CA2136234A1 - Enantiomeric dynemicin analogs, preparation and use thereof - Google Patents

Abstract

An enantiomer of 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 enantiomeric compounds have DNA-cleaving, antimicrobial and tumor growth-inhibiting properties that are enhanced over their racemates. Chimeric compounds having the enantiomeric 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 an enatiomeric compound or an enantiomeric chimer are disclosed, as are methods of preparing an enantiomeric compound.

Description

` WO 93/~3046 .~ ~ 3 6 2 ~ ~l PCl'/lJS93/04708 ENANTIOMERIC DYNEMICIN ANALOGS, PREPARATION AND llSE ~HERE~F

l~sc~' tlon :
The present inv~ntion relat s to novel DNA-cleaving, c:ytotoxic and anti-tumor compounds, and particularly to Qnantiomeric fused ring compound systems that cc~ntain ~n: en~diyne macr~cyclic ring and al50 an epoxide ring, as w~Il as chimeras that contain such a ~ ~:
fus~d ring compound sys~emO

B ckaroun~
r~ynemicin ~ ~Compourld 1 shown below), n whe~e Me is raethyl, is~ a potent antibactexial and anticanc~r agen~ r~centIy isolated i~rom ~li~3~ ;
che~sina ~ ~a): Konishi e~.~ al, J. ~ oc:, 3 0 ~ 37I5-37 16~ ~ ( 1990 )~; ~ (b): ~ Ronishi et al r, J Antib io~
4~ 1449-14;52 :(~1989) ] .~ ~ Its ~ striking molee~ular structure C~ombines; characteristics of~ oth the enediyne ~;olik; et 5h = 5~3461-3462 (1987); Golik et al.,:~ 2~:3462-3464 (1987); Lee et~
35~ a~~ 5O~Q~:3464-3466 (l98~; Ellestad~

~36~ -2- !

~t al., J. ~m. Chem. Soc., 109:3466 3468 (1987~] and the anthracycline ~I'Anthracycline ~ntibiotics':, H.S. El ~ adem, ed~, Academic Press, New York (1982) and "Recent Aspects in Anthracyclinone Chemis~ry", Tetrahedron Symposia-in-Print No. 17 ~ T~Ro Kelly, ed., Tetrahedron;
40:4537-4794 (1984)] clas~ of anti~iotios, and presents a con~iderable challenge ~o organic synthesis as well as a unique opportunity ~or the development of new synthetic technology and therapeutic agents.
The calicheamicin and esperamicin deriYatives are perhaps the best known of the enPdiyne compounds.
~ For a key paper describing the first synthesis of calicheamicinone, see: (a) Cabal et al., Soc., 112:3253 (1990). For other selected studies of lS model systems in the area of calicheamicins-esperamicins, see:~(b) Nicolaou et al, J. Am. ~hem.
Soc~, 110:4866-4868 (1988~; (c) Nicolaou et al., ~_ Chem. Soc., 10:7~47-724a (1988); ~d) Schoenen et al., Tetrahedron Lett., 30:376S-3768 (1989); (e) Magnus et al., J. ~ . Chem. Soc., 110:6921-69~3 (1988; (f) Kende et al., Tetrahedr n Lett., 29:4217-4220 (1988).

Brief Summar~ of_the In~n The present in~fentiorl relates to novel 2 5 enantiomeric fused ring compound systems that contain an epoxide rin~ and an: enediyne macrocyclic ring, and thus have structural features similar to dynemicin Ar A
contemplated enantiomeric fused ring compound is substantially free of the other enantiomer. The 30~ compounds have DNA-cleaving, anti~io~ic and antitumor :~ activities. :Compositions and mQthods of making and : usiny the compounds are disclosed.
An entiomeric fused ring compound of the invention has a structure that corresponds to the formula .

