, R5 is a hydrogen atom, Cr-Cs alkoxy, hydroxy, C, -C, acyl oxyethanol, oxyacetic acid, 0-nitrobenzyloxy and selected from the group consisting of rogen atoms, and A and the remaining R groups are as defined above. Ru.

More specifically, in the aspect (3), R2, R3, R5, Rf and P are hydrogen atoms. The compound of the present invention corresponds to the following structural formula (wherein R1 and P are (as defined above): More preferably, Rs is Cr-Cs alkoxy, hydroxy, Cr-Cm acyl oxy, oxyethanol or oxyacetic acid group, and P is a hydrogen atom (H ) or a hydroxyl group, and the fused ring compound has the structural formula shown below. : The present invention also contemplates chimeric compounds (also referred to as chimers or chimeras); The chimeric compounds have (i) oligosaccharide moieties or (ii) immunoreactivity with target tumor cells. a monoclonal antibody or an aglycone moiety bound to the binding site portion of the antibody; The above-mentioned fused ring compound is included.

The oligosaccharide moieties include ribosyl, deoxyribosyl, fucosyl, glucosyl, and galacyl. tosyl, N-acetylglucoseaminyl, N-acetylgalactoseaminyl , the sugar whose structure is shown below (in the formula, the wavy line adjacent to the bond indicates the bond position) containing a sugar moiety selected from the group consisting of

The monoclonal antibody or binding site portion thereof has an R4 oxyacetamide group or Through an ester bond, or an oxyacetamide group from W or an ester bond and is bonded to the fused ring compound, ie, the aglycone moiety. The oligosaccharide moiety is R Hydroxy group of 4 oxyethanol group or hydrogen of oxyethanol substituent of W It is glycosidic bonded to the aglycone moiety via a roxy group.

Pharmaceutical compositions are also contemplated by the present invention. This pharmacological composition is effective against DNA cleavage, antibiotics or or the above compound or chimera as an active ingredient in an amount effective to inhibit tumor cell growth. and is dissolved or dispersed in a physiologically acceptable diluent.

The compound, chimera or any pharmacological composition thereof may also be used to inhibit DNA cleavage, tumor cell Useful in growth inhibition methods or as an antimicrobial agent. In this way DNA to be cleaved, target tumor cells or target microorganisms to inhibit growth. Contacting the cells with the composition of the invention. This contact state can be changed to produce a predetermined result. to maintain for sufficient time. Performing multiple doses of the pharmacological composition to achieve a predetermined contact You can also do that.

Brief description of the drawing In the accompanying drawings forming part of the disclosure of the present invention, FIG. 1 is stained with ethidium bromide. This is a photograph of a 1% agarose gel containing phosphoric acid at pH 7.4 containing 20% by volume of THF. φX174 type I with compound 40 for 24 hours in buffer (50mM) Showing DNA cleavage. Lane 1 is for DNA alone as a control. , lanes 2-6 are 5000.2000.1000.500 and 100 respectively Results obtained using μM Compound 40 are shown. shown on the outside of the gel. ■, II and III indicate the DNA types ■, II and III, respectively. .

Figure 2 is a photograph of a 1% agarose gel stained with ethidium bromide, I) H8 ,0 in 50mM Tris-HCI buffer for 24 hours. 47. Cleavage of φ×174 type I DNA by 42.54.55.58 and 62 shows. Lane 1 is for DNA alone as a control, lane 2. 3.4.5.6.7 and 8 are respectively the compounds 40.47.42.54.5 5.58 and 62 (5 bacteria each) are shown. Type ■, II and The indication III is the same as in FIG.

Figure 3 shows MIA P over 4 days with various concentrations of compound 2 (DY-1). Figure 2 is a graph showing the results of two studies of % growth inhibition of aCa-2 human pancreatic cancer cells. .

Figure 4 shows that Compound 2 (DY-1) at various concentrations caused 4,920 Figure 2 is a graph showing the results of four studies of % growth inhibition of murine bladder cancer cells. two ICs for research. The values were 43 nlil and 91 nM.

Figure 5 shows MB49 over 4 days with various concentrations of Compound 21 (DY-2). Figure 2 is a graph showing the results of two studies of % growth inhibition of murine bladder cancer cells for 20 days.

Detailed description of the invention ■、Compound The compounds of the present invention have an enediyne macro attached to a fused ring corresponding to the following structural formula I. Contains rings.

Here, A represents a double bond or a single bond, and R1 is H, C, -C, alkyl, phenylene Noxycarbonyl, benzyloxycarbonyl, Cl-Cm alkoxycarbonyl , substituted CI-〇, alkoxycarbonyl (especially substituted ethoxycarbonyl) and represents a group selected from the group consisting of ;R1 is H, carboxyl, hydroxymethyl and carbonyloxy-〇+- represents a group selected from the group consisting of Cm alkyl, R3 is H and C, -CS alkyl; Represents a group selected from the group consisting of koxy groups, R4 is H, hydroxyl, C, -C , alkoxy, oxyacetic acid (-0CH*C0tH), C, -C, hydrocarbyl or benzyloxyacetate, oxyacetamide, oxyethanol (O CHtCHtOH), oximidadylthiocarbonyl and c, -cm acyl selected from the group consisting of oxy groups, R8 and R7 each represent H or an intervening bicarbonate group; Together with the nylene group, represents a 1.2 or 3 fused aromatic 6-membered ring system; 1.2 or is a substituted aromatic hydrocarbyl ring containing three 6-membered rings, and the fused ring compound is 3. Containing 4 or 5 fused rings, two of which are aromatic rings, In addition, in the aromatic hydrocarbyl ring system, W is bonded to the above structure as [a, b]. ; and 8 represent a hydrogen atom or a methyl group, and 9. W together with an intervening vinylene group. When IO~dioxoanthra group is represented, it represents a hydrogen atom.

Examples of R@ and R7 are shown in Scheme III and discussed in this connection. Please follow this.

As shown above, the bond between substituents R2 and Rs, A, is a double bond or a single bond. can be obtained. This bond A is preferably a single bond.

C, -C, alkyl groups included in the definition of R1 include methyl, ethyl, propyl, iso Propyl, butyl, 5eC-butyl, pentyl, 2-methylpentyl, hexyl , cyclohexyl, cyclopentyl, etc. Substituted C+-Ca alkyl Groups are also included in the definition of R1. Such substituted alkyl groups include 2-hydroxy Hydroxyl compounds such as ethyl, 4-hydroxyhexyl and 3-hydroxypropyl Sialkyl group, 2-chlorobutyl, 3-halopentyl group (e.g. 3-fluoropeyl group) and haloalkyl groups such as ethyl group). The above C, -C, alkyl group and and substituted Cl-Cm alkyl groups further include R2 carbonyloxy C, -C, alkyl Cl-Cs alkyl ester of R2 carboxyl group, and 'CFRI It is also the C, -C, alkyl part of the urethane group. These same alkyl groups are R 3 or the alkyl moiety of the Cl-Cm alkoxy group of R4. R4 again is a C, -C, acyloxy group (discussed below) included in the definition of R5, e.g. For example, except for cyclohexyl and isopropyl groups, suitable carboxyl groups of the above alkyl groups can be used. It is a bonic acid derivative, and the cyclopentane derivative is a cyclopentyl carbonate derivative. Limited to carboxyl groups. An example of such a C, -C, acyloxy group is form oxy, acetoxy, propionoxy, butyryloxy, isobutyryloxy cy, pentanonyloxy, 2-methylbutyryloxy, pivaloyloxy, hexa Sanonyloxy, etc.

The alcohol-carbonyl moiety of urethane R1 is typically the corresponding 710-form. meth derivatives, such as chloroformates such as phenyl chloroformate, and the 6-position of a fused ring system or the corresponding numbered position as shown in Scheme II of Nitrogen source of secondary amine formed by adding an acetylene group-containing moiety to Formed by reacting with children. Also, such groups are shown below. From carbamates formed using substituted ethyl alcohols, base-catalyst It can also be prepared by exchange using

C, -IJ alkoxycarbonyl group and substituted c, -cm alkoxycarbonyl Examples of groups include Cl-Cm alkoxy groups or substituents as described above bonded to a carbonyl group. Reaction of Cl-Cm alkyl chloroformates containing substituted C, -C, alkoxy groups It can be formed by

Particularly preferred substituted C,-CS alkoxycarbonyl group is substituted ethyl group. An example of a oxycarbonyl group is a group having a substituent other than a hydrogen atom at the 2-position of the ethoxy group. 2-trimethylsilylethoxycarbonyl, 2-phenylsulfonyl ethoxycarbonyl, α- or β-2-naphthylsulfonyl ethoxycarbo α- or β-2-anthracylsulfonyl ethoxycarbonyl, 2-propylene Lopenoxycarbonyl, 2-hydroxyethoxycarbonyl, 2-tripheny phosphonium ethoxycarbonyl halides (e.g. chloride, bromide or iodide) and 2-trimethylammonium ethoxycarbonyl halide (e.g., those listed above).

R1 is a group that can be removed enzymatically or otherwise within the cell, and the substituent is It is preferred to provide secondary amines that are free of secondary amines. R1 is represented by reaction formula III (wherein R 2-substituted-ethoxycarbonyl group as shown in ), e.g. For example, 2-phenylsulfonyl-12-naphthylsulfonyl- and 2-anthra Compounds that are silsulfonyl can be liberated via β-dissociation under relatively mild conditions. A secondary amine can be formed. Phenylsulfonyl ethoxycarbonyl, α-naph Naphthylsulfonyl and β-naphthylsulfonyl ethoxycarbonyl (collectively naphthylsulfonyl) phenoxycarbonyl) is a particularly preferred R1 group; Rubonyl group is preferred as RIM.

The R' group is a methyl or hydrogen atom, but the carbon atom of the vinylene group with W intervening Together 9. When forming a lO-dioxoanthra ring, the R8 group is a hydrogen atom. shall be When W forms a benzo ring, the Ra group is methyl. Particularly preferred.

R4 is a hydrogen atom, hydroxyl, oxyethanol (-CH, CH, OH), Oxyacetic acid (-0CH, C02H), oxyacetic acid C, -C, hydrocarbyl esters, such as ethyloxyacetate (-0CH, CO2CH, CH,), etc. Above (7) CI-Ca 7/14/14 Tono! Ste/Lz, 7+J/I/, C, -C, unsaturated esters such as propargyl, 2-butenyl, benzyl esters , and oxyacetamide, which constitute particularly preferred embodiments of the invention. Ru. Exemplary CI-Cm and benzyl ester prepared, namely compound 24a- g showed activity against MIA Paca-2 tumor cells.

In the present invention, pharmaceutically acceptable non-toxic salts of the oxyacetic acid, such as sodium, carbon Also contemplated are lium, ammonium, calcium and magnesium. oxy vinegar The acid amide corresponds to the chemical formula: -0CHtCONR' "R", where R13 is hydrogen atom (H) or CI-Cm alkyl (same as above), and R a4 is a hydrogen atom, CI-Cs alkyl, phenyl, ■- or 2-naphthyl, l- or 2-anthryl or having from 1 to about 6 amino acid residues or 1li14 and R13 together with an amide nitrogen atom, 5- or 6-membered rings found in pyrrolidine, piperidine, or morboline, etc. to be accomplished.

Peptides of particular interest herein are distamycin or Its derivatives are. Distamycin derivatives are themselves known DNA-cleaving agents. be. In fact, an N-promoacetyl distamycin adduct of compound 2 was prepared. . Other particularly preferred peptides are -Ala-Ala-Ala-[(-Ala-)i ].

R4 groups containing derivatized oxyacetic acid amides or esters also contain from 0 to about 6 residues. may include a peptidyl spacer, such as (-Ala-)s, which The compound is a monoclonal antibody or the binding site portion of the antibody (collectively referred to as “Milb ”), which is described in relation to Reaction Scheme 111 below (where is R,). The xhb used immunoreacts substantially only with target tumor cells. Immediately i.e., tumor cell specificity and, as a result, also confer specificity to the drug molecule. Ru. Such a Mab-linked fused ring enediyne is one type of chimeric molecule of the present invention. Ru.

the spacer portion of the compound-mb construct joins the two parts of the molecule together It works like this. When the number of peptide residues is zero, the lysine ε-amino group of b is An amide bond as shown in Reaction Scheme III is formed. The spacer peptide chain is present In some cases, this typically involves amino acid residues with small side chains, such as glycine or or alanine residues, or amino acid residues with relatively hydrophilic side chains, e.g. Contains phosphorus, glutamine and aspartate residues. Peptide spacers are typical does not contain a cysteine residue, or a bond between the two parts of the chimeric compound can have virtually any structure that does not interfere with Some of the well-known peptides It can be prepared by any of the following synthetic methods.

The Mab portion of the chimeric construct described above is an IgG or ■-intive complete antibody molecule. can be constructed, in which case multiple compounds can be present per antibody molecule. Antibody binding Moieties can also be used in which at least one compound is a proteinaceous antibody binder. Can be bonded to the joint part.

An antibody combining site portion is the part of an antibody molecule that can immunoreact with an antigen and is often called a paratope. An example of a binding site portion of an antibody is F(ab) of a complete antibody molecule. , F(ab'), F(ab')z and F7 moieties, and prepared by well-known methods. Can be manufactured. The complete monoclonal antibody and the portion containing the binding site portion of the antibody are Collectively they can be referred to as baratope-containing molecules.

Examples of anti-tumor mbs are listed in the table below with the names used in the publications and the same 12 Parklawn Drive, Rockville, Maryland 20852, USA 30+ American Type Culture Collection Deposit approval number (A TCC No.) and tumor antigens to which the mb paratope is reported to be reactive. Also posted. References for discussion of each mb and its immunoreactivity are listed for the antigen reference. Given by footnote below the text.

Examples of anti-tumor properties 83.6 HBl189 [) GD3’14.8 HB 9118 GD2” 11c64 --- GD3" 9.2.27---:] Condritin sulfate rotoglycan R24---GD3' HT29/26 HB 8247 Colon cancer, glycoprotein gp 29'HT29/ 36 HB 8248 Colon cancer, glycoprotein gp 29'CLT85 HB 8 240 Colon cancer 6F64.5 -m= Breast cancer 7 R38,l--1 Pancreatic cancer, 70Kd protein 7F36/22) fB 82 15 Human breast cancer 1T16 HB 8279 Human bladder tumor, glycoprotein gp48 "T43 HB 8275 Human bladder tumor 1 TIOI HB 8273 Human bladder Bladder tumor 9116-NS-19-I HB 8059 Colorectal cancer, monocya σ Ngulioside 1゜126 HB 8568 GD2” CLH6HB 8532 Colon cancer 1I CLG 479 HB 8241 Colon cancer +219.9 CRL 8019 C EA”CLNH5-m= Lung cancer +4 16-88 -m = colon cancer 15 1. Cheresch et al., Proc, Natl, Aca d, Sci, USA, 1985.82. pl).

5155-5159: Ib1d, 1984. 81. pl), 5767 -57712, Sheresh et al., Cancer Res, 1986.44 ．． pp, 5112-51183, Cielesh et al., J, Ce1l Bi ol,, 19g6.102. p, 6884, Bumol, etc. , Proc, Natl, Acad, Sci, USA, 1982.7 9. p, 1245; Harper et al., J. fmunol. , 1984.132. p, 20965, 0SPNo, 4.507,3 916, USP No, 4.579.827? , USP No. 4,522 ,9188, European Patent Application No. 84400420.0. Publication No. 011 8365. Published September 12, 1984 9, European Patent Application No. 8410251 7.4. Publication No. 0118891. ．． Published September 19, 1984 10, U SP No. 4,471,05711, Cielesh et al., J, Ce1l B iol,, 1986.102. I), 688; USP No. 4.4 71.05712, USP No, 4,579,82713, 0SP No, 4.349,52814, patent application PCT/US83100781; wo 8310431315, European Patent Application No. 85300610.4. Publication No. , 0151030. Published on August 7, 1985 Fused ring enediyne compounds of the present invention can also be glycosidic linked to sugar moieties to form a second type of chimeric molecule. can. In such chimers, the fused ring enediine compound is doxorubicin, calicheamicin antibiotics such as icin) or nisperamycin (esperaOIicin) The sugar moiety becomes an oligosaccharide moiety instead of an aglycone that is formed in the molecule. supersede. The bond between the aglycon and oligosaccharide of the fused ring enediyne compound is Typically through the hydroxyl group of the spacer group, the spacer group is itself reactive. is bonded to the fused ring enediyne via a hydroxyl group. preferred space The S group can be the R4 group discussed and exemplified below, or it can be a substituent of W. It is an oxyethanol group that can be

The oligosaccharide portion of the molecule typically comprises the fused ring enediyne compound (aglycone ) is added after the composition of the part is completed. However, other reactive molecules of the aglycone Remove any blocking groups on the functional group. These typically add the oligosaccharide moiety removed after Sugar moieties are added by standard methods as discussed below.

Glycosidic sugar moieties are monosaccharides, such as ribosyl, deoxyribosyl, and fucosyl. syl, glucosyl, galactosyl, N-acetylglucoseaminyl, N-acetyl The structure of the sugar moiety, which may be or is more preferably a tylgalactose aminyl moiety, is as follows: The wavy line shown below and adjacent to the bond in the formula below represents the position of the bond.

the position of the glycosyl bond to be formed on the sugar moiety used to form the chimeric compound; The moiety is typically activated prior to attachment to the fused ring enediyne compound. for example , other parts should be protected, for example, 'BuMezSi or Et, Si group, etc. TI (diethylaminosulfur in F) React with trifluoride (DAST) at -78°C in the presence of 4-person molecular sieves. In this way, a -fluoro derivative is formed. Enediyne with free hydroxyl group and then l-fluoro protected satskara in the presence of silver perchlorate and stannous chloride. A given protected, typically blocked, chimer molecule is reacted with obtain.

Similarly, in methylene chloride at about room temperature, the 1-position hydrochloride of the saccharide protected except for the xyl group) in sodium hydride and trichloroacetic acid. When treated with tonitrile, [Gerandler, etc., Car bohydr, Res, 1985゜Hydroxyl group of yne (aglycone) The saccharide is activated to the cup ring with. The cup ring is then A given chimera protected by boron trifluoride-etherate in tyrene form a compound.

Once the aglycon and oligosaccharide are cupped, any protecting groups present are removed and The desired compound is obtained, which is then recovered using standard methods. Synthesis example is below will be discussed.

If used, they can be benzo, naphtho and anthra rings. This invention intends to The ring derived from anthracene is a 9,10-dioxo group (anthracene). quinone), and therefore 9. It is called IO-dioxoanthra ring.

benzo ring, naphtho ring or 9. IO-dioxoanthra ring forms part of the fused ring system When formed, these fused rings have carbon atoms at the l- and 2-positions or (a, b) to the remaining fused ring system via. benzo, naphtho or 9. lO-dioxo Anthra-fused ring moieties contain one or more substituents at doctrine positions left open for substitution. be able to.

These substituents include hydroxyl, Cl-Cm alkoxy, oxygen C3-C, acyl selected from the group consisting of (chloro, bromo or iodo) I can f.

For benzo rings, l or 2 substitutions at one or two of the remaining positions of several groups groups can exist. Identical substituents because it reduces the possibility of isomer formation Symmetrical substitution by is preferred. If a single substituent is present on the benzo ring, its position The substituent is designated R5, a convenient definition of which includes a hydrogen atom. Thus, R5 is a hydrogen atom (i.e., has no substituent>, C, -C, alkoxy, benzyl group) oxy, 〇-nitrobenzyloxy, hydroxy, Cl-Ca acyloxy, oxy diethanol, oxyacetic acid, oxyacetic acid C+-Cm hydrocarbyl ester and and halogen. Hydroxyl group or hydroxy within the cell A group capable of forming a metal group is present, and the hydrogen atom is located at the met needle position with respect to the nitrogen atom of the adjacent ring. Preferably, a droxyl group is present. Two substituents on the benzo ring If present, these are denoted by RIG and general ■, c, -cm alkoxy, base acyloxy, oxo, C1-Ca acyloxy, hydroxy and halogen selected from the group consisting of

W is more preferably benzo containing one substituent R5. Particularly preferable 1! ! ! , R5 is the meta group of the benzo ring to the nitrogen atom bonded to RI. or in the para position. This Rs group is more preferably hydroxy, C,- C, alkoxy, benzyloxy, nitrobenzyloxy, Cl-Cs acyl Oxy, oxyethanol, oxyacetic acid and oxyacetic acid C, -C, hydrocal selected from the group consisting of biyl esters, in which R5 is in the meta position relative to the nitrogen atom. In this case, this R5 group is an electron-releasing group, e.g. hydroxy or hydroxyl in the cell. C, -CS acyloxy which can form a group is preferred. C, -C, reed The hydroxyl group is cleaved intracellularly by endogenous esterase, etc. to generate a hydroxyl group. It is thought that it is a prodrug type of the hydroxyl group that forms. This kind of electricity The presence of a child releasing group may help enhance the potency of the compound against target tumor cells. Conceivable. This enhanced ability is highly inducible for epoxide cleavage and cyclization reactions. It is believed that this is due to

When R3 is in the para position to the nitrogen atom, the RI group is O-nitroben Zyloxy, oxyethanol, oxyacetic acid or oxyacetic acid C+-Cshydro Preferably it is a carbyl ester. These groups are particularly useful for the preparation of chimeras. It is for use.

