CA2088499A1 - Dynemicin analogs: syntheses, methods of preparation and use - Google Patents

Abstract

A fused ring system compound is disclosed that contains an epoxide group on one side of the fused rings and an enediyne macrocyclic ring on the other side of the fused rings. The compounds have DNA-cleaving, antimicrobial and tumor growth-inhibiting properties. Chimeric compounds having the fused ring system compound as an aglycone bonded to (i) a sugar moiety as the oligosaccharide portion or (ii) a monoclonal antibody or antibody combining site portion thereof that immunoreacts with target tumor cells are also disclosed. Compositions containing a compound or a chimer are disclosed, as are methods of preparing a compound.

Description

2 PCT/US9~/05436 2 ~ ~ ~3 ~

DYNEMICIN ANALOGS:
SY~ITHESES, METHODS OF PREPARATION AND USE

De~ ription 5Cross-Reference - Relat d ADlplication This is a conti~uation-in-part of application Serial No. 67~,199, filed March 21, 1991, which is a continuation-in-part of application Serial No. 562,269, filed on August 1, 1990, both of whos~ disclosures are incorporated by reference.

Technical Field The present invention relates to novel DNA-cleaving, cytotoxic and anti-tumor compounds, and particularly to fused ring systems that contain an enediyne macrocyclic ring and also an epoxide ri.n~, as well as chimeras that contain such a fused ring system.

Backaround Art 20Dynemicin A (Compound l shown below), ~;~ OMe OH O OH

where Me i5 methyl, is a potent antibacterial and anticancer agent recently isolated from MicromonosPora chersina ~(a) Konishi et al, J. Am. Chem. Soc., 35112:3715-3716 (1990); (b) Konishi et al., J. A~ iot., 8 ~ 2 -42:1449-1452 ~1989)]. Its striking molecular structure com~ines characteristics of both the enediyne [Golik et al., J. Am. Chem. Soc., 109~3461-3462 (1987); Golik et al., J. Am. Chem. Soc., 109:3462-3464 (1987); Lee et al., J. Am. Chem. Soc., 109:3464-3466 (1987); Ellestad et al., J. Am. Chem. Soc., 109:3466-3468 (1987)] and the anthracycline ~"Anthracycline Antibiotics", H.S. El Khadem, ed., Academic Press, New York (1982) and "Recent Aspects in Anthracyclinone Chemistry", Tetrahedron lo Symposia-in-Print No. 17, T.R. Kelly, ed., Tetraheclron;
40:4537-4794 (1984)] classes of anti~iotics, and presents a considerable challenge to organic synthesis as well as a unique opportunity for the development of new synthetic technology and therapeutic agcnts.
The calicheamicin and esperamicin derivatives are perhaps the best known of the enediyne compounds.
For a key paper describing the first synthesis of calicheamicinone, see: (a) Cabal et al., J! Am. Chem.
Soc., 112:3253 (l990). For other selected studies of model systems in the area of calicheamicins-esperamicins, see: (b) Nicolaou et al, J. Am. Chem.
Soc., 110:4866-4868 (1988): (c) Nicolaou et al., J. Am.
Chem. soc., 110:7247-7248 (1988); (d) Schoenen et al., ~trahedron Lett., 30:3765-3768 (1989); (e) Magnus et al., J. Am. Chem. Soc., 110:6921-6923 (1988; (f) Rende et al., Tetrahedron Lett., 29:4217-4220 (1988).

Brief Summary of the Invention The present invention relates to novel fused ring systems that contain an epoxide ring and an enediyne macrocyclic ring, and thus have structural features similar to dynemicin A. The compounds have DNA-cleaving, antibiotic and antitumor activities.
Compositions and methods of making and using the compounds are disclosed.

W092/02~22 PCT/US91/05~36 2 ~ ù) U ~

A fusPd ring compound of the inventlon has a structure that corresponds to the formula wherein A is a double or single bond;
Rl is selected from the group consisting of H, C1-C6 alkyl, phenoxycarbonyl, benzyloxycarbonyl, C1-C6 alkoxycarbonyl, substituted C1-C~ alkoxycarbonyl (particularly substituted ethoxycarbonyl), and 9-fluorenylmethyloxycarbonyl;
R2 is selected from the group consisting of H, carboxyl, hydroxylmethyl and carbonyloxy Cl-C6 alkyl;
* is selected from the group consisting of H and C1-C6 alkoxy;
R4 is selected from the group consisting of H, hydroxyl, C1-C6 alkoxy, oxyacetic acid, oxyacetic C1-C~ hydrocarbyl or benzyl ester, oxyacetic amide, oxyimidazilthiocarbonyl and Cl-C~ acyloxy;
R6 and R7 are each H or together with the unsaturated carbon atoms of the intervening vinylene group form a one, two or three fused aromatic six-membered ring system;
W together with the carbon atoms of the depicted, intervening vinylene group forms a substituted aromatic hydrocarbyl ring system containing }, 2 or 3 ' .

W092/02522 PCT/US91t05436 ~ ~ 8 ~ 4 -six-membered rings such that said fused ring compound contains 3, 4 or 5 fused rings, all but two of which are aromatic, and in which that aromatic hydrocarbyl ring system, W, is joined ~a, b~ to the st:ructure shown (i.e., W is joined ~a,b] to the nitrogen-containing rings of the structure shown); and R8 is hydrogen or methyl, with the proviso that R8 is hydrogen when W, together with the carbon atoms of the intervening vinylene group is 9,lO-dioxoanthra.
In preferred practice, W together with the intervening vinylidene group forms a benzo ring so that a compound has the structural formula shown below.

RRs~

wherein R5 is selected from the group consisting of hydrogen, Cl-C6 a}koxy, hydroxyl, C1-C6 acyloxy, oxyethanol, oxyacetic acid, o-nitrobenzyloxy and halo, and A and the remaining R groups are as before described.
More particularly, in one embodiment, R2, R3, R5, R7 and R8 are hydrogen so ~hat a compound of the W~92/02522 2 @ ~ PCT/US91/05~36 invention corresponds to the structural formula shown below, where R1 and R4 are as previously defined.
~, More preferably, Rs is Cl-C6 alkoxy, hydroxyl, C1-C6 acyloxy, oxyethanol, or oxyacetic acid, and R4 is hydrogen (H) or hydroxyl so that a fused ring compound has the structural formula shown below.

Z o Also contemplated is a chimeric compound (also referred to as a chimPr or chimera) that is comprised of - a before-described fused ring compound as an aglycone portion bonded to (i) an oligosaccharide portion or ~ii) a monoclonal antibody or antibody combining site portion thereof that immunoreacts with target tumor cells.

W092/02522 PCT~US91~0~436 g ,'', 9 9 The oligosaccharide portion comprises a sugar moiety selected from the group consisting of ribosyl, deoxyribosyl, fucosyl, glucosyl, galactosyl, N-acetylglucosaminyl, N-acetylgalactasaminyl, a saccharide whose structure is shown be:low, wherein a wavy line adjacent a bond indicates thl3 position of linkage WO92/02522 ~ QU 3 ~ PCl/lJS91tO543b ,OMa H O~;~

UN~"O~ ~OH
PhCH20 OH t~ OMe H

~0~0~ ""..~.., ~ ~

PhC05~' ~J 's, OMa ~ OC

Me." ~O~O OMe 7Ma 7 HOlJ~'OH 5f~ OH

Me." 9~0 OMe OMe Ho~oH ~o~M~d ~'0 Me."", O ~ OM~ OH
~ ~ OMe HO~ `'OH
OMe A monoclonal antibody or binding site portion thereof is bonded to the fused ring compound aglycone portion through an ~4 oxyacetic acid amide or ester bond, or an oxyacetic acid amide or ester bond from W.
An oligosaccharide portion is glycosidically bonded to the aglycone portion through the hydroxyl of an R4 oxyethanol group or the hydroxyl of an oxyethanol-substituent of W.
A pharmaceutical composition is also contemplated. That pharmaceutical composition contains - a DNA cleaving, antibiotic or tumor cell growth-inhibiting amount of a before-defined compound or chimera as active agent dissolved or dispersed in a physiologically tolerable diluent.
A compound, chimera or a pharmaceutical composition of either is also useful in a method for cleaving DNA, for inhibiting tumor growth and as an antimicrobial. In accordance with such a method, the DNA to be cleaved, target tumor cells whose growth is to be inhibited or target microbial cells is (are) contacted with a composition of the invention. That contact is maintained for a time period sufficient for the desired result to occur. Multiple administrations of a pharmaceutical composition can be made to provide the desired contact.

Brief Descri~tion of the Drawin~s In the drawings forming a portion of this disclosure, Figure l is a photograph of an ethidium bromide stained l percent agarose gel that illustrates the cleavage of ~X174 form I DNA by Compound 40 after 24 hours in phosphate buffers (50mM) containing 20 volume ~ercent THF at pH 1.4. Lane l is the DNA alone as control, lanes 2-6 show the results obtained with 5000, W092/~2s22 PCT/US91/05436 2000, looO, 500 and lOO~M Compound 40, respectively.
The designations I, II and III outside the gel indicace forms I, II and III of the DNA, respectively.
Figure 2 is a photograph of an ethidium bromide stained 1 percent agarose gel that illustrates the cleavage of ~X174 from I DNA by Compounds 43, 47, 42, 5~, 5S, 58 and 62 after 24 hours in pH 8.0 50mM
Tris-HCl buffer. Lane 1 is the DNA alone as control, lanes 2, 3, 4, 5, 6, 7 and 8 show the results obtained with 5mM of each of compounds 40, ~7, 42, 54, 55, 58 and 62, respectively. The designations Form I, II and III
are as in Figure 1.
Figure 3 is a graph showing results from two studies of the percent growth inhibition of MIA PaCa-2 human pancreatic carcinoma cells over a four-day time period by various concentrations of Compound 2 (DY-l).
Figure 4 is a graph showing the results from four studies of the percent growth inhibition over a four-day time period of MB49 murine bladder carcinoma cells by various concentrations of Compound 2 (DY-l).
IC50 values for two of the studies were 43 nM and 91 nM.
Figure 5 is a graph showing the results from two studies of the percent growth inhibition over a four-day time period of MB4s murine bladder carcinoma cells by various concentrations of Compound 21 (DY-2).

Detailed DescriPtion of the Inventi.on I. The Com~ounds ~ A compound of the invention contains an enediyne macrocycle linked to a fused ring that corresponds to structural Formula I

W092/~2522 PCT/US91/05436 2~g~3 - 10 -F~6 R~F~2 wherein A is a double or single bond;
R1 is selected from the group consisting of H, C1-C6 alkyl, phenoxycarbonyl, benzyloxycarbonyl, C1-C6 alkoxycarbonyl, substituted C1-C6 alkoxycarbonyl (particularly a substituted ethoxycarbonyl) and 9-fluorenylmethyloxycarbonyl;
R2 is selected from the group consisting of H, carboxyl, hydroxylmethyl and carbonyloxy-C1-C6 a}kyl:
R3 is selected from the group consisting of and C,-C6 alkoxy;
R4 is selected from the group consisting of H, hydroxyl, C1-C6 alkoxy, oxyacetic acid (-OC~zC02H), C1-C6 hydrocarbyl or benzyl oxyacetic acid ester, oxyacetic amide, oxyethanol (-OCH2CH20H), oxyimidazylthiocarbonyl and C,-C6 acyloxy:
R6 and R7 are each H or together with the intervening vinylene group form a one, two or three fused aromatic six-membered ring system;
W together with the bonded, intervening, vinylene group (i.e., the unsaturated carbon atoms bonded to W) forms a substituted aromatic hydrocarbyl ring system containing 1, 2 or 3 six-memhered rinys such that said fused ring compound contains 3, 4 or 5 fused 6-membered rings all but two of which rings are W092/02522 ~ ~ 3 ~ ~ ~ PCT/U591/05436 aromatic, and in which that aromatic hydrocarbyl ring system, w, is joined [a, b] to the structure shown; and R8 is hydrogen or methyl with the proviso that R8 is hydrogen when W together with the intervening vinylidene group is 9,10-dioxoanthra.
Exemplary R6 and R7 groups are shown in Scheme III and are discussed in relation thereto, and thereafter.
As noted above, the bond, A, between the R2 and R3 substituents can be a double or single bond. The bond A is preferably a single bond.
A Cl-C6 alkyl group, as can be present in R1 is exemplified by methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, pentyl, 2-methylpentyl, hexyl, cyclohexyl, cyclopentyl and the like. A substituted Cl-C6 alkyl group is also contemplated as an Rl group. Such substituted alkyl groups include hydroxyalkyl groups such as 2-hydroxyethyl, 4-hydroxyhexyl and

3-hydroxypropyl, haloal~yl groups such as 2-chlorobutyl, 3-halopentyl such as 3-fluoropentyl, and the like. Tho above C1-C6 alkyl and substituted Cl-C6 alkyl groups are further contemplated as the C1-C6 alkyl portion of a carbonyloxy C1-C6 alkyl group of R2; i.e., a C1-C6 alkyl ester of a R2 carboxyl group, and o a R1 urethane group. Those same alkyl groups can constitute the alkyl portion of a C1-C6 alkoxy group Or R3 or R4. A C1-C6 acyloxy group as is present in R4 or R5 (discussed hereinafter) is a carboxylic acid derivative of an appropriate alkyl group, above, except for, for example, cyclohexyl and iso-propyl, and is limited to a cyclopentylcarboxyl group for the cyclopen~ane derivatives. Examples of such C1-C6 acyloxy groups include formyloxy, acetoxy, propionoxy, butyryloxy, iso-butyryloxy, pentanoyloxy, 2-methylbutyryloxy, pivaloyloxy, hexanoyloxy, and the like.

2 ~ 12 -The alcohol-carbonyl portion of a urethane is typically formed by the reaction of a corresponding halo formate derivative, such as a chloroformate like phenylchloroformate, with the secondary amine nitrogen atom that is formed by addition of an acetylenic group-containing moiety to the 6-position or a correspondingly numbered position of a fused ring system such as that shown in Scheme II hereinafter. Such groups can also be prepared by base-catalyzed exchange from a formed carbamate using the substituted ethyl alcohol as is llustrated hereinafter.
Exemplary C1-C6 alkoxycarbonyl groups and substituted C1-C6 aIkoxycarbonyl groups contain a before-described C1-C6 alkoxy group or substituted C1-C6 alkoxy group linked to the carbonyl group and can be formed by reaction of a C1-C6 alkylchloroformate.
Exemplary substituted ethoxycarbonyl groups that are a particularly preferred group of substituted C~-C6 alkoxycarbonyl group have a substituent other than hydrogen at the 2-position of the ethoxy group, and include 2-trimethylsilylethoxycarbonyl, 2-phenylsulfonylethoxycarbonyl, ~- or ~-2-naphthylsulfonylethoxycarbonyl, ~- or ~-2-anthracylsulfonylethoxycarbonyl, 2-propenoxycarbonyl, 2-hydroxyethoxycarbonyl, 2-triphenylphosphoniumethoxycarbonyl halide (e.g., chloride, bromide or iodide) and 2-trimethylammoniumethoxycarbonyl halide (as before).
It is particularly preferred that R1 be a group that can be enzymatically or otherwise removed intracellularly to provide the resulting secondary amine free of a substituent group. A compound where ~1 contains a 2-substituted-ethoxycarbonyl group such as a 2-phenylsulfonyl-, 2-naphthylsulfonyl- and 2-anthracylsulfonyl- as are shown in Scheme III tshown W092/02522 PCT/US91/0~36 2 $ $ ~?~ A~ 3 as R1 therein) can form the free secondary amine compound via a ~-elimination under relatively mi}d conditions. Phenylsulfonylethoxycarbonyl, ~-naphthyl-and ~-naphthylsulfonylethoxycarbonyl (collectively referred to as naphthylsulfonyletho~c:arbonyl) are particularly preferred R1 groups, with phenoxycarboxyl being a pre~erred R1 group.
An R8 group can be methyl or hydrogen with the proviso that R8 is hydrogen when w along with the intervening vinylene group carbon atoms forms a 9,10-dioxoanthra ring. It is particularly preferred that R8 be methyl when W forms a benzo ring.
R4 groups that are hydrogen, hydroxyl, oxyethanol (-OCH2CH20H), oxyacetic acid (-OCH2CO2H), o.~acetic C1-C6 hydrocar~yl esters such as the before-discussed C1-C6 alkyl groups such as ethyl oxyacetate (-OCH2C02CH2CH~), as well as C1-C6 unsaturated esters such as the allyl, propargyl, 2-butenyl and the like, as well as the benzyl ester and oxyacetic amides constitute particularly preferred embodiments of the invention.
Exemplary C1-C6 and benzyl esters that have been prepared: i.e., Compounds 24a-g exhibited activity against MIA PaCa-2 tumor cells.
A pharmac~ltically acceptable non-toxic salt of the oxyacetic acl~ such as sodium, potassium, ammonium, calcium and mag: sium is also contemplated.
An oxyacetic acid amide corresponds to the chemical formula -ocH2coNR13R14 wherein R13 is hydrogen (H) or C1-C6 alkyl ~as before) and R14 is independently hydrogen, C1-C6 alkyl, phenyl, l- or 2-napthyl, l- or 2-anthryl, or a peptide having l to about six amino acid residues;
or R13 and R14 together with the amido nitrogen atom fo~m a 5- or 6-membered ring as is present in pyrrolidine, piperidine or morpholine.

W092/02522 P~T/US91/05~36 A particularly contemplated peptide is distamycin, or a derivative thereof as discussed in Taylor et al., Tetrahedron, 40:457 tl984) and Baker et al., J. Am. Chem. Soc., 111:2700 (1989). Distamycin derivatives are themselves known DNA-cleaving agents.
Indeed, a N-bromoacetyldistamycin adduct of Compound 2 has been pre~ared. Another particularly preferred peptide is -Ala-Ala-Ala-, [(-Ala-)3].
- An R4 group that contains a derivatized oxyacetic acid amide or ester can also include a peptidyl spacer containing zero to about 6 residues such as (-Ala-)3 that links the compound to a monoclonal antibody or an antibody binding site portion thereof, collectively referred to herein as a "Mab", as is illustrated in relation to Scheme III hereinafter ~R or R3). The Mab utilized immunoreacts substantially only with target tumor cells; i.e., is tumor cell specific, and thereby provides further specificity to the drug molecules. Such a Mab-linked fused ring enediyne is one type of chimeric molecule of the invention.
The spacer portion of the compound-Mab construct serves to link the two portions of the molecule together. When there are zero peptide residues present, a lysine epsilon-amino group of the Mab forms the amido bond shown in Scheme III. The spacer peptide chain, when present, is typically comprised of amino acid residues having small side chains such as glycine or alanine, or relatively hydrophilic side chains such as serine, glutamine and aspartic acid. A peptide spacer is typically free of cysteine residues, but otherwise can have substantially any structure that does not interfere with bonding between the two portions of the chimeric compound. A peptide can be prepared by an one of several synthetic methods as are well known.

The Mab portion of the above chimeric construct can constitute an intact antibody molecule of IgG or IgM isotype, in which case, a plurality of compounds can be present per antibody molecule. The binding site portions of an antibody can also be utilized, in which case, at least one compound is linked to the proteinaceous antibody binding site portion.
An antibody binding site portion is that part of an antibody molecule that immunoreacts with an antigen, and is also sometimes referred to as a paratope. Exemplary antibody binding site portions include F(ab), F(ab'), F(ab')z and Fv portions of an intact anti~ody molecule, and can be prepared by well known methods. An intact monoclonal antibody and a portion that includes its antibody combining site portion can be collectively referred to as a paratope-containing molecule.
Exemplary anti-tumor Mabs are noted in the table below, listed by the name utilized in a publication, along with its deposit accession number at the American Type Culture Collection (ATCC No.), 12301 Parklawn Drive, Rockville, Maryland 20852 U~SoA~I and the tumor antigen with which the Mab paratope is -3ported to react. A citation to a discussion of each Mab and its immunoreactivity is provided by the footnote under the antigen listin~.

W092/02522 PCT/US91/0~436 Exem~lary Anti-Tumor Mabs Mab ATCC No. Antiqen B 3.6 HB 8890 GD31 14.8 HB 9118 GD22 liC64 -- GD33 9.2.27 -- Condritin ~sulfate proteoglycan4.

HT29/26 HB 8247 colon cancer glycoprotein gp 296 HT29/3~ ~ 8248 colon cancer glycoprotein gp296 CLT85 HB 8240 colon cancer6 F64.5 -- mammary carcinoma R38.1 -- pan carcinoma 7OKd protein7 ~36/22 HB 8215 human breast carcinoma8 T16 HB 8279 human bladder tumor, glycoprotein gp489 T43 HB 8275 human bladder tumor9 T101 HB 8273 human bladder tumor9 116-NS-19-1HB 8059 colorectal carcinoma monosialoganglioside10 CLH 6 HB 8532 colon cancerl2 CLG 479 HB 8241 colon cancerl2 19.9 CRL 8019 cEA13 CLNH5 ~~ lung carcinomal4 16-88 -- colon carcinomalS
.
Cheresch et al., Proc. Natl. Acad. Sci.,_USA, 82:5155-5159 (1985): Ibid, 81:5767-5771 (1984) 2 Cheresch et al. Cancer Res. 44:S112-5118 (1986) W092/02522 PCT/US91iO5436 2~ >~

3 Cheresch et al., J. Cell. Biol., 102:688(1986)

4 Bumol et al., Proc. Natl. Acad. sci. USA, 79:1245 (1982); Harper et al., J. Immunol.l 132:2096 (1984 U.5. Patent No. 4,507,391 6 U.S. Patent No. 4,579,827 7 U.S. Patent No. 4,522,918 8 European Patent Application No. 84400420.0, publication No. o 118 365, published September 12, 9 European Patent Application No. 84102517.4, publication No. 0 118 891, published September 19, U.S. Patent No. 4,471,057 Cheresch et al., J. Cell. Biol., 102:688 (1986);
U.S. Patent No. 4,675,287 25 12 ~.S. Patent No. 4,579~827 13 U.S. Patent No. 4,349,528 14 Patent Application PCT/US83/~0781, NO 83/04313 15 European Patent Application No. 85300610.4, publication No. 0 151 030, published August 7, 1985 A fused ring enediyne compound of the invention can also be glycosidically linked to a sugar moiety to form a second type chimeric molecule. In such a chimer, the fused ring enediyne compound takes the place of the aglycone as in an antibiotic molecule such as doxorubicin, calicheamicin or esperamicin, with the sugar moiety taking the place of the oligosaccharide portion. Bonding between the fused ring enediyne compound aglycone and oligosaccharide is typically via a hydroxyl group of a spacer group that is itself linked to the fused ring enediyne through a reacted hydroxy}
group. A preferred spacer group is an oxyethanol group W092~02522 PCT/US91/05436 2~3`~9 that can be an R4 group or can be a substituent of w as is discussed and illustrated hereinafter.
The oligosaccharide portion of the molecule is typically added after the synthesis of the fused ring enediyne compound (aglycone) portion i9 complete, except for any blocking groups on otherwise reactive functiona}ities of the aglycone that are typically removed after addition of the oligosaccharide portion.
A sugar moiety i~ added by standard techniques as are discussed hereinafter.
A glycosidically-linked sugar moiety can be a monosaccharide such as a ribosyl, deoxyribosyl, fucosyl, glucosyl, galactosyl, N-acetylglucosaminyl, N-acetylgalactosaminyl moiety or the more preferred saccharides whose structures are shown below, wherein a wavy line adjacent a bond indicates the position of linkage WO 92/02522 ~ 9 PCI/US91/05436 ~~ C ~ ~N~

C)Ma PhCH20 OH A .%, H
/~N~
~0~,0~ ,.~ o C3 .J C ~ OMe FMOC
1H ~ N~
M~ O ~ ~ ~N~J ~ o~oJ Me, ~S~ ~ D
Me.~o~OMo OH M ~ ,s H `OH e~ ~0 Me ' OH OMs ~ ~ OMe ~e~o~ ~N~
HO'~ ~ OH Me o , ~ 1 J Me OMe ~ " ~ " ~ and M~-" [~~oM~ OH ~ OM

HO~ ; `OH Me~,~~~N,~.,.O O Me OM~ I~S~ ~ ~ OH

Ms.,~ O O~OMe OH
f ~ OMe HO~ ~OH
OMe 208~99 The position of the glycosyl bond to be formed in the sugar moiety used for forming a chimeric compound is typically activated prior to linkage to the fused ring enediyne compound. For example, the l-position hydroxyl group of an otherwise protec:ted sugar (as with tBuMe~Si or Et3Si groups) is reacted with diethylaminosulfur trifluoride (DAST) in THF and in the presence of 4A molecular sieves at -78C to form the 1-fluoroderivative. The enediyne having a free hydroxyl group is then reacted with the 1-fluro-protected saccharide in the presence of silver perchlorate and stannous chloride to provide a protected desired, typically blocked, chimer molecule.
Similarly, treatment of l-position hydroxyl of an otherwise protected saccharide with sodium hydride and trichloracetonitrile [Grandler et al., C bohvdr.
E~ 203 (1985); Schmidt, Anaew. Chem. Int~_Ed., Engl., 25:212 (1986)] in methylene chloride at about room temperature provides a l-~-trichloroacetimidate group to activate the saccharide for coupling with the fused ring enediyne (aglycon) hydroxyl. Coupling is then carried out in boron trifluoride~etherate in methylene chloride to provide the protected desired chimer compound.
Once the aglycone and oligosaccharide are coupled, the protecting groups that are present are removed to provide the desired compound, which is then recovered using standard techniques. Exemplary syntheses are discussed hereinafter.
The l, 2 or 3 six-membered ring fused rings that along with the depicted vinylene group constitute the structure W are aromatic hydrocarbyl rings. Such rings can thus be benzo, naphtho and anthra rings, using fused ring nomenclature. The anthra (anthracene) derivative rings contemplated here contain 9,10-dioxo W092/02522 PCT/VS9l/05436 2 ~ ~, o !3 ~ ~ .

groups (are derivatives of anthraquinone) and are therefore referred to as 9,10-dioxoanthra rings.
Where a benzo, naphtho or 9,lQ-dioxoanthra ring forms part of the fused ring system, those fused rings are bonded to the remaining fused ring system through the carbon atoms of the 1- and 2-positions or are ta, b). A benzo, naphtho or 9,10-dioxoanthra fused ring portion can also contain one or more substituents at the ring positions remaining for substitution. Those substituent groups are selected from the group consisting of hydroxyl, C1-C6 alkoxy, oxo, C1-C6 acyloxy -and halo (chloro, bromo or iodo).
For a benzo ring, one or two substituents can be present at one or two of the remaining positions of ~5 the radical. Symmetrical substitution by the same substituent is pre~erred because of the lessened possibility for isomer formation. When a single substituent is present on a benzo ring, that substituent is referred to as ~5, which designation for convenience includes hydrogen. R5 is thus selected from the group consisting of hydrogen (no substituent), C1-C6 alkoxy, benzyloxy, o-nitrobenzyloxy, hydroxyl, Cl-C6 acyloxy, oxyethanol, oxyacetic acid, oxyacetic acid C1-C6 hydrocarbyl ester and halo. It is preferred that a hydroxyl group or a group that can form a hydroxyl group intracellularly be present, such that a hydroxyl group be present intracellularly at a position meta to the nitrogen in the adjacent ring. When two su~stituents are present on a benzo ring, they axe referred to as R~
and R11 and are selected from th~ group consisting of C1-C6 alkoxy, benzyloxy, oxo, C1-C6 acyloxy, hydroxyl and halo.
W is more preferably a benzo group that contains a single substituent Rs. In one particularly preferred embodiment, Rs is situated in the benzo ring -. . .