W093/2~6 2 1 3 6 7~ PCT/US93/04708 R~

wherein A is a double or single bond;
R1 is selected from the group consisting of H, ~ C1-C6 alkyl, phenoxycar~onyl, ben2yloxycarbonyl~ cl-C6 alkoxycarbonyl, ~ubstituted: C1-C6 alkoxycarbonyl ;(particularly ubs~ituted ethoxycarbonyl where ~he ; su`bstituent is: phenylsulfonyl or naphthylsulfonyl, with :phenylsu~lfonyI most particularly preferred), o~nitrobenzylo~c~rbonyl and : 9-~luorenylmethyl~oxycarhQnyl;
is s~lected ~rom the group consisting of H, carbo ~ l, hydxoxylmethyl and c~rbonyloxy Cl-C6 alkyl; ~
: R3 is selected ~rom:the gro~p consistin~ of H
and Cl-C6 a1koxy; ~
~ 4 is~selected from th~ group consist~in~ of H, hydroxyl,~ C1-C6~:~alkoxy, oxyacetic:acid, oxyacetic C1-C6 : hydrocarbyl or~b~nz~l e~ter, oxyacetic ami~e, xylmidazil~iocarbonyl and~C1- 6 acylo~y~
:::and R7:are each H or ~ogether with the 3~ unsatur~t~d~:oa~on~atoms ~o~ tha in~erv~ning vinylene ;:
gro~p~orm~a one,~two or ~hree fused aromatic six~
: ;m~mbered~ring~:system;~
W~togeth~r~with tha~ca~bon atoms of the;~
depi~ted,~intervening~;vinylene ~roup forms a~ ~romatic:
3;5~ `;hydrocarbyl ~ring:~system:containing l, 2 or 3 six-PCT/US~3/04708 membered rings such that the fused ring compound contains 3, 4 or 5 fused rings, all but two of which are aromatic, and in which tha~ aromatic hydrocarbyl ring system, W, is joined [a, b] to the structure 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 ~inylene group is 9,10-dioxoanthra.
In preferred practice, W together with the a intervening vinylidene ~roup ~crms a benzo ring 50 that a cont~mpla~ed ~nantiom~ric compound ha~ ~he structural ~rmula ~h~wn b~low.

wherein R5 is selected from the group ~consisting of hydrogen, C1-C6 alkoxy, hydroxyl, Cl~C6 acyloxy, oxyethanol, oxyacetic acid, oxyacetic acid amide, oxyacetic Cl-C6 hydrocarbyl ester, oxyethanol ter~iary amino- or quaternary ammonium-substituted C2-C~
alkyl carboxylate, 3-hydroxyprop-1-ynyl, o-nitrobenzyloxy and halo, and A and the remaining R
groups are as before described.

I` W093/23~6 ~? ~ 3 5 ,~ 3 4 PCT/US93/047~8 More particularly, in one embodiment, R2, R3, Rs, R6, R7 and R8 are hydro~en so that a contemplated enantiomeric compound of th~ invention corresponds to the structural formula shown bel~w, where Rl and R4 a~e S as previously defined.

o ~ R1~ ,~

.: ~. ~ ' F'f4 15~
More;preferably, R5 i~ Cl-C6 alkoxy; hydroxyl, :Cl-C6~acyloxy, carboxyl~, C1oC6:hydrocar~y} or benzyl carb~oxylate,~ oxyethanol, oxyacetio:acid, oxyacetic acid am~ide,~oxyethanol tertiary amino or quaternary 20~ ammonium-sub tituted~:C2-~ alkyl carboxylate~or : 3-hydroxyprop-1-ynyl~and F~ is hydrogen (H) or hydroxyl~
so that~a~fused~:ring compound~has the struetural formula shown below.