Particularly preferred compounds are those having a structure corresponding to formula Xlb described below. Ru.

The naphtho ring can have three substituents. This ring is hydroxy, Cl-Cs From alkoxy, benzyloxy, Cl-Cs acyloxy and halogen atoms a group R5 at the 4-position selected from the group consisting of hydroxy, Cl-Cs alkoxy , benzyloxy, Cl-Cs acyloxy, oxy and halogen atoms. with substituents at the 5-position (R1+) and 8-position (R'') selected from the group can. 9.10-Dioxoanthra ring has 4-position (R5), 5-position (R1) and and the 8-position (R1″), each of which is independently Hydroxy, C1-C@alkoxy, benzyloxy, Cl-Cm acyloxy and a halogen atom. Thus, R5, R1 and 12 are Defined as the same group, all these groups are either R5, RF or R12 However, they are shown separately here.

Exemplary structural formulas of such fused ring compounds are shown in Structural Formulas II to IX below. or where each of the R groups is as discussed above.

In addition to the above preferred description regarding R1 and that bond A is a single bond, some Certain other structural features and substituents are preferred.

That is, R2 and P are hydrogen atoms, and R6 and cFR7 are hydrogen atoms. is preferred. In addition, W is a substituted Ben group or an unsubstituted Ben group along with the vinylene group shown in the figure. A naphtho or 9,10-dioxoanthra ring is preferred. Furthermore, the applicable The fused ring compound contains a total of three fused 6-membered rings, resulting in W being the vinyl shown. It is preferable to form a benzo ring together with the ren group.

One particularly preferred group of compounds of the invention in which W is an R5-substituted benzo ring has the following formula: It corresponds to X.

Here, A is a double bond or a single bond, and R1 is H, C, -C, alkyl, or fluorine. phenoxycarbonyl, benzoxycarbonyl, Cl-Cm alkoxycarbonyl and 9-fluorenylmethyloxycarbonyl. R' is H, carboxyl, hydroxymethyl and carbonyloxy C+- selected from the group consisting of Cm alkyl, R'' is selected from the group consisting of H and C1 (, alkoxy); R4 is H, hydroxy, oxyacetic acid (-0CHtCO2H) , oxyacetic acid C+-Cm hydrocarbyl or benzyl ester, oxyacetic acid Amide, oxyethanol, oxyimidazylthiocarbonyl and C, -C, selected from the group consisting of acyloxy, R5 is a hydrogen atom, C, -C, alkoxy , benzyloxy, 0-nitrobenzyloxy, hydroxy, Cl-Cm acyl Oxyoxyethanol, oxyacetic acid, oxyacetic acid C+-Ca hydrocarbyl selected from the group consisting of ster and halogen atoms, and 6 and R7 are each H or together with intervening vinylene groups form a fused aromatic ring system of 1,2 or 3 and R6 is methyl or hydrogen atom.

Even more preferred groups of compounds of the invention have the following structural formulas XI, X[a and Xl This corresponds to b.

Here, R1, R4, R5 and P are as defined above.

Further preferred R1, R4 and P with respect to compounds corresponding to structure XI are Ru.

These preferences also relate to the compounds discussed above.

That is, R1 is most preferably phenoxycarbonyl, phenylsulfonyl ethoxy cyclocarbonyl, naphthylsulfonyl ethoxygalbonyl or hydrogen atom.

R8 is most preferably a hydrogen atom (+'l), which is represented by formula XIa or X gives lb of compound. R4 is most preferably H, hydroxyl, imidadylthi Ocarbonyloxy, benzyloxy acetate and C, -C, hydrocarbyl ethyloxyacetate group, such as ethyloxyacetate. The above formula 'XTa or Rs in Xlb is H, most preferably as in 11b above , hydroxy, Cl-Cs alkoxy, benzyloxy, Cl-Cm acyloxy C, oxyethanol, oxyacetic acid, oxyacetic acid C+-Cs, hydrocarbyl or or benzyl ester and O-nitrobenzyloxy. R5o-nitro Note that the benzyloxy group is not typically used in the pharmacological compositions discussed below. Should.

Structural formulas of particularly preferred compounds are shown below with compound numbers used in this specification. vinegar. In the formula below and elsewhere in this specification, ph is a phenyl group, Me is a methyl group, NBnO is an 0-nitrobenzyloxy group, and Nap h is a naphthyl group, and BuCO* is a pivaloyl group.

40°41 +1. pharmacological composition Compounds or chimeras of the invention include dynemycin A, calicheamycin, varnish peramicin and neocarzinostatin ), as well as a DNA cleaving agent, as well as an antibiotic and cytotoxic (antitumor) agent. It is useful. Accordingly, the compounds of the present invention are referred to as "active agents" or "active ingredients." can also be called.

DNA cleavage is carried out by the method described below and by Mantlo et al., J. Org, CheIL.

1989, 54. I), 2781; -j Nicolaou et al. , J, Aa Chea Sac, 1989.110@.

p, 7147; Nicolau et al., 1 bid, 19g8.110. p, 7247; or Zain ('1efn) et al.

5science, 19g8.240. p. 1198 and cited therein. The assay can be performed using methods described in various literatures.

Compounds or chimers of the invention, like dynemycin A, have Gram-positive bacteria. The activity can be measured using various known methods. For example, Konisi hi)'4. J. Antibiotics, 1989. XLII: 1 449 can be referred to. Antimicrobial and antitumor assays are also USP No. 4,837,206 and the procedures described below. Ru. This patent is incorporated herein by reference.

The above compounds are also described by Haseltine et al., J. Am. Chea Soc, 19g9゜III, p. 7638. benzyl mercaptan, triethylamine and 1,4-cyclohexylamine. The Bergman cycloaromatization reaction in the presence of sadiene can be carried out. It can be shown that This reaction is similar to that seen during DNA cleavage. This is a cyclic reaction and an auxiliary screen for selecting more active compounds. It can be used as a screen (Cco-screen).

Thus, the pharmaceutical composition comprises the above-described compound or chimer of the invention as an active agent. . Pharmacological compositions are well known in the pharmaceutical art, combining the active compound and a carrier therefor. Prepared by any method including compounding. For therapeutic use, the book The compounds or chymers of the invention can be administered in a series of known pharmaceutical compositions. like this The composition is suitable for oral, parenteral administration or can be formulated as a suppository. The composition of these In products, the active agent is typically dissolved or dispersed in a physiologically acceptable carrier. be scattered.

A carrier or diluent is a substance useful in administering the active compound and in the composition. Compatible with other ingredients and not harmful to the end of the recipe ``It must be pharmaceutically acceptable.'' As used herein, ``physiologically Tolerable (physiologjcally tolerable) J and and pharmaceutically acceptable e) The term J is used interchangeably and when administered to a mammal. , allergies or similar inappropriate reactions, such as gastric disorders, dizziness, etc. Refers to child-like entities and compositions. This physiologically acceptable carrier is may take a wide range of forms depending on the desired formulation and intended route of administration. Can be done.

As an example of a useful composition, a compound or chymer (active agent) of the invention may be present in a sterile suspension. Dissolved or dispersed in a liquid composition such as liquid or solution, or added with a suitable preservative. It can be used as an isotonic formulation containing. Particularly suitable for the purposes of the present invention are water-based buffered or unbuffered isotonic and sterile saline or glucose solutions. liquid, as well as water alone or an aqueous ethanol solution. These compounds or Additional liquid forms with which the drug may be formulated for administration include edible oils such as cottonseed oil, sesame oil, etc. Emulsion containing flavorings such as oil, coconut oil, peanut oil, etc. and elixirs and similar pharmaceutical vehicles. Replacement of liquid diluent An example of this is Remington's 7-Mathematical Sciences. on’s Pharmaceutical 5 Sciences), 77 Kupa Mack Publishing Co., Ltd. PA, Inc. Stone (1980).

The active agent can also be administered as liposomes. Well known in the field As such, liposomes are generally derived from phospholipids or other lipid materials. lipo saw The film is formed by monolayer or multilayer hydrated liquid crystals dispersed in an aqueous medium. Riposo Any non-toxic, physiologically acceptable and metabolizable lipid that can form a Wear. The composition of the present invention in liposome form contains stabilizers, preservatives, excipients, etc. It can be included in addition to drugs. Preferred lipids include synthetic and natural phospholipids and Sphatidylcholine (lecithin).

Methods for producing liposomes are known in the art. For example, Pre, 5 cott), Methods in Ce1l Biology, Vol. , XIV, Academic Press, New York (1976), p. See 33 et seq.

The active agent is further preferably a tablet containing a unit dosage of said compound or chymer. Alternatively, it can also be used in compositions such as pills. For this purpose, the drug (active ingredient) ) with known tablet-forming ingredients such as cornstarch, lactose, sucrose, and sodium chloride. Rubitol, talc, stearic acid, magnesium stearate, dicalcium phosphate gum, gum, or similar substances known as non-toxic physiologically acceptable carriers. Mix. Tablets or pills give unit dosages that give a sustained or delayed effect Can be formulated in layers or in other manners.

In addition to the carrier components described above, the pharmacological formulations described herein may contain one or more suitable additives. Optional carrier ingredients such as diluents, buffers, flavorings, binders, surfactants, enhancers, etc. Dosing agents, lubricants, preservatives (including antioxidants), etc., as well as blood at the intended end of the recipe. It can contain substances added to make the liquid isotropic.

The tablet or pill provides resistance to degradation in the membrane and contains the active ingredient. Envelope so that it is delivered intact to the duodenum or for sustained release. An enteric layer can also be provided. Various substances can be added to such enteric layers or can be used to form coatings, examples of which include polymeric acids or such acids and sheratz. Mixtures of shellac, cetyl alcohol, cellulose acetate phthalate, etc. It includes compounds. Particularly suitable enteric coatings include styrene-maleic acid copolymers. In combination with known materials that contribute to the enteric properties of the coating. enteric coated tablets The preparation method is USP No. 4.079.12 given to Sipos. 5.

This patent is incorporated herein by reference.

As used herein, the term unit dose refers to warm blood means a physically discrete unit suitable as a single dose for administration to a substance; Each such unit contains a predetermined amount and formulation of the drug calculated to provide a predetermined therapeutic effect. In combination with a pharmaceutically acceptable diluent. Unit dosage forms suitable for the present invention Examples of shapes are tablets, capsules, pills, powder sachets, granules, wafers, cashews, and tea. - Teaspoonfuls, dropperfuls perfuls) ampoules, vials, any separate combinations of the above, etc. .

The preferred or particularly preferred compounds or chymers listed above are useful for use in pharmaceutical compositions. preferred or particularly preferred for

The compounds or chymers of the invention are administered in an effective amount sufficient to achieve the desired result. present in pharmacological compositions. For example, in vitro DNA cleavage produces the desired result. , the compound or chimer of the invention has a DNA concentration of about 0.02 μg/ can be used in a sufficient amount to give a concentration of about 1.0 to about 5000 μM per μl. Wear. When used as cytotoxic (antitumor) agents, the compounds of the present invention or The effective amount is about 0.1 to about 15 mg per kg of body weight or per ml of blood The amount is sufficient to provide a concentration of about 0.01 to about 50 μg/nl. of the present invention The compound or chimer is about 0. Antibiotics in the concentration range of O1-1 to about 50 μml Shows activity. The above concentrations and dosages may vary depending on the particular compound of the invention used, as is well known. It varies depending on the substance and target, such as 1fDNA, tumor, microorganism, etc.

IIl, Banri The compounds or chimers of the present invention can be used as cytotoxic drugs in the cleavage of DNA. useful in inhibiting the growth of tumor cells and tumor cells, and used in the method. Compounds or chimers of the invention typically have the compositions described above. used in things.

According to such methods, the DNA or target cells to be killed or whose growth is to be inhibited are The cells are contacted with a composition comprising a compound or chymer (active ingredient) of the invention. child Here, the composition contains an effective or sufficient amount of the compound or compound of the present invention for the above purpose. The mer is dissolved in a physiologically acceptable (pharmaceutically acceptable) diluent as described above. or in a dispersed state. This contact has the desired result, i.e. cleavage of DNA, killing of cells. Maintain for sufficient time to achieve growth inhibition of wound or tumor cells.

Simply mix with the parabolite and set appropriate conditions i.e. temperature and untreated as a control. contact by keeping them together under suitable conditions that result in cell growth. Animals such as horses, mice or rabbits, livestock such as horses, cows or goats, or Primates such as monkeys, eizos or humans are treated. Here, the composition and should be killed Oral, nasal or anal administration, or contact with cells whose growth is to be inhibited. administers the composition to a mammal by intravenous, subcutaneous or parenteral administration. This is achieved by Thus, in vivo contact may be via the blood or lymphatic system. be achieved.

A single dose (or mixture) and the contact achieved therein should aim for a given contact condition. The contact between the active ingredient of the composition and the target cell will be at a certain level depending on the target cell. compounds of the invention over a period of time, such as days, weeks or months or more. It is maintained by repeated administration.

DNA cleavage and MIA PaCa-2 human pancreatic cancer cells (ATCC CRL 14 20) and former 4920 Murine Bladder Cancer Target Cells (Dana-Farber, Boston, MA) - Cancer Institute) (Dana Farber Cancer In Obtained from Dr. Lan Bo Chen of 5 titute) Examples of the method of the invention for the inhibition of 1) as well as 10 other tumor cell lines are provided below. Discussed below.

IV, compound synthesis Compounds of the invention can be prepared according to several routes. Some of them are shown below as reaction formulas. For example: A suitable synthesis plan for these syntheses is shown in the following reaction formula (2). Once again Reaction formula for general formal synthesis! I is shown below.

Reaction formula■: Briefly, a basic fused 3-14- or 5-fused 6-membered ring system is first formed. Ru.

As an example, compound 4 (W represents Benha's tetrahydro-phenanthridine fused ring system) According to Scheme II of the general synthesis method used, the oxygen-containing substituent R4 (the oxygen source (the child is bonded to the ring carbon atom at the 10-position) is produced as compound 5. the oxygen -The introduction of substituents is carried out by oxidation with monochloroperbenzoic acid (mPBA), etc., and then and reflux to combine the formed acyl ([ff1-oxide) with This is achieved by rearrangement to the 10-position of product 5 (steps a and b). This reed The alcohol group is removed to form the alcohol compound 6, step c), which is then converted into t-butyl Blocked with dimethylsilyl group (Sf　’　BuMe,; compound 7, step d) Ru.

A suitable acetylene group-containing compound also has the function of blocking the generated secondary amine. In the presence of a concomitant activating moiety, e.g. phenyl chloroformate, ethynyl It is added adjacent to the nitrogen atom (at the 6-position) by reaction with Grignard reagent, etc. to form substituent R1 of compound 8 (step e).

The epoxide ring is then modified as in compound 9 with (mCPBA) etc. It is oxidized (step f) and added between the 6- and 10-positions. epoxy to produce The do ring is on the side of the groove opposite to the acetylene group at the 6-position, and is preferably The id is in the α-configuration, while the acetylene group is in the β-configuration.

The oxygen bond R4 group Si' BuMe is replaced with hydrogen, and step gL is thus produced. The alcohol is then converted to the ketone, compound 11 (step III). (See also Moderation). The vinyl acetylene moiety of this enediyne-containing ring is necessarily If necessary, add as in compound 13. This process involves (Z) 1-chloro -4-trimethylsilyl-budo-en-3-yne (compound 12) and the ketone Compound If and butylamine, triphenylphosphine and palladium 1' This can be carried out by reacting in the presence of acetate (step i).

Equation III shows that the entire enediyne carbon skeleton is reacted in a single step, as discussed below. This shows that it can be bonded to the 6-position. Reaction formula III is also applicable to aniline compounds. Formation of benzo ring W by reaction with ethylchlorohexanone-2-carboxylate It explains the formation. Compound 41 was prepared from m-amicidine (R'=m-methoxy). Manufactured. On the other hand, in the preparation of compounds 59a and 59b, 1Ileta-pivaloy ester (R’yta-Cs acyloxy) to meta-benzyl ether Prepared from If R1 methyl group is required, ethyl 3-methylcyclohexano 2-carboxylate is used.

Derivatives of compounds having hydroxyl groups, such as compounds 2 or 21 (reaction formula 0 R1 or R4) of I is according to reaction formula III (R,=H,H) via step j , then according to Equation Vll, similar to that produced in step a of Equation III, meta-(2-nitrobenzyloxy)ani It can be prepared by using phosphorus. This meta-2-nitrobe The ndyloxy group (NBnO) is removed after step j of Reaction Scheme III and exposed to ultraviolet light. By irradiation, the meta-hydroxy- Substituted derivatives can be formed. meta-hydro of compound 2, compound 42 The xy-substituted derivative is formed via irradiated compound 43, with two molecules at a 2-concentration. It has been shown that stranded DNA cleavage occurs.

The trimethylsilyl group was removed to form a free (unbonded) acetylene group (reaction Formula II, after step j), the acetylene group is transferred to the carbonyl group in the 10-position with a base, e.g. The method of the present invention is obtained by inserting by reaction with lithium diisopropylamide (LDA). Forming a fused ring compound (where R4 is a hydroxyl group (OH)) degree k). This R4 group can be substituted with a hydrogen atom or derivatized as discussed below. can do.

As mentioned above, R@ and 7 are preferably hydrogen atoms (H). However, R @ and P together with the intervening vinylidene group form an aromatic l-12- or 3-membered ring system. Can be done.

The ring system may be a hydrocarbyl or heterocyclic group. If such a ring system exists When the enediyne-containing ring has an ethylenic bond, an unsaturated carbon-carbon may be one of the elementary bonds, and this all-enediyne carbon skeleton is typically As shown in There is.

Reaction formula III To explain reaction formula Ill in more detail, 2-ethoxycarbonylcyclohexano react with aniline (R' as shown below) and convert the reaction product into H,SO , to cyclize. This cyclized material is reduced with lithium aluminum hydride. and then air oxidation (step a) to form a 3-membered ring compound. this compound Oxidation, acetylation and rearrangement (step C) to form the acetoxy compound ( R=OAc) and its acetyl group is removed to form alcohol (step d, R= OH) and then convert this alcohol in step e to t-butyldimethylsilane. Reacting with lylutrifluoromethanesulfonate in the presence of 2,6-lutidine (R・O3i' BuMez).

The product of step e is first reacted with a chloroformate (of which R1 is shown below), Then, in step f, blocking with trimethylsilyl group (TMS) Diacetylide or aromatic diacetylide ring-based compounds (Rt is hereinafter referred to as (shown below) to form a partially bound macrocyclic ring precursor. to be accomplished.

This epoxide ring is formed in step g by reaction with mCPBA. The Si' BuMe.

The group is removed in the process by reaction with BuNF, and the alcohol produced is converted into molecules. Oxidation with pyridinium chlorochromate in step i in the presence of sieves gives the ketone form.

This macrocyclic ring can be reacted with lithium neuisopropylamide (LDA). The ring is closed in the "step". The hydroxyl group formed in the process In step k, react with a suitable 2-haloacetic acid derivative (Rs is shown below), Form the final product.

Rs　CHzCOOHR4”　OMeCHzCONH-Peptide OH CO(CHt)2cOOMe　OCOMeCHgCONJHPh　OCO’　B uCH2CONH-naphthyl 0NBn CH,C0NH-anthracil H CToCOt-Spacer Mab Spacer Satsuka Ride R with superscript numbers as defined elsewhere in this specification and R groups as used herein. Subscript numbered R groups were used in this reaction scheme to distinguish between.