W092/02~22 PCTIUS91/05~36 2~o ~ ~9 meta or ara to the nitrogen atom bonded to R1. That Rs group is more preferably selected from the group consistiny of hydroxyl, C1-C6 alkoxy, benzyloxy, o-nitrobenzyloxy, Cl-C6 acyloxy, oxyethanol, oxyacetic acid and an oxycacetic C1-C6 hydrocarbyl ester.
When Rs is meta to the above nitrogen atom, it is-preferred that the R5 group be an electron releasing group such as hydroxyl or a Cl-C6 acyloxy group that can provide a hydroxyl group intracellularly. A C1-C6 acyloxy group is believed to be a pro-drug form of the hydroxyl group that is cleaved intracellularly by an endogenus esterase or the like to provide the hydroxyl group. The presence of such an electron releasing group appears to assist in enhancing the potency of the compound against target tumor cells. It is believed that the enhanced potency is due to enhanced triggering of the epoxide opening and cyclization reactions.
When R5 is E3~a to the above nitrogen atom, it is preferred that the R5 group be an o-nitrobenzyloxy group, oxyethanol, oxyacetic acid or oxyacetic acid C1-C6 hydrocarbyl ester. Those groups are particularly useful for the preparation of chimeras.
A particularly preferred compound has a structure corresponding to Formula XIb, herein~fter.
A naphtho ring can have three substituents.
This ring can have a 4-position radica}, Rs, selected from the group consisting of hydroxyl, Cl-C6 alkoxy, benzyloxy, Cl-C6 acyloxy and halo, and substituents at the 5- (Rl) and 8-positions (Rll) that are selected from the group consisting of hydroxyl, Cl-C6 alkoxyJ
benzyloxy, Cl-C6 acyloxy, oxo and halo radicals. A
9,10-dioxoanthra ring can have three substituents at the 4- (R5), 5- (R9) and 8-positions (R12) that are independently selected from the group consisting of hydroxyl, Cl-C6 alkoxy, benzyloxy, Cl-C6 acyloxy and W092/02522 2 1~ ~ 3 ~ 3 ~ PCT/US91/05436 halo. Thus, Rs, R9 and R12 can define the same groups, and all three groups can be written as either Rs, R9 or R1Z, but they are shown separately herQin.
Exemplary structural formulas for such fused ring compounds are illustrated below by structural Formulas II-IX, wherein each of the R groups is as discussed before.

WO 92/û252~ PCT/US91/05436 2 ~ 9 9 `-:-,~ ~

N~ O R~Ni~ R~

,R~

R~s [~R

R" NJ~j'r R~ O
~R3 ~Fl s Vl~l 5 1%

In addition to the be~ore-stated preference reaarding R8 and that bond A be a single bond, several other structural features and substituents are preferred.
Thus, it is preferred that R2 and R3 be hydrogen, and that R6 and R7 be hydrogen. It is also preferred that the fused ring system W together with the depicted vinylene group be substituted benzo, or an unsubstituted benzo, naphtho or 9,10-dioxoanthra ringu It is further preferred that the fused ring compound contain a total of 3-fused six-membered rings so that w together with the depicted vinylene group for~s a benzo ring.
One particularly preferred group of compounds of the invention in which W is an Rs-substitu~ed benzo ring corresponds to structural Formula X.

~ ~5~

wherein A is a double or single bond;
R1 is selected from the group consisting of H, Cl-C6 alkyl, phenoxycarbonyl, benzoxycarb~nyl, C1-C6 alkoxycarbonyl and 9-fluorenylmethyloxycarbonyl;
R2 is selected from the group consisting of H, carboxyl, hydroxylmethyl and carbonyloxy Cl-C6 alkyl;

. .
2~8 ~ ~

R3 is selected from the group consisting of H
and Cl-C6 alkoxy;
R4 is selected from the group consisting of H, hydroxyl, oxyacetic acid (-OCH2C02H), oxyacetic Cl-C6 hydrocarbyl or benzyl ester, o~yacetic amide, oxyethanol, oxyimidazylthiocarbsnyl and cl-c6 acyloxy;
Rs is selected from the group consisting of hydrogen, C~-C6 alkoxy, ~enzyloxy, o-nitrobenzyloxy, hydroxyl, Cl-C6 acyloxy, oxyethanol, oxyacetic acid, oxyacetic acid Cl-C6 hydrocarbyl ester and halo: and R6 and R7 are each H or together form with the intervening vinylidine group form a one, two or three fused aro~atic ring system, and R8 is methyl or hydrogen.
A still more preferred group o~ compounds of the invention correspond to structural FormuIas XI, XIa and XIb.

R1~ R1a~
Xl Xla Xlb wherein R1, R4, Rs and R8 are as previously defined.
Of the compounds corresponding to structural Formula XI, there are further pre~erences for R1, R4 and R5. These preferences also relate to the previously discussed compounds.

W092/02522 ~ `7~ R PCT/US91/05436 Thus, R1 is most preferably phenoxycarbonyl phenylsulfonylethoxycarbonyl, naphthylsulfonylethoxycarbonyl or hydrogen. R8 is most preferably hydrogen (H) to provide a compound of Formulas XIa or XIb. R4 is most preferably H, hydroxyl, imidazylthiocarbonyloxy, benzyl oxyacetate and C~-C6 hydrocarbyl oxyacetate such as ethyl oxyacetate. RS in Formulas XI and XIa is H, but is more preferably hydroxyl, C~-C6 alkoxy, benzylo~y, C1-C6 acyloxy, oxyethanol, oxyacetic acid, oxyacetic acid C1-C6 hydrocarbyl or benzyl ester and o-nitrogenzyloxy as in Formula XIb. It is noted that an Rs o-nitrobenzyloxy group is not usually used in a pharmaceutical composition discussed hereinafter.
The structural formulas of particularly preferred compounds are shown below, along with compound numbers as utilized herein. In the formulas below and elsewhere herein, Ph = phenyl, ~e = methyl, NBnO =
o-nitrobenzyloxy, Naph = naphthyl and tBuCO2 = pivaloyl.
}1--0~ }h-O-~

}h-O-C~ O-~

21 ~CH2CO2C2Hs 24c WO 92/02522 PCl /lJS9l/05436 9~ - 28 -H~b PMhoO

PhOlN~ H"~
~W PhS~0)2(CH2)2 OMe 41a ~ 41b MeO

NaphS(012(CH2)20 41C & 41d HO

W092/02522 ~ PCT/US91/05436 _ ~9 _ S PhO ~ ~ PhO

OH N~nO OH

10 PhzO ~ H*~ PhO

15tBuCO 59aOH t~uCO2 59b II. Pharmaceutical Compositions A compound or chimera of the invention is useful as a DNA cleaving agent, and also as an antimicrobial and a cytoxic (antitumor) agent, as are dynemicin A, calicheamicin, esperamicin and neocarzinostatin. A compound of the invention can also therefore be referred to as an "active agent" or "active ingredient".
DNA cleavage can be assayed using the techniques dec ribed hereinafter as well as those described by Mantlo et al., J. or~. Chem., 54:2781 tl989): Nicolaou et al., J._Am. Chem. Soc., 110:7147 (1989): Nicolaou et al., J. Am. Chem. Soc., 110:7247 (1988) or Zein et al., Science, 240:1198 (1988) and the citations therein.

W092/02522 PCT/USg1/05436 A compound or chimer of the :in~ention is useful against Gram-positive bacteria such as s. aureus and e~idermis, Micrococcus luteus and ]3acillus subtillis as is dynemicin A. Such a compound or chimer also exhibits antimicrobial activity against E. coli, -Pseudomonas aeruainos, Candida albucans and Asperqillis fumiaatus. Activity of a compound of the invention against the above microorganisms can be determined using various well known techniques. See, for example, Konishi et al., J. Antibiotics, XLII:1449 (1989).
Antimicrobial and antitumor assays can also be carried out by techniques described in U.S. Patent No.
4,837,206, whose disclosures are incorporated by reference, as well as by the procedures described hereinafter.
A before-described compound can also be shown to undergo a 3ergman cycloaromatization reaction in the presence of benzyl mercaptan, triethylamine and 1,4-cyclohexadiene as discussed in Haseltine et al., J! Am.
Chem. Soc., 111:7638 (1989). This reaction forms a tetracyclic reaction as is formed during DNA cleavage, and can be used as a co-screen to select more active compounds.
A pharmaceutical composition is thus contemplated that contains a before-described compound or chimer of the invention as active agent. A
pharmaceutical composition is prepared by any of the methods well known in the art of pharmacy all-of which involve bringing into association the active compound and the carrier therefor. For therapeutic use, a compound or chimer of the present inYention can be administered in the form of conventional pharmaceutical compositions. Such compositions can be formulated so as to be suitable for oral or parenteral administration, or as suppositories. In these compositions, the agent is c~ Qi~s~ 3 typi~ally dissolved or dispersed in a physiologically tolerable carrier.
A carrier or diluent is a material useful for administering the active compound and must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. As used herein, the phrases "physiologically tolerable" and "pharmaceutically acceptable" are used interchangeably and refer to molecular entities and compositians that do not produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a mammal. The physiologically tolerable carrier can take a wide variety of forms depending upon the preparation desired for administration and the intended route of administration.
As an example of a useful composition, a compound or chimer of the invention (active agent) can be utilized, dissolved or dispersed in a liquid composition such as a sterile suspension or solution, or as isotonic preparation containing suitable preservatives. Particularly well-suited for the present purposes are injectable media constituted by aqueous injectable buffered or unbuffered isotonic and sterile saline or glucose solutions, as well as water alone, or an aqueous ethanol solution. Additional liquid forms in which these compounds or chimers can be incorporated for administration include flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, peanut oil, and the like, as well as elixirs and similar pharmaceutical vehicles. Exemplary further liquid diluents can be found in Remminaton' Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (1980).
An acti~e agent can also be administered in the form of liposomes. As is known in the art, 3 ~

liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono-or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be u~ied. The present compositions in liposome form can cont:ain stabilizers, preservatives, excipients, and the like in addition to the agent. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.
Methods of forming liposomes are known in the art. See, for example, Prescott, Ed., Methods in cell Bioloqy, Vol. XIV, Academic press, New YorX, N.Y.
(1976), p.33 et seq.
An active agent can also be used in compositions such as tablets or pills, preferably contai~ing a unit dose of the compound or chimer. To this end, the agent (active ingredient) is mixed with conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, gums, or similar materials as non-toxic, physiologically tolerable carriers. The tablets or pills can be laminated or otherwise compounded to provide unit dosage ~orms affording prolonged or delayed action.
It should be understood that in addition to the aforementioned carrier ingredients the pharmaceutical formulation described herein can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like, and substances included for the purpose of rendering the W092/02522 PCT/US91/0~436 2 v~ 3 formulation isotonic with the blood of the inten~ed recipient.
The tablets or pills can also be provided with an enteric layer in the form of an envelope tha~ serves to resist disintegration in the stomach and permits the active ingredient to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, including polymeric acids or mixtures of such acids with such materials as shellac, shellac and cetyl alcohol, cellulose acetate phthalate, and the-like. A
particularly suitable enteric coating comprises a styrene-maleic acid copolymer together with known materials that contribute to the enteric properties of the coating. Methods for producing enteric coated tablets are described in U.S. Patent 4,079,125 to Sipos, which is herein incorporated by reference.
The term "unit dose", as used herein, refers to physically discrete units suitable as unitary dosages for administration to warm blooded animals, each such unit containing a predetermined quantity of the agent calculated to produce the desired therapeutic effect in association with the pharmaceutically acceptable diluent. Examples of suitable unit dosage forms in accord with this invention are tablets, capsules, pills, powder packets, granules, wafers, cachets, teaspoonfuls, dropperfuls, ampules, vials, segregated multiples o~ any of the foregoing, and the like.
A previously noted preferred or particularly preferred compound or chimer is preferred or particularly preferred for use in a pharmaceutical composition.
A compound or chimer of the invention is present in such a pharmaceutical composition in an amount effective to achieve the desired result. For example, where in vitro DNA cleavage is the desired s~ 9~ - 34 -result, a compound or chimer of the invention can be utilized in an amount sufficient to provide a concentration of about l.0 to about 5000 micromolar t~M) with a DNA concentration of about 0.02 ~g~L. As a cytoxic (antitumor) agent, an effective amount of a compound or chimer of the invention is about O.l to about 15 mg per kilogram of body weight or an amount sufficient to provide a concentration of about O.Ol to about 50 ~g/mL to the bloodstream. A compound or chimer of the invention exhibits antimicrobial activity in a concentration range of about O.Ol mg to about 50 ~g~mL.
The above concentrations and dosages vary with the particular compound of the invention utilized as well as with the target, e.g., DNA, tumor, microbe, as is well known.

III. Methods A compound or chimer of the invention is useful in cleaving DNA, as a cytotoxic agent a~d also in inhibiting the growth of neoplastic cells, and is utilized in a method for effecting such a result. A
compound or chimer of the invention is typically utilized in a before-described composition.
In accordance with such a method, DNA or target cells to be killed or whose growth is to be inhibited are contacted with a composition that contains a compound or chimer of th~ invention (active ingredient~ present in an amount effective or sufficient for such a purpose, as discussed before, dissolved or dispersed in a physiologically tolerable (pharmaceutically acceptable~ diluent. That contact is maintained for a time sufficient for the desired result to be obtained; i.e., DNA cleaved, cells killed or neoplastic cell growth inhibited.

W092/02522 Sh ~ q~ 3 PCT/US91/OS436 Where the desired result is carried out in vitro, contact is maintained by simply admixing the DNA
or *arget cells with the composition and maintaining them together under the apyropriate conditions of temperature and for cell growth to occur, as for control, un~reated cells. Thus, a single admixing and contacting is typically su~ficient for in vitro purposes.
The above method is also useful in vivo, as where a mammal such as a rodent like a rat, mouse, or rabbit, a farm animal like a horse, cow or goat, or a primate like a monkey, ape or human is treated. Here, contact of a composition and the cells to be killed or whose growth is to be inhibited is achieved by administration of the composition to the mammal by oral, nasal or anal administration or by introduction intravenously, subcutaneously or intraperiton~a}ly.
Thus, contact in vivo is achieved via the blood or lymph systems.
Although a single administration (admixture) and its resulting contact is usually sufficient to maintain the re~uired contact and obtain a desired result in vitro, multiple administrations are typically . utilized in ~ivo. Thus, because of a body's breakdown and excreting pathways, contact between an active ingredient of a composition and the target cells is typically maintained by repeated administration of a compound o~ tha invention over a period of time such as days, weeks or months, or more, depending upon the target cells.
Exemplary methods of the invention for DNA
cleavage and inhibition of MIA PaCa-2 human pancreatic carcinoma (ATCC CRL 1420) and MB49 murine bladder carcinoma target cells (obtained from Dr. Lan Bo Chen of the Dana Farber Cancer Institute, Boston, MA) as well as 2 ~ ~ 8 ~l~r ~i 3 ~ 36 ten other neoplastic cell lines are discussed hereinafter.

IV. Compound S~ntheses A compound of the invention can be prepared by a number of routes, several of which are illustrated in the schemes hereinafter. The retrosynthetic plan fo these syntheses is illustrated below in Scheme I, with the general forward synthesis shown in Scheme II, thereafter.

Scheme I

PhO~¢~? ~ PhO~

W0 92/02522 ~ q 3 ~ PCl~US91/05436 Scheme ¢~0 a,b ¢5[~

4 cc5;R~Ac 6; R = H
d ~ ~; R - SitBuMe2 PhO

9; R ~ Si~BUMe2 9C10; R- H 8 J
11 kjrl3;R=siMe3 3; R ~ H

W092/02522 PCT/U~91/05436 2~ 38 -Briefly, the basic fused 3-, 4- or 5-fused six-membered ring system is first formed.
Following Scheme II for a general synthesis, using the tetrahydro-phenanthridine fus;ed ring system of Compound 4 (W is benzo) as exemplary, an oxygen-containing substituent R4 having an oxygen atom of that group bonding to the lO-position ring carbon atom is formed as Compound 5. Introduction of that oxygen-containing substituent can be accomplished by oxidation as with m-chloroperbenzoic acid (mCPBA), followed by acylation and reflux to rearrange the formed acylated N-oxide to the lO-position (steps a and b) of Compound

5. The acetyl group is removed to form the alcohol (Compound 6, step c), which is then blocked with a t-butyldimethylsilyl group (SitBuMe2; Compound 7 step d).
An appropriate acetylenic group-containing compound is added adjacent to the nitrogen atom (at the

6-position) as by reaction of an.ethynyl Grignard reagent in the presence of an activating moiety that also functions to block the secondary amine so generated, such as phenyl chloroformate to form an R~
substituent of Compound 8 tstep e).
The epoxide ring is added next between the 6-and lO-positions by oxidation as with (mCPBA) as in Compound 9 (step f). The formed epoxide ring is on the opposite side of the ring plane ~rom the 6-position acetylenic group, and preferably, the epoxide is in an ~-configuration, whereas the acetylenic group is ~.
The oxygen-linked R4 group SitBuMe2 is replaced with a hydrogen (step g) and the alcohol so formed is next converted to a ketone, Compound ll (see also step h, Scheme III). The vinyl.acetylene portion of the enediyne-containing ring is added as in Compound 13, when necessary. This step can be carried out by W092/025z2 PCT/US91/05436 9 ~

reacting (Z) l-chloro-4-trimethylsilyl-but-1-en-3-yne (Compound 12) with the ketone Compound 11 in the presence of butylamine, triphenylpho~phine and palladiumll acetate (step i).
Scheme III illustrates that the entire enediyne carbon skeleton can be bonded to the 6-position in a single step, as is discussed hereinafter. Scheme III also illustrates formation of a benzo ring W by reaction of an aniline compound with ethyl 10 cyclohexanone-2-carboxylateCompound 41 was prepared starting with m-anisidine (Rs = m-methoxy), whereas a meta-pivaloyl ester (R5 = meta-C5 acyloxy) was prepared from a meta-benzyl ether in preparing Compounds 59a and 59b. Where a R8 methyl group is desired, ethyl 3-methylcyclohexanone-2-carboxylate is utilized.
A derivative of compound such as Compounds 2 or 21 having a hydroxyl group ~Rs, or R4 of Scheme III) can be prepared following the synthetic route illustrated in Scheme III (R2=H,H) through step i and then Scheme VII, by utilizing meta-(2-nitrobenzyloxy)aniline for preparation of a tri-cyclic compound analogous to that formed in step a of Scheme III. The meta-2-nitrobenzyloxy (NBnO) group can be removed after step ; of Scheme III to form the meta-hydroxyl-substituted derivative of a compound such as Compounds 2 or 21 by irradiation with ultraviolet light.
A meta-hydroxyl-substituted derivative of Compound 2, Compound 42, has been so prepared via Compound 43, irradiated, and shown to cause cleavage of double stranded DNA at a 2mM concentration.
After removal of the trimethylsilyl group from the otherwise free (unlinked) acetylenic group (Scheme II, step j), the acetylenic group is inserted into the carbonyl group at position-10 by reaction with a base such as lithium diisopropylamide (LDA) to form a fused W092/~2522 PCT/US91/05436 2 ~ 3 ring compound of the invention where R4 is hydroxyl (OH) (step k). The R4 group can later be replaced with a hydrogen or derivatized as discussed :hereinafter.
As noted before, R6 and R7 are prPferably hydrogen (H). However, R6 and R7 along with the intervening vinylidene group can together ~orm an aromatic mono-, di- or tri-cyclic ring system that can be hydrocarbyl or heterocyclic. When such a ring system is present, the ethylenic bond of the enediyne-containing ring is also one of the unsaturated carbon-to-carbon bonds of aromatic ring system, and the entire enediyne carbon skeleton is typically bonded at the 6-position ~see Scheme III, R2) as a single unit, as i5 shown in Scheme XI.

WO ~2/02522 P~r/US91/~5436 2 ~

Scheme 111 ~; a, ~? TMS/;~Mg~r R2 b,c ~;~ R=H ¦ f d R = OAc O
e ~ R OSilBuMe2 R1O~5~p TMS
R2 R4 OSl~BuMe2 R oJ~ R,olS~TMS

OS1~BuMe2 ~ ~ R~

SLJ13$Tllt~llJTE SHEET

W092/02~22 PCT/US91/05436 2~ 93 - 4~ - .

Turning more specifically to Scheme I}I, 2-ethoxycarbonylcyclohexanone is reacted with the aniline ~R4 as shown hereinafter), and the reaction product cyclized by treatment with H2SO4. The cyclized ~aterial is reduced with lithium aluminum hydride and then air oxidized (step a) to form the tricyclic compound. That compound is oxidized, acetylated and rearranged (steps b and c) to form the acetoxy compound (R=OAc) whose acetyl group is removed to form the alcohol (step d, R=OH) that is then reacted with t-butyldimethylsilyl trifluoromethanesulfonate in the presence of 2,6-lutidine in step e (R=OSi~BuMe2).
The product of step e is reacted first with a chloroformate whose R1 group is shown hereinafter, and then with diacetylide or an aromatic diacetylide ring system compound (R2 as shown hereinafter) blocked with a trimethylsilyl group ~TMS) and cont~ining a mono-Grignard reagent in step f to form the partial}y linked macrocyclic ring precursor.

R1 = Me, Ph TMS ~/ R2 =
PhS02 ~
NaphthYI-s02--~/ ~ N
AnthraCyl-so2~J ~ J
HO ~ ~IAe H H
HO'--~ l Ph3P~ ~ OMe Me3N 1 ~, 2~ 3 - 43 ~

The epoxide ring is formed in step g by - reaction with mCPBA. The SitBuMe2 group is removed in step h by reaction with "BuNF, and the resulting alcohol is oxidized with pyridinium chlorochromate in the presence of molecular sieves in step i to form the ketone.
The macrocyclic ring is closed in step j by reaction with lithium diisopropylamide (LDA). The hydroxyl group formed in step j is reacted in step k with an appropriate 2-haloacetic acid derivative (R~ as shown hereinbelow) to form the final product.
R3 = CH2COOH
CH2CONH-peptide CO ( CH2 ) zCOOrIe CH2CONJHPh CH2CONH-Naphthyl C~2CONH-Anthracyl C~2CO2-spacer-Mab spacer-Saccharide R4 = OMe OH
OCOMe OCOtBU
ON8n H
Subscripted R groups are used tn this sche~e to distinguish R groups theroin ~rom the superscripted R
groups defined elsewhere herein.
The "spacer" noted for R3 is a peptide that typically contains zero to about six amino acid residues that links a monoclonal an~ibody, "Mab", to the compound. The R~ "spacer" linked to an oligosaccharide is typically an oxyethanol or oxyacetic acid group used to form the glycosidic bond to the saccharide, or bond to a paratope-containing molecule.

æ ~ ~ ~t~3 9 - 44 -A vicinal diyne aromatic cc~mpo~tnd suitable for introduction at the 6-position (basect on Compound 4) of a 3-, 4- or 5~fused six~mem~ered rinS~ system can be prepared by alkylation of a vicinal clihalide with trimethylsilylacetylene in the presence of diisopropylamine, dicyanophenylpallaclium chloride, triphenylphosphene and cuprous iodide to form the vicinal bis-trimethylsilylacetylene derivative.
Exemplary vicinal dihalo aromatic compounds commercially available from Aldrich Chemical Co. of ~ilwaukee, WI include 1,2-diiodobenzene, 1,2-dibromobenzene and 1,2-dichlorobenzene.
2,3-Dibromonaphthalene, 2,3-dibromoanthracene and 6,7-dichloroquinoxaline have the following Chemical Abstracts Registry Numbers (R.N.) 13214-70-5, 117820-97-0, and 19853-64-6.
After preparation o~ the vicinal bis-trimethylsilylacetylene derivative, one of the trimethylsilyl groups (TMS) is replaced with a hydrogen atom by reaction with silver nitrate and potassium cyanide. The resulting aromatic diacetylide is then reacted with the fused ring system as described previously.
An appropriate vicinal diacetylide can also be prepared via a vicinal, dihydroxymethyl compound. In one exemplary synthesis, 1,~-dimethoxy-6,7-dimethylnaphthalene (R.N. 73661-12-2) is reacted with N-bromosuccinimide (NBS) and azobisisobutyronitrile (AIBN) in a halogenated solvent such as carbon tetrachloride to form the corresponding vicinal dibromomethyl derivative. Reaction of that dibromo compound with hydroxide ion forms the vicinal dihydroxymethyl derivative. Mild oxidation of the dihydroxymethyl compound with pyridinium chlorochromate (PCC) provides the corresponding vicinal dialdehyde.