W~93~23~6 PCT/US93/04708 2~36?'~ 60 Any of the above fused ring enediyne compounds can bei prepared as a single enantiomer. Where the R~ ' group is also prepared from ~n indi~idual chiral compound, as where R1 is a 2-mono- or di~Cl C6 alkyl- ' sub~tituted-2-phenylsu~fonyl e~hoxycarbonyl group, any of t~e above compounds can be pre~en~ as a further en~ntiomer.
Al~o contemplated i~ a chimeric compound (also re~erred ~o as a chimer ox chimera) that is comprised of a before-des~rib~d enantiomeric fused ring compound as an aglycone portion bonded to (i) an oligosaccharide portion or (ii) a monoclonal antibody or antibody combining site portion thereof t~at immunoreiacts with target tumox cells.
The oligossccharide portion comprises a sugar moiety ~e~ected from the~group consisting of ribosyl, ` deoxyribosyl, fucosyl~ gluc~syl, galactosy~, : N-acetylglucosaminyl, N-acetylgalactasaminyl, a saccharide whose structure is shown below, wherein a wavy line adjacen~ a bond indicates the position of linkage.

:

:
`: : : :
.

, ~ ;;,: ` ~ ` ' :

! WO 93~23~46 PCI/US93/04708 35~2 O~ 0 HN~ ~ ~ F~N~J"OH
V~
PllcH~o C~ S ~ H
~N~

~O~O~N~ ~a~

co~ ;c ~~o ~5~

~O~ ~S O ~ I X~
H ~ ~ .O Y
~ ~N~ ~o~~O

~ ~ 7 ~o~O ~,.,,o ~

011~ ~N,~O10J

~OH ~ ~ o ~ ~NX~J O~
A OH o~

yO jh ~ ~N~

~o~J ~

: ~ 1H

WOg3/23046 PCT/U~93/Q~708 ~36î~4 A monoclonal antibody or binding site portion thereof is bon~ed to the enantiomeric fused ring compound aglycone po ~ ion through an R4 oxyacetic acid amide or ester bond, an o~yacetic acid amide or ester bond or o~yethanol ester bond from W such as from an Rs group. ~n oligosaccharide portion is glycosidically bonded to thQ aglycone portion through the hydroxyl of an R4 oxyethanol group or the hydroxyl o~ an oxyethanol-substituen~ o~ W, e.g. an R5 group.
A pharmaceutical composition is also ~ contemplated. That pharm~ceutical composition contains a DNA cleavin~, antibiotic or tumor cell growth-inhibiting amount of a before-de~ined enantiomeric : compound or chimera as activ~ agent dissolved or dis~ersed in a physiologica}ly tolerabl~ diluent.
~ n enan~io=eric compound, chimera or a pharmaceutical composition of either is also useful in a : ~ me~h~d for cleaving DNA, ~or i ~ ibiting ~umor ~rowth and as an antimicro~ial. In accordance with such a method, the DNA to be cleaved, target tumor cells whose growth is to ~e i~hibited or target microbial cells are : : contacted with a composition of the in~ention. That contact is maintai~ed ~or a time period sufi~icient for ~: t he desired result: to occur. Multiple administrations :
25 ~ of a pharmaceutical c~amposition can be made to provide the des ir~d contac~

' Br_f_esarie~il~the ~gs In the drawings forming a portion of this 3 0 : ,~isclosure, Fi~ure 1~ in two panel~ as Fi~s. la and lb are photo~raphs of ethidium: brc)mide stain~,~d 1 perc en~ agrose gel~ that illustrates the ~ffect on ~,~X~7~ Form I DNA by the following campourlds in 50 mM Tris-HCl buffer (Fig. la = pH 8.5, Fig" 1~ - pH 9.0) after ~8 hours at : :: ~ :: :
:: :

W093/23~6 2 1 ~ ~ 2 ~ ~1 PCT/VS93tO47~

37C. Lane 1 (loO mM) is the DNA control; lane 2 is DNA
plus ~ompound 21 (1.0 mM); lane 3 is DNA plus Compound 427 (5.0 mM); lan~ 4 is DNA plus Compound ~28 (5.0 mM);
lane 5 is DN~ plus Compound 42~ (5.0 m~); lane 6 is DNA

5 plu5 2-(phenylsulfonyl)propanol (5.0 mM); lane 7 is DNA
plus phenyl i60propenyl sulfone (500 mM); and lane 8 is DNA plus phenyl vi~yl sulfone (5.0 m~)~ Form I is sup~rcoiled DN~; Form II is nicked DN~; and Form III is linear DNA.
Detailed Descri~tion _ ~ nvention I. The_~y~pounds An enantio~eric compound of the invention cont~ins an enediyne macrocycle linked to a fused ring compound system that corresponds to structural For~ula I

~R?