The term "spacer" as indicated for R1 is typically 0 to about 6, monochrome A peptide containing amino acid residues that binds a null antibody Mab to the compound. O The R1 spacer attached to the oligosaccharide typically Oxyethanol used to bind to sid-bonded or paratope-containing molecules or oxyacetic acid group.

3-14- or 6-position of the 6-membered ring system (relative to compound 4) A suitable vicinal diyne aromatic compound is trimethyl vicinal civalide. Silyl acetylene causes diisopropylamine, dicyanophenyl paradichloride alkylated in the presence of um, triphenylphosphine and cupric iodide, It can be prepared by forming a vicinal bis-trimethylsilylacetylene derivative. Wear.

W+, Aldrich Chemical Co., Milwaukee Vicinal dihaloaromatic compounds available commercially from Cal Co, ) Examples are 1,2-dichlorobenzene, 1,2-dibromobenzene and 1,2-dichlorobenzene. Includes chlorobenzene. 2,3-dibromonaphthalene, 2,3-dibromore Nthracene and 6,7-dichloroquinoxaline are listed in the chemical abstract below. Registration numbers (R, N,) 13214-70 to 5.117820-97-0 and and 19853-64-6.

After the preparation of the vicinal bis-trimethylsilylacetylene derivative, the trimethylsilane Reacting one of the lyle groups (TMS) with silver nitrate and potassium cyanide Replace with hydrogen atom. The resulting aromatic diacetylide is then as previously described. react with a fused ring system.

Suitable vicinal dialdehydes are prepared via vicinal dihydroxymethyl compounds. It can also be manufactured. In one synthesis example, 1,4-dimethoxy-6,7-simethy Lunaphthalene (R, N, 73661-12-2) wo, N-Pro(-Sucsin) imide (NBS) and azohisisobutyronitrile (AIBN) and halogen The corresponding vicinal dibromomethyl derivatives can be obtained by reaction in a solvent such as carbon tetrachloride. form the body. The reaction between the dibromo compound and the hydroxyto ion is a vicinal reaction. Forms droxymethyl derivatives. The pyridinium of this dihydroxymethyl compound Mild oxidation by muchlorochromate (Pcc) leads to the corresponding vicinal dialde Give Hido. This dialdehyde, triphenylphosphine and carbon tetrabromide , then with butyllithium, and then with trimethylsilyl chloride. Upon reaction, it gives the vicinal di-TMS acetylene derivative. 1 TMS group After replacing the aromatic diacetylide with hydrogen as described above (as previously discussed), the aromatic diacetylide is with the fused ring system.

The above synthesis method also applies to unsubstituted aromatic compounds such as vicinal dicarboxylic acids, anhydrides, etc. Alternatively, it can also be applied to esters. For example, phthalic acid and naphthalene-2,3 -dicarboxylic acids are both available from Aldrich Chemical Company. what are these By reacting either or both with diazomethane, the corresponding dimethyl ester Can generate tell. Reduction of the diester to a vicinal dihydroxymethyl derivative can be achieved by reduction using diisobutylaluminum hydride (DIBAL). Wear. The resulting dihydroxymethyl compound is then reacted as described above to Forms certain compounds.

Aromatic diacetylides have two vicinanocetylene groups symmetrically bonded to the ring system. It is preferred to minimize the formation of isomers. Thus, 2.3-disubstituted- Naphthalene, anthracene or quinoxaline compounds are utilized, or 6, 7-disubstituted-quinoxalines and the like are used.

An example of the synthesis of a compound corresponding to Structural Formula V is shown in Reaction Scheme Iv below.

Reaction formula IV or, clonaphthazarine, compound 25 [Banvir (Banvil) e) et al., Can, J.

Chem, 1974.52. p, 80; Savard et al. Tetrahedron, 1984.40. p.

3455, and Echavarren et al., J. CheI LRes, (3), 364 (1986)) in step i. Compound 26 [Scmidt) etc., 5ynthesis, 95 8 (1982)] and ] by a Diels-Alder reaction to form an aminoamine. Thuraquinone (compound 27) is formed. DIBA in step ii of this quinone in the presence of cesium fluoride in step iii. Alkylation with methylene bromide in DMF yields 0-block compound 28. give.

Removal of urethane group with base in step iv, cycloaddition and reaction shown in Scheme III The following reactions were carried out for compound 29 (step v-viii) and compound 30 (step ix). and xii). Compound 30 is then reacted with lithium iodide in pyridine. and oxidized with 2,3-dichloro-5,6-dicyazbenzoquinone (DDQ), and pivaloyl chloride (Piv) to form compound 31 (steps xiii-xv ) to form.

Ethylmagnesium bromide was combined with compound 31 in the presence of phenylchloroformate. React (step xvi). The obtained compound was reacted with l[1cPBA, etc. Forming a poxide (step xvti). The 13 groups were removed with BuaNF, step xviii) to form the alcohol, which alcohol is then subjected to Jones) reagent to form the corresponding ketone (step xix). this The ketone is converted into (Z)-1-chloro-4-trimethylsilylbutol-l-en-3-y Compound 32 is formed by reacting with chlorine, palladium, Cul and butylamine. (Step XX). Compound 32 was treated with silver nitrate and potassium cyanide (step xxi ), LDA (step xxii), thiocarbonyldiimidazole (step xxii) i), butylstannane and AIB N (step xxiv) and then reacted with hydroxyto ions (step xxiv). step XXV), compound 33 is formed.

For the preparation of compounds corresponding to structural formula IX, ■-aminoanthraquinone (al Dritch Chemical Co.) can be used as a starting material. Here, the amino group is, for example, is blocked with a t-Boa group, and the quinone functionality is reduced with ζfDIBAL. The resulting phenolic hydroxyl groups are blocked with pivaloyl chloride.

The t-Boc group is then removed by reaction with trifluoroacetic acid (TFA) and the Cycloaddition of the aromatic ring with ethylcyclohexanone-2-carboxylate followed by Cyclization with sulfuric acid, reduction with potassium hydrogen lithium aluminum, air oxidation, Quinoid structures are reformed as baloyl groups are lost during these reactions.

The above reaction produces a fused ring system. This engineered macro ring part is then It is added like a solid.

As shown in structural formulas VL Vll, VIII and IX, the saturated 6- If a double bond is desired in the ring member, this functionality can be imparted as follows: can. i.e., forming the fused ring system and epoxide, all as discussed above. , generates one of the acetylene bonds, forming an amine block and a ketone. place Examples of compounds of specific structure are compounds 13 and 3 as shown in Schemes II and IV. It is 2. This compound is then oxidized to form a ketone at the 10-position of compound I3. A hydroxyl group is introduced into the α-position (9-position of compound I3). Then, This hydroxyl group can be replaced with triethylsilyl (TBS or EtsS+) chloride, for example. block with lido to close the macrocyclic enediyne ring. This ring closure results in Remove the hydroxyl group as described above or remove it during removal of the IES group. Block on groups that are not allowed.

The tTEs group is removed and the resulting hydroxyl group is converted to It is oxidized to a ketone. This ketone is then combined with LDA and methylchloride. Reacts with loloformate to form carboxyenol. that hydroxy The group can be methylated with diazomethane. Subsequent reactions with hydroxyto ions and neutralization of unsaturated methane represented by structural formulas VII, VIII and IX. Gives xycarboxyl.

In connection with compounds of structure VI having an R6 methyl group, the methyl group is β- It is in the configuration. Here, the ketone formed by the above-mentioned Swern oxidation was reacted with LDA, phenylselenyl bromide and hydrogen peroxide to obtain the enone. Form.

This enone is reduced with a hydrogenated steel that attacks from the α-face, and β - Form stereochemistry. This reduced enone is then reacted as described above. Provides a double bond and R1 and U'R3 groups.

General formula of compound 24: A compound with a suitable 2-haloacetic acid (compound 24a) or 2-haloacetic acid (compounds 24b-g) and a base of compound 2, such as cesium carbonate (C5z COx) and crown ethers such as 18-crown-6. It can be prepared more easily. An example of the synthesis of compound 24c is given below. R4 is C, -CS Al Compounds that are Koxy groups are CI-C@halo derivatives, such as iodide or Compound 2 can be obtained by alkylation in the presence of C5tCOs with riflate derivatives. It can be prepared in the same manner.

free amino acids by removal of the carbamate group from the nitrogen atom of a compound, e.g. Formation of ion-containing compounds, such as compound 40, is achieved by lithium aluminum hydride. This is achieved through reduction. Carbamate derivatives of dynemycin analogs Variants of the alcohol moiety include phenoxycarbonyl derivatives such as compound 2, As shown schematically in Figure 15, the substituted alcohol is added in the presence of alcohol sodium salt. It can be prepared by reaction with kohl.

■、Results The key synthetic steps leading to the synthesis strategy of the present invention are shown in reaction formula ① as described above. . Reaction formula II shows the quinoline derivative compound 4 [(a) 7 thumbnail (Masamune ) etc., J, Org.

1948, pp. 1537-1541]. Ru. Thus, m-chloroperbenzoic acid (mCPBA) in dichloromethane Treatment of compound 4 gives the corresponding N-oxide (step a), which is acetic anhydride. Step B) in which position-specific dislocation occurs when heated in kelheide) et al., J. Am, Chea Soc.

1954, 76, pp, 1286-1291], acetoxy derivative compound It gives product 5 (62% overall yield).

Compound 5 via standard methods (steps C and d) via hydroxy compound 6 , total yield 92! % of the corresponding silyl ether compound 7. Compound 7 and odor phenylchloroformate at −78°C into a mixture with ethynylmagnesium chloride. Addition of [Commins et al., J. Org. Chew, 19 90.55. pp, 292-298) led to the formation of compound 8 ((step e; yield 92%).

Treatment of compound 8 with mcPBA leads to the formation of epoxide compound 9 (step f ; yield 85%), which is desilylated in step g) and then oxidized (step h). It was converted to ketone compound 11 via alcohol compound 1O. Pd (OAct)-Cul catalyst to convert vinyl chloride derivative compound 12 and compound 11. Step i) of the cup ring, followed by treatment with AgNOs/KCN (step j) The required precursor compound 3 is obtained via compound 13 as a step ring product ( (overall yield 79%). Finally, treat compound 3 with LDA in toluene at -78°C. The desired gynemicin A model compound 2 is obtained (25% of the starting material). 80% yield based on recovery). Compound 2 is D in Figures 10 and 11. It is indicated by Y-1.

Compound 2 was crystallized from ether to obtain colorless prismatic crystals. mp 232 -235℃. X-ray crystallography (This X-ray crystallography It was carried out by Dr. Raj Chandha of the chemistry department of the state university. ) is its structure (ORTEP Drawing Oak Ridge Thermal Ellipso) ID plotter (drawing Oakridge Thermal Elli psoid (Plotter)) and some interesting structural features. revealed.

The acetylene portion is curved from linearity at the following angle: C14,160,4 °; C1 is found to be 3.63 people, and this value is equivalent to the MMX minimization structure of compound 2. Calculated values (3,63 people) and experimental differences in dynemycin A distance (3,54 people) [(a) Kolishi et al., J, Am , CheILSoc, 1990. -Li2. pp, 3715-3716; (aj Konishi et al., J. Antibiot, 1989.42. pp, +44 9-1452]. Calculated for these acetylene carbons. The calculated distance is found to be 3.40 people. Semelhack (Sean) elhack) et al., Tetrahedron, 1990.31.1) I), See 1521-1522. (IN 47402-3076, Bloomi Serena Software, P, O, BOX 3076 - (Serena S l+! in a computer program from software). MX87 Kaba (fo rce field) was used. ] Reaction formula V is a new transformation of model compound 2 This is a rough representation of the cascade.

Reaction formula V (then at 25°C for 24 hours, benzene/l, 4-cyclohexadiene (3: Treatment with p-toluenesulfonic acid (TsO) I-HzO) in l; 0.05M) In this case, compound 2 was probably converted via intermediates 14 and 15 in 82% yield. It was converted to compound 17. This transformation (2->17) is also carried out in benzene at 25°C. and 1.0 equivalents of TsOH-HtO and 50 equivalents of [! Run using tssiH (24 hours, yield 85%).

EtsS+H and some other hydrogen atoms to trap C-centered radicals. For research using ner, see Newcomb et al., J. Am, ChefLSac, 1986°108, pp, 4132-4 See 134. Compound 17 induced by this reaction with EtsSiH is Approximately 5% of products remain unidentified (detected by 'HNlllR spectroscopy). It was contaminated with

(Thus, the protonation of the epoxide group in compound 2 is the same as that of triol compound 14. This compound 14 undergoes spontaneous Bergman cyclization [( a) Berryman.

1971, 1ll), 1516-1517; (c) Wong et al. Tetrahedron Lett, 1980.21D pp.

217-2201 to form a benzenoid diradical, which in turn coexists with compound 15.15a as a cyclized product trapped by the hydrogen donor Or give 15b. Under this reaction condition, the triol compound 15 appears to have an upper peak. A Nacole-type rearrangement occurs to give compound 17 as the observed final product.

The structure of compound 17 is supported by spectroscopic data and strain X-ray crystallography. confirmed.

Additionally, trimethylsilyltrifluoromethylsulfone in the presence of EbSiH (Th!5OTf) induced a similar transformation (2->17) within 5 minutes at -78°C. (Reaction formula V; yield 78%). This means that this cyclization step This suggests that the activation energy is extremely low. Epoxide compound 2 (c Triol compound 14a (cd= The dramatic reduction in the CD distance when converting to 3.19 people, MMX) is worth mentioning. do. For comparison with other similar enediyne cyclizations and calculations, see (a)-: 7 Nocolaou et al., J. Am., CheIILSac, 1 988.110. I)I).

et al., 1bid, 1989. pp, 763B-7640 to 1(d) Kobal A similar stabilization of enediyne via complexation has been described by Magnus ) etc., J, k Chew Soc,, 1988゜110, J) I), 1626-1628 and Magnus et al., 1 bid, 19g8.110. p. p, 6921-6923; (e@) Semelhack (Seane 1hack) etc., Tetrahedron L ett,, 1990.32. pp, 1521-152Q; (f) Snyder et al., J, AOL Chea Soc, , 1989.1, I) I), 7630-7632; (g) Magnus et al. , Tetrahedron Lett, 1989.30. Imp, 1 See 905-1906.

In an attempt to prevent the pinacol rearrangement of triol compound 15, acetate-induced Conductor compound 2a was prepared from compound 2: acetic anhydride, 4-dimethylaminopyridine (DMAP), 84x) under the epoxide cleavage and cyclization reaction conditions described above. Ta. In fact, the acetate diol compound 15a is the starting material of compound 2a. and the final product of this cascade using TsOH HlO as initiator. (yield 84%, probably (via intermediate compound 14a)).

Additionally, the use of anhydrous HCl in r"cHtclt in the presence of Et, SiH (reaction described above) Formula V) is probably via intermediate compound 14b (cd=3.145, MMX) to initiate the cyclization cascade from compound 2 to produce compound 15b (yield 85x) gave. A similar conversion (2->t5b) also involves 3.0 equivalents of MgCl2 and 50 equivalents of of EtsSiH at 25°C using CH,C1 in is 1.2 equivalents of TiCl, and 50 equivalents of EtsS in CHtClt at -78°C. iH (0.5 hours, 60%).

Thus, only compound 15 undergoes further pinacol-type rearrangement to form compound 17. to be accomplished.

These cyclizations are similar to those seen for dynemycin A [(a) (Konishi) et al., J, Aa CheILSoC, 1990, Yu 12 ．． pp, 3715-3716; (b) Ko-shi et al.

Another method of initiating the cyclization of compound 2 is proposed based on cobalt complexation of the acetylene. Served. The initiation of this cyclization is shown in reaction formula Vl below. This route is Preventing the spontaneous cyclization of the acetylene during decleavage, the predetermined intermediate c It was devised to allow the isolation of is-diols.

Reaction formula Vl Thus, the reaction of compound 2 with Cot(CO)s (2,2 eq.) resulted in a yield of 96%. to produce dicobalt complex compound 18 (step a). 1 equivalent of Coy (CO) The use of monocobalt s together with the dicobalt derivative compound 18 and starting compound 2 Forms a complex. [Similar stabilization of enediynes via cobalt complexation Also, RAagnus et al.

Compound 18 was treated with a solution of trifluoroacetic acid in CHtCl (0°C) and then Treatment with water leads to the formation of a stable cis-cleavage product, compound 19 ((step b) , yield 92%).

Compound 19 was dissolved in CH2Cl, in the presence or absence of EtJ+H at 25°C. Exposure to trimethylamine N-oxide or ferric nitrate produces cyclization products and Compound 17 was obtained in a yield of 85-92℃%, and by liberating its acetylene group, Compound 14 is formed, which occurs spontaneously and sequentially as shown in Schemes V and Compounds 15 and 17 were subsequently produced. The same study conducted in CD, C1, (Reaction with Fe(NOs)z) is the incorporation of two deuterium atoms in compound 17 This indicates that methylene chloride is an effective hydrogen donor in these aromatization studies. It has been proven that it is a ner.

In order to obtain a model more similar to dynemycin A, the reaction formula VII below is shown. As shown, the tertiary and droxyl groups in Compound 2 were removed to form Compound 21.

Reaction formula VII Thus, the combination of compound 2 and thiocarbonyldiimidazole in the presence of DMAP The reaction resulted in the formation of compound 20 in 95% yield (step a). Compound 20 is Treated with 'Bu, SnH in the presence of a medium amount of AIBN (toluene, 75 °C). Upon reaction, the desired compound 21 is provided (step b, yield 72%). This model system compound Product 21 can also be produced by smooth cyclization and proper reaction initiation (reaction formula Vll) polycyclic compounds 23 (86%) and 23a (82%) give.

Dynemycin A model compound 21 (mp 25) determined by X-ray crystallography 1.2 - 52 °C, ether-recrystallized from petroleum ether) It was. The angles below indicate an arrangement of acetylene groups that deviates considerably from the preferred linear arrangement. Yes: C1? -C18-C19=170.2', C9-C19- C18・162.0°; C14-C15-C16=170.1°; and C 13-C14-C15・163.7°. Distance between carbons CI4 and C19 (cd distance) was found to be 3.59 people, which is the maximum/Jn structure of compound 21. (3,59A) and X-ray crystallography of dynemycin A (3,4) (9 people) obtained from Konisi (Konis). hi) et al., J. Aa CheILSoc, 1990. concave, pp, 3 715-3716].

Again, a significant reduction in the cd distance from compound 9 to cis-diol compound 10 ( cd=3.21 people.

MMX) and 1 Oa (cd = 3.19 people, l[).

In order to enable the production of the parent enediyne compound 40, the following reaction formula VIII is used. Compound 45 was designed and synthesized from compound 21 as shown.

Reaction formula VIII Thus, compounds 21 and 46 were prepared separately in acetonitrile for 3 hours. I in step a in the presence of equivalents of Cs, CO, and 0.5 equivalents of 18-crown-6 React with O equivalent of 2-phenylthioethanol [Ph5(CHz)tO)1] In this way, the corresponding phenylthioethoxy compounds 21a and 48a were produced, respectively. Next Process the product of the reaction in CH, CI at 0-25°C with 2.5 equivalents. were reacted individually with BPA to produce compounds 45 (overall yield 85%) and 47 (overall yield 85%). 2%). If the process step is carried out using 1 equivalent of m1BPA, the corresponding step Sulfoxides, compounds 21b and 46b, are prepared.

Compound 45 as step C to prepare unstable and non-isolated compound 40 in dioxane:l,4-cyclohexadiene (4:l) solution for 1 hour. Reacted with excess C5tCOs and 0.5 equivalents of 18-crown-6 at 25°C. . 1.2 equivalents of compound 47 as Step C in benzene at 5 °C for 1 h. 1. 8-diazabicyclo [s, 4. 01-’7-Undesen (DBU) and The reaction yielded compound 48 with a yield of 97%.

Freshly prepared compound 40 was prepared at 25 °C using 2 equivalents of nuclear reagent as step d. Phenol (i, Ph0H) or thiophenol (ii, Ph5H) to produce compound 49 (25% yield) or 50 (33% yield). They each got it. The reactivity of compound 40 and its ability to cleave DNA was demonstrated by epoxide cleavage. The concept of a ring-containing pathway and the action of dynemycin A as a partial mechanism It gives certainty to the mediation of iminoquinone methide species in the mode of use.