2 ~

Reaction of the dialdehyde with triphenylphosphine and carbon tetrabromide, ~ollowed by reaction with butyl lithium and then with trimethylsilyl c:hloride proYides the vicinal di-TMS acetylene derivative. Following replacement of one TMS group with hydrogen as discussed before, the aromatic diacetylide is reacted with the fused ring system as was also discussed before.
The above method of synthesis can also be applied to unsubstituted aromatic compounds, such as ~icinal dicarboxylic acids, anhydrides or esters. For example, phthalic acid and naphthalene-2,3-dicarboxylic acid are both available from Aldrich Chemical Co.
Either or both can be used to form the corresponding dimethyl esters by reaction with diazomethane.
Reduction of the diesters to vicinal dihydroxymethyl derivatives can be accomplished by reduction using diisobutylaluminum hydride ~DIBAL). The resulting dihydroxymethyl compounds are therea~ter reacted as described above to form a desired compound.
It is pre~erred that an aromatic diacetylide contain its two vicinal acetylenic groups symmetrically bonded to the ring system so as ~o minimize isomer formation. Thus, 2,3-disubstituted-naphthalene, anthracene or quinoxaline compounds are utilized, or a 6,7-disubstituted-quinoxaline, or the liXe.
An exemplary synthesis for compound$
corresponding to structural Formu}a V is illustrated in Scheme IV, shown below.

WO 92/02522 PCI`/US91/05436 j g L/~ ~ ~

schem@ IV

OH O NHCO2Me OH O NHCO2Me ~CI b. t ~ 11~113 OH O OPMB OH O OPMB

O~O NHCO2Me O~O N~
Iv-viil ~ ix-xiî

28 `~ 29 O~O NO~ Xill-xv OPlv O N
l ~
Y
1~ OTE~5 ~ OTE~S

~' 30P 31 J

PhO2C~
OPIv O N ~ OH O HN
11 1 O~ %XI~LXVI U 1 ~.
i~ ~

W092/02522 ~ ~s~ 3 PCT/US91/05436 Thus, chloronapthazarin, Compound 25, ~Banville et al., can J. Chem., 52:80 (1974); Savard et al., Tetrahedron, 40:3455 ~1984); and Echavarren et al., J. Chem. Res. (3), 364 (1986)] is reacted in step i with diene Compound 26 ~Schmidt et al., SYnthesis, 958 (1982)] in a Die~s-Alder reaction to form the aminoanthraquinone, Compound 27. Reduction of the quinone as with DIBAL in step ii, followed by alkylation with methylene bromide in the presence of cesium fluoride in DMF as s~ep iii provides the 0-blocked Compound 28.
Removal of the urethane group with base in step iv, annellation and subsequent reaction as illustrated in Scheme III provides Compounds 29 (steps v-v ii) and 30 (steps ix and xii). Compound 30 is then reacted with lithium iodide in pyridine, oxidized with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), and react~d with pivaloyl chloride tPiv) to form Compound 31 ~steps xiii-xv) .
Ethylmagnesium bromide is re~cted with Compound 31 in the presence of phenyl chlorofor~ate (step xvi). The resulting compound is reacted with mCPBA to form the epoxide (step xvii). The TBS group is removed with ~Bu4NF to form the alcohol (step xviii) that i5 then oxidized with Jones reagent (step xix) to ~orm the corresponding ketone. That keton~ is reacted with (Z)-1-chloro-4-trimethylsilylbut-1-en-3-yne, palladium, CuI and butylamine to form Co~pound 32 (step xx). Compound 32 is reacted with silver nitrate and potassium cyanide (step xxi), LDA (step x~
thiocarbonyldiimidazole (step xxiii), tri-n-butylstannane and AIBN (step xxiv), and then hydroxide ion (step xxv) to form Compound 33.
For preparation of a compound corresponding to structural Formula IX, l-aminoanthraquinone (Aldrich W092/0~522 PCT/US91/05436 3~ 48 -Chemical co.) can be used as a starting material. Here, the amino group is blocked as with a t-Boc group, the quinone function is reduced as with DI]3AL and the resu}ting phenolic hydroxyl groups are blocked with pivaloyl chloride.
The t-Boc group is then removed as by reaction with tri~luoroacetic acid (TFA) and thle aromatic ring is annelated with ethyl cyclohexanone-2-carboxylate, followed ~y cyclization with sulfuric acid, reduction with lithium aluminum hydride and then air oxidation to reform the quinoid structure as the pivaloyl groups are lost during the prior reactions.
The above reactions form the fused ring system. The enediyne macrocycle port~on is thereafter added as discussed be~ore.
Where a double bond is desired in the otherwise saturated six-membered ring as is shown ln structure Formulas VI, VII, ~III and IX, that ~ ~unctionality can be added as follows. The fused ring syste~ and epoxide are formed, one of the acetylenic linkages is made, the amine blocked and the ketone is formed, all as discussed previously. Exemp}ary compounds of the required structure are Compounds 13 and 3~ shown in Scheme II and IV. The compound is then oxidized to introduce hydroxyl group ~ (position 9 of Compound 13) to the ketone at po~ition 10 of Compound 13. That hydroxyl group is then blocked as with triethylsilyl (TES or Et3Si) chloride and the macrocyclic enediyne ring is closed. T~e hydroxyl group resulting from the ring closure is either re~oved as discussed before or blocked with a group that is no~
removed by removal of the ~ES group.
The TES group is re~oved and ~he resulting hydroxyl group is oxidized to a Xetone as by Swern oxidation. That ketone is then reacted with LDA and W092/02522 PCT/U~91/05436 ~ t7~ ~

methyl chloroformate to form a carboxy enol whose hydroxyl group can be me~hylated with diazomethane.
Subsequent reaction with hydroxide ion and neutralization provides the unsaturated methoxy carboxylic shown in structural Formulas VI, VII, VIII
and IX.
For a oompound of structural Formula VI, where the R8 methyl group is present, that mlethyl group is in the ~-configuration. Here, the ketone formed by the above Swern oxidation is react~d with LDA, phenylselenylbromide and hydrogen pero~.de to form an enone. That enone is reduced with copp2r hydride, which attacks from the ~-face to provide the ~-stereochemistry for the R8 methyl group. The reduced enone is then reacted as above to pro~ide the double bond and R2 and R groups.
A compound o~ the general ~ormula of Compound R6 ~ a) H;
ll ~ b) Msthyt;
25PhO--`1 t ;?.~ c) Ethyl;
fi'q~ s d) Iso-propyl;
2co2R6 e) Allyl;
24a~ f) Propargyl;
g) B~nzyl.

can be prepared by reaction of an appropriate 2-haloacetic acid (Compound 2~a) or 2~haloacetate ester W092/02522 PCT~US91/05436 C~ L~ 9 9 (Compounds 2~b-g) with Compound 2 in the presence of a base such as cesium car~onate (Cs2CO3) and a rrown ether such as lB-crown-6. An exemplary synt:hesis of Compound 2q¢ is provided hereinafter. A compound wherein R4 is a Cl-C6 alkoxy group can be similarly prepared ~rom Compound ~ by alkylation with a Cl C6 halo derivative such as iodo or a triflate derivative in the presence of Cs2CO3 .
Removal of the carbamate group from the nitrogen atom of a compound such as C~mpound 2 to ~orm a free-amine-containing compound such as Compound 40 is achieved by reaction with lithium aluminum hydride reduction. Variants in the alcohol portion of a carbamate derivative of a dynemicin analog can be prepared from a phenoxycarbonyl derivative such as Compound 2 by reaction with the replacing alcQhol in the presence o~ the sodium salt o~ the alcohol as is shown schematically in Figure 15.

V. Resul~s:
The key retrosynthetic step that led to the present synthetic strategy is shown in Scheme I, as noted previously. Scheme II outlined the construction of Compound 2 starting from the quinoline derivative Compound ~ ~(a) Masamune et al., J. O~q. Chem~ 29:681-685 (1964); (b) Curran et al., ~. Ora. Chem~, 49:2063-2065 (1984); (c) Hollingsworth et al., 2~ ora. Chem., 1537-1541 (1948)]. Thus, treatment of Compound 4 with ~-chloroperbenzoic acid (mCPBA) in dichloromethane gave the corresponding N-oxide (step a) which underwent regiospecific rearrangement tB~ekelheide et al., J. Am.
Chem. soc., 76:1286-1291 (1954)] upon heating in acatic anhydride tstep b) to give the acetoxy derivative Compound 5 (62 percent overall yield).

W092/02522 PCTJUS91/0~36 ~ ~ ~ &

Compound 5 was converted to the corresponding silyl ether Compound 7 in 92 percent overall yield by standard methods via hydroxy Compound 6 (steps c and d).
Addition of phenyl chloroformate tcomi~s et al., J O~q.
c~ , 55:292-298 (1990)] to a mixture of Compound ~ and ethynylmagnesium bromide at -7~C led to the formation of Compound 8 in 92 percent yield ~step e).
Treatment of Compound 8 with mCPBA led to epoxide Compound 9 (85 perrent) (step f), which was converted to ketone Compound 11 via alcohol Compound lo by desilylation (step g) followed by oxidation (step h).
Coupling Compound 11 with the vinyl chloride derivative Compol-nd 12 vi~ Pd(OAo2)-CuI catalysis (step i~ followed by AgNO~/KCN treatment (step j) resulted in the formation of the requisite precursor Compound 3 via coupling product Compound 13 (79 percent overall yield).
Finally, treatment of Compound 3 with LDA in toluene at -78C (step k) gave the target~d dynemicin A ~odel Compound ~ (80 percent based on 25 percent recovery of starting material). Compound 2 is also referred to as DY-l in Figures 10 and 11.
Compound 2 crystallized from ether in colorless prisms, mp 232-235C dec. X-ray crystallographic analysis (~his X-ray crystallographic analysis was carried out by Dr. Ra; Chandha, Department of Chemistry, University of California, San Diego) conf~rmed its structure (ORTEP drawing Oakridge Thermal Ellip~oid Plotter) and revealed some inter~sting structural features.
The acetylenic moieties are bent from linearity with the following angles: C14, 160.4; C15, 170.8; C18, 171.6 and Cl9, 162Ø The distance between carbons C14 and Cl9 ~cd distaAce) ~Nicolaou et al., J. Am. Chem. Soc., 110:4866-4868 (198~)~ was found to be 3.63A, a value that agrees well with the WO 92/02522 pcr/us91/os436 ~ - 52 -calculated one for the MMX minimized structure of Compound 2 (3.63A) and that of the experimentally derived ~(a) Konishi et al, J. Al;l. Chem. Soc~ 37~S-3716 (1990): (b) Konishi et al~, J. Anl;ibiot., 42:1449-1452 (1989)~ distance in dynemicin A (:3~54A). The calculated distance for these ace~ylen.ic carbons was found to be 3.40A. See: Se~melhack et al~, Tetrahed~Q~
Lett., 31:1521-1522 (1990). ~The ~87 force ~ield in computer programs ~MX and PC~ODE~ from Serena Software, P.O. Box 3076, Bloominqton, IN 47402-3076 was used.]
Scheme V outlines a cascade of novel transformations of model system Compound 2.

2 1~ o ~ ~ g Schem@ V

PhOJ~ _ PhOJ~

2:R=H t4:X=OH;R=H
2a~ Ac 14a :X=OH;R_Ac 14b:X=Cl;R_H

Bergman C:ycllzatlon PhJf~3 PhO

O Plnacol ~C
R~arrangement 17 . [t~:X=OH;R-H]
15a: X =OH; R - Ao 15b :X=CI;Fl-H

W O 92/02522 PC~r/US91/05436 ' Thus, upon treatment with ~-toluenesulfonic acid (TsOH H2O) in benzene-1,4-cyclohexadiene (3:1, 0.05 M~ at 25C for 24 hours, Compound 2 was converted to Compound 17 in 82 percent yield, presumably via intermediates 1~ and 15. This transfo~mation ~2~17) was also carried out using 1.0 equivalent of TsOH-H20 and 50 equivalents of Et3SiH in benzene at 25C (24 hoursj 85 percent).
For a study using E~SiH and a number of other hydrogen donors to trap C-centered radicals, see:
Newcomb et al., J. Am. Chem. Soc., 108, 4132-4134 (1986). Compound 17 derived from this reaction with Et3SiH was contaminated with about 5 percent of an, as yet, unidentified prod~ct (dstected by lH NMR
spectroscopy).
Thus, protonation of the epoxide group in Compound 2 initiates formation of triol Compound 14 which undergoes spontaneous Bergman cyclization E ~a) Bergman, Ac~. Chem. Res., 6:25-31 (1973); Jones et al., J. Am. Chem. Soc., 94:660-661 (1972); Lockhart et al., J. Am. Chem. Soc., 103:4091-4096; (b) Darby et al., J. Chem. Soc. Chem Commun., }516-1517 (1971); (c) Wong et al., Tetrahedron Lett., 21:217-220 (1980)] to for~ a benzenoid diradical which is, in turn, rapidly trapped by the hydrogen donor present to ~urnish cyclized product Compounds ~5, 15~, or lS~. Under the reaction conditions, triol Compound ~S apparently undergoe-q a pinacol-type rearrangement leading to the observed ~inal product Compound 17. The structure of Compound 17 was supported by its spectroscopic data and was confirmed by X-ray crystallographic analysis.
Furthermore, it was found that trimethylsilyl trifluoromethylsulfonate (TMSOTf) in the presence of Et~SiH induces the same transformation (2~17), (Scheme V) at -78C in less than 5 minutes (78 percent yie].d), W092/02522 PCT/US91tO5436 suggesting a very low energy of activation for the cyclization process. The rather dramatic shortening of the cd distance in going ~rom epoxide Compound 2 (cd=
3.63A, X-ray and MMX) to triol Compound l~a (od= 3.19A, MMX) is noteworthy. For comparison with other similar enediyne cyclizations and calculations, see~
Nicolaou et al, ~. Am. Chem. Soc., 1a~0:4866-4868 (1988);
(b) Nicolaou et al., J. Am. C~Lem SocO, 110:7247-7248 (1988); (c) Hazeltine et al., J. Am. Chem. Soc., 111:7638-7640 (1989): (d) for similar stabilization of enediynes via co~alt complexation,-see: Magnus et al., J. Am. Chem. Soc., 110:1626-1628 (1988) and Magnus et al., J. Am._Chem. Soc., llQ:6921-6923 (1988); (e) Semmelhack et al., Tetrahedron Lett., :1521-1522 (1990); (f) Snyder et al J. Am. Chem. Soc., 111:7630-7632 ~1989); ~g) Magnus et al., Tetrahedron Lett., 30:1905-1906 ~1989).
In an attempt to prevent the pinacol rearrangement of triol Compound 15, the acetate derivative Compound 2a wa~ prepared from Compound 2 ~acetic anhydride, 4-dimethylaminopyridine ~DMAP), 84 per~ent], and was subjected to the epoxide opening and cyclization reaction conditions as described above.
Indeed, the acetate diol Compound 15~ was obtained (84 percent yield) as the final product of this cascade starting with Compound 2a and using TsOH-H20 as the initiator ~presumably via intermediate Compound 14~).
The use of anhydrous ~Cl in CH2Cl2 in the presence of Et3SiH ~Scheme V hereinbefore) also resulted in triggering of the cycliza~ion cascade leadi-~ from Compound 2 to Compound 15b ~85 percent yield) presumably via the intermediacy of Compound l~b (cd= 3.145 A, MMX).
The same conversion (2~15b) was also e~fected by the use of 3.0 equivalents of MgCl2 and 50 equivalents of Et3SiH
in CH2Cl2 at 25C (12 hours, 87 percent) or 1.2 s~,p~

equivalents of TiC14 and 50 equivalents of Et3SiH in CH2Cl2 at -7RC (0.5 hour, 60 percent).
Thus, on~y Compound 15 underwent-the further pinacol-type rearrangement to form Compound 17.
These cyclizations are analogous to those observed for dynemicin A~ ~ta) Konishi et al., J.
Am.Chem. Soc., 112:3715-3716 (1990); (b) Konishi et al., J. Antibiot., 42:1~49-1452 (1989); (c) Sugiura et al., Proc. Natl. Acad. Sci. USA, 87:3831-3835 (1990)].
An alternate mode of triggering the cyclization of ~ompound 2 bàsed on cobalt complexation of the acetylenes was devised. This triggering cyclization is illustrated in Sc~eme VI, below. This pathway was designed so as to prevent the acetylenes from spontaneously cyclizing upon epoxide opening and thus allow the isolation of the postulated intermediate c is-diol .

WO 92/02522 PCT'/US91/05436 Scheme Vl (O1:~)3Co~ ~
~ ~o~)3cl~j ~ o2(C)d p (och PhO~

[ ~ ~ C

Thus, reaction of Compound 2 with Co2(C0)8 (2.2 equivalents) resulted in the formation of the dicobalt complex Compound 18 (step a) :in 96 percent yield. Use of one equivalent of Co2(CO)8 resulted in the formation of a monocobalt complex :in addition to dicobalt derivative Co~pound 18 and starting material Compound 2. [For similar stabilization of enediynes via cobalt complexation, see: ~agnus et al., J. Am. Chem.
Soc., 110:162~-1628 (1g88); and Magnux et al., J. Am.
Chem. So~., 110:6921-6923 (1988)].
Treatment-of Compound 18 with trifluoroacetic acid in CH2CI2 tzero degrees C) followed by aqueous workup led to the formation of the stable cis opening product Compound 19 (step b: 92 percent yield). Upon exposure of Compound 19 to ferric nitrate, or trimethylamine N-oxide in C~2Cl2 in the presence, or absence, o~ Et~SiH at 25C, the cyclized product Compound 17 was obtained in 85-92 percent yield via liberation of the acetylenic groups to afford Compound 1~ followed by spontaneous and sequential generation of Compounds 15 and 17 as shown in Schemes V and VI. The same study ~reac~ion with Fe(N03)3] carried out in CD2C12 resulted in the incorpora~ion of two deuterium atoms in Compound 17 con~irming methylene chloride as an effective hydrogen atom donor in these aromatization studies .
To obtain a closer model to dynemicin A, the tertiary hydroxy group in Compound 2 was removéd to form Compound Zl as shown in Scheme VII below.

WO 92t0252~ P'CI`/US91/05436 $ ~

Sch~me Vll a C2 R=OH I--~20: R~~ N~N Bergman b S Cycllzatlon ~21 :R-H

[
23: OH 22: Y=OH
23a: Cl 22a: X-CI

W092/02~22 PCT/US91/05436 Thus, reaction of Compound 2 with thiocarbonyldiimidazole in the presencle of DMAP resulted in the formation of Compound 20 in 9S lpercent yield (step a). Compound 20, upon treatment with "Bu~SnH in the presence of a catalytic amount of ~IBN (toluene, 75C), led to the desired Compound 21 (step b; 72 percent yield). This model system Compound 21 also underwent smooth cyclization to polycycles Compounds 23 t86 percent) and 23n (82 percent) upon suitable triggering, (as summarized in Scheme VII).
An ORTEP drawing of dynemicin A model Compound 21 (mp 2Sl.2 52C dec, from ether - petroleum ether) as determined by X-ray crystallographic analysis was prepared. The following angles revealed considerable deviation of the acetylenic groupings from their preferred linear arrangement: C17-C18-C19 - 170.2:
C9-C19-C18 - 16~.0; C14-C15-C16 - 170.1; and C13-C14-C15 = 163.7. The distance between carbons C14 and C19 (cd distance) was found to be 3.59A, which agrees well with the values derived for the MMX
minimized structure of Compound 21 (3.59A) and from the X-ray crystallographic analysis of dynemicin A (3. 54A) .
~Konishi et al., J. Am. Chem. Soc., 112:3715-3716 (1990)]. Again, the considerable shortening of the cd distance i9 noted in going from 9 to the cis dlols 10 (cd- 3.21A, MMX) and 10a (cd- 3. lgA, MMX) .
In order to allow for the generation of the parent enediyne system Compound ~0, Compound ~S was designed and synthesized from Compound 21 as shown in Scheme VIII, below.

.
, WO 92/V2~22 ~ ~ ~ 3 1r ~ 9 PCI /US91/05436 Scheme Vlll PhO

21 :R=H 4~:R=H
46:R_OMe 47:Fl=OMe ~c ll PhOH
Rli) PhSH
lii) TSoH-H2o ~ R
49:X_OPh;R=H 4û R-H
~J : X = SPh; R = H 4~ ~ R _ ONle 51 :X_OH;R=OMe Thus, Compounds 21 and 46 were separately reacted with ten equivalents of 2-phenylthioethanol [PhS(CH2~20H~ in acetonitrile for 12 hours in the presence of three equivalents of Cs2CO~; and O.5 5 equiva}ents 18-crown-6 in step a to form the corresponding phenylthioethoxy Compounds 21~ and ~6a, respectively. The products of that reaction were then individually reacted with 2.5 equivalents of mCBPA in CH2Clz at 0-25C to provide Compounds 45 (85 percent -overall) and 47 (82 percent overall) as step b. When step b is carried out with one equivalent of mCBPA, the corresponding sulfoxides, Compounds 21b and 46b are prepared.
To prepare Compound ~0 that was too labile for isolation, Compound 4S was reacted with an excess of Cs2C03 and 0.5 equivalents of 18-crown-6 in a dioxane:l,4-cyclohexadiene (4:1) solution for one hour at 25C as step c. Compound 47 was reacted with 1.2 equivalents of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in benzene at 5C for one hour to provide a 97 percent yield o~ Compound ~8 as step c.
Freshly prepared Compound ~0 was reacted with either phenol (i, PhOH) or thiophenol (ii, PhSH) using two equivalents of either nucleophile at 25C for two hours to provide Compound~ 49 (25 percent yield) or 50 (33 percent yield, respectively, as step d. The reactivity of Compound ~0 and its ability to cleave DNA
add credibility to the notion of a pathway including epoxide ring opening and the intermediacy of an iminoquinone methide species in the mode of action of dynem~cin A, at least as a partial m chanism.
Interestingly, the methoxy derivative, Compound 48 proved to be quite stable under basic or neutral conditions. Treatment of Compound ~8 with 0.5 equivalents of ~-toluenesulfonic acid-H20 (step cl iii, .

TSOH) in dioxane:water:l,4-cyclohexadiene (4:1:1) at 60C for ten minutes provided Compound 51 (20 percent yield).
An X-ray crystallographic analysis of Compound ~8 confirmed its molecular structure and revealed a number of interesting parameters including a cd distance of 3.63A [Calcd. cd=3.6}A, NNX~ and the non-linearity of the acetylenic groupings ~angles at acetylenic carbons:
C-14, 163.3; C-15, 173.0: C-18, 169.3: C-19, 160.5~].
Enediyne model system Compo~md 43 (Scheme IX) was designed for its potential to generate species ~2 under photolytic, neutral conditions. Following the strategy developed earlier for the synthesis of Compound 21, Compound 43 was constructed in good overall yield.
Reactions of Compound 43 are shown in Scheme IX.

WO 92/02522 . PCI/US91/05~36 S~8 ~ 64 -Scheme IX

I) EtOH
b Il) EtSH
111) n-PrNH2 H J[~

44a: R = PhO; Nu = EtO + 54: R ~ PhO
44b: R = PhO; Nu O EtS
Mc: R Nu - NHn~Pr ' : .: , , 9 ~

Irradiation in step a of Co~pound 43 in THF-H20 (10:1) for 40 minutes [Hanover mercury lamp, pyrex filter] at ice-cooling temperature resulted in clean conversion to Compound 42 as observed by TLC and lH NMR
spectroscopy (THF-d8: D2O 10:1, 300 MH;~). Attemp~ed isolation of Compound 42 1Pd to decomposition, whereas treatment of crude Compound ~2 in a p~I 8.0 buffer-THF
solution containing either ethanol (EtOH), mercaptoenthanol (EtSH) or n-propylamine (nPrN~2) as nucleophiles (Nu) at 25C under argon atmosphere for 1.5 hours provided a mixture of Compounds 4~ and 54 (31 percent yield), ~b (34 percent yield), or ~o (46 percent yield), respectively, as step b.
Noteworthy was the isolation of Compound 54 (Scheme IX) in about 5-10 percent yield ~rom the reaction of 42 with ethanol, in air, along with Com~und 44a (33 percent yield), as a s~nsitive but stable molecule under neutral conditions. The iso}ation of Compound 54 provides strong 2vidence for the intermediacy of quinone methide Compound 52, [a) Dyall et al., J. Am. Chem. Soc., 94:2196 (1972); b) Zanarotti, Tetrahedron Lett., 23:3815 (1982): c) Zanarotti, J._Orq.
Chem., 50:941 tl985); d) Angle et al., J. Am. Chem.
Soc., 111:1136 (1989); e) Angle et al., ~etrahed~on Lett., 301193 (1~89); f) Crescenzi et a}., ibid 31:6095 ~1990); g) Barton et al., ibid 31:8043 (1990); h) Angle et al., ibid 11~:~524 ~19gO)], trapping of which with molecular oxygen would provide Compound 5~ as is shown in Scheme X, below.

WO 92t02522 PCTIUS91/05436 s~ 3g~ ~ 6S -Scheme X

11~\ p~oc(o3~
PhOC(~ THF buffer (1~

HO 42~ H 56- R1 Me t~2 ~1 [Ph~

54: R~ = H; R2 = PhOC(O) 1 58: R~ - lUle; R~ = H 53: Rl = H
62: R1 - Me; R2 ~ PhOC(O) 57: R~, Me . ., . , :.
' 2 ~

Remarkably, treatment o~ Compound 55 under similar conditions (THF-pH 9.O buffer in air) resulted in the isolation of Compounds 58 and 62 in 32 percent and 25 percent yields, respectively, as stable molecules. Quinone methide intermediates have been . implicated in the mode of action o~ anthracycline antibiotics tfor recent postulated quinone ~ethide intermediates in the area of anthracycline antibiotics, see: a) Boldt et al., ~_5a~ hY~r 52:2146 (1987);
b) Gaudiano et al., J. orq. Chem., 54:5090 (1989);
c) Egholm et al., J. Am Chem. Soc., 111:8291 (1989);
d) Ramakrishnan et al., J. Med Chem., 29:1215 (1986);
e) Boldt et al., J. Am. Chem. Soc., 1t1:2283 tl989):
f) Gaudiano et al., J. Am. Chem. Soc., 11~:9423 (1990);
g) Karabelas et al., J. Am. Chem, Soc., ~ 5372 (1990);
h~ Gaudiano et al., J. Am. Chem. Soc~, 112:6704 ~1990)~
and dynemicin A.
The pivaloate ester Compounds 59a and ~9b wer~
also synthesized according to the previously discussed general strategy for ~heir potential to liberate phenolic species of type of Compound 42 (Scheme IX), thus presenting yet another triggering mechanism to initiate the dynemicin A-type cascade. Indeed, upon exposure to four equivalents of LioH in ethanol:water (3:1) at 25C for 4-~ hours, Compounds 59~ and 5gb were smoothly converted to products 60~ (56 percent) and 60b (42 percent~, respectivelyt presumably via a sequence involYing concomitant carbamate exchange ~EtO- for PhO-].