Importantly, the methoxy derivative, compound 48, is highly reactive under basic or neutral conditions. It was found to be stable. Dioxane: Water: 1,4-cyclohexadiene ( 4:1:1) at 60°C for 10 minutes. Treatment of compound 48 with -HtO (step diii, TSOH) results in compound 51 (yield 20%).

X-ray crystal analysis of compound 48 confirmed its molecular structure; 3. ．． CD distance of 63 people [ calculated cd = 3.61 people, MMX] and nonlinearity of acetylene group [acetylene carbon Angle at: C-14°163.3°, C-15,173,0°; C-18 , 169,3°; C-19,160,5°. revealed.

Compound enediyne model compound 43 (reaction formula IX) under photodegradable neutral conditions. It was designed with respect to its potential to yield 42. Regarding the synthesis of compound 21 Compound 43 was made in good overall yield according to the strategy developed in . of compound 43 Reaction formula Reaction formula IX Compound 43 in THF-HtO (10:1') for 40 min at ice-cold temperature The irradiation in step a (1-tube mercury lamp, virex filter) is TLC and 'HNMR spectroscopy (THF-da:Dto 10:1.300 MHz ) resulted in complete conversion to compound 42 as observed by . Compound 42 Attempted isolation of resulted in decomposition, while ethanol (E tOH), mercaptoethanol (EtSH) or n-propylamine (' PrNHt) in a pH 8.0 buffer-THF solution at 25°C under argon atmosphere. The treatment of crude compound 42 for 1.5 hours below is as step A mixture of 4a and 54 (31% yield), 44b (34% yield), or 44C ( Yield: 46%).

Compound 44a (33% yield) as a sensitive but stable molecule under neutral conditions. - together with compound 54 in about 5-10% yield from the reaction of 42 with ethanol in air. The isolation of (Scheme IX) was noteworthy. Isolation of compound 54 is based on the quinone methane Mediation of compound 52 [a) Dyall et al., J. Am, Che m, Sac, 94:2196 (1972); b) Za-n arotti), Tetrahedron Lett, 23:3815 (1 982);c) Zanarotsuchi, J, Org, Chea@, 50 : 941 (1985); d) Angle et al., J. Am. Chem. , Soc, 111:1136 (1989); e) Angle et al., Tetrah edron Lett, 30:1193 (1989); f) Cresenji (Cre scenzi) et al., supra 31:6095 (1990); g) Barton (B arton) et al., supra 31:8043 (1990); h) Angle et al., supra. 112:4524 (1990)] and The trapping of the compound by molecular oxygen is shown in the reaction formula X below. Compound 54 is given.

Reaction formula Remarkably, under similar conditions (in air, THF-p buffer at 19.0) Treatment of compound 55 produced the compound in yields of 32% and 25%, respectively, as a stable molecule. This resulted in the isolation of products 58 and 62. Quinone methide intermediates are anthracycline antibiotics Substances [quinone methide intermediates recently postulated in the area of anthracycline antibiotics] Regarding a) Boldt et al., J. Org. Chem., 5 2:2146 (1987); b) Gaudiano et al., J. 　nrg.

Chea, 54:5090 (1989); c)-T-Egholm et al., J. Am., Chem. Soc., 111:8291 (1989); d) La. Ramakrishnan et al., J. Med. Chem., 2 9+1215 (1986); e) Bolt et al., J. Am. Chem. Soc. 111:2283 (1989); f) Galagiano et al., J. Am, ChenhS. oc, 112:9423 (1990); g) Karabe Ias) et al., J. AIL Chea Soc, 112{5372 (1990); h) Galagiano et al., J. Am. Chem, Soc., 112: 6704 (1990) and the mode of action of diomycin A. Was.

Additionally, pivaloate ester compounds 59a and 59b were added to compound 42 as a compound. Furthermore, it liberates the nol compound and thus initiates the dynemycin type A cascade. The general strategies mentioned above with respect to their potential to provide alternative incentive mechanisms (countermeasures) Synthesized according to formula IX). In fact, at 25°C in ethanol and water (3:1) After exposure to 4 equivalents of LiOH for 4-6 hours, presumably simultaneous carbamate Compounds 59a and 59 were obtained via a sequence involving a 2-to-exchange [EtO- in place of Ph0-]. b was smoothly converted to products 60a (56%) and 60b (42%), respectively.

We also synthesized methoxy compound 41, which showed significant results under neutral and basic conditions. showed stability. However, as expected, this compound under acidic conditions It was quickly cyclized. For example, benzene:l,4-cyclohexadiene (1:1) Upon treatment with TsOH-H,0 for 1 hour at 25°C in The aromatized product compound 61 (32% yield) was given.

The treatment of compound 41 in steps a and b of Reaction Scheme V yields the corresponding methoxyphenyl derivative compound 41a with hydrogen instead of . 2-phenylthioene under basic conditions as described for Scheme VIII. Tanol, 2-α-naphthylthioethanol or 2-β-naphthylthioethano Treatment of compound 41a with a polymer followed by oxidation resulted in compounds 41b and 41c, respectively. and 41d.

The synthesis of compounds 70 and 80 proceeded as summarized in Scheme XI below.

Reaction formula XI Thus, readily available compounds using palladium(0)-copper(1) catalysts 11 [Nicolaou et al., J, AIL CheILSoc ,, 112 Near 416 (1990); Nicolaou et al., J.Aa.CheaSo C., 113:3106 (1991)] and compound 71 in step a. to give compound 72 (55% yield). Due to lithium hydroxide in the process Desilylation of compound 72 followed by base induction (lithium diisopropylamide; L DA) Ring closure yielded compound 74 via compound 73 in step C (75% overall yield).

Compound 7 to thionimidazolide Compound 75 described anywhere herein 4 (84% based on 67% conversion of starting material), followed by step e. The deoxygenation by tri-n-butyl stannane (''BusSnH) was 76 (9 4%). to 2-phenylthioethoxy (PhSCHICH, 0) The exchange of the phenoxy (Phi) group of compound 76 was carried out under basic conditions (2 equivalents of Ph5 CH,CH,ONa, TI(F) smoothly occurs to give compound 77 (harvested) in step f. 92%) and from this compound 2.5 equivalents of mcPBA are used in step g. Oxidation produced sulfone compound 70 (81% yield).

A similar chemical reaction using naphthalene ditriflate compound 81 is represented by reaction formula ■I As shown in the figure, compound 11 is converted to arene diyne via intermediate compounds 82 to 84. Compounds 85-80 were produced in fair yields. The process uses benzene and compared to step a of the mechanism, which used a 3.5-h reaction. Yields of 55% and 56% were obtained using tonitrile and a reaction time of 1 hour, respectively. . Step i is 5 equivalents of trimethyl in acetonitrile (which was present in steps a and b) silylacetylene, 0.05 equivalents of Pd(PPhs), 4.0.2 equivalents of CuI and and 2 equivalents of triethylamine at 25°C for a reaction time of 20 hours. 2 was obtained in 76% yield. Steps C-g for both reactions were the same. Compound 77 Alternatively, the reaction of 88 with 1 equivalent of dPBA is the reaction of the corresponding sulfoxide, compound 77a and and 88a.

Free amine compounds 76a and 87a below, corresponding to compounds 76 and 87, Under basic conditions their corresponding phenylsulfonylethyl carbamates 70 and and 80. Both these compounds are stable at 25°C, while chemical Compound 40 decomposed at that temperature.

Treatment of compounds 76a and 87a with silica gel in benzene results in epoxide ring opening. was started to produce the corresponding diols 76b and 87b, respectively. diol compound 76b spontaneously cyclizes at 25°C in the presence of 1,4-cyclohexadiene to form cyclohexadiene. The aromatized diol, compound 76c, was produced. In contrast to compound 76b , diol compound 87b is 1. Stable in the presence of 4-cyclohexadiene However, it was cycloaromatized at high temperature (65 °C, 2 h) to form compound 87c. .

Enzynylation is then performed to measure the precise structural and spectroscopic changes that occur. The cycloaromatization of compounds 76.70.87 and 80 was investigated. Thus, acidic conditions [1,2 equivalents of TsOH-HtO, benzene-cyclohexadiene (4:1) , 25°C, 4 hours] Cycloaromatization of Compound 76 (about 0.02M solution) smoothly produced the corresponding naphthalene derivative (78%), but the W spectrum and Significant changes in the fluorescence spectra were observed between the starting arene diyne and the cycloaromatized product. No observations were made regarding the product.

However, in contrast, a similar acid-derived base of naphthaleneenediyne compound 87 Bergman cycloaromatization, as expected, converts the starting arene to Strong and distinctive characteristics different from the fluorescence profile and fluorescence profile of diyne compound 87 Anthracene-containing epoxy-containing anthracene-induced It gave 89 (49%) of conductor compounds. [UV(EtQH), 87:λ,,, (log e) 304 (3, 47, 294 (4, 01), 284 (S, 26 ).

267-240 (4,53-4,55), 214 (4,50 t+m; 89: λ, 1, (Iogε) 390 (3, 74), 369 (3, VB), 351 (3,66), 333 (3°45), 318 (3,20), 267-24 4 (4,43-4,46), 214 (4,43), fluorescent iEtOH, 1 μM, Excitation at 260 generations, Compound 87: λ, 435.412.393. 374.357 circles; 89: λ14.520°466, 442.413.392 nm].

Cycloaromatization of compounds 70 and 80 attempted under various basic conditions probably Decomposed by free amine and diradical species generated in situ, while acid treatment results in low yield. percentage (20% and 15%, respectively) of epoxy ring-opened (dihydroxy) compounds 78 and 89a. Importantly, both compounds 70 and 80 is DNA cleavage under basic conditions (supercoiled Φ174 DNA 5pi(9,0) It showed potent anti-cancer activity against various cell lines as described below.

Chimera containing satucalide Fused ring enediyne as aglycone and the above monosaccharide or as oligosaccharide moiety Chimeric compounds containing both oligosaccharides are also contemplated as described above. Said The saccharides relate to calicheamicin oligosaccharides.

Said saccharides include calicheamicin oligosaccharides (structures F and G), Corresponding to the xime precursor (structures and E) and its fragments (structures A-G) Ru. More specifically, trisaccharide structure A has a hydroxylamine bond to the B ring analog. Corresponds to calicheamicin A ring and E ring. Monosaccharide structure B is hydroxyl Corresponds to the A ring alone with the B ring analog attached. Trisaccharide thiobenzoate Structure C corresponds to rings A, E and B, as well as C ring analogs. The five-membered ring structure is complete corresponds to the FMOC-blocked oxime precursor of the calicheamicin oligosaccharide. , while the five-membered ring structure E is its FMOC-unblocked variant. Structure F and G is an epimer at the 4-position of the A-ring hydroxylamine bond syn-oligosaccharide, and structure F is the natural calicheamicin oligosaccharide stereochemical structure. has. These saccharides are described in more detail below.

The above saccharide of calicheamicin oligosaccharide is a derivative of a known compound. , their suitably protected precursors, their complete synthesis is provided herein. There is no need to explain it in detail. These syntheses were described by Nicolau et al., J. Am. CheaSoc, 112:4085-4086 (1990); Nicolau et al. cit., 112:8193-8195 (1990); Nicolau et al., J.C. hea Sac, Chea Coanun, 1275-1277 (29 90), as well as U.S. Patent Application No. 071520, 245 (May 7, 1990). 07/695.251 (filed on May 3, 1991) , the entire disclosures of which are incorporated herein by reference. And that too? However, the saccharide used here is slightly different from the reported saccharide. , thus their synthesis is explained below at least the relevant parts of the trisaccharide linked Hydroxylamine compounds were prepared by Nicolau et al., J. Am. Chew Sac.

112:13193-8195 (1990) for the preparation of compound 12. Prepare with However, instead of the methyl glycoside precursor, i.e. compound 9 of that paper, . -Nitrobenzyl (shown as NBnO or 0NBn in the reaction formula) Use Kosido. The synthesis of the trisaccharide is shown in Reactions III and XIII and is explained below. Ru.

Reaction formula Xll In this way, the D-7 cos compound 90 was beroacetylated in step a and tetraacetylated. yield compound 91, which is converted to anomeric promide compound 92 in the process. , glycosylated with 0-nitrobenzyl alcohol in step C to obtain compound 93. (63% overall yield). In step d, compound 93 was deacetylated to obtain compound 94, which This is selectively reacted with carbonyldiimidazole in step e to form the necessary ring intermediate. 95 was obtained in 86% overall yield.

Reaction formula X1l1 Referring to Scheme XIII, intermediate compound 95 is then converted to AgClO in step a. Glycodyl fluoride compound 96 (Mukaiyama et al., Ch. eDLLett, 431 (1981); Nicolau et al., J. Au Chem. Soc, 106:4159 (1984)) compound 8; Me = methyl). trisaccharide compound 97 (yield 80%, A ratio of anomers of approximately 5:1) was produced stereoselectively. Carbonate protecting group in process Compound 97 was chromatographed by removing (NaH-HOC)ItCHtOH, 90%). Graphical purification produced compound 98, which in step C was converted to -Bu2SnO-Brt [ David et al., J. Chem, Soc, PerkinTra ns 1.1568 (1979)] to form hydroxyl via intermediate compound 98. Siketone compound 99 was obtained (65%+17% yield of compound 98).

In step d, oxime is formed with 0-benzylhydroxylamine under acidic conditions. Compound 100 (90%, single geometric isomer with unassigned stereochemistry, ph = f phenyl), which was silylated under standard conditions in step e to yield compound 101 (90% ) was obtained. Photolytic cleavage of the 0-nitrobenzyl group from compound 101 [Zenhabi ( Zenhavi et al., J.

Org, Chea 37:2281 (1972); Zenhabi et al., supra, 37 :2285 (1972); Otsuka et al., J. AIL Chem, Soc, 1 00:8210 (1978); Pillai, 5ynthesis, 1 (1980) n is engineering In step f, lactol compound 102 was produced with a yield of 95%. Compound 10 in step g 2 in CHvClt at 25°C for 2 hours as NaH-C1sCC=N [Ge. Grandler et al., Carbohydr, Res, 135:203 (1985); Schmidt, An gew Chea I A Ed, Bngl.

25:212 (1986)] with a yield of 98%. Midate compound 103 was produced. Schmidt conditions [Gerandler et al., Carb ohydr, Res, 135:203 (1985); Schmidt, Angew CheILfnt, Ed, Engl, 25:21 2 (1986) Benji crying under J A Reacting alcohol (2,0 equivalents) with trichloroacetimidate compound 103 Te [BF.

・Ebb, CHtClt, -60--30°C], β-glycoside compound 10 4 (79% yield) with its anomer (16%, separated by chromatography) - Both were produced stereoselectively.

['HNMR, 500MHz, cs[1m, 104:J+, t=6. 5Hz, shrimp compound 104:J+, t"2.4Hz] Meanwhile, in step g° Glucosyl fluoride was obtained by treating compound 102 with DAST in 90% yield. Compound Io2a (approximately 1:1 anomer mixture) was obtained. In step h°, silver silicate [bottom] Paulsen et al., CeuBer, 114:3102 (198 1)] Reacting compound 102a with benzyl alcohol in the presence of -5nCIt β-glycoside compound 104 and its anomer were obtained in 85% yield (approximately 1; l anomer mixture).

The production of intermediate compound 106 via compound 105 is carried out in steps i and j by conventional debinding. The process progressed smoothly under protective conditions. Finally, in the step, compound 106 was added to TL(0'Pr a) to produce the desired product as the only detectable product upon exposure to Physics in the presence of a. Target compound 107 was produced (92% yield). Importantly, NaCNBHs- Reduction of compound 106 with H+ preferentially yielded the 4-epimer of compound 107 (Yield 90%). Attribution of stereochemistry of compound 107 and shrimp-107 at C-4 The genus is based on the 'HNMR coupling constant ['HNMR, so.

1i1Hz, C5Ds, 107:Js, 4"9.5.J4, s:9.5Hz; Lp107: Js, 44.9Hz, J4. s4.5Hz @.

The hydroxylamine-linked A-ring derivative (structure B) was prepared by Nicolau et al., J. AIL C. hetn.

Starting from compound 9 of Sac, f12:1t293-8195 (1990) It can be prepared by Therefore, 2-hydroxyl was replaced with t-butyldimethyl as described above. Block with silyl ('BuMe, St) group and etch the carbonate group at room temperature. Removed by reaction of sodium hydride in tyrene glycol-THF. Keto group was prepared by oxidation with dibutyltin oxide ('Bu, 5nO) at 65°C. It is possible. The hydroxyl at position 3 is similarly blocked with Me, Si groups, and Oximes are produced as described above.

The trisaccharide cm analogue (structure C) is di=+ Lau et al., J, k CheaSoc, 11 2:8193-8195 (1990). like this The compound 19 was then treated with triethylamine and a catalytic amount of DMA in methylene chloride. The blocked oxime-containing trisaccharide is prepared by reacting with benzoyl chloride in the presence of P. obtain.

The use of Compounds 2 and 103 in preparing exemplary chimeras is described below in Reaction-1 HIV and described below.

Reaction formula xl■ Reaction formula Thus, as shown in Reaction Scheme XIV, compound 2 and ethylbutylene under basic conditions Coupling of lomoacetate yields derivative compound 24C (60% yield) , this is processed by (i) ester hydrolysis; (ii) 2-pyridylthioester production (synthesis). In addition, the reduction in step b) and (fit) step C produces a primary alcohol compound. It was converted into a substance Ill (total collection: 05%). The above BF, under the influence of EttO A cup of compound 111 (1,2 equivalents) and trichloroacetimidate compound 103 The ring contains two major products (70%, ratio of approximately tit) and two minor products (14%, ratio of about 1:1) and these can be chromatographically analyzed. It was separated.

The main isomer is the diastereomeric β-glycoside compound 112a (R+ = 0 ．． 12, silica, 20% ethyl acetate in petroleum ether) and 112b (R,= 0.11, silica, 20% ethyl acetate in petroleum ether) ['H1lIli lR, 500MHz, C@D@, 112a:Jl, t”6.5Hz, 11 2b:JP1 = 6.5 Hzl, while a minor isomer is present at C-1 in compound 11 2a and 112b α-ano v-['HNMR, 500111H2, C5Ds, Shrimp-112a: J,, t=2.4Hz, Shrimp-112b: Jl,! =2. 4Hz]. Compound 1 as described above for Compound 104 Sequential deprotection of 12a and 112b yields intermediate compounds 113a and 113b, respectively. Oxime compounds 114a and 114b were produced via this process.

Finally, compounds 114a and 1 under PhzSiHt-Ti(0'Pr)* condition Reduction of 14b exclusively yielded target compounds 115a and 115b, respectively (90% yield). ). The C-4 stereochemistry of compounds 115a and 115b is -4 coupling constant J4. s”9.5 and Ja, s”9, 5Hz It was based again on. Structures 112a-115a and 112b-115b are interchangeable. be. This is because the absolute stereochemistry of the aglycone has not been determined. Ru. Physical data for compounds 115a and 115b are shown below.

glycosidically linked to the intact calicheamicin oligosaccharide or its analogs Additional chimeras containing fused ring enediynes were prepared. An exemplary synthesis step is shown below. Outlined in Oshiki V and U I.

Thus, a compound such as compound 111 of Scheme from the corresponding carbamate by reaction with phenylthioethanol in the presence of 2-(phenylsulfonyl)ethyl carbamate, yielding compound 120 did.

Racemic compound 120 was reacted with compound 123 as shown in Scheme xv.

Compound 123 was prepared from compound 121 via compound 122, and and with respect to Scheme XIII for the preparation of compound 103. It is explained as follows. Compound 121 itself is described by Nicolau et al., J. Am., Che II Similar to the method described in LSoc, 112:8193-8195 (1990). , except that instead of compound 12 in that published paper, used compound 99 and in their published paper 'Bulil t! instead of etSi blocking group. A tsSi blocking group was used.

Thus, compound 121 was prepared in THF-HtO (9:1.v/v) at 0°C in step a. Compound 122 was obtained with a yield of 95%. The compound was treated with a catalytic amount of NaH and React with trichloroacetonitrile (1:12.v/v) in methylene chloride. Compound 123 was obtained in a yield of 98% through the process. Compounds 120 and 123 in step C benzyl alcohol, BF in methylene chloride, -EttO at 0°C → - Coupled at 30°C as a ratio of one β-anomer to one α-anomer of approximately 5=1. Compound as a mixture of diastereomeric anomers 124a and 124b present I got 124. The β-anomer is shown as compound 124a, and the α-anomer is also shown as compound 124a. -, compound 124b is not shown.