6~ L~ 6~-t-BuJ~O~ EHoO~oEtl 59a- R - OH 60a~ _ OH

PhO~ ~hO~

M~O 41 OH MeO OH

The methoxy Compound ~l was also synthesized and exhibited reasonable stability under neutral and basic conditions. As expected, however, this compound cyclized rapidly under acidic conditions. For example, upon treatment with TsOH-H2O in banzene:l,4-cyclohexadiene (1:1) at 25C for one hour, Compound 4~
afforded the aromatized product Compound 61 (32 percent yield).

W092/02522 PCT/US9l/05436 2~

Treatment of Compound 41 as per steps a and b of Scheme V yields the corresponding methoxyphenyl derivative Compound 41~ having a hydrogen in place of the hydroxyl of Compound ~1. Treat~aent of Compound ~la with 2-phenylthioethanol, 2-a-naphthylthioethanol or 2-~-naphthylthioethanol under basic conditions as discussed in regard to Scheme VIII, followed by oxidation led to Compounds 41b, ~1~ and 41~, respectively.
The synthesis of Compounds 70 and 80 proceeded as summarized in Scheme XI, shown below.

g~CJ~

Scheme Xl ,~ H~ ,)n ,~
~J-- .t d ~ 74: R = OH pN ~ 77: n O
b ~ 73. R _ H r 75: R a --o,~N~ ~ 4- 70: n _ 2 1~ 76: R - H 8 1~ .
H ~ I
PhOJ~ ~ TtO~

11 ~l 71 81 ~hOJ~ ~hOJ~ h, O -- R -- -- I

; 82~ ~ OT~ Sl I; a6- R 3 --O N~ ~ ~80. n ~ 2 M~ --u l_ 84: R - = H ~ _ 87: R ~ H 8 2 ~u ~

Thus, coupling of the readily available Compounds 1~ [Nico}aou et al., J. Am. Chem. soc., 112:7416 (1990); Nicolaou et al., J. ~a~_~3~ 9~, 113:~106 (1991)] and 71 using palladium (O) copper(l) catalysis afforded produc~ Compound 72 (55 percent yield) as step a. Desilylation of Compound 72 with lithium hydroxide in step b followed by base-induced (lithium diisopropylamide; LDA) ring closure led to Compound 74 via 73 (7S percent overall yield) in step c.
Conversion of Compound 7~ to the thionoimidazolide Compound 75 t84 percent based on 67 percent conversion of staring material) as discussed elsewhere herein, followed by deoxygenation with tri-n-butylstannane (nBu3SnH) in step e resulted in the formation o~ 76 t94 percent). Exchange of the phenoxy (PhO) group of Compound 76 with 2-phenylthioethoxy (PhSCH2CH20) took place smoothly under basic conditions (two equivalents of PhSCH~C~zONa, T~F) leading to Compound 77 (92 percent yield) in step f, from which the sulfone Compound 70 was generated by oxidation using 2.5 equivalents mCPBA (81 percent yield) in step g.
Similar chemistry employing the naphthalene ditrifla~e Compound 81 led from Compound 11 to arenediyne Compounds 85-~0 via intermediate Compounds 82-~4 in comp~rable yields as depicted in Scheme XI.
Step h utilized acetonitrile as a solvent and a one hour reaction time as compared with step a o~ the scheme that used benzene as solvent and 3.5 hours of reaction to obtain yields of 55 and 56 percent, respectively. Step i utilized five eguivalents of trimethylsilylacetylene, 0.05 equivalents of Pd(PPh3) 4, O.2 eguivalents of CuI
and two equivalents o~ triethylaminP in acatonitrile (as were present in steps a and b) with a reaction ti~e of 20 hours at 25C to provide Compound 82 in 76 percent yield. Steps c-g of both reactions were ident cal.

Reaction of Compounds 77 or 88 with one equivalent each of mCPBA leads to preparation of the corresponding sulfoxides, Compounds 77~ and ~8a.
The free amine Compounds, 76~ and 87a, shown below, corresponding ~o Compounds 76 and 87 were prepared from their corresponding phenylsulfonylethyl carbamates 70 and 80 under basic conditions. Those compounds were both stable at 25c, whereas Compound 40 decomposed at that temperature.
10. Treatment of Compounds 76~ and 87a with silica gel in benzene initiated epoxide opening to form the corresponding diols 76~ and 87b, respectively. Diol Compound 76b cyclized spontaneously at 25C in the presence of 1,4-cyclohexadiene to ~orm the cycloaromatized diol, Compound 76c. In contrast to Compound 76b, dlol Compound 87b was stable in the presence of 1,4-cyclohexadiene, but cycloaromatized at elevated temperature (65C, two hours) to form Compound 87O.

H, 76a H
87a The cycloaromatization of enediyne Compounds 76, 70, 87 and 80 was then studied in order to determine the precise structural an~ spectroscopic changes taking W092/02522 2 ~ PCT/US91/05436 place. Thus, whereas cycloaromatization of CQmPOUnd 76 (about 0.02M solution) under acid conditions [1.2 equivalents of TsOH.~20, benzene-cyc}ohexdiene (4:1), 25C, four hours] produced smoo~hly the corresponding naphthalene derivative (78 percent~ no dramatic changes in the W and fluorescent spectra were observed for the starting arenediyne a.~d cycloaromatized product.
In contrast, however, similar acid-induced Bergman cycloaromatization of the naphthalene enediyne Compound 87 furnished the epoxy-containing anthracene derivative Compound 89 (49 percent) that exhibited, as expected, strong and characteristic W and fluorescence pro~iles that were distinct from those of the starting arenediyne Compound 87 ~ W (EtOH), 87 A~X (log ~) 304 ~3.47, 294 (4.01), 284 (4.26~, 267-240 ~4.53-4.55), 214 ~4.50) nm; 89 A~X (log ~) 390 (3.7~), 369 (3.78), 351 (3.66), 333 (3.45), 318 (3.20), 267-244 (4.43-4.46)r 21 (4.43) nm. Fluorescence (EtOH, 1 ~M, excitation at 260 nm, Compound 87:A,,,,,X 435, 412, 393, 374, 357 nm; 89:A,,,,~X
520, 466, 442, 413, 392 nm].
Attempted cycloaromatization of Compounds 70 and 80 under a variety of basic conditions led to decomposition, presumably via the in situ generated free amines and diradical species, whereas acid treatment resulted in the formation of epoxy-opened (dihydroxy) Compounds 78 and 89a albe.t in low yields (20 percent and 15 percent, respectively). Interestingly, both Compounds 70 and 80 exhibited DNA cleaving properties under basic conditions (supercoiled ~174 DNA, pH 9.0) and potent anticancer activity against a variety of cell lines as discussed hereinafter.

Saccharide-Containinq Chimeras Chimeric compounds that include both a fused ring enediyne as the aglycone and a beforP-discussed W092/02522 PCT/US91/0~36 9~ - 74 -mono- or oligosaccharide as the oligosaccharide portion is also contemplated, as noted earlier. The previously depicted saccharides are related to the calichea~icin oligosaccharide.
The before-depicted saccharides correspond to the calicheamicin oligosaccharide (Structures F and G), the oxime precursor thereto (Structures D and ~l, and fragments th~reof (Structures ~-C). More specifically, the disaccharide Structure A corresponds to th~
}o calicheamicin A and E rings with the hydroxylamine link - to a B ring analog. The monosaccharide Structure B
corresponds to the A rinq alone with the hydroxylamine-linked B ring analog. The trisaccharide thiobenæoate Structure C corresponds to rings A, E and B, and a C
ring analog. The 5-ring Structure D corresponds to the FMOC-blocked oxime precursor to the complete calicheamic~n oligosaccharide, whereas 5-ring Structure is the FMOC-debloc~ed version thereof. Structures F
and G are the complete calicheamicin oligosaccharides that are epimeric about the 4-position of the A ring hydroxylamine linkage, with Structure F having the native calicheamicin oligosaccharide stereochemistry.
These saccharides are discussed in more detail hereinafter.
Inasmuch as the prev ously depicted saccharides of the calicheamicin oligosaccharide are derivatives of known compounds, as are their suitably protected precursors, their complete synt~eses need not be discussed in complete detail herein. Those syntheses are described in Nicolaou e~ al., J._~m. Chem. Soc., 112:4085-4086 (1990); Nicolaou et al., Ibid., ~1~:8193-819S (1990); Nicolaou et al., J. Chem. Soc. Chem C~mmun~, 1275-1277 (1990) as well as in U.S. Patent Application Serial No. 07/520,245 filed ~ay 7, 1990 and Serial No. 07/695,251 filed May 3, 1991, all of whose W092/02522 PCT/US9~/05436 disclosures are incorporated by reference herein.
Nevertheless, the saccharides used h~ereln differ somewhat from the reported saccharides, so their syntheses will be discussed, at least in pertinent part herein below.
The disaccharide-linked hydroxylamine compound is prepared in a manner analogous to that of Compound 12 of Nicolaou et al., J. ~m. Chem. Soc., ~ 8193-8195 (1990), except that an o-nitrobenzyl (shown as NBnO or ONBn in the schemes) glycoside is utilized instead of the methyl glycoside precursor, Compound 9 of that paper. The synthesis for the disaccharide is illustrated in Schemes XII and XIII, and is discussed below.
Scheme Xll Me~O~X c r r n 0 Me ~ O ~ ONBn RO ~ "~O RO ~ ~OR O

~ 1 ~ - Ac;X_OAc d ~ .g_H 95 b G 2:R=Ac;X=Br 2~

Thus, D-fucose Compound 90 was peracetylated in step a to form tetraacetate Compound 91 which was converted to the anomeric bromide Compound 92 in step b, and glycosylated with o-nitrobenzyl alcohol to afford Compound 93 in step c ~63 percent overall yield).
Deacetylation of Compound 93 in step d led to Compound 94j which reacted selectively with carbonyl diimidazole in step e to afford the requisite ring A intermediate 95 in 86 percent overall yield.

WO 92/02522 PCI/US91/05~36 2~'~`3 ~99 - 7~ - .

Scheme Xlll 02N~

OMe I oc ¦ QMe FblOC
- a Me~O.~O ~
F~OJ R o~J~oloJ M6 b~ 97 R, - R2 - O~O

OMe FMOC I O2N~
Me~O~X ~ ¦ .QMe Frloc PhCH20~N~J4~"oloJ M~ Me~orO ~
OSlEt3 ~ X ~OJ~OJ
~ OR
102:X_OH ~ 102a~X-F I d~99:X-O;R=H
nC103: X = OCCI3 1 e 100: X = N-O-CH2Ph; R, H
h~¦ I C~101: X, N~CH2Ph; R = SlEt3 OMe R2 PhCH2O-N~4O~¢f Mo~3 OR~ OMe 104: R~ = SiEt3; R2 = FMOC Me~O~,~b ~ H~
105: Rl = H; R2 = FMOC~ J". ¦ I Ma J C~106: R1 = R2 ~ H PhCH2O N~ r ~o~o~
OH

;UBSTITUTE 5HEET

2~ J _~; 3 Turning to Scheme XIII, int~rmediate Compound 95 was then coupled [Mukaiyama et al.l Chem. Lett., 431 (1981); Nicolaou et al., J Am. Chem._Soc., 106:4159 (1984)] to glycosyl fluoride Compound 96 ~Compound 8 of the above paper: Me=methyl) with AgClOb-SnCl2 catalyst in step a, leading stereoselectively to disac~haride Compound 97 as the major anomer (80 percent yield, about 5:1 ratio of anomers). Chromatographic purification of Compound 97 with removal of the carbonate protecting group (NaH-HOCH2CH20H, 9o percent) in step b to for~
Compound 98, and treatment in step c with "Bu2SnO-Br2 ~David et al., J. Chem. Soc. Perkin Trans 1. lS68 (1979)~ led to hydroxyketone Compound 99 (65 percent yield plus 17 percent Compound 98) via intermediate Compound 98.
Oxime formation in step d with O-benzyl hydroxylamine under acid conditions led to Compound 100 (90 percent, single geometrical isomer of unassigned stereochemistry; Ph-phenyl) which was silylated in step e under standard conditions to furnish Compound 101 (90 percent). Photolytic cleavage ~Zenhavi et al., J. Or~.
Chem., 37:2281 (1972); Zenhavi et al., ibid, 37:2285 (1972~; Ohtsuka et al., J. Am. Chem Soc., 1~:8210 (197~,; Pillai, Synthesis, 1 (1980)] of the Q-nitrobenzyl group from Compound 101 (THF-H2Q, 15 minutes) produced lactol Compound ~02 in 95 percent yield in step ~. Treatment o~ Compound 102 with NaH-Cl3CCsN [Grandler et al., Car~ohvdr. F~ 5:203 (1985); Schmidt, Anaew Chem. Int. Ed.,_Enql., 2~:212 (1986)] in CH2C12 for two hours at 25C in step g resulted in the formation of the ~-trichloroacetimidate Compound 103 in 98 percent yield. Reaction of benzyl alcohol (2.0 equivalents) with trichloroacetimidate Compound ~03 under the Schmidt conditions ~Grandler et al., Car~oh~dr Res., 135:203 (1985); Schmidt, An~ew W092/U2522 PCT/U~91/~5436 ~ 8'~ 78 -Chem. Int. Ed., Enal,., 25:2i2 (1986)] ~BF3-Et20, CH2Cl2, -60 - -30C] resulted in stereoselective formation of the ~-glycoside Compound 104 (79 percent yield) together with its anomer (16 pe~rcent, separated chromatographically) ~1H NMR, 500~Xz, C6D6, 104: Jt2=6.5 Hz, epi-Compound 104: J-2= 2.4 Hz].
On the other hand, treatment of lactol Compound 102 with DAST in step g' led to the glycosyl fluoride Compound 102a in 90 percent yield (about 1:~
anomeric mixture). Reaction of Compound 102a with benzyl alcohol in step h' in the presence of silver silicate [Paulsen et al., C,hem. Ber., 114:3102 (1981)l - SnC12 resulted in the formation of the ~-glycoside Compound 104 and its anomer in 85 percent (about 1:1 anomeric mixture).
Generation of intermediate Compound 106 via Compound 105 proceeded smoothly under standard deprotection conditions in steps i and j. Finally, exposure of Compound 10~ to Ph2SiH2 in the presence of Ti(OiPr)4 in step k resulted in the formation of the desired target Compound 107 as the only detectable product (92 percent yield~. Interesting}y, reduction of Compound 106 wi~h NaCNBH3-H~ led predominantly to the 4-epimer of Compound 107 (90 percent yield). The, ~5 stereochemical assignments of Compound 107 and epi-107 at C-4 were based on ~H NMR coupling constants [1H NMR, 500MHz~ C6D6, 107: J3,4=9.5, J4,5q9.5 Hz: epi-107 J3~-l.g Hz, J~5=1.5 Hz].
The hydroxylamine linked A ring derivative (Structure B) can be prepared starting with Compound 9 of Nicolaou et al., J. Am. Chem. Soc., ~ 8193-8195 (1990). There, the 2-hydroxyl is blocked with a t-butyldimethylsilyl (tBuMe2Si) group as before, and the carbonate group removed by reaction of sodium hydride in 3S ethylene glycol-THF at room temperature. The keto group W092/02522 PCT/VS91~0~36 3 `~ ~ ~

- 7~ -can be prepared by oxidation with dibutylstannic oxide (nBu2SnO) in methanol at 65. The 3-position hydroxyl is similarly blocked with a tBuMe2Si group, and the oxime formed as above.
The trisaccharide plus C r~ng analog (Structure C) can be readily prepared from Compound 19 of Nicolaou et al., J. Am. Chem. Soc., ~ 81g3-8195 (1990). ~hus, that Compound S~ is reacted with benzoyl chloride in the presence of triethylamine and a catalytic amount of DMAP in methylene chloride to provide the blocked oxime-containing trisaccharide.
The use of Compounds 2 and 103 in preparing an exemplary chimera is illustrated in Scheme XIV, below, and discussed hereinafter.

æ ~

~\ Scheme XIY

PhO N~ ~

b--~ ~4c: R - CH2COOEt 110: R CH2COS
111: R = CH2C:H2OH

PhOJ~ OPh ~ OM~ R2 M~l~~X~ Me~O~O~
PhCH2O N~ 440 0 PhCH20'NO~4'0 0 OR, ORl e 112a R, = SIE~3, R2 - FMOC ~? ~112b C;~113a R~ = H, R2 = FMOC----113b f ~14a R~ R2 s H 1 ~1~4b PhO~ OFh ~ OM~ OMc H
Me~,oro~,, Me~OrO~

PhCH2O-N""~"'O O ~ PhCH o N"`~`~y~4"o 0 115b 115a .: .. :.-.

, W092/02522 PCT/~S91/05436 ~ P~d ~ g ,,1~

Thus, as shown in Scheme XIV, coupling of Compound 2 with ethyl bromoacetate under basic conditions led to derivative Compound 2-~c (60 percent yield), which was converted to prima~y alcohol Compound 111 (80 percent ov rall yield~ by: (i) ester hydrolysis;
(ii) 2-pyridyl thiolester formation (collectively step b) and (iii) reduction in step c. Coupling of Compound 111 (1.2 equivalent) with trichloracetimidate Compound 103 under the influence of BF3 Et20 as described before 10 led to the formation of two major products (70 percent, about 1:1 ratio) and two minor products (14 percent, about 1:1 ratio), which were chromatographically separated.
The major isomers were shown to be the 15 diastereomeric ~-glycoside Compounds 1~2a (Rf-0.12, silica, 20 percent ethyl acetate in petroleum ether) and 1~2b ~R~-0.10, silica, 20 percent ethyl acetate in petroleum ether) [~ NMR, 500MHz, C~D6, 112a: J12=6.5 Hz;
112b; J12=6.5 Hz], whereas the minor isomers were shown 20 to be the ~-anomers of Compounds 112a and 112b at C-i ~1H NMR, 500MHz, C6D6, epi-112a, J12=2.4 Hz: epi-112b, J12=2.4 Hz]. Sequential deprotection of Compounds 112a and 1~2b as described above for Compound 10~ led to oxime Compounds 114n and ll~b via intermediate Compounds 25 113h and 113b, respectively.
Finally, reduction of Compounds 11~ and 1~4b under the Ph2SiH2-Ti~OIPr)4 conditions led exclusively to the targeted Compounds llSa and 115b respectively (90 percent yield). The C-4 stereochemistry of Compounds 30 ~15a and 115b was again based on the coupling constants J43=9.5 and J45=9.5 Hz for the new}y installed H-4.
Structures 112a-115a and ~12b-115b are interchangeable, since the absolute stereoche~istry of the aglycons has not been determined. Physical data for Compounds 115a 35 and ll5b are provided hereinafter.

W092/0252~ PCT/US91/05436 ~ ~ ~ " ~ ~ 3 Still further chimeras have been prepared that contain a fused-ring enediyne glycosidically-linked to the complete calicheamicin oligosaccharide or an analog thereof. Exemplary synthetic steps are outlined in Schemes XV and XVI, below.

WO 92/02522 PCl/US91/05436 2 ~

Scheme XV
OMe FMOC
Me~o~X ~N~
Me~O~N~J"",OlOJ
~~S** OSI~t3 Me"" O--OJ~OM OSlEt3 OMe 12t: X_ CH2 NO2 Et3SlO~"~OSlEt3 a [~ .
- OMe . 122: X = OH

~ OMe FMOC

Me o ~J `N~o~N~Me ~~S~ OSIES3 J~OM OSiE~3 1 24a and 1 24b ~ I OMe E~3SIO~ "OSiEt3 OMe Thus, a compound such as Compound 111 of Sche~e XIV was reacted with phenylthioethanol in the presence of a base as discussed elsewhere to exchange out the phenoxide moiety from the corresponding carbamate. That compound was then oxi.dized with m-chloroperbenzoic acid to form the corresponding 2-(phenylsulfonyl)ethyl carbamate, Compound 120.
Racemic compound 120 was reacted with Compound 123 as shown in Scheme XV. Compound 123 was prepar~d from Compound 121 via compound 122, and shown in the scheme and discussed in regard to Scheme XIII as to the preparation of Compound 103. Compound 121 was itsel~
prepared in a manner analogous to that discussed in Nicolaou et al., J. Am. Chem. Soc., 112:8193-81g5 (1990), except that Compound 99 herein was utilized instead of Compound 12 of the published paper, and Et3Si blocking groups were used instead of ~BuMe2Si b}ocking groups that were used in the published paper.
Thus, Compound 121 was irradiated in T~F-~2O
(9:1, v~v) at zero degrees C in step a to provide a 95 percent yield of Compound 122. That compcund was reacted with a catalytic amount of NaH and trichloroacetonitrile in methylene chloride (1:12, v/v) at 25C to provide a 98 percent yield of Compound 123 in step b. Compounds t20 and 123 were coupled in step c in benzyl alcohol, BF3-Et2O in methylene chloride at -60 - -30C to provide Compound 12~ as a mixture of diastereomeric anomers 124a and 124b present as a Q- to ~-anomer ratio of about 5:1, the Q-anomer beinq shown as Compound 12~, and the ~-anomer, Compound 12~b, not being shown.
. Reaction of Compounds 12~ and 12~b with "BuNF
using standard conditions removed the triethylsilyl groups. The oxime- and FMOC-containing, hydroxyl deblocked compound that resulted, Compounds 125a and W092~02522 ~ ~ ~ 3 q ~ ~ PCT/US91/~5436 l2sb~ thus contained the oligosaccharide portion disc~ssed previously as oligosaccharide Structure D.
Subsequent reaction with diethylamine under usual conditions re~oved the FMOC group to form Compounds 126a and 126b. The completely deblocked oxime-containing oligosaccharide portion of Compounds 126a and 126b is the oligosaccharide discussed prev.ously as oligosaccharide Structure E.

Scheme XVI

~ ~ l H

¦ BF3-0Et2-NaCNBH3 -50C(65%) Me O O~ OMe Me o ~ O ~ O~N~
~ S~` ~ H ~ OH
Me"" O ~ ~ OH
l ~ ~ OMe HO~ ~" ""OH 127a and 127b OMe . Scheme XVI shows only the oxime-containing ring of Compound l26a, with the remaining portions indicated by the wavy lines. As is shown in Scheme XVI, reduction of Compound ~26a wi~h sodium cyanoborohydride in BF3-Et2O at -50C provided a 65 percent yield of ~W13STITUTE SHE'r æ~?~ 9~

epimeric Compounds 127a and 1~7b, that were present in about equal amounts. Those compounds were epimeric a~
the 4-position of the A ring as indicated by the number 4 and the arrow. The epimeric portions of Compounds 127a and ~27b provide the oligosaccharide portions previously identified as oligosacchari.de Structures and G, wi~h only the epimer present in calicheamicin (Structure F) being shown in Scheme XVI.
As prepared, Compounds 125-727 were mixtures of diastereomers. Those diastereomers have been separated, but the absolute configurations of the fused-ring enediyne portions are presently unknown, and only one is illutrated, whereas both were shown in Scheme XIV.
Preparation of other chimeras using other appropriate fused ring enediyne aglycons and oligosaccharide portions.
As noted earlier, an R5 group such as that shown in Formula S is preferably a hydroxyl group or a group convertible thereto intracellualarly. The presence of an Rs hydroxyl group also pro~ides an atom (oxygen) that can be used to link a hydroxyl-containing spacer group that itself can be glycosidically linked to a before-described saccharide. A generalized synthesis is illustrated in Scheme VI. A more specific partial synthesis i9 illustrated in Scheme XVII, below, that was used to prepare Compound 59.

PCl lUS91/05436 WO 92t02522 S~ 3 Scheme XVII
xJ~f~
R~

130 R,R-O, XcOEt ~131 R,R=O(CI 12)2O. X,OEt S[~ 132 R,R-O(CH2)2O, X=OH ,~

134 R,R_O~C~2)2O. R
133X=Thlazolidlne ~ 135 R=OH
1 j 136 R=O~n BnOJ~O 3~1 138 137a 3-OBn ~ 137b 1-OBn ~ (oNBnBr) ,~uMe251 HO O~BuMe2SI NBnO 1 43a t41a uco.~Q

W092/02522 PCT/US91/05~36 2 ~ 9 3 88 -Thus, in accordance with Scheme XVII, ethyl cyclohexanone-2-car~oxylate, Compound 130 was reacted in step a with 1.2 equivalents of ethylenle glycol and 0.1 equivalents of TsOH H20 in benzene at reflux for ten hours to form Compound 13~. Compound 131 was reacted in step b with sodium methoxide-methanol ;~t reflux for eight hours to ~orm Compound 132 in 78 percent yield from Compound 130.
Compound 132 was then reacted in step c with Compound 133 in the presence of 1.2 equivalents of DCC
and 0.1 equivalents of DMAP in methylene chloride at 25C for 14 hours to form Compound 134 in ~6 percent yield. Compound 134 was reacted with m-aminophenol in T~F at reflux for 96 hours to provide Compound 135 in 87 percent yield in step d. Compound 135 was reacted with benzylbromide, 1.05 equivalents of NaH and 0.1 equivalents of "Bu4NI in THF at 25C in step e to form Compound 136 in 72 percent yield, Compound 1~6 was cyclized in step f in 37 percent HCl-THF (1:2.7) at reflux for three hours to provide a mixture of the cyclized product Compounds 137a and 137b in 100 percent yield. Compounds 137a and 137 were formed in about a 4:1 ratio in the order named.
Compounds 137a and 137b as a mixture were treated with DIBAL and two equivalents of LiAlH4 in ~HF at reflux for three hours and then with oxygen and SiO2 at 25C for 24 hours to form Compound 138 in 50 percent yield.
A similar reaction sequence can be used with ~-anisidine in place of the m-aminophenol to form the corresponding methoxy compound. That compound when treated with two equivalents of sodium ethylthiolate in DMF at 160C, followed by acylation with acetic anhydride and then reaction with sodium methoxide provides the substituted phenol para- to the nitrogen W092/02522 P~T/US91/05436 ~c~ ~3 3 atom. That phenol, when treated as in step e for~s the benzyl ether, Compound 139.
Either of Compounds 138 or 139, when treated as in steps a and b of Scheme II, or c-e of Scheme III
provides the corresponding benzyloxy Compoundq 140a or 1~0b. Hydrogenation of either of Compounds 140a or ~40b usiny 10 percent Pd/C in ethanol at 25~C provides the corresponding phenol dérivatives Compound~ l~la and 14~.
Reaction of either phenol with 1.05 equivalents of NaH and mercaptothiazolyl pivolate in THF
provides the corresponding pivaloyl esters Compounds 142a and 142b. The pivaloyl ester Compound 142a was prepared as discussed above after a five minute reaction time at 25C in 99 percent yield.
Similarly, reaction of either of Compounds l~la and l~lb with 1.05 eguivalents of NaH and of o-nitrobenzyl bromide and 0.1 equivalents of "Bu4NI in THF provides the corresponding photolabile ONBn group and Compounds 143a and ~43~. Compound 1~3a was prepared in 90 percent yield after a one hour reaction time.
. Any of Compounds 1~2a, 1~2b, 1~3a or ~3b can be used to form a ~used-ring enediyne compound of the invention using the steps outlined in the prior reaction schemes, such as Compounds 59~ and 59b.