The reaction of compounds 124a and 124b with "BuNF using conventional conditions is trivial. The ethylsilyl group was removed. Hydroxyl debromination containing the resulting oxime and 1C The checked compounds, compounds 125a and 125b, thus contain oligosaccharide structures. It contained an oligosaccharide moiety previously described as . Diethylamide under normal conditions Subsequent reaction with phosphorus removed the group and produced compounds 126a and 126b.

Completely unblocked oxime-containing oligosaccharides of compounds 126a and 126b The moiety is an oligosaccharide previously described as oligosaccharide structure E.

Reaction formula XVI Reaction formula XVI shows only the oxime-containing ring of compound 126a, and the remaining part is Indicated by a line. As shown in Reaction Scheme XVI, -5 in BF, .EtwO Reduction of compound 126a with sodium cyanoborohydride at 0°C yields yielding 65% of epimeric compounds 127a and 127b, which were present in approximately equal amounts. It existed. These compounds, as shown by number 4 and the arrow, have a The 4th place is an epimer. The epimer portion of compounds 127a and 127b is an oligosaccharide. giving the oligosaccharide moieties previously identified as structures F and G, calicheamycin ( Only the epimers present in structure F) are shown in reaction scheme ■v■.

Compounds 125-127 prepared were a mixture of diastereomers. these diastereomers have been separated, but the absolute charge distribution of the fused ring enediyne moiety is currently unknown. and only one is shown, while both are shown in Scheme XVI. .

Others using other suitable fused ring enediyne aglycones and oligosaccharide moieties Preparation of chimeras of As noted earlier, the R5 group, such as the group shown in formula X, may be a hydroxyl group or or a group that can be converted into it within cells. Also, Rs hydroxy The presence of the hydroxyl group itself indicates that the hydroxyl group can be glycosidically bonded to the saccharide mentioned above. Provides an atom (oxygen) that can be used to attach a sil-containing spacer group. General A synthetic synthesis is shown in reaction formula Vl. More detailed partial synthesis is shown in reaction formula XVII below. and was used to prepare compound 59.

Reaction formula XVII Thus, according to reaction formula XVO, ethylcyclohexanone-2-carboxylene Compound 130 was refluxed in benzene in step a for 10 hours at 1.2 React with equivalents of ethylene glycol and Q, 1 equivalent of TsOH-HtO to form a compound. Product 131 was produced. Compound 131 was refluxed in the process for 8 hours. Um methoxide - Compound from compound 130 in 78% yield by reacting with methanol Product 132 was produced.

Compound 132 was then dissolved in methylene chloride in step C for 14 hours at 25°C. ．． Reacted with compound 133 in the presence of 2 equivalents of DCC and 0.1 equivalents of DMAP. Compound 134 was produced in a yield of 96%. Compound 134 was refluxed in nW to give 96 The compound was reacted with m-aminophenol over a period of time in step d with a yield of 87%. 135 was obtained. Compound 135 was added in step e to benzyl bromide, 1 in n1 at 25°C. ．． 05 equivalents of NaH and 0.1 equivalents of "Bu, NI etc. were reacted with a yield of 72%. Compound 136 was produced.

Compound 136 was refluxed in 37% HCI-THF (1:2.7) in step f. cyclized over time to yield 100% cyclized products compounds 137a and 137b A mixture of was obtained. Compounds 137a and 137b are in the specified order in a ratio of about 4=1. was generated. Compounds 137a and 137b as a mixture were refluxed in THF. treated with DIBAL and 2 equivalents of LiAIHa for 3 hours at 13 g of compound in 50% yield upon treatment with oxygen and 5 ins over 24 h at °C. was generated.

Using a similar reaction sequence using p-anisidine instead of m-aminophenol to produce the corresponding methoxy compound. The compound has 1 in χ Treated with 2 equivalents of sodium ethyl thiolate at 60°C, followed by acetic anhydride. Nitrogen atoms when cetylated and then reacted with sodium methoxide gives a phenol substituted at the para position. The phenol is added in step e. When processed, it produces the benzyl ether, compound 139.

Compound 138 or 139 can be prepared in steps a and b of Scheme II or in Scheme III. When treated as in ce, the corresponding benzyloxy compound 140a or gives 140b. Compound using 10% Pd/C at 25°C in ethanol Hydrogenation of compound 140a or 140b results in the corresponding phenol derivative compound 141 a and 141b are given.

Any of the phenols in 1 and 1.05 equivalents of NaH and mercaptothiacylyl The reaction of the viborate was performed with the corresponding pivaloyl ester compounds 142a and 142b. give.

When pivaloyl ester compound 142a was reacted as described above at 25°C for 5 minutes. It was prepared in 99% yield after a while.

Similarly, either compounds 141a and 141b in THF and 1.05 equivalents of N The reaction of aH and 0-nitrobenzyl bromide and 0.1 equivalent of "Bu, N1 is , giving the corresponding photoactive 0NBn groups and compounds 143a and 143b. combination Product 143a was prepared in 90% yield after 1 hour reaction time.

Any of compounds 142a, 142b, 143a or 143b was The fused ring enediyne compounds of the present invention, e.g. 59a and 59b.

Reaction formula XVIII Condensation with a spacer moiety in the benzo ring in the para position to the carbamate nitrogen atom The use of compound 143b to prepare ring enediyne compounds is shown in Scheme XVH. explained. Thus, compound 143b can be synthesized as described elsewhere herein. and react to produce fused ring enediyne compound 150. These previous reactions is illustrated by two arrows between compounds 143b and 15(l).Compound 150 is the para-hydroxy analogue of the compound formed in the step of reaction formula Vllll .

Compound 150 was then mixed with 2-bromoethanol and 01111L in THF in step a. Compound 151 is produced by reaction with H. Compound 151 in step b r-mCPB Oxidation with A produces compound 153, whose ethoxyethanol hydroxyl group is It can be used to form a glycosidic linkage to saccharide as described above.

Alternatively, compound 150 was prepared with ethyl bromoacetate in acetonitrile as in Step C. and cesium carbonate to produce the corresponding ethylcarboxymethyl derivative. can do. Hydrolysis of its ester with lithium hydroxide in THF-water Solution and neutralization gives the free acid compound 152 in step d. free acids such as Oxidation gives compound 154. Using the carboxylic acid group of compound 154 to Ester linkages to tucharide or esters to the monoclonal antibodies mentioned above. Alternatively, chimeras can be generated via amide bonds.

The results of an exemplary ONA M cleft study using Compound 40 in the method of the present invention are shown in Figure 1. show.

In addition, compound 41 [formula X (wherein R1 is phenoxycarbonyl, R1, R 1.Ra, R? , Ra are all hydrogen, A is a saturated bond, and R4 is hydrogen. and R8 is methoxy at the "e" position of the benzo ring, such as J. Other compounds cleaved the DNA. Compound 41 at a concentration of 2 mmol at pH 5.0 used.

Furthermore, compounds 40.42 and 54 interacted with supercoiled φX174 DNA at pH 8.0. Significant DNA cleavage occurs when incubated with 5 mmol of co-J. (Figure 2).

Double-stranded and single-stranded cuts, as in the case of dynemycin A (compound 1) observations are noteworthy in these studies. Methoxy induction pieces 7.55.58 and 62 showed reduced activity towards DNA.

On the other hand, these compounds showed antibacterial activity, and all the compounds analyzed were active against tumor cells. and demonstrate the activity of the species in vitro. MIA using compound 2 (DY-1) A graph showing inhibition of PaCa-2 human pancreatic cancer cell proliferation is shown in duplicate in FIG.

Inhibition of MB-49 Murine Bladder Cancer Cell Growth with Compounds 2 and 20 (DY-2>) Graphs illustrating this are shown in quadruplicate and double in FIGS. 4 and 5, respectively. Shown in Figure 4 IC, obtained from extensive research. The values were 43 nM and 91 nM. comparative In the study, compound 2 was tested in non-transformed CV-1 African green monkeys. Kidney cells (ATCCCCCL 70) or Wl-38 human lung cells (ATCCCC L75) is more active against l1lB-49 cancer cells than against l11B-49 cancer cells. It was also shown.

Each of the analog compounds 24a-g to dynemycin was introduced into MIA PaCa-2 cells. The esters (compounds 24b-g) exhibited antitumor activity against the free acids (compounds 24b-g). It is more effective than a).

Compounds 24a-g are also active against MB-49 cells, CV-1 cells and It was inactive against B-38 cells. Compounds 2b-d shown below are respectively It showed weaker antitumor activity against Zen-49 cells than Compound 2.

-S%10H (2c); \Ha0H (2d).

The above chemistry is based on a Peter atom that is strategically placed on the aromatic ring with respect to the epoxide moiety. The lone pair of electrons of the child (N or 0) is available to initiate the cycloaromatization reaction By showing that there are two mechanisms for inducing the dynemycin type A cascade, Support the feasibility of the pathway. These reactive species are generated within cells by enzymatic reactions. can be released from suitable precursors under mild conditions in the laboratory, as described above. can. In addition, the relative stability of compounds 42, 54, 58 and 62 and the observations , which enables bioreduction scenarios before intercalation as well as the possibility of DNA interacting nuclearly with quinone methide species. This is important in terms of Thus, dynemycin A has a dual mechanism (nucleophilic and radical The proposal that cleavage of adenine and guanine can interact with DNA by Not only because of the superiority observed for (in view of the chemical properties of compound 42) It is clear that it is attractive.

Further biological evaluation data is shown in Tables 1-3 below.

The compound produced by LtAIHa reduction of compound 21 (39 , o, io mmol) The ether solution of compound 40 was evaporated to dryness under reduced pressure and the residue was dissolved in T)IF (4 ml). A 25 mmol solution of compound 40 (assuming complete conversion of compound 2I) in obtained). Supercoiled covalently closed circular state I) φX174DN Analysis of compound 40-induced damage to A. Ink at various concentrations of compound (100 μM to 5000 μM) over ~24 hours. 1) agarose gel to separate the various 1) IIIA products. This was done by electrophoresis.

Thus, base pair equivalents were added to 2.0 μl of phosphate buffer (50 mmol) at pH 7.4. The same buffer was added to a vial containing a solution of 50 μmol of heavy chain DNA in the φX174 form. Add 6.0 μl of solution and 2.0 μl of 5.0 mmol ethanol solution of compound 40. added.

The vials were then placed in a 37°C oven for 12-24 hours. Bromothymol Add a 2.0 μm portion of glycerol-containing buffer containing blue indicator to each well. Added. A 10 μl aliquot was then withdrawn from each. aliquot ge Electrophoretic analysis was performed using a 1.0% agarose gel containing ethidium bromide. I drove for an hour at 115 volts. DNA cleavage has a nick ed) Relaxed circular DNA (2) Form II) or linear DNA (Form III) This is shown by the production of 310 nm ultraviolet light using ethidium bromide. was detected by visual inspection of the gel.

Procedure for 6-well cytotoxicity assay MIA PaCa-2 cells, MB-4 9 cells, CV- cells or Wl-38 cells were incubated at 100°,000 cells in 3 ml of medium. Cells/well density was plated into each well of a 6-well plate. them at 4 o'clock (37°C, 7% C08). then contain the compound to be analyzed. Add 6 μl of the solution to medium (5% fetal bovine serum and 1% 3 ml of RPMI-1640 containing glutamine, thus making one Ethanol was added in the wells to create a 0.2% ethanol control. next Plates were incubated for 4 days at 37° C., 7% ω. Then drain the medium. and added crystal violet dye (Hucker formulation). Cover the bottoms of the wells with water and then rinse them with tap water until the solution is clear. Suida. Add 3 ml/well of Sarkosyl to the stained cells. Solubilized in solution (N-laurylsarcosine, 1% in water) for quantification. next The absorbance of the solution was read on 590-650 dishes.

Large-Scale Screening for Cancer Cell Lines In addition to the screens described above, A panel of 10 cancer cell lines targeting some of the compounds and chimeras or all.

This screen used the sulforhodamine B cytotoxicity assay described below.

6 Sulforhodamine B cytotoxicity assay 1. Target cells in 96-well plate Preparation a. Drain the medium from the Tts flask of one or more target cell lines and separate the cells. Carefully wash the sublayer twice with sterile PBS (approximately 5 ml per wash).

b. Add 5 ml of trypsin/EDTA solution and wash the monolayer for approximately 15 seconds. .

C. Drain all but 1 ml of trypsin/EDTAJ from the flask and remove from the flask. Cap the tube tightly and incubate at 37°C for approximately 2-5 minutes until the cells become loose. to

d. Fill the flask with tissue culture (T, C,) medium (RPM11640 + 10% Add 10-15 ml of fetal calf serum and 2 mmol of glutathione and pipette Wash the cells by gently moving the tube up and down.

e. Remove a 1/2 ml aliquot of the cell suspension and place it in a counting glass. Transfer to a 75m x culture tube.

f, count cells with a hemocytometer using trypan blue and measure viability. do.

g, adjust the volume of cell suspension with T, C, medium to l x 10' cells/m Obtain the density of l.

h, T, C, 100 μl of medium was added to AI and Bl of a 96-well plate as a blank. and add.

i. Add 100 μm of cell suspension to the remaining wells of the 96-well plate.

j, Plates were incubated in a humidified incubator at 37 °C and 5-10% CO2 for 24 h. In the meantime, the drug stock solution is dissolved by dissolving the drug in a suitable solvent (determined during chemical characterization studies). , the drug-solvent solution is sterile filtered through a sterile 0.2μ filter unit. Prepared by Aliquots were taken from each filtered drug solution and 0. Measure D. The drug concentration was measured using

b. Add the previously prepared drug stock solution to the desired initial concentration (101 to 10-') in T.C. medium. M). A maximum tJ) volume of 220 μm of diluted drug is used in the assay96 Required for wel plate.

C. Doxorubicin stock solution is diluted to 10-7 to 10-9 M in T.C. medium. Toxicity controls were prepared by: Minimum volume of 300μm for 96-well plate Needed.

3. Addition of sample drug, compound, chimera and control to 96-well platea, multi Columns in a 96-well plate using a channel pipettor and sterile tips Remove 100 μl of T,C, medium from wells in non-2 and discard.

b. Add 100 μl of initial compound dilution to adjacent duplicate wells in column 2. do. (Four substances can be tested in duplicate in a 96-well plate).

C, Remove lOμl of diluted compound from the well in column #2 and transfer to column #2. Transfer to corresponding wells in 3. by gently pipetting up and down for approximately 5 minutes. Mix.

d. Transfer 10 μI to appropriate wells in column #4 and plate in column #12. Continue making 1:10 dilutions of the compound across the plates.

e. Remove 100 μl of medium from wells Fl, Gl, and wells and discard. toxicity Control (T, C, doxorubicin diluted in medium) 100μ Add to each well.

Incubate plates f0 for a total of 72 hours (moisture incubation at 37° C., ω of 5-10%). Test the plates for signs of cytotoxicity at 24 hour intervals. Examine the symptoms using a microscope.

4. Cell fixation a. Adherent cell system: 1. Place cold (4°C) 50% trichloroacetic acid ( Fix the cells by gently layering 25 μl of 10% TCA). Generate the final TCA concentration.

2. Incubate the plate at 4°C for 1 hour.

b. Suspension of cell lines: 1. Let the cells settle out of the solution.

2. 2 Sμl of cold (4°C) 80% TCA on top of the growth medium in each well Fix the cells by gently layering.

3. Leave the culture undisturbed for 5 minutes.

4. Place the culture in the refrigerator at 4°C for 1 hour.

C1 Wash all plates 5 times with tap water.

d. Air dry the plate.

5. Cell staining a, 0.4% (wt/vol) sulforhodamine BCSR dissolved in 1% acetic acid B) Transfer 100 μl into 96-well plate using a multichannel pipettor. Add to each well.

b. Incubate the plate for 30 minutes at room temperature.

After incubating for 0.30 minutes, shake the plate to remove the SRB solution. Ru.

d. Wash the plate twice with tap water and once with 1% acetic acid, after each wash. Shake the solution. Wipe the plate with a clean, dry absorbent towel after the final wash. Ugh.

e. Air dry the plate until no moisture is visible.

f, 10 mmol unbuffered Tris base (pH 1 O, 5) 100 Add μm to each well of a 96-well plate and shake on an orbital shaker. Incubate for 5 minutes.

g, plate in a microtiter plate reader at 540n! read on il take.

Then ICs. value, i.e. of the compound required to kill half of the treated cells. The concentration was calculated.

The cell lines analyzed are listed below along with their respective sources.

Cell line source and type N) IDF Normal human dermal fibroblasts as a control - Kroanthus Co. Poration (C1anetics Corporation) C4pan-1 American Type Culture Collection (ATCC) Molt-4 (all OVCAR-3 is a human cancer cell line described by the ATCC. OVCAR-4 Sk-Mel-28 UCLA P-3 Originally Nori, Morton C of the University of California in Los Angeles Obtained from Dr. Morton. M-14 and n-21 are human melanoma cell lines; On the other hand, P-3 is a human non-small cell lung cancer cell line.

Also, the following IC regarding NHDF cells and cancer cells. The following known anti- A control study was conducted using a cancer drug.

Average IC,. Range of values (molar concentration) Medicine NHDF Cancer cells Dexorubicin-1,6XlO-IO-9,8XlO-' Dynemycin A 1 G-'　1.6XIQ-'-9,8XIO-""Karichiz7Fish:/　2. 5X10-@5X10-'-10-''Morpho IJ/Doxorubicin-1,6 X10-’-9,8X10-@Taxol 10-’1G’″1-to-* Methotrexate 5X1G -1>10-' -1o-"Sis Brach"/ 5X10-’ 10-’-10-” Melpheran 10-’ 10-4-1 0-'IcLA P-3 cells were sensitive at 10-12M. all other Cells were sensitive at concentrations of 1.56110-''M and higher.

HMalt-4 cells were sensitive at 10-"M. All other cells were sensitive at 3. 9XIO-'' was sensitive at concentrations above the IR.

Tables 1 and 2 below provide the above information regarding the fused ring enediyne compounds disclosed herein. Average ICs from all 5 to 10 cancer cell lines listed above. Show data. These tables are The description of the R group in the general structural formula and -membrane structural formula of each compound is shown. Also, the above The compound number is shown. Table 3 shows two types of fused ring enediyne compounds (compound 47 and 120) and six chimeras.

Table 3 Anticancer activity of enediyne compounds (IC,.) Zero Zero MOCT-4 (leukemia) cell line? ICs obtained from =1.0X10-'As seen from the data in these tables , these compounds and chimeras had activities similar to those of known anticancer drugs.

Compounds with R4 hydrogen include R4 hydroxyl groups or other oxygen-containing groups. tended to be more active than compounds with Carbamate part is phenylsulfate Compounds containing a honylethoxy group or a naphthylsulfonylethoxy group are About 10-100 times more than similar compounds with phenoxy group as the mate moiety It was active. Donating electrons corresponding to hydrogen at para position to carbamate nitrogen atom Compounds with groups have an activity equal to or lower than compounds with hydrogen. On the other hand, there is a tendency for hydrogen to be present in the cell at the meta position relative to the nitrogen atom. Compounds with electron donating groups (e.g. 59a and 59b) produced in They tended to be more active than compounds with hydrogen atoms in position.

Regarding the chimeras, the data in Table 3 shows that these chimeras are effective.

These data also indicate that the presence of the FMOC group suppresses the activity, but the presence of the oxime does not. This indicates that the presence of the active substance does not suppress the activity. Additionally, these data are A chimera with an oligosaccharide stereochemistry has an epimeric stereochemistry at C-4 of the A ring. This indicates that the chimera is more active than the chimera with the structure.

The data in these tables also apply to the compounds and chimeras described herein. Shows particular activity against t-4 leukemia cells. Thus, these cells Regarding compounds 59b, 41b, 41c and 41d in other cell lines. It showed an IC8° value that was 10.000 times more potent than the efficacy observed for the drug. Mo1t The activity of chimeric compound 127a against -4 cells was compared to that of other cell lines tested. It was about 100 times the average. In addition, these IC1o values for Molt-4 cells is the IC! of these compounds against NHDF cells! . value than 10,000-1 It was 00.000 times smaller.

Solution of 4 (1.42 g, 7.furmimoles) in dichloromethane (50 ml) The solution was heated at 25°C with rdPBA (1.58 g of 85% sample, 7.76 mmol). Treated and stirred for 1 hour. The solution was dissolved in saturated sodium bicarbonate solution (50ml). Pour and extract. The aqueous layer was further extracted with dichloromethane (2.50ml) and combined. The organic layer was dried (Na1SO4), evaporated under reduced pressure, and the residue was flushed with silica. Purified by chromatography, eluting with 25% MeOH in EtOAc. Pure N-oxide compound 4a (1,24 g) as an off-white crystalline solid , 80%). R+□0.34 25% Meoll in CEtOAc); Point 131.7°C (recrystallized with EtOAc); Engineering R (CDCI) V-2950, 15110, L 90° 1 koo, 1210. 1140 cra”+’HN 'M]'l (500) Q4z, CDC1,): 6 B, 72 (d.

Co 8.3 Hz, l H, H-41, 11, co L (s, L H, H-6), 7.91 (d,, I-8,] H!, l M, H-1), 7.61 (t, S- , Hz, IH, H-2or H-3), 7.61(t, Jmll, 3H z, l　H,H-2or　H-ko), ko+02　(t, J-6,'3Hz, 2 M.

H-10), 2.79 (t, J-6, Ko R2, 2H, H-7), L9B-C 84 (m, 4 H) iMS (TAB+) ll7J (relative intensity), nt! n! 1ty) 200 (M+H, 1ool, 184(12)j HRIJS:C+s)I+aNG and site calculation value (M+H) 200.1075 , actual value 200.1055° Example 2: Compound 2 Engiyne 3 (205 μ, 0.50 mmol) in dry THF (10 ml) A solution of lithium diisopropylamide (in cyclohexane) was cooled to -78°C. 0.37 ml of a 1.5 M solution (O, Sa mmol). 1 at -78℃ After stirring for an hour, the reaction was quenched with saturated ammonium chloride solution (2 ml) and Warm to warm, pour into saturated sodium bicarbonate solution (30 ml) and dichloromethane (30 ml). 50+nl). The combined organic extracts were dried (Na1SO4), Evaporated under reduced pressure and prepared by flash chromatography on silica, petroleum 3 (48, 23%) recovered by elution with 50% ether in ether was followed by 1. lO-membered enediyne compound 2 (,120i, 59% ) autopsy.

R+ lIO, 42 (50% ether in petroleum ether); melting point 228-230 °C decomposition (ether ((dd, J-8, 1, x, 3Hz, 1 town aromatic), 7.47-7.10 Yu 51.0. 1 piece 5. B, L 1. Ko, 129.3.　 128. o, 127.8. 126. Ko, 125.8°125.3. 124. 0. 122.2. 121.6. 100.4. 94. ko, 9 ko, 9.　 8!1.11゜74.1. Fuco, 2. 64.4. 50.5. 5.4. 2 pieces, 2. 19. lj ME2 m/l (relative strength) 409 (2g, M+1, 36B (18), 2 koro (ill.

162 (11), 131 (1001; HRMS: as CtsH+5NO4 Calculated value: 409.1314, actual value: 409.1314.

Elemental analysis, calculated value as CzeHuNO4, HzO: C,73,06,H, 4,95,N, 3.28 Actual value: C,? 3.44; H, 5,04; N, 3 ．． 26 Compound 2-d was combined with Compound 2 and a suitable alcohol and as shown in Figure 15. It was prepared from compound 2 by reaction with a mixture of its sodium salts.

Example 3: N-phenyloxycarbonyl-6-(3(Z)-hexene-1,5 -cyinyl)-6a+10a-epoxy-1O-oxo-5,6,6a, 7. 8.9.10.10a-Octahydrophenanthridine (Compound 3) Silver nitrate (5.28 g, 31.2 mmol) was dissolved in H2O-BtOH-THF at 25°C. Silyl acetylene compound 13 (4,50 g) in 100 ml of mixture (1:1:1) , 9.36 mmol) and -TLC analysis (30% in petroleum ether). The mixture was stirred until 13 % of ether) had been consumed (approximately 1 hour). next Potassium cyanide (4.32 g, 57.6 mmol) was added to the mixture. was stirred for 10 minutes. The mixture was dissolved in saturated sodium bicarbonate solution (100ml). Poured and extracted with dichloromethane (3x100ml). Dry the combined organic layer Na25O4), evaporated under reduced pressure and flash chromatographed on silica. Refined by petroleum engineering Js + lO, l Hz, 2 M, olefin), co, 6 (d, Jml, 2 Hz, LH, cmc-150,9,115,9, 130,0,C29,ko,12B,8. 127.6. 125.9. 125 ．． 8゜12 pieces, 0. 12L4. 120.4. 120.2. 90+6.　 115.1. 82.8. 80.1゜75.1. 57.4. 4g, 1.　 ko8.9. 2 pieces, 9. 18. R5m/sl relative strength) 409 (2 , M+), 2s2 (x5), 212 (1B), 162 (59), 58 (1 ool.

HRMS: Calculated value as CtaH + wNO< + 409.1314, Actual value: 409.1308゜Example 4: lO-acetoxy-? , 8.9.10-tet Lahydrophenanthridine (compound 5) 7.8.9.10-Tetrahydrophenanthridine N in acetic anhydride (50ml) - A solution of oxide (compound 4aX, 1.23 g, 6.18 mmol) at 10 Heat to 0 °C for 20 h, evaporate to dryness, and dissolve in dichloromethane (50 ml). and washed with saturated sodium bicarbonate solution (50ml). Remove the aqueous layer from dichloromethane. (2x 50 ml) and dry the combined organic layers with NatSO<). The residue was purified by flash chromatography on silica and evaporated under pressure. Pure compound 5 (1.15 g, 1 7211, 1241 eim”j’! (MMR (5001ml, CDC1, ): 611.70 (s+1H.

H-6), 51.0s (d, 3wa 9.5 Hz, l Ii, H-4), 7+ 76 (d, co-i, 5 Hz + 1 (bd, J-17, 5 Hz, l H,!- 7), 2+aa-2,80 (!11.1)! , H-7), 2.27 (bd, , 7-13.8 Hz, l H, H-9), 2.05 (g, 3 H, 0AC), 2.01-1. Jig(!11.3M); 13CNMIR( 125mz, CDCl, ) + 6 170.2. 152.3°14f, 5.1 core,! I, ljl, 8. 1 piece 0.1. 127.9. 127.0. 12 6.8.　122.2゜64.5. 29.1. 27.11. 21+7.　 1B, 4; Ks (FAB+3 2Mj (relative strength) 242 (M+H, 1 oo), LB2 (2 pieces); HRMS: Calculated value as C15H + 1NOt ( M+H): 242.1181, actual value: 242.1181; elemental analysis, Calculated value as C15H+1NOt: C, 74, 67, H, 6, 27, N, 5.80 Actual value: C, 74,59; H, 6,31; N, 5.82 Example 5: lO-hydroxy-7,8,9,10-tetrahydrophenanthridine (compound 6) Solution of compound 5 (1.15 g, 4.77 mmol) in methanol (50 ml) The solution was treated with potassium carbonate (200 ml, catalytic amount) and stirred for 1 hour. the solution Pour into saturated sodium bicarbonate solution (100 ml) and dichloromethane (IK 1 00ml x 50ml). Dry the combined organic layers with NatS. O4), evaporated under reduced pressure and the residue was purified by flash chromatography on silica. Purified and eluted with ethyl acetate to give alcohol compound 6 as a white crystalline solid ( 0.95 g, 100%) was obtained. R, = 0.20 (ether); melting point 176-1 77°C (recrystallized with ether) Engineering R (CDC1x) -360Or Example 6: 1O-tert-butyldimethylsilyloxy-7,8,9,10-tetrahyde Drophenanthridine (compound 7) Compound 6 (3,70 g, 1111. A solution of 2°6-lutidine (3.4 ml, 27.9 mmol) and and tert-butyl-dimethylsilyl triflate (5,35ml 1.22.3 mmol). After stirring at 25°C for 1 hour, methanol (2 ml) was added. and stirring continued for an additional 5 minutes (9 minutes), then the solution was diluted with saturated sodium bicarbonate solution (1 00ml) and extracted. Refresh the aqueous layer with dichloromethane (2x 50 ml). The combined organic layers were dried with Natso4), evaporated under reduced pressure, and the residue was Purified by flash chromatography on silica, 50% in petroleum ether. Pure silyl ether compound 7 (5°38 g, 92%) was obtained.

7: Colorless oil; R, = 0.50 (70% ether in petroleum ether) DC1,) V-,2970,2930,21160cm-';'HNMR( 500MRz, CDCl, ) = 68.68 (s, l H, H-6), 8. OB (d, J-4, 7Hz, IH, H-17 or H-li, 11.05 (d,, 7-4.7 M!, 1!, H-10r H-ri.

7.62 (t, J-4, 7Hz, engineering H,!-2t or toko), fu, 5 (t , J-4,7Hz.

1 K, H-2 also: tH-3), 5.45 (t, J-2, 8Hz, L H, H-40), 3.00 (dd, 5m5.5.16.6 Hz, LH, CB- Person r), 2.81 (rrr, l H, CH-person r)tCDC, ): 615 2+8. Yu 47-0. Yu4yu+2. Yu29J, Yu29. Ko, Yu 27.9 ゜126.9. 126.1. i2 co+6.6 co, 2. KoLil, 27. 0. 25+8. LB, 2. 16.4゜-16,-4,5: KS (FA B+) - ((relative strength i) 314 (M+H.

Yu007. 256 (7), 182 (ee); HRMS: C+aHtsNO Calculated value as 5i (M+H) 314.1940, actual value 314.1951° Example 7: N-phenyloxycarbonyl-10-tert-butyldimethylsilyl Ryloxy-6-ethyl-5,6,7,8,9,10-hexahydrophenanth Lysine (compound 8) Quinoline compound in dry THF (166 ml)? (5.20g, 16.6mm A solution of ethynylmagnesium bromide (mol) in THF was cooled to -78°C. 36.5 ml of 0.5 M solution, 18.3 mmol) followed by phenylchlorophos lumate (2.30+1.18.3 mmol).

The solution was gradually warmed to 25 °C over 1 h and saturated ammonium chloride solution Stop the reaction with (10+nl) and pour into saturated sodium bicarbonate solution (150ml). and extracted.

The aqueous layer was extracted with dichloromethane (2x 100ml) and the combined organic layers were dried. NatS(L), evaporated in vacuo and flash chromatographed on silica. Further purification yields the pure product as a colorless oil, eluting with lO% ether in petroleum ether. Carbamate compound 8 (6.96 g, 92%) was obtained (approx. 3=1 mixture of isomers).

R, = 0.85 (30% ether in petroleum ether) (27), 75 (29); fmMs: Calculated value as CtsHssQsNSi (M”): 459.223 0, actual value: 459.2233° Example 8: N-phenyloxycarbonyl-1 0-tert-butyldimethylsilyloxy-6a:10a-epoxy-6-eth Chill-5,6,6a, 7.8.9.10.10a-octahydrophenanthri Gin (Compound 9) Jig 00me9: Compound 8 (8.60g, 18.8 in z (120ml) mcPBA (8.08 g of 60% sample, 37.a) and stirred at 25° C. for 3 hours. Saturate the solution with sodium bicarbonate Pour into the solution (loO[01), extract, and remove the aqueous layer with dichloromethane (2x 100 m 1) was further extracted. The combined organic layers were dried (NatSO4) and evaporated under reduced pressure. The residue was purified by flash chromatography on silica and purified with petroleum ether. The epoxide compound 9 (8 , 20 g, 92%) (mixture of two isomers, ratio approximately 3:l). R,=0 ．． 73 (30% ether in petroleum ether) (500FHz, CDCl,): l f, 8B (d,, 7m7.1 R2, IH, H-41,7,50-7,1o (m, B Ml, 5.58 (b s,　IH,! (-61,4,82(dd,'J-10,o.

5.7 R2, L H, H-10), 2.34 (dd, J-14, 8, 5, 6 Hz, LH), 2.09151.1.　lco5.5. 129.2. 129 ．． 2. 129.1. '12B, 3. 128.1.　127.9゜12f, 1. 125.5. 121.6. 7B, 5. 7, 8. F2.8. 6 9.9. 60.4. 4B, 0゜ko1.0 and F (29, 6, 26, 0 and hi 25 ．． 8.24.0. andhi26.5.1B,2 and'(20,ko, -0,28,- 0,21! , -0, core; Hs: &j (relative strength) 475 (bi, 2), 4 19 (10011ko25 (28), 26g (10), 222 (14), 151 (1B), 7 (42): HRIIIS: Calculated value as CnHssO4NSi (M”) +475.21 79, Actual value: 475.2175° Example 9: N-phenyloxycarbonyl- 6a: 1Oa-epoxy-6-ethyl-1O-hydroxy-5,6,6a, 7 ．． 8.9. true 0.10a-octahydrophenanthridine (compound 10) Epoxide compound 9 (8.20 g, 17.4 mmol) in THF (100 ml) 20.9 ml of a solution of IM in TBAFCTHF, 20.9 mmol ) was heated to 42°C for 3 hours. Evaporate the solution in vacuo and flush with silica Purified by chromatography, eluting with 50% BttO in petroleum ether. Pure alcohol (6.OOg, 100%) was obtained as a white crystalline solid ( approximately 3:1 mixture of isomers as shown by NMR).

R1 = 0.31 (50% ether in petroleum ether); melting point 78-79°C (B Recrystallized with ttO) H-4), 7.50-7.08 (m, 8 Ml, 5. 62 and 5.59 (d, J-L.O Hz, IH, H-6), 4. 89 and 4.70 (mouth, 1 H, H-10), 2.47-Lko 5 (-8 Ml; 13C NKR (125 MNz, CDCl,): 6 150.9 , 135.5, 129. Ko.

1251, 7, 128.6, 128.4, 127.7, 127. Ko, 126.1, 125. B, 121.5.

and 1 ko-8; MS ma (relative strength) koro 1 (pi, 651, 224 ( 00), 196 (24), 180 (29), 167 00), 94 (4 01. 77 (45) iHR) AS: Calculated value as C2tH+eNO<(M ”): 361. 1314, Actual value: 361. 131?.

Example 10: N-phenyloxycarbonyl-6a:10a-epoxy-6 -ethyl-lO-oxo-5. 6. 6a, 7. 8, 9, 10, l Oa-octahydrophenanthridine (compound 11) Alcohol compound 10 (6.00 g, 17.4 mmol) was dissolved in dry dichloromethane. (180 ml), powdered active 4-person molecular sieve (1 g) and Treated with pyridinium chlorochromate (6.25 g, 29.0 mmol) did. The suspension was stirred at 25° C. for 1 h, filtered through Celite, and concentrated under reduced pressure to give a residue. was purified by flash chromatography on silica, 30% in petroleum ether. Ketone 74, 2, 5 as a white form when eluted with EtzO. 4, 4 centre. Ko, 3g. 9.2 pieces. B, 111. ko; R5 ma (relative strength) 3 59 (Tomari Woo), 266 (52), 222 (65), 194 (5 4).

:I80 (5:t), 146 (45), 69 (80);'ass:C Calculated value (M'') as nHnN04: 359.1158, actual value: 359. 1154 elemental analysis, calculated value as CxtHuN04: C, 73.53.　 H, 4.7? ．． N, 3.90 Actual value: C. 73.27. H. 4.79;N, 3.91.

Example 11: N-phenyloxycarbonyl-6-[6-drimethylsilyl-3 (Z)-hexene-t,S-cyinyl]-6a: loa-epoxy-IO-o Kiso-5. , 6. 6a,? ．． 8. 9.

10, 10a-octahydrophenanthridine (compound 13) dried and degassed Palladium acetate (IIX192, 0.86 mmol) in benzene (10 ml) ) and triphenylphosphine (832 mmol, 3.17 mmol) in an argon atmosphere. Heated at 60° C. for 1 hour under ambient air. The resulting dark red solution was cooled to 25°C and dried. (Z)-chloroenine compound 12 (2) in dry degassed benzene (20 ml) ．． 88 g, 18.2 mmol) was added followed by n-butylamine (1.92 mmol). 19.4 mmol) was added. The solution was stirred at 25°C for 15 minutes, Acetylene 11 (4 ．． 49 g, 12.5 mmol) followed by copper(I) iodide (512 mg , 2, 69 mmol) were added. The solution was stirred at 25°C for 2 hours until saturated. Poured into sodium bicarbonate solution (100ml) and extracted. Remove the aqueous layer from dichloromethane. Extract with (2 x 50a + 1) and dry the combined organic layers. Evaporate under reduced pressure and purify the residue by flash chromatography on silica. Coupling produced as a colorless gum eluting with 20% ether in petroleum ether. Compound 13 (4.50 g, 74%) was obtained. R. =0.51H, H-4 ), 7.52-7.09 (machi tJH, aromatic), 5.99 (d, , 7 rin 1.6Hz, lH, H-63, 5.82 (d, J-11.2 Hz, IM, olefin), 5.66 (dd, J Maro 112, 1.6 H z, LH, olefin), 2.76 (dt, , 7■15. Co.

4, 7 Hz, l H, H-93, 2+71 (dad, J-15.3 , 10.8, 6.1 Hz, lH.

150, 9, lko5.8, 129.9, 129+2, I211.9, 128.4, 127.7, 126.0.

125,B,! 22.9, 121.4, 120.8, 118+9, 1 03.6, 10 Yu. 5, 90.4.

a:1. o, 74.9, 57.5, 411.3, 3B. 9, 2:1. 9, 111.2, 0.00; MS ml supervision (relative strength) 481 (M' , 11), 360 (100), 146 (xo);t(Rlils:C■ Calculated value as HotO4Nsi (M"): 481.1709, actual value + 48 1.　1705.

Example 12: Compound 2a Endiyne compound 2 (100.1■, 0.22 A solution of acetic anhydride (0.50 ml, 5.31 mmol) was added at 25°C. ) and MAP (10 cm, catalyst amount).

After 2 hours, the reaction mixture was poured into saturated sodium bicarbonate solution (25 ml) and diluted. Extract with lolomethane (3 x 25 ml) and dry the combined organic layers with Na2 SO2), evaporated under reduced pressure and the residue purified by flash chromatography (silica, stone). 30% ether in oil ether) to give acetate compound 2a (11 0. 7 mg, 100%) was obtained. 2a: White crystalline solid, melting point 212-2 Decomposed at 14°C (recrystallized with ether), R, = 0.55 (petroleum ether 7, 9 2 (d, J=8.1 Hz, LH, aromatic), 7.50-7.09 (!11, 8 If.

aromatic), 5.83 (d, JmlO.l Hz, LH, olefin) , 5.65 (d.

yrHz, CDCI,): 6 169.1, 150.8, 130.0, I29. ), 1211.4, 12s. l.

12!1.0, 127.2, Engineering 26. B, L25.7, L25.2, 124 ．． Ko, 122.9, 121+5.

97, 9, 96.5, 93.7, 8B. 9. 7B. 0, 7, 5, 62.6, 50. Ko, 29. B.

22, 7. 21.8, 1!1.8; R5 (FAB+) m7j (relative strength degrees) 452 (M+H, 52), 410 (37), 392 (1ool, Ko 16 (32), 272 (43), 242 (ko 0), Engineering 54 () 7), 1 Coro (70); HRIilS:Cz. Calculated value as H□NOs (M+H) :452. 1498, actual value: 452.　1469.

Endiyne compound 2a (93.0 mg, 0 ．． 206 mmol) and l.

A solution of 4-cyclohexadiene (1.0 ml) was added to p-toluene-sulfonic acid. (39μ, 0.23 mmol) and stirred at 60° C. for 2 hours. Remove solvent under vacuum and the residue was purified by flash chromatography (silica, 50% in petroleum ether). % ether) to give diol acetate compound 15a (80.2 , 83%). 15a: White crystalline solid, melting point 198-200°C (A (recrystallized with tell).

Example 13: Compound 15 Colorless solid. R, = 0.22 (50% ether in petroleum ether) (d, J-7.8)! z, l i (, aromatic), 7.40-7. OL (-luck) multiplet @ts, 12H, aromatic), 6. B co(bs, L H, O H), 5.59 (s, l H, kl-C H(C)-C), Ko. 1 F (m, l He C) I2), 2.28 (m, I K.

Ml (z, CDCI,): 6 174.9, 150J, l here. 7, 134.8, L here, 5, 129+7.