æ~'~gl~g Scheme XVIII

O~BuMezSl phS(CHz)2 a / ¦ c,d PhS~C- ~0 ~ PhS~CHz)2~

Phs(o)2(cH2)2 153 ~ PhS~0)2~CH2)20 OCH2CH20H Q~ 154 OCH2cc~2H

.

WO g2/02522 PCI/lJS91/05436 ~J~

The use o~ Compound 1~3b to prepare fused-ring enediyne compounds having a spacer portion in the benzo ring ~ara to the carbamate nitrogen atom is illustrated in Scheme XVIII. Thus, Compound q43b is reacted as discussed elsewhere herein to form fu~s~d-ring enediyne Co~pound 150. Those prior reactions are indicated schematically by the two arrows betwelen Compounds 1~ ~
and 150. Compound 150 is a Dara-hydroxy analog o~ the compound for~ed in step b of Scheme VIII.
Compound ~50 is then reacted in step a with 2-bromoethanol and NaH in the T~F ~o form Compound 15~.
Compound 151 is oxidized in step b as with mCPBA to form Compound 153, whose ethoxyethanol hydroxyl group can be used to form a glysidic link to a saccharide as discussed previously.
Compound 150 can also be reacted with ethyl bromoacetate and cesium carbonate in acetonitrile as in step c to form the corresponding ethyl carboxymethyl derivative. Hydrolysis of that ester with lithium hydroxide in THF-water and neutralization provides the free acid Compound 152 in step d. Oxida~ion of ~he Sree acid as in step b provides Compound 15~. The carboxylic acid group of Compound 15~ can be used to form a chimera via an ester link to a before-discussed saccharide, or an ester or amide link to a before-discussed monoclonal antibody.
The results o~ an exemplary DNA-cleavi~g study using Compound ~0 in a method of the invention are illustrated in Figure 1. Other compounds such as Compound 41 [Formula X wherein Rl is phenoxycarbonyl, R2=R3=R6=R7=R8=H, A is a saturated bond, R4 i5 hydroxyl and R5 is methoxy at the "e" position of the ~enzo ring]
also cleaved DNA. Compound 41 was utilized at a 2 mM
concentration at pH 5Ø

W092/02~22 PCT/US91/0~36 Compounds 40, ~2 and 54 were further found to cause significant DNA cleavage when incubated at S~M
with supercoiled ~X174 ~NA at pH 8,0 (Figure 2).
Noteworthy in these studies is the observation of double strand cuts as well as single strand c:uts, as is the case with dynemicin A ~Compound 1). The methoxy derivatives 47, 55, 58 and 62 exhibited diminished activity against DNA.
On the other hand, the compounds exhibit anti-microbial activity and all of those assayed exhibit some activity n ~itro against tumor ce~ls. A graph illustrating the inhibition of MIA PaCa-2 human pancreatic carcinoma cell growth using Compound 2 (DY-1) is shown in duplicate in Figure 3. Graphs illustrating the inhibition of MB-49 murine bladder carcinoma cell growth for Compounds 2 and 20 ~DY-2) are shown in Figures 4 and 5, in quadruplicate and duplicate respectively. Values of IC50 obtained from the two wider range studies shown in Figure 4 were 43 nM and 91 nM. In a comparative study, Compound 2 was also shown to be more active against the cancerous MB-49 cells than against non-transformed CV-l African green monkey kidney cells (ATCC CCL 70) or WI-38 human lung cells (ATCC CCL
75).
Each of the dynemicin A analog Compounds 2ia-g exhibited anti-tumor acti~ity against MIA PaCa-2 cells, with the esters (Compounds 24b~g) being more potent than the free acid (Compound 2~a). Compounds 24n-g were also active against MB-49 cells and inactive against CV-1 and WI-38 cells. Compounds 2~-~ shown in below each exhibited weaker anti-tumor activity against NB-49 cells than did Compound 2.

2 ~

RsOH O
¢~ RsONa Rs~

2 R5_ -~ H3(2b); 2b-d 3H (2c);

~H(2d)-The described chemistry supports the viability o~ two paths as triggering mechanisms for the dynemicin A-type cascade by showing that a lone pair of elec~rons on a heteroatom (N or 0) strategically positioned on the aromatic ring in relation to the epoxide moiety serves to initiate the cycloaromatization reaction. Such reactive species can be genera~ed within the cell by enzymatic reactions, or as shown above, be release~ from suitable precursors under mild conditions in the laboratory. In addi~ion, the relative stability and observation of Compounds ~2, 5~, 58 and 62 is interesting in that it allows ~or the scenario of bioreduction prior to intercalation as well as for the pos~ibility o~ DNA interacting nuclecphilically against quinone methide species. Thus the propos~tion that dynemicin A may be interacting with DNA by a dua~
mechanism (nucleophilic and radical~ appears attractive, not only because of the obser~ed preference for cleavage of adenine and guanine, but also in view of the chemistry of Compound ~2.
Further biological evaluation data are provided hereinafter in ~ables l-3.

~ ~ 3'~

~est Mode for carrvin~ out the Invention Methods DNA Cleava~e Studies 5The ethereal solution of Compound ~o produced by the LiAlH4 reduction of Compound 21 (39 mg, O.lo mmol) was evaporated in vacuo to dryness and the residue dissolved in THF (4 m~) to give a 25 ~M solution of Compound 40, assuming complete conversion of Compound 21. Analysis of Compound ~0-induced damage to - supercoiled, covalently closed, circular (form I) ~X174 DNA was performed by incubation at varying concentrations of Compound 4n ( lOO ~N - 5000 ~M) in phosphate buffered aqueous solution at 37C for 12-24 hours followed by agarose gel electrophoresis to 3eparate the various DNA products.
Thus, a vial containing a 50 micromolar per base pair solution o~ ~X174 Form I double stranded DNA
in 2.0 microliters of pH 7.4 phosphate (50 mM) buffers were added 6.0 microliters of th-e same buffer solution and 2.0 microliters of a 5.0 millimolar ethanol solution of Compound ~0.
The vials were then placed in a 37C oven for 12-24 hours. A 2.0 microliter portion of glycerol loading buffer solution containing bromothymol blue indicator was added to each vial. A 10 microliter aliquot was then drawn from each. Gel electrophoresis analysis of the aliquots was performed using a 1.0 percent agarose gel with ethidium bromide run at 115 volts for 1 hour. DNA cleavage was indicated by the formation of nicked relaxed circular DNA ~for~ II) or linearized DNA (form III), which was detected by ~isual inspection of the gel under 310 nanometer ultraviolet light using ethidium bromide.

Procedure for 6-Well CYtotoxicity Assay MIA PaCa-2 cells,- MB-49, CV-l or WI-38 oells were loaded into each well of a 6-well plate at a density of 100,000 cells/well in 3 ml culture medium.
They were incuba~ed ~or 4 hours (37C, 7 percent CO2).
Then 6 microliters o~ solution containing a compound to be assayed were added into 3 ml of medium (RP~I-1640, with 5 percent fetal bovine serum and 1 percent glutamine) in a 500X dilution so that in one well ethanol was added to make a 0.2 percent ethanol control .
The plates were then incu~atsd ~or 4 days (37C, 7 percent CO2). The medium was then drained, crystal violet dye ~Hucker formula) was added to cover the well bottoms and then they were rinsed with tap water until rinses were clear. The stained cells were solubilized for quantitation with Sarkosyl solution tN-Lauryl sarcosine, 1 percent in water) at 3 ml/well. The absorbance of ~:~e solution was then read at 590-650 nm.

Larae Scale Screenina Aaainst Ca~cerous Cell Lines In addition to the screening already discussed, several of the before-described compounds and chimeras were scre~ned against several or all of a panel of ten cancerous cell lines as target cells. This screening utilized a sulforhodamine B cytotoxidity assay as discussed below.

SULFOR~ODAMINE B CYTOTOXICITY ASSAY
1. Preparation of target ~ells in 96-well plates a. Drain media from T~ flask of target cell line(s) and carefully wash cell monolayer two times with sterile P8S (approximately 5 mL per wash) b. Add 5 mL trypsin/EDTA solution ancl wash monolayer for ap~roxima~ely 15 seconds WO 92/02522 PCI`/US91/05436 c. Drain all but approximately 1 mL of trypsin/EDTA from flask, cap flask tightly, and incubate at 37C for approximately two to five minutes until cells come loose.
d. Add 10-15 mL tis~ue culture (T.C.) medium ~PMI 1640 plus 10 per~e~t fetal calf serum and 2 m~q L-glutathione) to flask.
and pipet gently up and down to wash cells.
e. Remove a 1/2 mL aliquot of the cell suspension and transfer to a glass 12 X
75 mm culture tube for counting.
f. Count cells on a hemacytometer using trypan blue, and determine percent viability.
g. Ad~ust volume of cQll suspension with T.C. media to give a density of l X 105 cells/mL.
h. Add 100 ~L of T.C. medium to wells Al and Bl of a 96-well plate for blanks.
i. Add lO0 ~L of cell suspension to the remaining wells of the 96-well platPs.
j. Incubate plates for 24 hours at 37C, 5-lO percent C0z in a humidified incubator.

2. Preparation of sample drugs and toxic control a. Stock drug solutions were prepared by dissolving drug in the appropriate solvent (determined during chemical - characterization studies) and sterile filtering the drug-solvent solution through a sterile 0.2 ~ filter unit. An aliquot was taken from each filtered drug W092tO2522 PCT/US91/05436 3 ~ ~ ~

solution and the O.D. was measured to determine the drug concentration.
b. Dilute the stock drug solution prepared above with T.C. medium to the desired initial concentration (10-2-104M). A
minimum volume of 220 ~L of diluted drug is required per 96-well plate used in the assay.
c. Prepare toxic control by diluting stock doxorubicin solution to lO-7 to 109M in T.C. medium. A minimum volume of 300 ~L
is required per 96-well plate.

,, 3. Addition of Sample Drugs, Compounds, Chimeras and Controls to 96-well Plates a. Remove and discard lO0 ~L of T.C. medium from the wells in Column #2 of the 96-well plate using a multi-channel pipettor and sterile tips.
b. Add 100 ~L of the initial compound dilution to adjacent duplicate wells in Columns #2. ~Four materials can be tested in duplicate per 96-well plate.) c. Remove 10 ~L of diluted compound from the wells in Column #2 and transfer to the corresponding wells in Column #3. ~ix by pipetting up and down gently approximately five times.
d. Transfer 10 ~L to the appropriate wells in Column #4 and continue to make 1:10 dilutions of compound across the plate throu~h Column #12.
e. Remove and discard 100 ~L of medium from wells Fl, G1, and ~1. Add 100 ~L of 2 ~

toxic control (Doxort~)icin diluted in T.C. medium) to each of these wells.
f. Incubate (37C, 5-10 percent C02 in humidified incubator) plates for a total of 72 hours. Check plates at 24 hour in~ervals microscopically for siyns of cytotoxicity.

4. Cel} Fixation a. Adherent cell lines:
- 1. Fix cells by gently layering 25 ~L
of cold (4C) 50 percent trichloroacetic acid (TCA) on top of the growth medium in each well to produce a final TCA concentration of 10 percent.
2. Incubate plates at 4C for one hour.
b. Suspension cell lines:
1. Allow cells to settle out of solution.
2. Fix cells by gently layering 25 ~L
of cold (4C) 80 percent TCA on top o~ the growth medium in each well.
3. Allow cultures to sit undisturbed for Xive ~inutes.
4. Place cultures in 4C refrigerator for one hour.
c. Wash all plates five ti~es with tap water. -d. Air dry plates.

5. Staining Cells a. Add 100 ~L of O .4 percent (wt./vol.) Sulforhodamine B (SRB) dissolved in 1 W09~/02~22 PCT/US91/OS436 _ 99 _ percent acetic acid to each well of 96-well plates using multichannel pipettor.
b. Incubate plates at room temperature for 30 minutes.
c. After the 30 minute incubation, shake plates to remove SRB solution.
d. Wash plates two times with tap water and 1 x with 1 percent acel:ic acid, shaking out the solution after each wa~h. Blot plates on clean dry absorbent towels after last wash.
e. Air dry plates until no standing moisture is visible.
f. Add 100 ~L of lOmM unhuffered Tris base (ph 10.5) to each well of 96-well plates and incubate ~or f iV2 minutes on an orbital shaker.
g. Read plates on a microtiter plate reader at 540 nM.
IC50 values; i.e., the concentration of Compound required to kill one-half of the treated cells, were then calculated.
The cell lines assayed are listed below along 25 with their respective sources:
Cell Line Source and TY~e NHDF Normal human dermal fibroblasts, as controls - Clonetics Corporation O Capan-l American Type Culture Collection (ATCC) Molt-4 (All are human cancer cell lines as OVCAR-3 described by the ATCC) OVCA~-4 - --Sk-Mel-28 W092/02522 PCT/US9l/flS436 2 ~ 3 UCLA M-14 Dr. R. Reisfeld of The Scripps Research UCLA M-21 Institute, and ori~inally obtained from UCLA P-3 Dr. D. Morton, University of California, Los Angeles. M-14 and ~-21 are h~man melanoma cell lines, whereas P-3 is a human non-small cell lung carcinoma ce}1 line.
Control studies were also carried out using the fol}owing well known anticancer dr~gs with the following ICso values for NHDF and cancer cells.

Ranqe of Averaae IC5a Values ~Molarit ~y~ NHDF c~ 5~lls Dexorubicin -- 1.6X101~ - 9.8XlO~
Dynemicin A lo~ 1.6XlO~ - 9.8Xl0 Calicheamicin 2.5Xl0^9 5X105 1o-1~
Morpholinodoxorubicin -- 1.6Xl07 - 9.8Xl09 ~axol 1o8 10~7 ~ 109 Methotrexate 5XlOs >104 - 10 8 Cis-Platin 5Xl05 lo~6 _ lo6 Melphelan 104 104 ~ 106 * UCLA P3 cells were susceptible at lOl2~. All other cells were susceptible at 1.56X101 M or higher concentrations.
** Molt-4 cells were susceptible at 10l2M. All other cells were susceptible at 3.9X109~ or higher concentrations.

Tables 1 and 2 herein below provide average IC50 data from five to all ten of.the above cancer cells lines for fused ring-enediyne compounds disclosed herein. Those tables provide a generic structure and a description of the R group of that generic structure for each compound. Compound numbers as provided hereinbefore are also provided. Table 3 contains data WO92/02522 ~ PCr/US91/05436 for two fused-ring Pnediyne compounds (Compounds ~.7 and 120) as well as data for six chimers.

WO 92/02522 PCr/US91/05436 g !~ ~ 9 -- 102 --Table 1 Anticanc~r Activity (IC50) Of En~diyrl~ Compounds .. , ,_ _ .
Structure Number of Compound IC50 (M) . . . . _ . . -, ~ 2- X = OH 6.3x1 o-6 H, ~\ d,6; X = OM~ 6.3x1~ 5 PhO~N' ~--1 24b: X = OCH2CO2Me 5.0x10'6 ~lJ 24a: X = O~ H2CO2H 6.3x~
W x 21: X = H 5.0x10'6 . ~
o ~
,, H~ 74- X = OH 2.0x10'5 PhO~N~0 76 X = H 7.9x10 ~x O H,~, ~ 85: X = OH 2.0x10'5 PhO~N~ IB7: X = H 3.2x10 ~x PhoJ~ 41: X = OH 2.0x10'5 ~J 41a: X _ H 2.0x10 5 ~x . - _ ¦~\ 59a: X = OH 7 ~xl0'7 ,I~Hy ~! (9.8x10'~
~\~1 59b: X - H 2.0x10'7 ~x (1 .0xt 0'~
IEuCO2~ . . . ` .
*Average dats obtalnsd from 5~10 eell lines.
*~Data obtalned from MOLT 4 (Leukemia) cell llne.

WO 92/02522 PCl`/US91 /05436 ~t~ S ''`~

Table 2 Antican~er ActiYi~y (IC~O) Of En@diyne Compourlds Structure Number of Compound ICso ~M) _ . _ 1~\ 21: R a Ph 5.0x10 6 J~H.. ~ ~ 21a: R = CH2CH2SPh 6.3x1û 5 RO N--~' ~ 21b: R = CH2CH2SOPh 3.2x10 5 ~ ~ 4~: R - CH2Ctl2SO2Ph 2.0x10 7 _ ~I~H,,~ 76: R = Ph7.9xlO 6 RO N~ 77 R = CH2CH2SPh 3.1;x10 5 ~ 70: R = CH2CH2SO2Ph 3.2x10'6 _ 87: R = Ph 3.2x10 6 ~H~ 88: R = CH2CH2SPh~i.0x10 5 RO ~j 80: R = CH2CH2SO2P h 1 .6xl o 6 . . . _ ROJ~N~'~ 41b- R ~ CH2CH2SO2Ph 2 0x10 5 ~J 41c: R = CH2CH2SO2Ar" 2.0xlO 7 H 41d: R = CH2cH2so2Arb 1.3x10 7 M~O .
. .. . . .................... .
Ar~= I Arb=
¢~ ~
*Average data obtained from 5-10 cell ~ines.
**lC50 = 1.0x10 l1 obtained from MOLT-4 (Leukemia) cell line.

$~

Table 3 Anticancer Activity (IC50) Of Enediyn~ C:ompounds O~O~N~ I
Number ot Compound ~ IC50 (1~A) 47: X = OMe 5.0x10 6 120: X = OCH2t:H20H 4.0%10'5 125a: X = OCH2CH20R1 ~ 10'~
126a: X - OCH2CH20R2 4.4x10'5 125b: X = OCH2CH20R1 ~ 10'4 126b: X = OCH2CH20R2 8.7x10'6 127a: X z OCH2CH20R3 1.4x10'7 127b: X = OCH2CH20R4 7.6x10 6 0111~ FMOC

~ O ~`N~o~Q
R1= ~ S ~ OH
~fr~~ 0~ H
HO~'`OHll-~,o_~ ' N~

~1 ~ N~oH ~
R2 = ~, _oJ~o~ OH

"~1 `-- ~ ,Q,X

rr ~ ~ Y
HO~`'OH ~ N~
f b OH

0~
.. ... ..
*Av~rage data obtalned trom 5~10 cell linas.
**1050 = 1.0x109 obtained from MOLT-4 (Leukemia) cell line.

~J t,i `'~ 3 As will be seen from the data of the tables, the compounds and chimeras had activities similar to those of the well known anticancer dnlgs.
Compounds having an R4 hydrogen tended to be 5 more active than those having an R~ hydroxyl or other oxygen-containing group. Compounds whose carbamate portion contained a phenylsulfonylethoxy or naphthylsulfonylethoxy group were about 10 to ~00 times more active than similar compounds having a phenoxy group as F~rt of the carbamate. Compounds having an electron releasing group relative to hydrogen E~ to the carbamate nitrogen atom tended to be equal to less active than those with hydrogen, whereas compounds with an intracellu}ar-formed electron releasing group (e.g.
Compounds 59a and 59b) relative to hydrogen m~a to that nitrogen atom tended to be more active than compounds having hydrogen at that position.
Turning to the chimeras, the data Table 3 indicate that the chimeras are effective. Those dat,a also indicate that the presence of the FMOC grsup inhibits activity, but that presence of the oxime does not. Those data also indicate that chimeras having the stereochemistry of the calicheamicin oligosaccharide are more active than those having the epimeric stereochemistry at C-4 of the A ring.
The data of the tables also show compounds and chimeras described herein to be particularly active against Molt-4 leukemia cells. Thus, for those cells, Compounds 5g~, ~lb, ~1c and ~1~ exhibited IC50 values 10,000 times more potent than the potency observed against the other cell lines. The activity of chimeric Compound 127a against Molt-4 cells was about 100-times that of the average of the other cell lines examined.
Those ICs~ values against ~olt-4 cells were also 10,000-C~ 9 ~ -- l o 6 100,000 times smaller than the ICso values for those compounds against ~HDF cells.

Coml~ound Pre~aratio~ and Data Example 1: 7,8,9,10-Tetrahydrophe~nanthridine N-oxide (ComPound 4aL
A solution of ~ (1.42 g, 7.76 mmol) in dichloromethane (50 mL) was treated Zlt 25C with mCPBA
(1.58 y of an 85 percent sample, 7.76 mmol) and stirred for 1 hour. The solution was poured into saturated sodium bicarbonate solution (~0 mL) and extracted. The aqueous layer was extracted with further dichloromethane (2 x 50 mL), the com~ined organic layers were dried (Na2SO4), evaporated in vacuo, and the residue was purified by flash chromatography on silica eluting with 25 percent MeOH in EtOAc to give the pure N-oxide Compound ~ (1.24 g, 80 percent) as an off-white crystalline solid: Rf=0.34 (25 percent MeO~ in EtOAc~;
mp 131.7C (from EtOAc): }R (CDCl3) v~ 2950, 1580, 1390, 1300, 1210, 1140 cm1: 1H NMR (5U0MHz, CDCl3): ~ 8.72 ~d, J=8.3 Hz, 1 H, H-4), 8.31 (s, 1 H, H 6), 7.91 (d, J=8.3 Hz, 1 ~, H-1), 7.68 (t, J=8.3 Hz, 1 H, H-2 or H-3), 7.61 ` (t, J=8.3 Hz, 1 ~, H-2 or H-3), 3.02 (t, J=6.3 Hz, 2 H, Z5 H-10), 2.79 (t, J-~.3 Hz, 2 H, H-7), 1.98-1.84 (m, 4 H);
MS (FA~+) m~ (relative intensity) 200 (M~H, 100), 184 ~12); HRMS calcd for C13~14NO (M+H) 200.1075, found 200.1055.

Example 2: Com~ound 2 A solution of enediyne 3 (205 mg, 0.50 mmol) in dry THF (10 mL) was cooled to -78C and treated with lithium diisopropylamide t0.37 m~ of a 1.5 N solution in cyclohexane, 0.56 mmol). A~ter stirring 1 hour at -78C, the reaction was quenched with sa~urated am~onium chloride solution (2 mL), allowed to warm to room W092/02522 2 ~ 9 Pcr/~s91/os436 temperature, poured into saturated sodium bioarbonate solution (30 mL), and extracted with dichloromethane (3 x 50 mL). The combined organic extracts were dried (Na2SO4), evaporated in vacuo, and purifi~d by flash chromatography on silica eluting with 50 percent ether in petroleum ether to give recovered 3 (48 mg, 23 percent), followed by the ten-membered enediyne Compound 2 (120 mg, 59 percent) as a white crystalline solid:
Rf=0.42 (50 percent ether in petroleum ether); mp=228-230C dec. (from ether); IR (CDC13) V~x 3420,2360,2330,1720 cml; lH NMR (500 MHz, CDCl~ 8.60 (dd, J-8.1, 1.3 Hz, 1 H, aromatic), 7.47-7.10 (series o~
multiplets, 8 H, aromatic), S.83 (d, J-10.1 Rz, 1 ~, olefinic), 5.67 (dd, J~10.1, 1.6 Hz, 1 H, olefinic), 5.53 (d, J-1.6 Hz, 1 H, C ~-N), 2.35-1.71 (series of multiplets, 6 H, C H2); 13C NMR (125 MHz, CDCl3): ~
151.0, 13S.8, 131.3, 129.3, 128.0, 127.8, 126~3, 125.8, 125.3, 124.0, 122.2, 12~.6, 100.4, 94.3, 93.9, 88.8, 74.1, 73.2, 64.4, 50.5, 35.4, 23.2, 19.1; MS: m/e (relative intensity) 409 (26, M~), 368 (18), 236 (11), 162 (13), 131 (100); HRMS: calcd. for C26Ht~NO4: 409.1314, found: 40~.1314; Anal. calcd. ~or C26Hl~NO4.H20: C, 73.06;
~, 4.95; N, 3.28. Found: C, 73.44; H, 5.04; N, 3.26.
Compounds 2-d were prepared from Compound 2 by reaction of Compound 2 wi~h a mixture of the appropriate alcohol and its sodium salt as shown in Figure 15.

Example 3: N-Phenyloxycarbonyl-6-(3(Z)-hexene-1,5-diynyl)-6a:10a-epoxy-10-oxo-5,6,6a,7,8,9,10,10a-octahydro~henanthridine tCom~ound 3) Silver nitrate (5.28 g, 31.2 mmol) was added to solution of the silyl acetylene Cbmpound ~3 (4.50 g, 9.36 mmol) in 100 mL of a H 20-EtOH-THF mixture (1~
at 25C, and the mixture was stirred until TLC analysis 2 ~ 108 -(30 percent ether in petroleum ether) indicated the consumption of 13 (approximately 1 hour). Potassium cyanide (4.32 g, 57.6 mmol) was then added and the mixture was stirred for 10 minutes. The mixture was poured into saturated sodium bicarbonate solution (loo mL) and ex~racted with dichloromethane (3 x loO mL). The combined organic layers were dried (Na2SOb), evaporated in vacuo, and purified by flash chromatosraphy on silica eluting with 30 percent ether in petroleum ether to give enediyne Compound 3 (2.30 g, 60 percent) as a colorless gum: R~=0.38 (30 percent ether in petroleum ether): IR
(CDC1~) V~X 3304, 2940, 2240, 1720, 1492, 1378, 1321, 1206 cm1; lH NMR (500 MHz, CDCl~ 8.87 (dd, J=7.8, 1.4 HZ, 1 H, H-4), 7.53-7.09 ~m, 8 H, aromatic), 5.93 (d, J=1.2 Nz, 1 H, H-6), 5.78 and 5.79 (AB quartet, J-10.1 Hz, 2 ~, olefinic), 3.16 (d, J-1.2 Hz, 1 H, cac-H), 2.79-2.66 (m, 2 H, H 9), 2.38-2.29 (m, 2 H, H7), 2.04-1.89 (m, 2 H, H-8); 13C NMR (125 MHz, CDCl3): ~
15~.9, 135.9, 130.0, 129.3, 128.8, 127.6, 125.9, 125.8, 123.0, 121.4, 120.4, 120.2, 90.6, 85.1, 82.8, ~0.1, 75.1, 57.4, 48.1, 38.9, 23.9, 18.3: MS m/e (relative intensity) 409 (2, M+), 262(15), 212(18), 162(59), 58~100); ~RMS: calcd. for C26Hl~N04: 409.1314, found:
409.1308.
Example 4: lO-Acetoxy-7,8,9,10-tetrahydro~henanthridine (Com~ound 5) A solution of 7,8,9,10-tetrahydrophenanthridine N-oxide (Compound 4a) (1.23 g, 6.18 mmol) in acetic anhydride t50 mL~ was heated to 100C for 20 hours, evaporated to dryness, dissolved in dichloromethane (50 mL) and washed with saturated sodium bicarbonate solution (50 mL). The aqueous layer was extracted with dichloromethane (2 x 50 mL), the combined organic layers were dried (NazSO4), evaporated in vacuo, h ~ 9 and the residue was purified by flash chromatography on silica eluting with Et20 to give pure Compound S ~ 1.15 g, 77 percent) as a white crystalline solid: R f=0.33 (ether); mp=128-129C ~from ether); I~ (CDCl3) Y"", 2970, 1728, 1241 cm 1; lH N~IR (500 MHz, CDC13): ~ 8.70 (s, 1 H, H-6), 8.08 (d, J=9.5 Hz, 1 H, H-4), 7.76 (d, J=9.5 Hz, H, H-1), 7.63 (t, J=7.2 Hz, 1 H, H-2 or H-3), 7.52 (t, J=7.2 Hz, 1 H, H-2 or H-3), 6.57 (bs, 1 H, CH-OAc) ~ 3.02 (bd, J=17.5 Hz, 1 H, H-7), 2.88-2.80 (m, 1 H, H-7), 2.27 (bd, J=13.8 Hz, 1 H, H-9), 2.05 ~s, 3 H, OAc), 2.0L-1.88 (m, 3 Hl; C Nr~ (125 I~z, CDC13): ~ 170.2, 152.3, 147.5, 137.8, 131.8, 130.1, 127.9, 127.0, 126.8, 122.2, 64`.5, 29.1, 27.8, 21.7, 18.4; MS (FAB+) ~ (relative intensity) 242 (M+H, 100), 182 (23); HRNS calcd. for C15H~,sN02 (Mt~) 242.1181, found 242.1181; Anal. calcd. for C15Hl5NO2: C, 74.67; H, 6.27J N, 5.80. Found: C, 74.59;
II, 6.31; N, 5.82.

Example 5: 10-Hydroxy-7,8,9,10-tetrahvdro~henanth~dine (ComDound_6L
A solution of Compound 5 (1.15 g, 4.77 mmol ) in methanol (50 mL) was treated with potassium carbonate (200 mg, catalytic) and stirred for 1 hour. The solution was poured in~o ~aturated sodium bicarbonate solution (100 mL) and extracted with dichloromethane ~1 x 100 mL, 2 x 50 mL). The combined organic layers were dried (MgSO4), evaporated in vacuo, and tXe residue was purified by flash chromatography on silica eluting with ethYl acetate to give alcohol Compound 6 (O.g5 g, 100 perc_nt) as a white crystalline solid: Rf=0.20 (ether): mpal76-177C (from ether); IR (CDCl3) Y",x 3600, 2950, 1510 cml; lH NMR (500 MHz, CDC13): ~ 3.51 (s, 1 H, H-6), 8.20 (d, J-9.1 Hz, 1 H, H-4), 8.00 (d, J=9.1 Hz, 1 H, H-l), 7.61 (t, J=6.8 Hz, 1 H, H-2 or H-3), 7.55 (t, J=6.8 Hz, 1 H, H-2 or H-3~, 5.39 ~bs, 1 H, CH-OHl, 2.89 W092/02~22 P~T/US91/05436 2 ~

(bd, J=16.1 Hz, 1 H, H-7), ~.80-2.72 Im, 1 H, H-7), 2.80-2.60 (bs, 1 H, OH), 2.24 (bd, J=:L2.5 Hz, 1 H, H-8 or H-9), 2.07-1.88 (m, 3 H): MS (FAB+) m/e ~relati~e intensity) 200 ~M~H, 100), 154 (41), :L36 (37), 109 (24);
HRMS calcd. for C~3H14NO (~+H) 200.1075, found 200.1085.

Example 6: lO-tert-Butyldimethyls:ilyloxy-7,8,9,10-tetrah~dro~henanthridine (ComPound ?~
A solution of Compound 6 (3.70 g, 18.6 mmol) in dry dichloromethane (100 mL) was treated with 2,6-lutidine (3.4 mL, 27.9 mmol) and tert-butyl-dimethylsilyl triflate (5.35 mL, 22.3 mmol~. After stirring ~or 1 hour at 25C, methanol (2 mL) was added, stirring was continued for a Purther 5 minutes, and then the solution was poured into saturated sodium bicarbonate solution (100 mL) and extracted. The aqueous layer was extracted with further dichloromethane (2 x 50 mL), the combined organic layers were dried (Na2SO4), evaporated in vacuo and the residue was purified by ~lash chromatography on silica eluting with 50 percent ether in petroleum ether to give pure silyl ether Compound 7 (5.38g, 92 percent) as a white so}id.
7: colorless oil: Rf=0.50 ~70 percent ether in petroleum ~5 ether); IR (CDCl3) V~X 2970, ~930, 2860 cm1; lH NMR ~500 M~z, CDCl3): ~ 8.68 ~s, 1 H, H-6), 8.08 (d, J=4.7 Hz, 1 H, H-l or H-4), 8.05 ~d, J-4.7 ~z, 1 H, H-l or ~-4), 7.62 (t, J-4.7 Hz, 1 H, H-2 or H-3), 7.53 (t, J-4.7 Hz, 1 H, H-2 or H-3), 5.45 (t, J-2.8 Hz, 1 H, H-10), 3.00 (dd, J=5.5, 16.6 Hz, 1 H, CH-Ar), 2.81 (m, 1 H, CH-~r), 2.23-2.10 (m, 2 H, CH2), 1.88-1.78 (m, 2 H, CH2), 0.84 (s, 9 H, tBu), 0.22 (s, 6 H, SiMe2); 13c NMR (125 XHz, CDCl3): ~ 152.8, 147.0, 141.2, 129.8, 129.3, 127.9, 126.g, 126.1, 123.6, 63.2, 31.8, 27.0, 25.3, 18.2, 16.4, -3.6, -4.5; MS (F~B+) m~e (relative intensity~ 314 (M+H, 2~ G g ~9 9 -- 111 -- .

100), 256 t7), 182 t11~; HRMS calcd. for Cl~28NoSi (M+H) 314.1940, found 314.1951~

Example 7. N-Phenyloxycarbonyl-10-te~t-butyldimethylsilyloxy-6-ethyl-- 5~6~7~8~C~lO-hexahydrophenanthridine - tCompound 8~ _ A solution o~ ~uinoline Compound 7 (5.20 g, 16.6 mmol) in dry THF (166 mL) was cooled to -78C and treated with ethynylmagnesium bromide t36.5 mL o~ a 0.5 M solution in THF, 18.3 ~mol) followed by phenyl chloroformate t2.3 mL, 18.3 mmol~. The solution was allowed to wàrm up 510wly t 25C over 1 hour, quenched with saturated ammonium chloride solution (10 mL), poured into saturated sodium bicarbonate solution ~150 mL) and extracted. The aqueous layer was extractsd with dichloromethane ~2 x 100 mL), the combined organic layers were dried (Na2S0~), evaporated in vacuo, and purified by flash chromatography on silica eluting with 10 percent ether in petroleum ether to give pure carbamate Compound 8 (6.96 g, 92 percent) as a colorless oil (about 3:1 mixture of isomers as judged by NMR).
Rf=0.85 (30 percent ether in petroleum ether); IR
(CDCl3) V~X 3300, 2952, 2858, 2250, 1715, 1473, 1204 c~ H NMR (500 MHz, CDC13): ~ 7.68 (d, J=7.5 Hz, 1 H, H-4), 7.40-7.12 (m, 3 H), 5.6~, 5.61 (2 x 5, 1 H, H-6), 5.00, 4.69 t2 x bs, 1 H, H-10), 2.50-1.50 (m, 7 H), 0.80, 0.92 (2 x s, 9 H, tBu), 0.28, 0.19, 0.10, 0.09 (singlets, 6 H, SiMe2)7 13C NNR (125 MHz, CDCl3): ~
151.1, 136.3, 132.9, 129.8, 129.3l 127.2, 126.0, 125.4, 125.1, 124.2, 124.1, 123.9, 122.0, 80.-2, 72.3, 65.0 a~d 64.2, 48.7 and 48.2, 32.3 and 31.4, 28.0, 26.1, 18.4 and 16.3, -4.1 and -4.8: MS m/e ~relative intensity) 459 (M~, 10), 402 (100), 366 tlO), 308 t24), 206 t26), 151 W09~/02522 PCT/US91/05436 2~ 112 -(27), 75 t29); HRMS calcd. for Cz8~3303NSi (M ): 459.2230, found: 4S9.2233.

Example 8: N-Phenyloxycarbonyl-lo-tert-butyldimethylsi}yloxy-6a:1 oa-epoxy-6-ethyl-5,6,6a,7,8,9,10,10a-octahYdro~henanthridine ~Com~nd g ~
A solution of Compound 8 (8.60 g,. 18.8 mmol) in dichloromethane (120 mL) was treated with mCPBA (8.08 .
g of a 60 percent sample, 37.6 mmol) and stirred a~ 25C
for 3 hours. The solution was poured into saturated sodium bicar~onate solution (lOOmL), extraçted, and the aqueous layer extracted with further dichloromethane (2 x 100 mL). The combined organic }ayers were dried (Na2SO4), evaporated in vacuo, and the residue was purified by flash chromatography on silica eluting with 10 percent ether in petroleum ether to give epoxide Compound 9 (8.20 g, 92 percent) as a white foam (mixture of two isomers, about 3:1 ratio~; R~=0.73 (30 percent ether in petroleum ether); IR V~X (C~Cl3) 3307, 2953, 2250, 1721, 1494, 1384, 1322, 1250, 1207 cml; lH NMR
(500 MHz, CDCl3): ~ 7.88 (d, J=7.1 Hz, 1 H, H-4), 7.50-7.10 (m, 8 H), 5.58 ~bs, 1 H, H-6), 4.82 (dd, J=10.0, 5.7 Hz, 1 H, H-10), 2.34 (dd, J=14.-8, 5.6 Hz, 1 H), 2.09 (bs, 1 H), 1.95-l.Q5 (m, Z H), 1.78-1.62 (m, 2 H), 1.40-1.30 (m, 1 H); 13C NMR (125 MHz, CDCl~ 153.9 and 151.1, 135.5, 129.2, 129.2, 129.1, 128.3, 128.1, 127.9, 127.1, 125.5, 121.6, 78.5, 73.8, 72.~, 69.9, 60.4, 48.0, 31.0 and 29.6, 26.0 and 25.8, 24.0, and 26.5, 18.2 and 20.3, 0.28, -0.28, -0.37; M~: m/e (relative intensity) 47S(M~,2), 419 (100), 325 (28), 268 (10), 222 (14), 151 (18), 73 (42); HRMS: calcd. for Cz8H3304NSi (M~):
475.2179, found: 475.2175.

. g ~

Example 9 N-Phenyloxycarbonyl-6a:10a-epoxy-6-ethyl-10 hydroxy-5,6,6a,7,8,9,10,10a-octahvdrophenanthridine LCom~Qund 10) A solution of epoxide Compoun* 9 (8.20 g, 17.4 mmol) in THF (lOOmL) was treated With TBAF (20.9 mL of a 1 M solution in THF, 20.9 mmol) and heated to 42C for three hours. The solution was evaporated iD~Y99~ and purified by flash chromatography on silica eluting with 50 percent Et20/petroleum ether to give pure alcohol Compound lo (6.00 g, 100 percent) as a white crystalline solid (about 3:1 mixture of isomers as shown by N~).
Rf=O .31 (50 percent ether in petroleum ether); mp 78-79C (from Et20); IR (CDCl3) ~x 3580, 3306, 2951, 2250, 1720, 1595, 1494, 1382, 1322, 1206 cm1; lH NMR
(500 MXz, CDCl3): ~ 7.91 and 7.88 (d, J=8.0 Hz, 1 H, H-~), 7.50-7.08 ~m, ~ H), 5.62 and 5.59 (d, J=1.0 Hz, 1 H, H-6), 4.89 and 4.70 ~m, 1 H, H-10), 2.47-1.35 (m, 8 H); 13C NNR (125 NNz, CDCl3): ~ 150.9, }35.5, 129.3, 128.7, 128.6, 128.4, 127.7, 127.3, 126.1, 125.8, 121.5, 78.7 and 78.2, 74.8 and 70.8, 73.2, 66.6, 65.9 and 64.4, 60.9 and 58.2, 47.8, 30.3 and 27~0, 24.1 and 19.0, 15.2 and 13.8; MS m/e (relative intensity) 361 (M~,65), 224 (100), 196 (24), 180 (29), 167 (30), 94 (40), 77 (45);
HRMS: calcd. for C2~ N04 (M~); 361.1314, found:
361.1317.

Example 10: N-Phenyloxycarbonyl-6a:~Oa-epoxy-6-ethyl-10-oxo-5,6,6a,7,8,9,10,10a-octahvdro~henan~hridine tCom~ound 11) A}cohol Compound 10 (6.009, 1704 ~mol) was dissolved in dry dichloromethane (180 mL) and treated with powdered, activated 4A molecular sieves (1 g) and pyridinium chlorochromate (6.25 g, 29.0 mmol). The suspension was stirred for 1 hour at 25C, filtered through celite, concentrated in vacuo, and the residue W0~2t02522 PCT/VS91/05436 was purified by flash chromatography on silica eluting with 30 percent EtzO/petroleum ether to give ketone Compound 11 (4.49 9,75 percent) as a white foam: Rf=0.5 (50 percent ether in petroleum ether); IR (CDCl~) V~X
3306, 2259, 1721, 1491, 1321, 1206 cm l: lH N~R (500 ~Hz, CDC13): ~ 8.50 (d, J=7.8 Hz, 1 H, ~-4), 7.53-7.10 (m, 8 H, aromatic), 5.73 (d, J=2.4 ~z, 1 H, ~-6), 2.76 (dt, J=15.2, 4.9 ~z, 1 H, ~-9), 2.60 (ddd, J=15.2, 10.4, 6.1 Hz, 1 H, H-9), 2.37-2.28 (m, 2 H, H-7~, 2.21 (bs, 1 H, C-C-H), 2.04 1.90 (m! 2 H, H-8); 13C NMR: (125 MXz, CDCl3): ~ 201.0, 153.9, lS1.0, 135.8, 129.9, 129.3, 129.0, 127.6, 126.1, 125.9, 123.0, 121.5, 77.7, 74.9, 74.2, 57.4, 47.3, 38.9,23.8, 18.3; ~S m/e (relative intensity) 359(M~,100), 266 (52), 222 (65), 194 ~54), 180 (51), 146 (45), 69 (80); HRMS calcd. for C22~17N04 (M ): 359.1158, found: 359.1154: Anal. calcd. for C22H1~N04: C, 73.53; H, 4.77; N, 3.90. Found: C, 73.27;
~, 4.79: N, 3.91.

Example 11: N-Phenyloxycar~onyl-6-~6-trimethylsilyl-3(Z)-hexene-1,5-diynyl]-6a:10a epoxy-10-oxo-5,6,6a,7,8,9,10,10a-octah~d~oPhq~anthridine ~Commound 13) Palladiuml~ acetate (192 mg, 0.86 mmol) and triphenylphosphine (832 mg, 3.17 mmol) in dry, degassed benzene (10 mL) were heated under argon at 60C for 1 hour. The resulting dark red solution was cooled to 25C, and the (Z)-chloroenyne Compound '2 (2.88 g, 18.2 mmol) in dry, degassed benzene (20 m~) was added, followed by n-butylamine (1.92 mL, 19.4 mmol). The solution was stirred for 15 minutes at 25C, cooled to zero degree C, and the acetylene }1 ~4.49g, ~2.5 mmol) in dry, de~assed benzene (50 mL) was added, followed by copper (I) iodide (512 mg, 2.69 mmol). The solution was ~ s~irred for 2 hours at 25C, poured into sa~urated W092t02522 2 ~ PCT/US91/0~436 sodium bicarbonate solution (100 mL) and extracted. The aqueous layer was extracted with dichloromethane (2 x 50 mL), the co~bined organic layers were dried (Na2SO4), evaporated in vacuo, and the residue was purified by flash chromatography on silica elutinq with 20 percent ether in petroleum ether to give the coupled product Compound 13 (4.50 g, 74 percent) as a colorless gum:
Rf=0.51 (30 percent ether in petroleum ether); IR
(CDCl3) V~X 2962, 1~20, 1492, 1378, 1322, 1252, 1206, 846 cm-1; 1H NMR (500 MHz, CDCl3): ~ 8.36 (d, J=8.5 Hz, 1 H, H-4), 7.52-7.09 (m, 8 H, aromatic), 5.99 (d, J=1.6 Hz, 1 H, H-6), 5.82 (d, J=11.2 Hz, 1 H, olefinic), 5.66 (dd, J-11.2, 1.6 Hz, 1 H, ole~inic), 2.76 (dt, J-15.3, 4.7 Hz, 1 H, H-9), 2.71 ~ddd, J-~5.3, 10.8, 6.1 Hz, 1 H, H-9), 2.39-2.30 (m, 2 H, H-7), 2.07-1.89 (m, 2 H, ~-8), 0.25 (s, 9 H, SiMe3); 13C NMR (125 MHz, CDCl3): ~ 201.1, 150.9, 135.8, 129.9, 129.2, 128.9, 128.4, 127.7, 126.0, 125.8, 122.9, 121.4, 120.8, 118.9, 103.6, 101.5, 9~.4, 83.0, 74.9, 5775, 48.3, 38.9, 23.9, 18.2, 0.00: MS m/e (relative intensity) 481 (M',11), 360 (100), 146 (10);
HRMS: calcd. for C2~H270~NSi (N~): 481.1709, found:
481.1705.

Example 12: mpound 2~
A solution of enediyne Compound 2 (100.1 mg, 0.224 mmol) in pyridine (2 mL) was treated with acetic anhydride (0.50 mL, 5.31 mmol~ and DMAP (10 mg, catalytic) at 25C. After 2 hours, the reac~ion mixture was poured into saturated sodium bicarbonate solution (25 mL), extracted with dichloromethane (3 x 25 mL), the rombined organic layers were dried (NazSQ4), e~aporated in vacuo, and the residue was purified by flash chromatography (silica, 30 percent ether in pe~roleum ether) to give acetate Compound 2a ~}10.7 mg, 100 percent). 2a: white crystalline solid, mp 212 214C

W092t02522 P~T/US91/0~36 2 ~ 9 ~ -dec. (from ether); Rf=0.55 (50 percent ether in petroleu~ ether): IR (CDC13) ~x 3075, 2950, 2215, 1742, 1720, 1500, 1216, 769 ~ml; 1H NMR (soo M~z, CDCl3): ~
7.92 (d, J-8.1 Hz, 1 H, aromatic), 7.50 7.09 (m, 8 H, aromatic), 5.83 (d, J=10.1 Hz, 1 H, ole~inic), 5.65 (d, J=10~1 ~z, 1 H, olefinic), 5.53 (s, 1 H, N-CH(C)-C~, 2.51-1.70 (m, 6 H, CH2), 2.18 (s, 3 H, OAc); 13C NMR (125 MHz, CDCl3): ~ 169.1, 150.8, 130.0, 129.3, 128.4, 128.1, 128.0, 127.2, 126.8, 125.7, 125.2, 124.3, 122.9, 121.5, 97.9, 96.5, 93.7, 88.9, 78.0, 73.5, 62.6, 50.3, 29.8, 22.7, 21.8, 18.8; ~S (FAB+) m/e (relative intensity) 452 (M+H, 52), 410 (37), 392 (100), 316 (32), 272 ~43), 242 (30), 154 (77), 136 (70); HRMS calcd. for C28H28NO5 (M~H) 452.1498, found 452.1469.
A solution o~ enediyne Compound 2a (93.0 mg, 0.206 mmol) and 1,4-cyclohexadiene (1.0 mL) in benzene (3.0 mL) was treated with ~-toluene-sulfonic acid (39 mg, 0.23 mmol) and stirred at 60C for 2 hours. The solYent was removed ~n_y~çg~ and the residue purified by flash chromatography (silica, 50 percent ethPr in petroleum ether) to give diol acetate Compound 15a (80.2 mg, 83 percent). 15~: while crystalline solid, mp 198-200C (from ether).

Example 13: ComPound 15 Colorless solid: Rt-0,22 (50 p~rcent ether in petroleum ether); IR (CDCl3) V~X 3360, 3072, 2950, 1738, 1715, 1500, 1192 cml; lH NMR (500 MHz, CDC13): ~ 7.57 (d9 ~=7.8 Hz, 1 H, aromatic), 7.40-7.01 (series of multiplets, 12 H, aromatic), 6.83 (bs, 1 H, O H), 5.59 (s, 1 H, N-C H(C)-C), 3.17 (m, 1 H, CR2), 2.28 tm, 1 H, CH2), 2.26 (s, 3 H, OAc), 1.80-1.40 (series o~
multiplets, 3 H, C~z), 0.72 (m, 1 H, CH2); 13C NMR (125 MHz, CDC13): ~ 174.9, 150.8, 137.7, 134.8, 133.5, 129.7, 129.3, 129.2, 129.0, 128.8, 12~.5, 128.2, 127.8, 127.7, o~3 125.5, 124.8, 123.4, 121.8, 93.8, 75.1, 70.6, 61.4, 32.5, 31.4, 22.6, 19.8; MS: m~e (relative intensity) 471 (M+, 19~, 245 (loo), 162 (loo), 94 (42): HRMS: calcd.
for c28~2506N (M ): 471.1682, found: 471.1683: Anal~
calcd. for C28H2sO6N: C, 71.33; ~, 5.34; N, 2.97. Found:
C, 71.36: H, 5.54: N, 2.84.

Example 14: Compound lSb Dry HCl gas was bubbled through a solution of .lo acetate Compound 2a (32 mg, 0.071 mmol) and 1,4-cyclohexadiene (40 mg, 0.32 mmol) in dichloromethane (4 mL) at 25C for 30 seconds. The solvent was removed i~
y3g~Q and the residue purified by flash chromatography (silica, 50 percent ether in petroleum ether) to give the c~loride, Compound lS~ (25 mg, 80 percent).
Colorless gum: Rf=0.21 (50 percent ether in petroleum ether); IR (CDCl3) V~X 3500, 2945, 1710, 1492, 1400, 1225, 789 cm1; lH NMR (500 MHz, CDCl~): O 7.72 (d, J=8.1 Hz, 1 H, aromatic), 7.4S-6.36 (m, 12 H, aromatic), 5.85 (s, 1 H, benzylic), 2.56 (bs, 1 H, OH), 2.37 (bs, 1 H, OH), 2.34-1.42 (m, 6 H, C~2): 13C NMR (125 MHz, CDCl3): ~ 151.2, 134.7, 132.6, 130.4, 129.4, 129.3, 128.6, 128.5, 128.2, 128.~, 128.1, 127.5, 125.7, 124.5, 124.2, 124.0, 121.7, 81.2, 80.4, 70.2, 62.7, 35.4, 33.7, 18.~; MS (FAB+): ~/e (relative intensity) 580 (M+Cs, 100), 419 (42~, 286 tlOO), 154 (37): HRMS: Calcd. for C2~H2204NClCs ~M+Cs): 580.0291, found: 580~0286.