Engineering 29. Ko, 129.2, 129.0. 12B. 8,128.5,128.2 , Engineering 27.8, 127.7.

125.5. 124.8. L23.4. 121. B, 9: lJ, 75.1 ．． 70.6. 6L4゜Here. 5. Ko1.4.22.6.19.8; Mg: m/@(relative intensity) 471 (M+, 19), 245 (Woo), 162 ( 1oo), 94 (42);) tRlils: Calculated value C as CuHuOsN M"): 471.1682, actual value: 471.1683 elemental analysis, CtsHx Calculated value as sOmN: C,? 1.33. H, 5, 34; N, 2.97 Actual value: C,? 1.36. H, 5,54; N, 2.84° Example 14: Compound Thing 15b Dry HCI gas was added to the acetate compound 2a in dichloromethane (4 ml) at 25 °C. (32■, 0,071 mmol) and 1,4-cyclohexadiene (40■, 0.32 mmol) for 30 seconds. The solvent was removed under reduced pressure and the residue For flash chromatography (silica, 50% ether in petroleum ether) Further purification gave a chloride, Compound 15b (25μ, 80%).

Colorless gum; R1 = 0.21 (50% ether in petroleum ether), 4 19 (42), 286 (1oo), 154 (core); 1 (RMS: Ct, Calculated value (Hoes) as Htt04NCIC5: 5g0.02'Jl, actual Measured value: 580.0286° Example 15: Compound 17 Compound 17 was prepared by several methods described below.

Method (i): Cobalt complex compound 19 (42 mg, 0.060 mmol) of EtsN”-0-(32.7 μm, 0.06 mmol). The mixture was treated with a 29 mm slurry and stirred at 25° C. for 4 hours.

The solution was poured into saturated sodium bicarbonate solution (25 ml) and Ct(tclt( 3K (25 ml). Dry the combined organic layer and remove Na*504). The aromatized product is evaporated under pressure and purified by flash chromatography. The product Compound 17 (17.7μ, 83%) was obtained.

Method (ii): Endiyne compound 2 (57.8m) in benzene (1.5ml) g, 0.141 mmol) and 1,4-cyclohexadiene (0.5 ml) ) was treated with p-toluenesulfonic acid (29,6μ, 0.155 mmol) The mixture was stirred at 25°C for 24 hours. The solvent was removed under reduced pressure and the residue was subjected to flash chroma Purification by tography (silica, 50% ether in petroleum ether) to give the keto Compound 17 (53.7 mm, 92%) was obtained.

Method (iii): Trimethylsilyl triflate (15 μm, 0.08 mm di-nediyne compound 2 (32 ml) in dichloromethane (2 ml) at -78°C. g, 0.078 mmol) and triethylsilane (40 μ, 0.32 mmol) ) was added to the solution. After 5 minutes, the mixture was diluted with saturated ammonium chloride solution at 48°C. (1 ml), diluted with ether (10 ml) and water (2X 3 ml). ), brine (3 ml) and dried (MgSO, ).

The organic solvent was removed under reduced pressure and the residue was purified by flash chromatography (silica, petroleum 50% ether in ether) to give ketone compound 17 (22 mg, 68%).

Method (iv): 11a JICl gas in dichloromethane (4 ml) at 25°C. Dinediyne compound 2 (32■, 0.078 mmol) and 1,4-cyclohexane Bubbled into a solution of diene (40 μ, 0.32 mmol) for 30 seconds. Reduce solvent pressure and the residue was purified by flash chromatography (silica, 5% in petroleum ether). Purification with 0% ether) gave compound 17 (25μ, 78%).

White crystals; R, = 0.63 (70% ether in petroleum ether); melting point 19 1-193℃ (recrystallized with methylene chloride/ether); (!d, Komaro 7.ri, 1.3 Hz, iM, aromatic), 51.09 (d, J-7, s Hz, IC l2); “CNyR (125KHz, CDCl, ): & 20f, 5. 1 5, 0.　150.9゜14!1.2. 1 core, i, 1 core 4.2. 129 J, 129.5. 1211.5. 128.2. Engineering 27.8゜Y 27.1. ! 26+1,126.0,124. Ko, 122. a, 121.1! , Engineering 21. Ko , 82.5°21f (55), 94 (29)i HRMS: Calculated value (M”) as CtsH2+04N: 411.1471, Actual value: 411.1468 elemental analysis, C! IH! Calculated value as 104N: C ，? 5.90. H, 5, 14, N, 3.40 Actual value: C,? 5.66;H , 5, 45; N, 3.14° Example 16: Compound 18 Endiyne compound 2 (124 mg, 0.30 mmol) in CHtCI2 (4 ml) A solution of Cot(CO)s (260 μm, 0.76 mmol) was The mixture was stirred at 5°C for 5 minutes. The solution was concentrated under reduced pressure and the residue was flash chromatographed. The cobalt complex compound 18 (291 cm, 98%) was obtained by purification by roughy.

Green crystalline solid; Melting point: 300°C (recrystallized with ether); R+ □0.8 0 (50% ether in petroleum ether) xR H1 aromatic), 7.61-71 ro2 (m, 8 M, aromatic), 6.47 [bs, IH, N-CM(C)-Cl, 6.38 (bd, J-1o, 7 Hz, LH, olefin), 6.19 (bd, J-10, 7Hz, IH , OL/Fui:/), 3.50 (bg, LH, OH).

2.70-1.71 (m, 6H, Cl2): 13CNMR (125K Hz, CDCl, ): 6199.1. "r9B, 6. Yu97.8. 151. ko, 1 ko 4.9. 132.9. lJ0.9. 129.4゜i2 B, 8. 127.2. 125.8. 125. Ko, 125. i, 124.8 ．． L2 co, 4.　121.6゜951.5. BB, 9. B1.5. 80 ．． 1. 7B, 0. 73.9. 63.1. 59.0. 44.2゜24. 9. Near, IH wet (FAB+) ma (relative strength) 1 unit 14 (Hoes, engineering) 1), 1086 (M+C5-co, 113), 1058 (Hoes-2C0 , 6), 11030 (+Cs-koC mouth, 19), 1002 (Hoes-4CO , ml), 94 Hoes-4CO-Co.

Yuro), 91B (Yul), 890 (24), 862 (34), B13 ( 1oo); HRMS: Calculated value as C5aH+sO+5NCOaCS (Hoe s): 1113.7086, actual value: 1113.70010 Example 17: Compound Thing 19 Cobalt complex compound 18 (291 mg, 0, A solution of trifluoroacetic acid (68,6 μ+, 0.89 mmol) was added at 0°C. mol). After 5 minutes, the mixture was dissolved in saturated sodium bicarbonate solution (25 ml ) and extracted with CHtClt (3K 25 ml).

The combined organic layers were dried (Mg5O4), evaporated under reduced pressure, and subjected to flash chroma. Purification by tography (silica, 50% ether in petroleum ether) to give the keto Compound 19 (167.4 mm, 81%) was obtained.

Brown crystalline solid: melting point > 300°C (recrystallized with ether), R, = 0.25 (50% ether in petroleum ether) Engineering R 202,9,198,9,19B,1. 154.3. 150.9. 144 ．． 0. L33.2.　132.8゜129.6. 128. Ko, Z2B, l, 126. F, 126.0. 125.8. lJ, Ko, 12L8゜108.7. 912. 92.5. 112. L, 84.0. 6L5. 56.2. 3 g, 0. Q, 2゜21.6 + Mg (FAB+) Mu (relative strength) 82 g (Hoes, i)).

800 (ill), 651B ()4), 6ko9 (20), 555 (: 12), 527 (fool; HRMS: CuH + sNO + oC02C5 Calculated value (M+C3): 827.8727, actual value: 827.8730 elements Analysis, calculated value as CuHnNO+oCOt: C, 55, 27, H, 2, 75 ; N, 2.01; Co, 16.97 Actual value: C, 54,98; H, 2,7 9, N, 1.86°; Co, 15.22.

Example 18: Compound 20 Thiocarbonyldiimidazole (180 μ, 0.99 mmol) was added to diimidazole at 25°C. Alcohol compound 2 (137 μ, 0.335 mmol) in chloromethane (2 ml) ) and 4-dimethylaminopyridine (DMAPX25, 0.18 mmol) was added to the solution. After 48 hours, the solution was concentrated under reduced pressure and the residue was flash-crushed. Purified by chromatography (silica, 80% ether in petroleum ether) Thionimidazolide 20 (160 μm, 95%) was obtained. 20: White crystalline solid , melting point 178-179°C decomposition (recrystallized with ether/dichloromethane); R, = 0.62 (70% ether in petroleum ether) 8.49 (!, 1 H,N -CM-N), 7.71-7.05 (m1j, 11 M, aromatic).

5.93 (d, j=IQ, 3Hz, 1M, old fin), 5.73 (d d, , 7-10.3゜1-6 Hz, L Hlf L/7 in) + 5 -60 (d+ Jml, 6 R2, l H, N-CH-1coff, 0.1 co5. 9.　lco0.9. 129.4. 129. Ko, 12B, 2. 127.0. 　126.4゜! 25.51. 125.4. 12 pieces, 9. L2 co, 2.　 121. Ko, 1!7.7. 100.6. 94. Ko.

9 pieces, 9. 8B, 9. 115.4. 74.5. 65.9. 6 pieces, 2. 50. Ko, 2E1.0. 22.7゜yua, a:P; (FAB+lma (Relative strength) 65 (M+Cs, 21).

419 (19), core 9 (15), 286 (1ool, 154 (ko.) ;) IRMS: Calculated value as C3oHt+N5OaSC5 (M+Cs): 65 3.0385, actual value: 653.0360 elemental analysis, C3oHt+N5OaS Calculated values: C, 69, 35, H, 4, 07, N, 8.09. S, 6.1 7 Actual measurement value: C, 69,01, H, 4,17; N, 7.91; S, 6.19゜actual Example 19: Compound 21 Imidazolide compound 20 (112■, 0.24 mmol), azobisisobutyro Nitrile (ATBN; 3 mg) and tri-n-butylstannane (n-BusSn A solution of H) (94 μm, 0.36 mmol) was heated at 75°C for 2 hours to remove the solvent. The residue was purified by flash chromatography to remove the deoxygenated product. The product Compound 21 (71μ, 75%) was obtained.

White crystals; R, = 0.82 (30% ether in petroleum ether); melting point 248 ~250’C decomposition (recrystallized with ether) Engineering R (CDCl3) -, 2945, 2B72. 22 pieces 2.2205. 17 12. Yu465. l here5゜uni B5 cm”r’HkrKR (500K Hz, CDCl, l: 6 7.67 (d,, 7-7.5 1 (z.

1, aromatic), 7.6-7.14c town 8, aromatic), 5.84 (dd .

J-10,5,1,6Hl, 1H, olefin) / 5.72 (dd, J’l10.5. 1.6Hz, lH, OL/Fin), 5.57( d, J-1,5i Hz, LH, N-CM-Cm).

(125’KEz, CDCl,): 6! 51. Mouth, here 5.5. 129 ．． 4. 129.4.　128.2゜127, ko, 125. li, 125. B, Engineering 25.4. ! 25.0. 122.0. Engineering 22.0.　1215゜101 ．． 8. Ta 4.9. 91.4. 511! , 51. 70.5. 61.1. 50.0. 29. B, 22.9°22.5. L5.5: Mg: - (Shiang vs. strength) 393 (20, M”).

294 (9), 252 (15), 212 (Yul), 149 (42); HRM S: C□H, calculated value as m0sN (M"): 393.1365, actual measurement value :393.1332 Example 20: Compound 23 Endiyne compound 21 (30 μ, 0.0 furmmol) in benzene (2 ml) A solution of [,4-cyclohexadiene (0.5 ml)] was dissolved in TsOH, HtO ( 18 mg, 0.09 mmol) and stirred at 25° C. for 24 hours. solvent Removed in vacuo and purified the residue by flash chromatography for diolization. Compound 23 (26 ml, 85%) was obtained.

Colorless gum, R, = QJ5 (50% ether in petroleum ether) (bd, J” -,4,4Hl, LH, aromatic), 7.47 (bd, Jm7.8 Hz, UH1 aromatic), 7.40-7.09 (m, 10 M, aromatic), 6.81 (d, Jml, 11!z, L Hr aromatic), 5.78 (!1 ．． LH, N-benzyl), j, oo (bs.

2 II, OK) r ko, 24 (s, l II, benzyl), 2. 42-0.72 (m1j, 6H.

Cl21; 13CNAG (125)'Q(z, CDC1,) = 6151. 0.　lcoB, 2.　lco4. centre.

12ri, 4.129.4. 129. Ko, 12B, 8. 12B, 4. 128 ．． Ko, Yu27. i, 126.9°125.7. 125.0. 124.4. 121,8. 121+8. 121.5. 8 pieces, 0. 6g, 2゜65. 1. 51.2. Here, 5. 27.1. 1B, 7; Ms (FAB+): ml supervision (relative strength) 546 (15, M+C8L core 9 (ko 1), 12 (koO), 286HRMS: Calculation as Ct mHz zOaNcs Value (M+C3): 546.0681, actual value = 546.0691.

Example 21: Compound 23a El gas was added to enediyne compound 21 (30■, 0. 3 in a solution of 1 flo mmol) and 1,4-cyclohexadiene (0,5 ml) It was blown for 0 seconds. The solvent was removed under reduced pressure and the residue was subjected to flash chromatography. Purification was carried out to obtain chloride compound 23a (27μ, 84%).

Pale yellow crystals; melting point 114-116°C; R1 = 0.62 (50% in petroleum ether) % ether)OH), 2.50-1.60 (m, 6H, Cl2); KS:　m7j (relative strength)　564　(5,M+Csl,　4k9(k00) , Core 9 (587: HRMS: Calculated value as CteTotOsNCIC9 ( M+C5): 564.0343, actual value: 564.0351.

Ethyl bromoacetate (27 mg, 0.16 mmol) was added to anhydrous D at 60°C. Alcohol compound 2 (34 mg, 0.08 mmol) in MF (2 ml) and and C5tCO, (67 mg, 0.2 mmol). the mixture Heat the mixture for 3 hours, dilute with ether (10 ml) and add NH2Cl (2x 3 m 1), water (2 x 2 ml), and brine (2 ml).

The organic layer was dried (Mg5O+) and purified by flash chromatography. Ester compound 24c (37.5 mg, 91%) was obtained.

ImMS: Calculated value as C5oHtsOaNCs (M+Cs): 628.07 36, Actual value: 628.0750° Compound 24a, b, d, e , f and g are prepared similarly.

Example 23: Separate preparation of compound 5 (a) Add toluenesulfonic acid (TsOL HtO; 8 mmol, O, OS mmol). Alcohol compound 2 (18 mg, 0.045 mmol), 1,4-cyclohexyl Once in a solution of sadiene (0.2 ml) and benzene (0.5 ml) at 25°C. and the solution was stirred for 24 hours. The organic solvent was removed under reduced pressure and the residue was Ketone compound 5 (15μ, 90%) was purified by flash chromatography. Obtained.

(b) Trimethylsilyltrifluoromethylsulfonate (TMSOTf; 15 μm, 0.08 mmol) in -78°C cH*c1t (2 ml) (7)7/L /: 7-yl compound 2 (32 mg, 0.078 mmol) and triethyl Added to a solution of silane (EtsSiH) (40 μm, 0.32 mmol).

After 1 minute, the mixture was quenched at -78°C with saturated Nl and CI solution (1 ml). Stopped, diluted with ether (10 ml), washed with water (2x 3 ml) and diluted with saline ( 3 ml) and then dried (MgSO,). Remove organic solvent under reduced pressure , the residue was purified by flash chromatography to obtain ketone compound 5 (22 , 75%).

(c) Alcohol compounding of HCI gas in dichloromethane (4 ml) at 25°C compound 2 (32 μ, 0.078 mmol) and 1,4-cyclohexadiene (40 μ , 0.32 mmol) for 30 seconds. Remove the solvent under reduced pressure and remove the residue. The residue was purified by flash chromatography to obtain ketone compound 5 (25■, 8 0%) was obtained.

Example 24: Compound 2b Phenyl carbamate compound 2 (42■, 0.1 0.3 mmol) in sodium methoxide (17 mg, 0.31 mmol) ) and heated at 60°C for 2 hours. The reaction mixture was purified with dichloromethane (25 ml), washed with sodium bicarbonate solution (25 ml) and dried SO4), evaporated under reduced pressure and the residue purified by flash chromatography (silica, stone). 40% ether in oil ether) to give methyl carbamate 2b (28 , 5■, 80%). 2b: White crystalline solid, melting point 126-127°C (E R, = 0.43 (50% in petroleum ether); R, = 0.43 (50% in petroleum ether) Etenori aromatic), 7.25-7. Engineering O (m, Ko is also aromatic), 5.8”l (d, J-10, 1Hz, lM, olefin), 5.69 (d, , 7-1 0.1 Hz, 1 M, O’i 7 in L5.45 (s, l H, CH- N), Ko, B2 (s, Ko H1 mouth Ma), 2+79 (s, l M.

OH), 2.27-1.72 (rn, 6 H, CM,); 13C) fMR ( m2j MHz, CDCl, ): Jl here, ri, here 1. Ko, 127. g, 127.5. Here 6. Ko,! 24.9. 123.9. Yu22.1゜100 ．． 7. 94.1. BB, 3. 74.2. 7 pieces, 1. 65.8. 64 ．． 2. 5, 7. 50.1゜ko5.2. 2 pieces, 2. 19.2. 15. 2; 5nls (relative intensity) Ko4f (M", 100) 2ri1 (35), 204 (501; HRlilS: C□H1? Calculated value as NO4 (M”) :347.1158, Actual value: 347.1159 Elemental analysis, Cf1HI□NO4 Calculated value: C, 72,61, H, 4,93; N, 4.03 Actual value: C ，? 2.63. H, 5, 24, N, 3.79° (1.0M solution in ether 0 ．． 25ml 1.0.25 mmol). After stirring for 30 minutes, the reaction Stopped with sodium bicarbonate solution (1 ml), diluted with ether (20 ml), , 5 ml of 0M LiO solution solution) to remove phenol, and dry. (NatSO4), filter and store under argon at -78°C until required. Stored.

MS (FAB+) m/e (relative intensity) 290 (97), 278 (75), 27 4 (M+H, 98), 235 (100); HRMS: CnH + sNO toshii ” Calculated value (M+H): 274.1232, actual value: 274.1247 Physical data on selected compounds of: Compound 42 R1=0.83 (50% diethyl ether in benzene) 2.35-1.55 (m, 6 H, CH,).

Compound 43 R, = 0.78 (50% diethyl ether in benzene) Jw7.7 Hz, III, aromatic), 7.13-7.01 (m, 5 H, aromatic).

6.97-6, Bfu (m, 2 M, aromatic), 6.81 (bc!, 7 -11.9 Hl, i! (.

aromatic), 6.66 (t, J'-), Hz, lH, aromatic), s, so (bs.

L H, CHN), 5.31 (d, , 7-LO, l Iiz, L H, O L /Fui7),! 5. L7((id, J-10,1,1,]Iiz, LH, x reflex), s, 1 core and 5.04 (λB.

j dew 16. o Hz, 2 M, λrc! %O), 2.29 (bs, IH , Mouth H), 2.15-1.115 (m, 4 H, CH2), 1.70-1.6 0 (!11. 1 H, CI (1), 1. Core 129 (!11. L” c! 1”xR (to C6)’4 Ko554.2954.292fu, 172B, 1615°HRMS:Cs5H□N! Calculated value as 07 (M+H) :561.1162, Actual value: 561.1162 Compound 44C R, = 0.33 (5% methanol in methylene chloride) Hz, LH, aromatic) , 5.511 (s, IH, cHM), 5.46 (t, J=5.6 Hz, L H, O5, NHC Kuchimachi, Ko, L9-2.95 (+! l, 2 H, l :H, 81(CON), 2.110(dt, J-u, Ko, 6.3HE, LH, 3, CM2NM), 2.53-2.26 (Tomi, 5H.

C51, CH, FJ l:H,, OH), 1114 (s, L H, 0 town, 1.68 (ad, J-12, 4° 4.7 Hz, LH, CM,), 1. 61-L Colo (m, 511. 3.CH, NHCON.

Mouth (2CH2N! (, CI,), 0.94 (t, S-7, Ko mz, Ko H, OR, CH, 3-1'M (:ON).