Example 15: Com~ound 17 Compound 17 has been prepared by several methods as indicated below.
Method (i): A solution of the cobalt complex Compound ~9 (42 mg, 0~060 mmol) in C~zC12 (1 mL~ was treated with Et3N~-O- (32.7 mg, 0.29 ~mol) and stirred at 25C for 4 hours. The solution was poured into W092/02522 PCT/U~91/05436 fJ ~ 118 - .

saturated sodium bicarbonate solution (25 mL) and extracted with CH2Cl2 (3 x 25 mL). The combined organic layers were dried (MgS04), evaporated in vacuo and purified by flash chromatography to give the aromatized product Compound S7 (17.7 mg, 83 percent).
~ethod (ii): A solution of enediyne Compound 2 (S7.8 mg, 0.141 mmol) and 1,4-cyclohexadiene (0.5 mL) in benzene (l.S mL) was treated with D-toluenesulfonic acid (29.6 mg, 0.155 mmol) and stirred at 25C for 24 hours.
The solvent was removed in_vacuo and the residue purified by flash chromatograph (silica, 50 percent ether in petroleum ether) to give keton Compound 17 (53.7 mg, 92 percent).
Method (iii): Trimethylsilyl tri~late (15 ~L, 0.08 mmol) was added to a solution o~ enediyne Compound 2 (32 mg, 0.078 mmol) and triethylsilane (40 mg, 0.32 mmol) in dichloromethane (2 mL) at -78C. After five minutes, the mixture was quenched at -78C with saturated ammonium chloride solution (1 ~L), diluted with either (10 mL), washed with water (2 x 3 mL), brine (3 mL) and dried (MgS04). The organic solvent was removed in vacuo and the residue was purified by flash chromatograph (silica, 50 percent ether in petroleum ether) to give ketone Compound 17 (22 mg, 68 percent).
Method (iv): Dry HCl gas was bubbled through a solution of enediyne Compound 2 (32 mg, 0.078 mmol) and 1,4-cyclohexadiene (40 mg, 0.32 mmol) in dichloromethane (4 mL) at 25C for 30 seconds. The solvent was removed in vacuo and the residue purified by flash chromatography (silica, 50 percent ether in petroleum ether) to give Compound 17 ~25 mg, 78 percant).
White crystals: R~sO.63 (70 percent ether in petrol~um ether); mp=l91-lg3C ~from methylene chloride/ether); IR (CDCl3) v~ 3480, 3080, 2935, 1712, 1490, 1264, 1192 cm1; lH NMR (500 MHz, CDC13): ~ B.33 W092/02522 P~T/US91/05436 ? ~1 ~

(dd, J=7.9, 1.3 Hz, 1 H, aromatic), 8.09 (d, J=7.5 Hz, 1 H, aromatic), 7.54-7.02 (m, 11 H, aromatic), 5.65 (s, 1 H, benzylic), 2.75 ~bs, 1 H, OH), 2.69-1.80 (m, 6 H, CH2), 13C NMR (125 MHz, CDCl3): ~ 207.5, 153.0, 150.9, 5 148.2, 137.1, }34.2, 129.8, 129.5, 128.5, 128.2, 127.8, 127.1, 126.1, 126.0, 124.3, 122.8, 121.8, 121.3, ~2.5, 65.0, 64.1,40.0, 30.2, 23.5, MS~ (relative intensity) 411 (M~,100), 318 (58), 274 (49), 246 (12), 217 (55), 94 (29); HRMS: Calcd. for C26H21O4N (M~):
411.1471, found: 411.1468; Anal. Calcd. for C26H21O4N: C, 75.90: H, 5.14; N, 3.40. Found: C, 75.66: H, 5.45; N, 3.14.

Example 16: Com~ound 18 A solution of the enediyne Compound 2 (124 mg, 0.30 mmol) in CH2C12 (4 mL) was treated with Co2(CO)8 (260 mg, 0.76 mmol) and stirred at 25C for 5 minutes.
The solution was concentrated in vacuo and the residue was purified by flash chromatography to give the cobalt complex Compound 18 (291 mg, 98 percent).
Green crystalli~e solid; mp>300C (from ether);
Rf-0.80 (50 percent ether in petroleum ether); IR
(CDC13) V~X 3500, 2950, 2872, 2095, 2070, 2025, 172S, 1492, 1207 cm1; 1H NMR (500 MHz, CDC13): ~ 8.87 (bs, 1 H, aromatic~, 7.61-7.02 (m, 8 H, aromatic), 6.47 ~bs, 1 H~ N-CHtC)-C~, 6.38 (bd, J-10.7 Hz, 1 H, ole~inic), 6.19 (bd, ~-10.7 Hz, 1 H, ole~inic), 3.50 (bs, 1 H, OH), 2.70-1.71 ~m, 6 H,CH2); ~3C NMR (125 MHz, CDCl~
lg9.1, 198.6, 197.8, 151.3, 134.9, 132.9, 130.9, 129.4, 128.8, 127.2, 125.8, 125.3, 125.i, 124.8, 123.4, 121.6, 98.5, 88.9, 81.5, 80.1, 78.0, 73~9, 63.1, 59.0, 44.2, 24.9, 17.~: MS (FAB~) m/e (relative intensity) 1114 (M+Cs, 11), 1086 (M+Cs-CO, 18), 1058 ~M+Cs-2CO, 6), 1030 (M+Cs-3CO, 193, 1002 (M+Cs-4CO, 11), 943 (M~Cs-4CO-Co, 10), 918 (11), 890 (24), 862 (34), ~13 (100): HRMS

calcd. for C38H19O~6NCo~Cs (M+Cs) 1113.7086, found 1113.7001.

Example 17: Compound l9 A solution of the cobalt complex Compound 18 (291 mg, 0.30 mmol) in CH2Cl2 (4 mL) was treated at zero degree C with trifluoroacetic acid (68.6 ~L, 0.89 mmol).
After 5 minutes, the mixture was poured into saturated sodium bicarbonates solution (25 mL) and extracted with CH2Cl2 (3 x 25 mL). The combined organic layers were dried (MgSO4)~ evaporated n_vacuo, and purified by flash chroma~ography (silica, 50 percent ether in petroleum ether) to give the ketone Compound 19 (167.4 mg, 81 percent).
Brown crystalline solid: mp ~300C (from ether);
R~-0.25 (50 percent ether in petroleum ether); IR
~CDC13) V~x 3408, 2945, 2100, 2065, 2032, 1875, 1735, 1680, 1512, 1217 cm1; 1H NMR (500 MHz, CDC13): ~ 7.81 (d, 3=8.2 ~z, 1 H, aromatic), 7.42-7.11 (m, 8 H, aromatic), 7.00 (d, J=10.2 Hz, 1 H, ole~inic), 6.39 ts, 1 H, N-C H(C)-C], 5.52 (d, J=10.2 Hz, 1 H, olefinic), 3.35-1.82 (m, 7 H, CH2, OH); 13C NMR (125 MHz, CDCl3): ~
202.9, 198.9, 198.1, 154.3, 150.9, 144.0, 133.2, 132.8, 129.6, 128.3, 128.1, 126.7, 126.0, 125.8, 123.3, 121.8, 108.7, 93.2, 92.5, 82.1, 81.0, 68.5, 56.2, 38.0, 30.2, 21.6; MS (FAB~) m~ (relative intensity) ~8 (~S+Cs, 17.), 800 (18), 6~8 (74), 639 (20), 555 (3~), 527 (100~; ~RMS
calcd- for C32Hl~NOl0co2cs (M+Cs) 827.8727, found 827.8730; Anal. calcd. for C~2Hl~NOloCo2: C, 55.27; H, 2.75; N, 2.01; Co, 16.97. Foun~: C, 54.98; H, 2~79; N, 1.~6: Co, 15.22.

Example 18: Compound 20 Thiocarbonyldiimidazole (180 mg, 0.99 mmol) was added to a solution of the alcohol Compound 2 (137 mg, W092/025Z2 ~ PCT/US91/05436 0.335 mmol) and 4-dimethylaminopyridine (DMAP) (25 mg, 0.18 mmol) in dichloromethane (2 mL3 at 25C. After 48 hours, the solution was concentrated ln vacuo and the residue purified by flash chromatography (silic~, 80 percent ether in petroleum ether) to give thionoimidazolide 20 (160 mg, 95 percent). 20: white crystalline solid, mp 178-179C dec. (from etherJdichloromethane); Rf=0.62 (70 percent ether in petroleum ether); IR (CDCl3) V~X 3042, 2912, 2195, 1710, ~1500, 1495, 1212, 1105 cm1; H NMR (500 MHz, CDCl3):

8.49 (s, 1 H, N-CH=N), 7.71-7.05 (m, 11 H, aromatic), 5.93 (d, J=10.3 Hz, 1 H, olefinic), 5.73 (dd, J=10.3, 1.6 Hz, 1 H, olefinic), 5.~0 (d, J=1,6 Hz, 1 H, N-CH-C3C), 3.08 (d, J=ll.l Hz, 1 H, C~2), 2.46-1.70 (m, 5 ~, CH2); t3c NMR (125 MHz, CDCl3): ~ 179.1, 153.4, 151.0, 137.0, 135.9, 130.9, 129.4, 129.3, 128.2, 127.0, 126.4, 125.8, lZ5.4, lZ3.9, 123.2, 121.3, 117.7, 100.6, 94.3, 93.9, 88.9, 85.4, 74.5, 65.g, 63.2, 50.3, 28.0, 22.7, 18.4; MS (FA8+) m/e (relative intensity~ 653 ~M~Cs, 21), 419 (19), 379 (15), 286 (100), 154 (30); HRMS Calcd. for C30H2lN3O4SCs (M+Cs) 653.0385, found 653.0360; Anal.
calcd. for C30H21N30~S: C, 69.35; H, 4.07; N, 8.09; S, 6.17. Found: C, 69.01; H, 4.17; N, 7.91; S, 6.19.

Example 19: Com~ound 2~
A solution of the imidazolide Compound 20 (112 mg, 0.24 mmol), azobisisobutyronitrile (AIBN; 3 mg) and tri-n-butylstannane (~-Bu3SnH) (94 ~L, 0.36 mmol) was heated at 75C for 2 hours, the solvent was removed n vacu~, and the xesidue was purified by flash chromatography to give the deoxygenated product Compound 21 t71 mg, 75 percent).
White crystals; Rf-0.62 (30 percent ether in petroleum ether); mp 248-250C dec. (from ether);
IR(CDCl3) V~X 2945, 2872, 2232, 2205, 1712, 1465, 1325, W092t~2522 PCT/US91/OS436 c~ 122 - ' 1185 cm1; 1H NMR (500 MHz, CDCl3): ~ 7.67 (d, J=i.5 Hz, 1 H, aromatic), 7.6-7.14 (m, 8 H, aromatic), 5.84 (dd, J=10.5, 1.6 Xz, 1 H, olefinic), 5.72 (dd, J=10.5, 1.6 Hz, 1 H, olefinic), 5.57 (d, J=1.6 Hz, 1 H, N~CH-C-~, 3.85 ~d, J=1.6 Hz, 1 H, C~C-CH-C), 2.49 (m, 1 H, CH7 2.30 (m, 1 H, CHz), 2.12-1.60 (m, 4 H, C X2); 13C NMR
(125 MXz, CDCl3): ~ 151.0, 13$.5, 129.4, 129.4, 128.2, }27.3, 125.8, 125.8, 125.4, 125.~, 122.0, 122.0, 121.5, 101.8, 94.9, 91.4, 8R.8, 70.5, 61.1, 50.0, 29.8, 22.9, 22.5, 15.5; MS: m!e (relative intensity) 393 (20,M~), 294(9), 262(15), 212 (11), 149 (42); HRMS: Calcd. for Cz6Hl903N (M~): 393.1365, found: 393.1332.

Example 20: Com~ound 23 A solution of the enediyne Compound 21 (30 mg, 0.076 mmol) and 1,4-cyclohexadiene (0.5 mL) in benzene (2 mL) was treated with TsOH.H20 (18 mg, 0.09 mmol) and stirred at 25C for 24 hours. The solvent was removed in vacuo and the residue was purified by flash chromatography to give the diol Compound 23 (26 mgj 85 percent).
Colorless gum; Rf=0.35 (50 percent ether in petroleum ether); IR (CDCl3) V~x 3310, 3082, 292S, 1705, 1592, 1395, 1200 cm1; 1H NNR (500 MHz, CDC13): ~ 7.58 ~bd, J-4.4 Hz, 1 H, aromatic), 7.47 (bd, J-7.8 Hz, 1 H, aromatic), 7.40-7.09 (m, 10 ~, aromatic), 6.81 (d, J=8.1 Hz, 1 X, aromatic), 5.78 (s, 1 H, N-benzylic), 4.00 (bs, 2 H, OH), 3.24 (s, 1 H, ben2ylic), 2.42-0.72 ~m, 6 H, CH2); 13C NMR (125 MHz, CDCl3): ~ 151.0, 138.2, 134.7, 129.4, 129.4, 129.3, 128.8, 128.4', 128.3, 127.1, 126.9, 125.7, 125.0, 124.4, 121,8, '121.8, 121.5, 83.0, 66.2, 65.1, 51.2, 33.5, 27.1, 18.7: MS(FAB+): m/e (relative intensity) 546 (15, MtCs), 379 t31), 312 (30), 286 (100); HRMS: Calcd. for C26H2304NCs (M+Cs): 546.0681, found: 546.0691.

W092/02522 ~l? ~ 9 Pcr/vs9t/05436 Example 21: C~ound 23a HCl gas was bubbled through a solution of the enediyne Compound 21 (30 mg, O.076 mmol1 and 1,4-cyclohexadiene (0.5 mL~ in CH2Clz at 25c for 30 seconds.
The so}vent was removed in vacuo and the residue was purified by flash chromatography to give the chloride Compound 23~ (27 mg, 84 percent).
Pale yellow solid; mp=114-116"C: Rt=0.62 (50 percent ether in petroleum ether); IR (CDCl3) V~X 3500, 3065, 2932, 1712, 1495, 1382, 1200 cm1; 1H NMR (500 MHz, CDCl3): ~ 7.71-6.73 (m, 13 H, aromatic), 5.87 (s, 1 H, N-benzylic), 3.62 (s, 1 H, benzylic), 2.52 (bs, 1 H, OH), 2.50-1.60 (m, 6 H, CH2); MS: m/e (relative intensity) 564 (5, M+Cs), 419 (100), 379 (58); HRMS:
Calcd. for C26H2203NClCs (M+Cs): 564.0343, found:
564.0351.

Example 22: ComPound 24c Ethyl -omoace~at~ ~27 mg, 0.16 ~mol) was added to a mixture of the alcohol Compound 2 ~34 mg, 0.08 mmol) and Cs2C03 (67 mg, 0.2 mmol) in anhydrous DMF (2 mL) at 60C. The mixture was heated for 3 hours, diluted with ether (10 mL), washed with NH4C1 (2 x 3 mL), water (2 x 2 mL), and brine (2 mL). The organic layer was dried (MgSO4) and purified by flash chromatography to give the ester Compound 2~c (37.5 mg, 91 percent.
Colorless oil; R~=0.55 (50 percent ether in petroleum ether): lH NMR (500 MHz, CDCl3): ~ 8.63 (d, J=10.3 H-, 1 H arc-.~t$c), 7.45 7.10 (m, 8 H, aromatic), 5.82 (d, J=10.5 H , 1 H, olef^z.ic), 5.68 (dd, J=10.5, l.S Hz), 1 H, ole~inic), 5.51 td, J=1.5 Hz,.l H, CsC(C)CHN], 4.34 td, J=15.6 Hz, 1 H, C(O)CH20~, 4.28 ~d=15.6 Hz, 1 H, C~O)CH20], 4.24 (m, 2 H, OCH2C~3), 2.35-1.70 (m, 6 H, CH2), 1.30 (5, J=8.2 Hz, 3 H, OCH2CH3); 13C

'2 ~

NMR (125 MHz, CDCl3): ~ 169.5, 150.9, 135.6, 131.0, 129.3, 129.3, 128.1, 127.7, 125.7, 125.6, 123.9, 122.4, 121.6, 121.6, 98.2, 96.3, 94.5, as.o, 80.1, 73.2, 68.1, 63.2, 61.S, 50.6, 30.0, 29.8, 19.2, 14.5: HRMS calcd.
C30H2sO6NCs (M~Cs), 628.0736: found: 628.0750.
Compounds 24a, ~, d, e, f and ~ are similarly prepared.

Example 23: Alterna~ive Preparation of Compound 5 (a) ~oluenesulfonic acid (TsOH.H20; 8 mg, 0.05 mmol) was added in one portion to a solution of the alcohol Compound 2 (18 mg, 0.045 mmol), 1,4-cyclohexadiene ~0.2 mL) and benzene (0.5 mL) at 25C, and the solution was stirred for 24 hours. The organic solvent was removed in vacuo and the residue was purified by flash chromatography to give the ketone Compound 5 (15 mg, 90 percent).
(b) Trimethylsilyl trifluoromethylsulfonate (TMSOTf: 15 ~L, 0.08 mmol) was added to a solution of the alcohol Compound 2 (32 mg, O.078 mmol) and triethylsilane (Et3SiH) (40 mg, 0.32 mmol) in CH2C12 ~2 mL) at -78C. After 1 minute, the mixture was quenched at -78c with saturated NH4Cl solution (1 mL), diluted with ether (10 mL), washed with water (2 x 3 mL), brine 2S (3 mL) and then dried (MgS04). The organic solvent was removed in vacuo, and the residue was puri~ied by ~lash chromatography to give the ketone Compound 5 (22 mg, 75 percent).
(c) HCl gas was bubbled through a solution of the alcohol Compound 2 (32 mg, 0.078 ~mol) and 1,4- j cyclohexadiene (40 mg, 0.32 ~mol) in d chloromethane (4 mL) a~ 25C for 30 seconds. The solYent was removed i vacuo and the residue was purified by flash chromatography to give the ketone Compound 5 (25 mg, 80 percent).

W092/02522 . PCT/US91/05436 2~o~

- 1~5 -Example 24: ComPound 2b A solution of the phenyl carbamate Compound 2 (42 mg, 0.103 mmol) in dry methanol (4 mL) was treated wi~h sodium methoxide (17 mg, 0.31 mmol) and heated at 60C for 2 hours. The reaction mixture was diluted with dichloromethane (25 mL), washed with sodium bicarbonate solution (25 mL), dried (Na2SO4), evaporated in vacuo and the residue purified by flash chromatography (silica, 40 percent ether in petroleum ether) to gi~e methyl carbamate 2b (28.5 mg, 80 percent~. 2~: white crystalline solid, mp 126-127C (from ether/petroleum ether)~ Rf=0 . 43 (50 percent ether in petroleum ether);
IR tCDC13) v~ 3600, 3450, 2957, 2257, 2250, }706 cm1;
lH NMR (500 MHz, CDCl3): ~ 8.65 (d, J=8.0 Hz, 1 H, aromatic), 7.25-7.1Q (m, 3 ~, aromatic), 5.81 ~d, J=lo.1 Hz, 1 H, olefinic~, 5.69 (d, J~10.1 Hz, 1 H, olefinic), 5.4S (s, 1 H, CH-N), 3.82 (s, 3 H, OMe), 2.79 (s, 1 H, OH), 2.27-1.72 ~m, 6 H, CH2); 13C NMR (125 MHz, CDCl3):
135.9, 131.3, 127.8, 127.5, 126.1, 124.9, 123.9, 122.1, 1~0.7, 94.1, 88.3, 74.2, 73.1, 65.8, 64.2, 53.7, 50.1, 35.2, 23.2, 19.2, 15.2; MS ~ relative intensity) 347 (~, 100) 291 (35), 204 (50); ~RMS calcd. ~or C2,~,~O4 (k~) 347.1158, found 347.1159. Anal. calcd. for C2,H17NO4:
C, 72.61; H, 4.93; N, 4.03. Found: C, 72.63; ~, 5.24;
N, 3.79.

Example 25: ComDound ~0 Carbamate Compound 21 ~39 mg, 0.10 mmol) in THF
~3 mL) was treated at zero degrees C with LiAlH~ ~0.25 mL of a 1.0 M solution in etherr 0.25 mmol). After stirring for 30 minutes, the reaction was quenched with saturated sodium bicarbonate solution (1 mL), diluted with ether (20 mL), washed with 1.0 M aqueous LiOH
solution (2 x 5 m~) in order ~o remove phenol, dried ~Na2SO4~, filtered and stored under argon at -78C until 29~34~`~

required. MS (FAB+) m/e (relative intensity) 290 (97) r 278 (75), 274 (M~H, 98), 235 (100); Hl~MS calcd. for C19H1~NO (M+H) 274.1232, found 274.1247.
Physical data for further selected compounds:

Com~ound ~2 Rf=O. 63 (50 percent diethyl ether in benzene);
1H NMR (300 MHz, d8-THF/D20, 10~ 8.53 (d, J=8.8 Hz, 1 H, aromatic), 7.45-7.10 (m, 5 H, aromatic), 6.88 (dd, J=2.5 Hz, 1 H, aromatic), 6.63 (dd, J=8.8, 2.5 Hz, 1 H, aromatic), 5.97 (d, J=10.0 Hz, 1 H, olefinic), 5.78 (dd, J=10.0, 1.6Hz, 1 H, olefinic), 5.46 (bs, 1 H, CHN), 2.35-1.55 (m, 6 H, CH2).

CQm~ound 4 3 Rt'0.78 (50 percenk diethyl ether in benzene);
1H NMR (300 MHz, C6D6): ~ 8.97 (d, J=9.0 Hz, 1 H, aromatic), 7.72 (d, J=8.3 Hz, 1 H, aromatic), 7.52 (d, J=7.7 Hz, 1 H, aromatic), 7.13-7.01 (m, 5 H, aromatic), 6.97-6.87 tm, 2 H, aromatic), 6.81 (bd, J-8.9 Hz, 1 H, aromatic), 6.66 (t, J=7.9 Hz, 1 H, aromatic), 5.90 (bs, 1 H, C HN), 5.31 (d, J=10.1 Hz, 1 H, o}efinic), 5.17 (dd, J=10.1, 1.7 Hz, 1 H, olefinic), 5.13 and 5.04 (AB, J=16.0 Hz, 2 H, ArCH2O), 2.29 (bs, 1 H, OH), 2.15-1.85 ~m, 4 H, CH2), 1.70-1.60 (m, 1 H, CH2), 1.37-1.29 (m, 1 H, CH2); IR (C~) Y~x 3554, 2954, 2927, 1728, 1615, 1579, 1529, 1506, 1494, 1378, 1343, 1302, 1280, 1252, 1239, 1202 cm~l; HRMS Calcd for C33H25N2O~ (M+H) 561-11 found 561.1162.
Com~ound ~c Rf=O . 33 (S percent methanol in methylene chloride); lH NMR (300 MHz, CDC13):~ 7.43 (dd, J=5.3, 3.5 Hz, 1 H, aromatic), 7.25-7.10 (m, 4 H, aromatic), 3S 6.56 (dd, ~=8.4, 2.2 Hz, 1 H, aromatic), 6.53 (d, J=2.2 W092/025Z2 PCT~US91/05436 ~ ~, Hz, 1 H, aromatic, 5.58 (s, 1 H, CHN), 5.46 ~t, J=5.6 Hz, 1 H, CH2NHCON), 3.19-2.~5 (m, 2 H, CH2NHCON), 2.80 (dt, J=11.3, 6.3 Hz, 1 H, CH2CH2NH), 2.53-2.26 (m, 5 H, CH2CH2NH, CH2, OH), 1.84 (s, } H, OH), 1.6~ (dd, J=12.4, 4.7 Hz, 1 H, CH2), 1.61-1.36 (m, 5 H, CH2CH2NHCON, CH2CH2NH~ CH2), 0-94 (t, J=7.3 Hz, 3 H, CH3cH2cH2NHcoN)~
0.89 (dd, J=4.8, 2.4 Hz, 1 H, CH2j, 0.83 (t, J=7.3 Hz, 3 H, CH3CH2CH2NH), 0.78-0.59 (m, 1 H, CH2); IR (CHCl3) Y~x 3591, 343g, 3270, 2964, 2935, 1645, 1613, 1500, 145g, 1416, 1252, 1188 cml; HRMS Calcd. f~r C26H34N304 (M+H~:4s2.254s, found: 452.2549.

ComPo~~d 45 Rt-0.22 ~silica, 70 percent diethyl ether in petroleum e~her) lH N~R (500 MHz, CDC13):~ 7.92-7.1 (~, 9H, aromatic), 5.73 (d, J-10.1 Hz, 1 H, ole~inic), 5.63 (d, J~10.1 Hz, 1 H, olefinic), 5.37 ~bs, 1 H, NC H), 4.65-4.22 (m, 2 H, SO2CH2CH2), 3.73 (S, 1 H, C~CH2), 3.48 (m, 2H, SO2CH2CH2), 2.43-1.52 (m, 6 H, CH2~; 13C NMR (125 MHz, CDCl3): ~ 134.01, 129.3, 128.5, 128.1, 127.9, 127.1, 125.2, 124.8, 121.9, ~01.7, 93.7, 91.2, 88.6, 70.1, 60.9, 59.3, 55.0, 49.4, 29.3, 23.1, 22.3, 15.6; IR
(C~C13) Y~x 2975, 2950, 1715, 1360, 1300, 1150 cm~; HRMS
Calcd. for C2~H~SNCs (M~Cs~):618.0351, found: 618.0352.
Compound ~8 Rt~0.61 (silica, 70 percent diethyl e~her in petroleum ether) ~H NMR t500 MHz, CDC13):~ 8.32 (d, J=7.34 Hz, 1 H, aromatic), 7.11 (t, J=7.34 Hz, lH, aromatic), 6.82 ~t, J=7.34 Hz, 1 H, aromatic), 6.55 (d, J-7.34 Hz, 1 H, aro~atic), 5.83 (d, J=8.8 Hz, 1 H, olefinic), 5.73 (dd, J=8.8, ~.74 Hz, olefinic), 4.32 ~d, J=1.74 Hz, 1 H, NC H)~ 4.00 (bs, 1 H, NH), 3.50 (s, 3 H, 0C H3), 2.36-1.68 (m, 6 H, CH2); 13C NMR (125 MHz, CDC13):
142.2, 131.0, 128.3, 123.~, 122.5, 122.0, 119.3, 115.9, 2~8~3~

100.2, 96.9, 94.6, 87.3, 79.6, 72.9, 62.9, 29~0, 24.6, 19.0; IR (CHCl3) ~x 3400, 2950, 2850, 1100, 1080 cm~;
HRMS Calcd. for C20H~702N (~): 303.1337, found 303.1348.