0.19 (dd,, 7-4.Ii, 2.4 Hz, LH, (4), 0.11 3 (1+ Coff, CHz, CH, 3,0!2CM, N'H), O+711 -0.59 (11, l 11. mouth); ! , 3439.　3270.　2964. 29 pieces 5. 1645. R61 , 1500.　1459゜1416, engineering 252.11B80゜ HRMS: Calculated value as CtsHsJs04 (M+H): 452.2549 , actual value +452.2549 compound 45 R1=0.22 (silica, 70% diethyl ether in petroleum ether) HRMS : Calculated value as CtsHtsSNC5 (M+Csゝ)61B, 0351, actual Measured value: 618.0352 compound 48 R1=0.61 (silica, 70% diethyl ether in petroleum ether) J-7, 34Hz, LH, aromatic)', s, ako (01, JM8.B Hz, L H.

tshifin), 5.73 (dd, J-8,8,1,74Hz, olefin) , 4.32 (d.

J-1, 14H1, LH, NCMl, 4.00 (bs, IH, NM), Ko, 50 (m, ko H1QCH,), 246-1.68 (m, 6 H, CM,); “C NAG (125MM, CDC1,): 142.2. iComi, o, 12B, Ko, 12 Ko,! , 122.5. 122+mouth, 119. Ko, 115.9°100 ．． 2. 9g, 9. 94.6. 8f, ko, f9.6. 72.9. 62 ．． 9. 29.0. 24.6゜19.0; Engineering R (CHC engineering,) v-340 0,2950,2a50.　1100. 1080cm”r) IRMS:Ct oHItOtN and site calculation value (M”) 303.1337, actual value: 303 ．． 1348R+ = 0.3 (50% diethyl ether in petroleum ether) Fi 7L 5.6 Fu (dd, J-mlo, L) HE, L H, fL- Fi 7) , 5.48Cf) C1,): 6 155.1. 150.9. 136.8 ．． 133+2. l) 1.0. l) 0.1゜129, ko, 125.1! ,! 24.2. 124. Yu, 122.2, 12th, 6.　11 pieces, 0.99.5゜ 94.9. 9 pieces, 9. 8B, 4. 79.3. 72. i, 6, 2. 5 2.1. 50.5. 2B, 5゜R1: 0.31 (diethyl ether) 1□ TS(5゜.8□.

CDC1,): 67. Tips (d,, 7 meals 10.3Hz, l)! , Olefi ), 6. Koro (dd.

J-10,4,2,0Hz, IM, olefin), 5.85 (d, J -9.8Hz, LH.

Oref In), 5.10 (d.

! -2.0Hz, 1kl, olefin), 46 Yuko (d, J=4.0H z, l M, ) Jl (114,07 (d, 5m3.0 Hz, H, Olef (Fin), Ko, F2 (dt1..7-4.2.1.7Hz, IH, CIot), 3.41 (S, KoH, Oguchi), 3.20 (m, LH, Lochi).

1B4.4. 157.2.1coff, 7.1coff 4.8. 123. Ko, Engineering 22 ．． 6.　11ko, ko, 911J.

9B, 4. 90.7. R7,2,7B,1. 74.8. F4.0. 5 a+4. 57.1! l, 51.5゜coni lO (3,09), 285 (ko, co f), 256 (ko, 7), 244 (ko, 5); HRMS: Cl. H1?04 Calculated value as NC3 (M+Cs): 468.0212, actual value: 468.0 254 compounds 76 R1 = 0.44 (33% ethyl ether in petroleum ether)'HNKR (300 MIHz, CDCl5) 6 7.611 (dd, J -1, 8, 1, 1Hz , l　I(, aromatic)　, 7.52　(br　s, IF!, aromatic i=1 ．． 7.37 (t.

, T San 7.7 ML 2 M, aromatic), 7. Ko O (dd, J-7, 4, L lHz, IH, aromatic! , 7.26-7.13 (m, 8N, aromatic), 5.61 (s, IH, CKN), l! 17 (t, J-2,!l 1 4z, l H, CM, CM-), 2.49 (dd, JHR1I!S: C. Calculated value as Hr + N0sC8: 576.0576 (M+Cs") , Actual value: 576, 0611 (M+C5”j Compound 87 R+; 0.52 (33% ethyl ether in petroleum ether) HRMS: Cs4 Calculated value as HtsNOsC5: 626.0732 (M+Cs"), actual measurement Value: 626.0?32 (M+Cs”) Compound 89 R, = 0.53 (50% ethyl ether in benzene) , CDCl, ) 6 g, 40 (s, 111. aromatic), l! , 2 pieces ( s, 1M, aromatic), L22 (s, LH, aromatic), 7.99-7 ．． 88 (m, 21 (, aromatic), 7.75 (dd, J-7,7,1,7 Hz, LH.

1.94 (dd, 3-1, 4.6 Hz, LH, CM,), 1. sco (br t, J”13.5)!z, 2 14. CM, l, 0.9! l (dddd,, 7-13.9.1, 9.1, 9°4, 4. 3 Hz, LH, cH2).

4.2. 0.7 Hz, Dyn-human rL 7.52 (m, l H, Dyn- human r), 7.41-6.98 (J 12 Hr 11 krr propargyl) l), 6.98 (dd, LH,, 71117, 1°), l Hz+ ) width - person r), 5.89 (bs, 1H, OH), 5.83 (bs, LH, 0-N-H), 5.713 (s, l H, E-1), 5+211 (d , lH,j -10,0Hz.

0CH2CH20)r　ko, 9koko, 90　(!II, L　H,Σ-ko], +77 (dd, L H, J -2,44 (dd, I H, J m 9.5. 9.5 Hz, input -4), 2+col (dd, IM,, 7-HRMS:C Calculated value CM+Cs as a5HisNsO++): 994.2891, Actual value: 994.2904Ar), 7. :12-7.02 (m, 12H, 11 people r, Probargi JL, H), 6.90 (dd.

1 M,, 7-7.1. 7.1az; oyn-person r), 5.90 (bs, IH, 0-N-H).

5.88 (bs, l 14. OH), 5.82 (s, IH, 5-ml, 5.29 (d, 1)! , J-11,1,9,0Hz, E-5ax), 4. 4+1 (d, H, Ko ■), 4 Hz, A-1) +4.22 -4.17 (m, 2 H, OCR, CH20), 4.14 (dd, L H, J -11 ,1゜4.7 Hz,! -5aq), 4.09 (dd, LH,;! -9, 5,9,5Hz, A-3).

4.06-4.01　(me　　Ht　0(1(z(:)iz’)r　ko, 9 Coco, am (! It, l H.

OCR2CM20), Ko, 8 Fuco, 81 (!11. LH, Σ-3), Ko, 751 (dd, LM,, 7-9.5. 7.1 Hz, input-2), 3 ．． 58 (dq, LH, S -9, 5, 6, 1Hz, input -5).

Ko, 2fu ("r Ko H, mouth CM,), 2. JI4 (ddd, L H, Jm 9.0, 9.0, 4.7Hz,! -4), 2.77 (rrr, 2H , N-Q (2), 2.54 (dd, l H, J■12.2゜2.51 (z ,! -2@q), 2.42 (dd, LH, J-9+5.9.5 Hz , A-4).

2, KoO (dJ I Ht J-14-6r 10.5 Hz, Dyn-CH z) r 2-06 (ddr 1M, 7m 14.6.), I Hz, D yn-CH2), 1.97-1.8 (!It, 4H, DYI’l-0chi) , 1.51 (dd, l 14. .7 am 12.2. 9.2) 1z, ! -2ax), L here (d.

3H, S w-6, 1Hz, A-6), Eng, zo (t, Ko 14. J m 6.5 Hz, N-CH, -cH,); Engineering R (CMCI,): v-, - 2962, 2957, 2929, L Fucoco, 1 piece 86° 1 piece 2 pieces, 1146. 1097.　10fu0mouth゛1; ImMS: Calculated value as Ca*HssNsO+ + (M+Cs　''): 99 4.2891, actual value +994.2904 In a preferred embodiment of the species of the present invention Although described and illustrated with respect to the same, those skilled in the art will appreciate that various improvements, changes, and It is readily recognized that modifications, omissions and substitutions may be made without departing from the spirit of the invention. .

+23456 Day GURE 1 FIGLJRE, 2 Growth inhibition (%) international search report PC? /US911054:16 Continuation of front page (31) Priority claim number (32) Priority mill July 23, 1991 (33) Priority claim country United States (US) (81) Designation-EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, NL, SE), AU , CA, FI, JP, N.

(72) Inventor Smith Alien L United States California state 92121 Saint DA Genesee Avenue 9545 (72) Inventor: Wendeborn, Sebastian Vui, United States of America, Calif. Fornia 92037 La Giyou Via Maloka (72) Inventor Nicolau Kyria California, United States of America 92037 La Jiyou Black Gold Road 9625 (72) Inventor: Schreiner Erwin P. California, USA nia state 92126 Sun DA Gold Coast Drive 9589 (72) Inventor: Stahl Wilhelm, California, United States 92079 Solana Peach R8 South Nato Avenue 661 (72) Inventor Dai Wei Ming California, United States 92122 Sun DA Nobel Drive 4089

Claims (1)

[Claims] 1. Structural formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, A is a double bond or a single bond; R1 is H, C1-C6 alkyl, phenoxycarbonyl, benzyloxycarbonyl) R2 is selected from the group consisting of H, carboxyl, hydroxymethyl and carbonyloxy-C1-C6 alkyl; R3 is selected from the group consisting of H and C1-C6 alkoxy; R4 is H, hydroxyl; sil, C1-C6 alkoxy, oxyacetic acid, C1-C6 hydrocal of oxyacetic acid selected from the group consisting of biyl or benzyl esters, oxyacetamide, oxyethanol, oxyimidazylthiocarbonyl and C1-C6 acyloxy; R6 and R7 are each H, or one with an intervening vinylene group; 2 or more forms a 6-membered fused aromatic ring system; one, two, or three six-membered molecules together with the bonded vinylene group, in which W is [a, b] bonded to the nitrogen-containing ring of the structure shown. Substituted aromatic hydrogen containing rings and R8 is hydrogen or methyl, provided that W is an intervening When R8 is 9,10-dioxoanthra together with the vinylene group, R8 is hydrogen. 2. R6 and R7 are H, or together with an intervening vinylene group, a benzo ring system or The fused ring compound according to claim 1, wherein the fused ring compound forms a naphtho ring system. 3. The condensation according to claim 1, wherein the substituted aromatic hydrocarbyl ring system W is selected from the group consisting of a substituted or unsubstituted benzo ring, a substituted or unsubstituted naphtho ring, and a substituted 9,10-dioxoanthra ring. ring compound. 4. 4. A fused ring compound according to claim 3, wherein the aromatic hydrocarbyl ring system formed is an otherwise unsubstituted benzo ring. 5. The aromatic hydrocarbyl ring system formed contains hydrogen at one or two of the remaining positions. Droxyl, C1-C6 alkoxy, o-nitrobenzyloxy, benzyloxy The condensed ring according to claim 3, which is a benzo ring substituted with a group selected from the group consisting of Ring compound. 6. The aromatic hydrocarbyl ring system formed is hydroxyl, C1-C6 alkoxy hydroxyl, C1-C6 alkoxy, benzyloxy, and halo at the 5- and 8-positions. 4. The fused ring compound according to claim 3, which is a naphtho ring having a group selected from the group consisting of acyloxy, C1-C6 acyloxy, oxo, and halo. 7. The aromatic hydrocarbyl ring system formed contains hydrogen at one or more of the 4-, 5-, and 8-positions. consisting of droxyl, C1-C6 alkoxy, C1-C6 acyloxy, and halo The fused ring compound according to claim 3, which is a 9,10-dioxoanthra ring substituted with a group selected from the group consisting of: 8. The fused ring compound according to claim 1, wherein A is a single bond. 9. Formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, A is a double bond or a single bond; R1 is H, C1-C6 alkyl, phenoxycarbonyl, benzyloxycarbonyl) selected from the group consisting of C1-C6 alkoxycarbonyl, substituted ethoxycarbonyl and 9-fluorenylmethyloxycarbonyl; the group consisting of hydroxymethyl and carbonyloxy-C1-C6 alkyl R3 is selected from the group consisting of H and C1-C6 alkoxy; R4 is H, hydroxy sil, oxyacetic acid, C1-C6 hydrocarbyl ester or base of oxyacetic acid selected from the group consisting of acyl ester, oxyacetamide, oxyethanol, oximidadylthiocarbonyl, and C1-C6 acyloxy; R5 is H, selected from the group consisting of loxyl, C1-C6 alkoxy, o-nitrobenzyloxy, benzyloxy, and C1-C6 acyloxy; R6 and R7 are each H, or together with an intervening vinylene group, one, two or A fused ring compound structurally conforming to three fused aromatic six-membered ring systems; and R6 is methyl or hydrogen. 10. The contraction according to claim 9, wherein R2, R3, R5, R6 and R7 are H. Ring compound. 11. R1 is phenoxycarbonyl, phenylsulfonylethoxycarbonyl or The fused ring compound according to claim 10, which is naphthylsulfonyl ethoxycarbonyl. 12. R4 is H, hydroxyl, C1-C6 alkoxy, oxyacetic acid, imidazylthiocarbonyloxy, oxyacetic acid amide and C1-C6 hydro of oxyacetic acid 12. The fused ring compound according to claim 11, which is selected from the group consisting of carbyl esters and benzyl esters. 13. Formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R1 is H, phenylsulfonyl ethoxycarbonyl, naphthylsulfonylethoxycarbonyl selected from the group consisting of cycarbonyl, phenoxycarbonyl and benzyloxycarbonyl; and R4 is H, hydroxyl, oxyacetic acid, C1-C6 hydrocarbyl ester or benzyl ester of oxyacetic acid, oxyacetic acid amide, oxyethanol, (selected from the group consisting of ximidazylthiocarbonyl and C1-C8 acyloxy). 14. R1 is phenoxycarbonyl, phenylsulfonylethoxycarbonyl or The fused ring compound according to claim 13, which is naphthylsulfonylethoxycarbonyl. 15. 15. The fused ring compound according to claim 14, wherein R4 is selected from the group consisting of H, hydroxyl, and oxyethanol. 16. Formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R1 is H, phenoxycarbonyl, benzyloxycarbonyl, phenylsulfonyl selected from the group consisting of nylethoxycarbonyl and naphthylsulfonylethoxycarbonyl; R4 is H, hydroxyl, oxyacetic acid, oxyacetamide, C1-C6 hydrocarbyl or benzyl ester of oxyacetic acid and oxyethanol; selected from the group consisting of; and R5 is arranged meta or para to the nitrogen atom bonded to R1, and xyl, C1-C6 alkoxy, benzyloxy, C1-C6 acyloxy, o oxyethanol, oxyacetic acid, C1-C6 hydrocarbyl ester of oxyacetic acid, or a fused ring compound selected from the group consisting of benzyloxyester and o-nitrobenzyloxy). 17. 17. The fused ring compound according to claim 16, wherein R1 is phenylsulfonylethoxycarbonyl. 18. 18. The fused ring compound according to claim 17, wherein R4 is H. 19. The fused ring compound according to claim 18, wherein R5 is hydroxyl or C1-5 acyloxy. 20. A condensate having the following structural formula dissolved or dispersed in a physiologically acceptable diluent: A pharmaceutical composition comprising a DNA-cleaving amount or a cytotoxic amount of a fused ring compound. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, A is a double bond or a single bond; R1 is H, C1-C6 alkyl, phenoxycarbonyl, benzyloxycarbonyl) R2 is selected from the group consisting of H, carboxyl, hydroxymethyl and carbonyloxy-C1-C6 alkyl; R3 is selected from the group consisting of H and C1-C6 alkoxy; R4 is H, hydroxyl; sil, C1-C6 alkoxy, oxyacetic acid, C1-C6 hydrocal of oxyacetic acid selected from the group consisting of biyl or benzyl esters, oxyacetamide, oxyethanol, oxyimidazylthiocarbonyl and C1-C6 acyloxy; R6 and R7 are each H, or one with an intervening vinylene group; 2 or more forms a 6-membered fused aromatic ring system; one, two, or three six-membered molecules together with the bonded vinylene group, in which W is [a, b] bonded to the nitrogen-containing ring of the structure shown. Substituted aromatic hydrogen containing rings and R8 is hydrogen or methyl, provided that W is an intervening (provided that R8 is hydrogen when combined with the vinylene group to form 9,10-dioxoanthra)21. 21. A composition according to claim 20, wherein R6 and R7 are H or together with intervening groups form a benzo or naphtho ring system, and R2, R3 and R8 are H. 22. The substituted aromatic hydrocarbyl ring system W is a substituted or unsubstituted benzo ring, A group consisting of a substituted or unsubstituted naphtho ring and a substituted 9,10-dioxoanthra ring The composition according to claim 21, selected from the following. 23. The aromatic hydrocarbyl ring system formed is hydroxyl, C1-C6 alkoxy, benzyloxy, C1-C6-acylo 22. The composition of claim 21, wherein the benzo ring is substituted with a group selected from the group consisting of oxy, oxyethanol, oxyacetic acid, and halo. 24. 22. The composition according to claim 21, wherein A is a single bond. 25. R1 is phenoxycarbonyl, phenylsulfonylethoxycarbonyl or The composition according to claim 21, which is naphthylsulfonyl ethoxycarbonyl. 26. 26. The composition according to claim 25, wherein W is substituted or unsubstituted benzo. 27. 27. The composition of claim 26, wherein R6 and R7 are both H. 28. The benzo group, W, is meta or para to the nitrogen atom bonded to R1; hydroxyl, C1-C6 alkoxy, benzyloxy, C1-C6 acyloxy Claim 27, wherein the substituted compound is substituted with a moiety selected from the group consisting of: composition. 29. (i) oligosaccharide moieties or (ii) monochromes immunoreactive with target tumor cells. antibody or a key containing an aglycon moiety attached to the antibody binding site portion thereof. Mera compound, where the aglycon moiety has the structural formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, A is a double bond or a single bond; R1 is H, C1-C6 alkyl, phenoxycarbonyl, benzyloxycarbo R2 is selected from the group consisting of H, carboxyl, hydroxymethyl and carbonyloxy-C1-C6 alkyl; R3 is selected from the group consisting of H and C1-C6 alkoxy; R4 is H, hydroxyl; sil, C1-C6 alkoxy, oxyacetic acid, C1-C6 hydrocal of oxyacetic acid selected from the group consisting of biyl or benzyl esters, oxyacetamide, oxyethanol, oxyimidazylthiocarbonyl and C1-C6 acyloxy; R6 and R7 are each H, or one with an intervening vinylene group; 2 or more forms a 6-membered fused aromatic ring system; one, two, or three six-membered molecules together with the bonded vinylene group, in which W is [a, b] bonded to the nitrogen-containing ring of the structure shown. Substituted aromatic hydrogen containing rings and R8 is hydrogen or methyl, provided that W is an intervening When combined with the vinylene group is 9,10-dioxoanthra, R8 is hydrogen), and the oligosaccharide moiety is a ribosyl, deoxyribosyl , Fucosyl, Glucosyl, Ga Lactosyl, N-acetylglucosaminyl, N-acetylgalactasamyl, structure Saccharides whose structure is shown below (where the wavy line adjacent to the bond indicates the position of the bond) contains a sugar moiety selected from the group consisting of There are chemical formulas, tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, and ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ The above monoclonal antibody or its binding site part If R4 oxyacetic acid amide is bonded to the aglycone moiety of the fused ring compound via an ester bond or an oxyacetamide or ester bond from W, and the oligosaccharide moiety is hydroxyl of R4 oxyethanol group or hydrogen of oxyethanol substituted W The chimeric compound described above is characterized in that the aglycone moiety is glycoside-bound by droxyl. 30. A The chimeric compound according to claim 29, wherein is a single bond, R2, R3, R6, R7 and R8 are hydrogen, and W is benzo. 31. 31. The chimeric compound of claim 30, wherein said aglycone moiety is tethered to said oligosaccharide moiety. 32. The glycosidic bond between the aglycone moiety and the oligosaccharide moiety is R4 oxyethano 32. The chimeric compound according to claim 31, which is formed from a group. 33. R1 is phenoxycarbonyl, phenylsulfonylethoxycarbonyl or The chimeric compound according to claim 31, which is naphthylsulfonyl ethoxycarbonyl. 34. 33. A chimeric compound according to claim 32, wherein said oligosaccharide moiety is selected from the oligosaccharide groups indicated.