Compound 50 R~=0.40 (silica, 70 percent d$ethyl ether in petroleu~ ether) lH NMR (500 MH~, CDCl3): ~ 7.63 (d, J=7.5 Hz, 1 H, aromatic), 7.35 (m, 2 H, aromatic), 7~18 (d, J=7.0 Hz, 1 H, aromatic), 7.14 (t, J=7.0 Hz, 1 H
aromatic), 7.09 (t, J=~.O Hz, 1 H, aromatic), 7.03 (m, 3 H, aromatic), 6.87 ~d, J=7.0 Hz, 1 H, aromatic), 6.80 (t, J=7.0 Hz, 1 H, aromatic), 6.61 (t, J-7.0 Hz, 1 H, aromatic), 6.31 (d, J=7.5 Hz, aromatic), 4.12 (2s, 2H, NH and N-CH), 3.73 (s, 1 H, OH), 3~62 (t, J=2.82 Hz, 1 H, CH-CH2), 2.80 (ddd, J=12.8, 12.8, 5.64 Hz, 1 H, CH2), 2.41 (ddt, J=12.8, 12.8, 4.5 Hz, 1 H, CHz), 1.75 (dd, J=13.16, 4.88 Hz, 1 H, CH2), 1.54 (m, 1 H, CEI2), 1.39 (bd, J=13.16 Hz, 1 H, CH2), 0.9 (m, 1 H, CH2); 13C NMR
(125 MHz, CDC13): 141.6, 139.2, 137.1, 135.1, 133.7, 130.6, 128.1, 127.8, 127.4, 127.0, 126.8, 126.4, 119.9, 115.8, 70.4, 62.1, 55.6, 33.3, 29.8, 28.0, 18.8: IR
(C~Cl3) V~X 3450, 3390, 3070, 2930, 2~70, 1490, 1470 c~1; HRMS Calcd. for C2sH23OSN (M~):385.1500, found 385.1500.
Compound 55 Rt~0.3 (50 percent diethyl ether in petroleum ether); 1H NMR (500 MHz, CDC13): ~ 8.21 ~d, J-8.8 Hz, 1 H, aromatic), 7.32 (t, J=7.6 Hz, 2 H, aromatic), 7.18 (t, J=7.6 Hz, 1 H, a~omatic), 7.10 (bd, J=6.9 Hz, 2 H, aromatic), 6.89 (bs, 1 H, aromatic), 6.65 tdd, J=8.8, 2.7 Hz, 1 H, aromatic), 5.82 (d, J=10 Hz, 1 H, olefinic), 5.67 (dd,~J=10, 1.7 Hz, 1 H, olefinic), 5.48 (s, 1 H, OH), 5.28 (bs, 1 H, CHN), 3.47 (s, 3H, OCH3), 2.28 (dd, J=15.1, 8.2 Hz, 1 H, CH2), 2.15 (m, 2 H, CH2), ~ ~ ~1 ,q 4~

1.92 (m, 2 H, CR2), 1.75 (m, 1 H, CH2): 13C NMR (12S MNz, CDC13): ~ 155.1, 150.9; 136.8, 133.2, 131.0, 130.1, 129.3, 125.8, 124.2, 124.1, 122.2, 12~L.6, 113.0, g9.5, 94.9, 93.9, 88.4, 79.3, 72.1, ~3.2, 5;'.1, 50.5, 28.5, 5 23.2, 18.9: IR(CHC13) V~x 3400, 3004, 2979, 2936, 2876, 1719, 1384 cml; XRMS Calcd. for C2~21NO5Cs (M+Cs):
57200474, found 572.0429.

Com~ound S8 Rf=0.3i (diethyl ether); 1H NMR (500 MHz, CDC13): ~ 7.37 ~d, J=10.3 Hz, 1 H, olefinic), 6.36 (dd, JolO.4, 2.0 Hz, 1 H, olefinic), 5.85 (d, J=9.8 Hz, } H, ole~inic), 5.81 (dd, J~1.7 Hz, 1 H, olefinic), 5.10 (d, ~=2.0 Hz, 1 H, olefinic), 4.13 (d, J=4.0 Hz, 1 ~, NH), lS 4.07 (d, J=3.0 Hz, H, ole~inic), 3.72 (dd, J=4.2, 1.7 Hz, 1 H, CHN), 3.41 (s, 3H, OCH3), 3.20 (m, 1 H, CH2), 2.36 (m, 1 H, CHz), 2.10 (m, 1 H, CH2), 1.88 (m, 1 H, CH2), 1.74 (m, 1 H, C~2); C NMR (125 MHz, C6D6): ~
184.4, 157.2, 137.7, 134.8, 123.3, 122.6, 113.3, 98.~, 98.4, 90.7, 87.2, 78.1, 74.8, 74.0, 58.4, 57.8, 51.5, 27.5, 27.4, 14.3; IRtCHC13) V~X 3527, 3385, 2956, 2928, 2855, 1656, 1597 cm1; W (CHC13, c=2.2 x 10-4): A(log 330 (3.09), 285 (3.37), 256 (3.7), 244 (3.5); HRMS
Calcd. for C20H1704NCs (M~C~): 468.0212, found ~68.0254.
ComPound 76 Rf = 0.-44 (33 percent ethyl ether in petroleum ether~: 1H NMR (300 M~z, CDC13) ~ 7.68 (dd, J = 7.8, 1.1 Hz, 1 H, aromatic), 7.52 ~br s, 1 H, aromatic~, 7.37 (t, J = 7.7 Hz, 2 H, aromatic), 7.30 (dd, J = 7.4, 1.1 Hz, 1 H, aromatic), 7.26-7.13 (m, 8 H, aromatic), 5.61 (s, 1 H, CHN), 3.87 (t, J - 2.8 Hz, 1 H, CH2CH-), 2.49 (dd, J
= 15.0, 7.8 Hz, 1 H, CH2), 2.32-2.19 (m, 1 H, CH2), 2.08-1.98 (m, 2 H, CH2), 1.90~1.82 (m, 1 H, CH2), 1.68-1.55 (m, 1 H, CH2): 13C NMR (125 MHz, CDC13) ~ 150.9, W092/02522 PCT/US91/0~436 c~ 130 135.6, 130.0, 129.2, 129.2, 12~.9/ 128.8, 128.6, 128.3, 128.3, 128.1, 128.1, 127.7, 127.2, 126.8, 125.6, 125.2, 121.5, 121.5, 96.9, 90.7, 90.6, 88.8, 70.1, 60.7, 49.7, 28.9, 22.7, 22.6, 15.5; W (EtOH) A~x (log ~) 282 (3.55), 260 (sh, 3.74), 237-210 (br, 4.36-4.32) nm; HRMS
CalcdO for C30H21NO3Cs: 576.0576 (~+Cs~), found 576.0611 (M+Cs~).

Com~ound 87 Rf z 0.52 (33 percent ethyl ether in petroleum ether); lH NMR (300 MHz, CDC13) ~ 7.75 (s, 1 H, aromatic), 7.73 (s, 1 H, aromatic), 7.72-7.66 ~m, 3 H, aromatic), 7.53 (br s, 1 H, aromatic), 7.50-7~42 (m, 2 H, aromatic), 7.38 (t, J = 7.7 HZ, 2 H, aromatic), 7.30-15 - 7.13 (m, 5 H, aromatic), 5.64 (s, 1 H, CHN), 3.92 (br s, 1 H, C~2CH~, 2.53 (dd, J = 15.4, 7.5 Hz, 1 H, CH2), 2.34-2.22 (m, 1 H, CH2), 2.12-2.00 (m, 2 H, CH2), 1.91-1.84 (m, 1 H, CH2), 1.70-1.60 (m, 1 H, CH2); 13C NMR tl25 NHz, CDC13) ~ 150.9, 135.8, 132.3, 131.7, 130.3, 129.3, 129.3, 128.7, 128.4, 128.3, 128.1, 127.7, 127.6, 127.6, 127.3, 127.2, 125.6, 125.2, 123.8, 122.7, 121.5, 96.4, 90.7, 90.5, 88.8, 70.2, 60.8, 49.8, 28.g, 22.7, 22.6, 15.5; HRMS Calcd. for C34H23N03Cs: 626.0732 (M+Cs~), found 626.0732 ~M+Cs~).
ComDound ~
Rf = 0.53 (50 percent ethyl ether in benzene);
H NMR (300 MHz, CDCl~) ~ 8.40 (s, 1 ~, aromatic), 8.23 (s, 1 H, aromatic), 8.22 (s, 1 H, aromatic~, 7.99-7.88 (m, 2 H, aromatic), 7.75 (dd, J - 7.7, 1.7 Hz, 1 H, aromatic~, 7.54 (s, 1 H, aromatic), 7.50-7.40 (m, 5 H, aromatic), 7.30 (d, J = 7.4 Hz, 3 H, aromatic), 7.14-7.00 (m, 2 H, aromatic), 6.11 (s, 1 H, CHN), 3.62 (br s, 1 H, CH2CH), 2.46 (dddd, J = 10.8, 10.8, 3.3, 3.3 Hz, 1 H, CH2), 2.30 (ddd, J = 13.6, 13.6, 6.0 Hz, 1 H, CH2), W092/02522 PCT/US9t/0543~
2 ~ 3 c~

- 13~ -1.94 (dd, J = 13.3, 4.~ Hz, 1 H, CH2), 1.53 (br t, J =
13.5 Hz, 2 H, CH2), 0.98 (ddddd, J = 13.9, 13.9, 13.9, 4.3, 4.3 Hz, 1 H, C~2)-Compound llSa Pale yel}ow oil: Rf= 0.39 ~silica, 10 percent methanol in dichloromethane), [a]D~ = -87.3 ~c = 0.48, CHCl3), lH NMR, (500MHz, C6D6): ~ = 8.97 (dd, 1 H, J =
4.2, o.7 Hz, Dyn-Ar~, 7.52 (m, 1 H, Dyn-Ar), 7.41-6.98 Cm, 12 H, 11 Ar, propargylic H), 6.98 (dd, 1 H, J = 7.1, 7.1 Hz, Dyn-Ar), 5.89 (bs, 1 H, OH), 5.83 (bs, 1 H, O-N-H), 5.78 (s, 1 H, E-l), 5.28 (d, 1 H, J = 10.0 Hz, vinylic H), 5.10 tdd, 1 H, J = 10.0, 1.7 Hz, vinylic H), 4.58-4.51 (m, 2 H, CH2-Ph), 4.50 (d, 1 H, J = 7.4, A-l), 4.48-4.40 (m, 1 H, E-5ax), 4.25-4.17 (m, 1 H, E-5eq), 4.13-4.02 (m, 3 H, A-3, OCH2CH2O), 4.01-3.94 (m, 1 H, OCH2CH2O), 3.93-3.90 (m, 1 ~, E-3), 3.77 ~dd, 1 ~, J =

9.5, 7.3 Hz, A-2), 3.69-3.52 ~m, 1 H, A-5), 3~26 (s, 3 H, OCH3), 2.78-2.66 ~m, 2 H, E-4, N-CH2), 2.65-2.57 ~m, 1 H, N-CH2), 2.47 ~dd, 1 H, J = 12.0, 2.2 Hz, E-2eq), 2.44 (dd, 1 H, J = 9.5, 9.5 Hz, A-4), 2.31 (dd, 1 H, J =
}4.5, 6.5 Hz, Dyn-CH2~, 2.04 ~d, 1 H, J = 6.7 Hz, Dyn-- CH2), 1.9501.83 ~m, 4 H, CH2), 1.43 (dd, 1 H, J = 14.5, 9.3 Hz, E-2ax), 1.33 (d, 3 H, J = 6.1 Hz, A-6), 1.04 (t, 3 H, J = 6.5 Hz, N-CH2-CH3): IR (CHCl3) ~x = 2965~ 2931 1733, 1380, 1323, 1146, 109~, 1071 cm1; HRMS calcd. for C49H55N311 (M+Cs ) 994-2891; found 994.2904.

Compound 1~5b Pale yellow oil; Rf = 0.38 (silica, 10 peroent methanol in dichloromethane), [a]D25 - +12S.7 (c- 0.68, CHCl3), lH NMR (500MHz, C~D6): ~ a 8.93 ~dd, 1 H, J =
4.2, 0.7 Hz, Dyn-Ar), 7.56 (dd, J = 7.4, 1.4 Hz, Dyn-Ar), 7.32-7.02 (m, 12 H, 11 Ar, propagylic H), 6.90 (dd, 1 H, J = 7.1, 7.1 Hz, Dyn-Ar), 5.90 (bs, 1 H, O-N-H), W092/02522 PCT/US9t/0~436 s~
5.88 (bs, 1 H, OH~, 5.82 (s, 1 H, E-l), 5.29 (d, 1 H, J
= 1002 Hz, vinylic H), 5.11 (dd, 1 ~, J = 10.2, 1.7 Hz, vinylic H), 4.56-4.51 (m, 2 H, CH2Ph), 4.49 (dd, 1 H, J
= 11.1, 9.0 Hz, E-5ax), 4.48 (d, 1 H, J = 7.4 Hz, A~
4.22 -4.17 (m, 2 H, OC~2CHzO), 4.14 ~dd, 1 H, J = 11.1, 4.7 ~z, E-5eq), 4.09 (dd, 1 H, J = 9.~, 9.5 Hz, A-3), 4.06-4.01 (m, ~ H, OCH2C~20), 3.93-3.88 (m, 1 H, OCH2C~20), 3.87-3.81 (m, 1 H, E-3), 3.78 (dd, 1 H, J -9.5, 7.1 Hz, A-2), 3.58 (dq, 1 H, J = 9.5, 6.1 Hz, A-5), 3.27 (s, 3 H, OCH3), 2.84 (ddd, 1 H, J - 9.0, 9.0, 4.7 - Hz, E-4), 2.77 (m, 2 H, N-CH2), 2.54 (dd, 1 H, ~ = :L2.2, 2.5 Hz, E-2eq), 2.42 (dd, 1 H, J = 9.5, 9.5 Hz, A-4), 2.30 (dd, 1 H, J = 14.6, 10.5 Hz, Dyn-CH2), 2.06 (dd, 1 H, J = 14.6, 7.1 Hz, Dyn-CH2), 1.97-1.83 (m, 4 H, Dyn-CH2), 1.51 (dd, 1 H, J = 12.2, 9.2 Hz, E-2ax), 1.33 (d, 3 H, J - 6.1 Hz, A-6), 1.10 (t, 3 ~, J - 6.5 Hz, N-CH2-CH3); IR (CHC13) : V~x ~ 2962, 2957, 2929, 1733, 1386, 1323, 1146, 109~, 1070 cml; HRMS calcd. for C4~55N30~1 (M~Cs ~ 994.2891: found 994.2904.

Although the present invention has now been described in terms of certain preferred embodiments, and exemplified with respect thereto, one skilled in the art will readily appreciate that various modifications, changes, omissions and substitut$ons may be made without departing from the spirit thereo~.

Claims (34)

CLAIMS:

1. A fused ring compound corresponding to the structural formula wherein A is a double or single bond;
R1 is selected from the group consisting of H, C1-C6 alkyl, phenoxycarbonyl, benzyloxycarbonyl, C1-C6 alkoxycarbonyl, substituted C1-C6 alkoxycarbonyl, and 9-fluorenylmethyloxycarbonyl;
R2 is selected from the group consisting of H, carboxyl, hydroxylmethyl and carbonyloxy-C1-C6 alkyl;
R3 is selected from the group consisting of H and C1-C6 alkoxy;
R4 is selected from the group consisting of H, hydroxyl, C1-C6 alkoxy, oxyacetic acid, oxyacetic C1-C6 hydrocarbyl or benzyl ester, oxyacetic amide, oxyethanol, oxyimidazilthiocarbonyl and C1-C6 acyloxy;
R6 and R7 are each H or together form with the intervening vinylene group form a one, two or three fused aromatic six-membered ring system;
W together with the bonded vinylene group forms a substituted aromatic hydrocarbyl ring system containing 1, 2 or 3 six-membered rings such that said fused ring compound contains 3, 4 or 5 fused rings, all but two of which are aromatic, and in which W is joined [a, b] to the nitrogen-containing ring of the structure shown; and R8 is hydrogen or methyl, with the proviso that R8 is hydrogen when W together with the intervening vinylene group is 9,10-dioxoanthra.

2. The fused ring compound according to claim 1 wherein R6 and R7 are H, or together with the intervening vinylene group form a benzo or naphtho ring system.

3. The fused ring compound according to claim 1 wherein said 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.

4. The fused ring compound according to claim 3 wherein the formed aromatic hydrocarbyl ring system is an otherwise unsubstituted benzo ring.

5. The fused ring compound according to claim 3 wherein the formed aromatic hydrocarbyl ring system is a benzo ring substituted at one or two of the remaining positions by a radical selected from the group consisting of hydroxyl, C1-C6 alkoxy, o-nitrobenzyloxy, benzyloxy, C1-C6-acyloxy, oxyethanol, oxyacetic acid and halo.

6. The fused ring compound according to claim 3 wherein the formed aromatic hydrocarbyl ring system is a naphtho ring having a 4-position radical selected from the group consisting of hydroxyl, C1-C6 alkoxy, C1-C6 acyloxy, benzyloxy and halo, and radicals at the 5- and 8-positions selected from the group consisting of hydroxyl, C1-C6 alkoxy, benzyloxy, C1-C6 acyloxy, oxo and halo.

7. The fused ring compound according to claim 3 wherein the formed aromatic hydrocarbyl ring system is a 9,10-dioxoanthra ring substituted at one or more of the 4-, 5- and 8-positions by a radical selected from the group consisting of hydroxyl, C1-C6 alkoxy, C1-C6 acyloxy, and halo.

8. The fused ring compound according to claim 1 wherein A is a single bond.

9. A fused ring compound corresponding in structure to the formula wherein A is a double or single bond;

R1 is selected from the group consisting of H, C1-C6 alkyl, phenoxycarbonyl, benzyloxycarbonyl, C1-C6 alkoxycarbonyl, substituted ethoxycarbonyl and 9-fluorenylmethyloxycarbonyl;
R2 is selected from the group consisting of H, carboxyl, hydroxylmethyl and carbonyloxy-C1-C6 alkyl;
R3 is selected from the group consisting of H and C1-C6 alkoxy;
R4 is selected from the group consisting of H, hydroxyl, oxyacetic acid, oxyacetic C1-C6 hydrocarbyl or benzyl ester, oxyacetic amide, oxyethanol, oxyimidazilthiocarbonyl and C1-C6 acyloxy;
R5 is selected from the group consisting of H, hydroxyl, C1-C6 alkoxy, o-nitrobenzyloxy, benzyloxy, and C1-C6 acyloxy;
R6 and R7 are each H or together form with the intervening vinylene group form a one, two or three fused aromatic six-membered ring system; and R8 is methyl or hydrogen.

10. The fused ring compound according to claim 9 wherein R2, R3, R5, R6 and R7 are H.

11. The fused ring compound according to claim 10 wherein R1 is phenoxycarbonyl, phenylsulfonylethoxycarbonyl or naphthylsulfonylethoxycarbonyl.

12. The fused ring compound according to claim 11 wherein R4 is selected from the group consisting of H, hydroxyl, C1-C6 alkoxy, oxyacetic acid, imidazylthiocarbonyloxy, oxyacetic amide and oxyacetic C1-C6 hydrocarbyl or benzyl esters.

13. A fused ring compound corresponding in structure to the formula wherein R1 is selected from the group consisting of H, phenylsulfonylethoxycarbonyl, naphthylsulfonylethoxycarbonyl, phenoxycarbonyl and benzyloxycarbonyl; and R4 is selected from the group consisting of H, hydroxyl, oxyacetic acid, oxyacetic C1-C6 hydrocarbyl or benzyl ester, oxyacetic amide, oxyethanol, oxyimidazilthiocarbonyl and C1-C6 acyloxy.

14. The fused ring compound according to claim 13 wherein R1 is phenoxycarbonyl, phenylsulfonylethoxycarbonyl or naphthlsulfonylethoxycarbonyl.

15. The fused ring compound according to claim 14 wherein R4 is selected from the group consisting of H, hydroxyl, and oxyethanol.

16. A fused-ring compound corresponding to the formula wherein R1 is selected from the group consisting of H, phenoxycarbonyl, benzyloxycarbonyl, phenylsulfonylethoxycarbonyl and naphthylsulfonylethoxycabony:
R4 is selected from the group consisting of H, hydroxyl, oxyacetic acid, oxyacetic amide, oxyacetic C1-C6 hydrocarbyl or benzyl ester and oxyethanol; and R5 is situated meta or para to the nitogen atom bonded to R1 and is selected from the group consisting of hydroxyl, C1-C6 alkoxy, benzyloxy, C1-C6 acyloxy, oxyethanol, oxyacetic acid, an oxyacetic C1-C6 hydrocarbyl or benzyloxy ester and o-nitrobenzyloxy.

17. The fused ring compound according to claim 16 wherein R1 is phenylsulfonylethoxycarbonyl.

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-C6 acyloxy.

20. A pharmaceutical composition that comprises a DNA-cleaving or cytotoxic amount of a fused ring compound having the structural formula shown below dissolved or dispersed in a physiologically tolerable diluent wherein A is a double or single bond:
R1 is selected from the group consisting of H, C1-C6 alkyl, phenoxycarbonyl, benzyloxycarbonyl, C1-C6 alkoxycarbonyl, substituted C1-C6 alkoxycarbonyl, and 9-fluorenylmethyloxycarbonyl;
R2 is selected from the group consisting of H, carboxyl, hydroxylmethyl and carbonyloxy-C1-C6 alkyl;
R3 is selected from the group consisting of H and C1-C6 alkoxy;
R4 is selected from the group consisting of H, hydroxyl, C1-C6 alkoxy, oxyacetic acid, oxyacetic C1-C6 hydrocarbyl or benzyl ester, oxyacetic amide, oxyethanol, oxyimidazilthiocarbonyl and C1-C6 acyloxy:
R6 and R7 are each H or together form with the intervening vinylene group form a one, two or three fused aromatic six-membered ring system;
W together with the bonded vinylene group forms a substituted aromatic hydrocarbyl ring system containing 1, 2 or 3 six-membered rings such that said fused ring compound contains 3, 4 or 5 fused rings, all but two of which are aromatic, and in which W is joined [a, b] to the nitrogen-containing ring of the structure shown; and R8 is hydrogen or methyl, with the proviso that R8 is hydrogen when W together with the intervening vinylene group is 9,10-dioxoanthra.

21. The composition according to claim 20 wherein R6 and R7 are H, or together with the intervening group form a benzo or naphtho ring system, and R2, R3 and R8 are H.

22. The composition according to claim 21 wherein said 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.

23. The composition according to claim 21 wherein the formed aromatic hydrocarbyl ring system is a benzo ring substituted at one or two of the remaining positions by a radical selected from the group consisting of hydroxyl, C1-C6 alkoxy, benzyloxy, C1-C6-acyloxy, oxyethanol, oxyacetic acid and halo.

24. The composition according to claim 21 wherein A is a single bond.

25. The composition according to claim 21 wherein R1 is pehnoxylcarbonyl, phenylsulfonylethoxycarbonyl or naphthylsulfonylethoxycarbonyl.

26. The composition according to claim 25 wherein W is subsituted or unsubstituted benzo.

27. The composition according to claim 26 wherein R6 and R7 are both H.

28. The composition according to claim 27 wherein said benzo group, W, is substituted meta or para to the nitrogen atom bonded to R1 with a moiety selected from the group consisting of hydroxyl, C1-C6 alkoxy, benzyloxy, C1-C6 acyloxy, oxyethanol, oxyacetic acid and an oxyacetic C1-C6 hydrocarbyl ester.

29. A chimeric compound comprised of an aglycone portion bonded to (i) an oligosaccharide portion or (ii) a monoclonal antibody or antibody binding site portion thereof that immunoreacts with target tumor cells, wherein said aglycone poriton is a fused ring compound corresponding to the structural formula wherein A is a double or single bond;

R1 is selected from the group consisting of H, C1-C6 alkyl, phenoxycarbonyl, benzoxycarbonyl, C1-C6 alkoxy carbonyl, substituted C1-C6 alkoxycarbonyl, and.
9-fluorenylmethyloxycarbonyl;
R2 is selected from the group consisting of H, carboxyl, hydroxylmethyl and carbonyloxy-C1-C6 alkyl;
R3 is selected from the group consisting of H and C1-C6 alkoxy;
R4 is selected from the group consisting of H, hydroxyl, C1-C6 alkoxy, oxyacetic acid, oxyacetic C1-C6 hydrocarbyl or benzyl ester, oxyacetic amide, oxyethanol, oxyimidazilthiocarbonyl and C1-C6 acyloxy;
R6 and R7 are each H or together form with the intervening vinylene group form a one, two or three fused aromatic six-membered ring system;
W together with the bonded vinylene group forms a substituted aromatic hydrocarbyl ring system containing 1, 2 or 3 six-membered rings such that said fused ring compound contains 3, 4 or 5 fused rings, all but two of which are aromatic, and in which W is joined [a, b] to the nitrogen-containing ring of the structure shown; and R8 is hydrogen or methyl, with the proviso that R8 is hydrogen when W together with the intervening vinylene group is 9,10-dioxoanthra;
said oligosaccharide portion comprising a sugar moiety selected from the group consisting of ribosyl, deoxyribosyl, fucosyl, glucosyl, galactosyl, N-acetylglucosaminyl, N-acetylgalactasaminyl, a saccharide whose structure is shown below, wherein a wavy line adjacent a bond indicates the position of linkage , , , , , and said monoclonal antibody or combining site portion thereof being bonded to said fused ring compound aglycone portion through an R4 oxyacetic acid amide or ester bond or an oxyacetic acid amide or ester bond from W, and said oligosaccharide portion being glycosidically bonded to the aglycone portion through the hydroxyl of an R4 oxyethanol group or the hydroxyl of an oxyethanol-substituted W.

30. The chimeric compound according to claim 29 wherein A is a single bond, R2, R3, R6, R7 and R8 are hydrogen, and W is benzo.

31. The chimeric compound according to claim 30 wherein said aglycone portion is bonded to said oligosaccharide portion.

32. The chimeric compound according to claim 31 wherein the glycosidic bond between the aglycone and oligosaccharide portions is formed form an R4 oxyethanol group.

33. The chimeric compound according to claim 31 wherein R1 is phenoxycarbonyl, phenylsulfonylethoxycarbonyl or naphthylsulfonylethoxycarbonyl.

34. The chimeric compound according to claim 32 wherein said oligosaccharide portion is selected from the group of oligosaccharides shown.