~o ~ ~

' :~
: wherein ~ is a double or single bond;
: R1 is sel~ted from the yroup ~onsisting of ~, C6 alkyI~, phenoxycar~onyl, benzyloxyca`rbohyl~ Cl-¢6 :
alkoxycar~onyl:, substitut~d Cl-C6 alkoxycarbonyl (particulsr1y~a sub:stituted ethoxycarbonyl wher~ the i~
s~bstituent is phenylsulfonyl or naph~hylsulfonyl with ~:: : pheny1sulfony1~most particularly preferred), Q-nitrobenzyloxycarbonyl and ~; 35 9-fluorenylmethyloxycarbonyl;

:~ ~

W093~23~ ~3~3 ~ PCT/US93/~470~ ~

1~--~2 is selected fr~m the group consisting of H, carboxyl, hydro~ylmethyl and carbonyloxy-C1-C6 a}Xyl;
R3 is selected from the group consisting of H
and Cl-C6 alkoxy;
R4 is sele~ted from the group consisting of H, hydroxyl, C1-~6 alkoxy, oxyacetic acid (-OCHzCO2H), Cl-C6 hydrocarbyl or benzyl oxyacetic acid es~er, oxyacetic amid~, oxyethanol (-OC~2C~2OH), oxyimidazylthiocar~onyl and Cl-C6 acyloxy;
R~ and R7 are each ~ or together with the interv2ning ~inylene group ~orm a one, two or three fused aromatic six-membered ring system;
W together with the bo~ded, intervening, ~inylene g~oup (i.e., the unsaturated carbon atoms bonded to W) forms a substituted aromatic hydrocarbyl ring system containing 1~ 2 or 3 six-membered rings such that sald fused ring compound con~ains 3, ~ or 5 fused .

6-me ~ ered rings all but ~wo of which rings are aromatic, and in which that aromatic hydrocar~yl ring system, W, i5 joined ~a, bJ to the structure shown; and R8 is~hydrogen or methyl with the proviso that Ra is hydrogen wh~n W together with the interYening ~: vinylidene group is 9,lO-~ioxoanthra.
A compound o~ Formula I and the other fused ring enediyne compounds disclosed herein are chiral, and are prepared:as a s~ingle or individual enantiomer that is substantially free o~ the other enantiomer. Only one of the enantiomeric pair is shown in Formula X and most o~ the other formu~as depicted herein. For ease in 30 ~ d~piction, the depicted enantiomeric~fu~d ring e~ediyne compounds are shown having the absolute ~tereochemistry o~ dynemicin A [Landley;et alO, ~ _~o~ Yo~ 9~, 33:4395 (1~91~ and ~ender, Pro~ __atl. Acad. Sci. U$A, 8~:8835 (199lj3, which absolute stereochemistry is ~; 35 preferred.

: ~ .
; ~ ~

wo 93,23046 2 1 3 ~ ~d ~ l PCI /US93/047û8 .

Th~ synth~s~s and assays using the racemic forms o~ the comlpound~; disclosed herein are disclosed in Int~rnatlonal P.pplic:ation WO 92/02522, published on February 20, 1992 and in the p~ablished literaturP. See, for example, Nic:c~laau e~ al., fis~s~ 1172-1178 ( 1992 ) and th c:itations therein . A compoun~
contemplated her~ i5 one or t~e oth2r or both of the two separate ~nantios~l~rs ~hat constitute those racemates.
A contemplated enantiomer i5 synthesized using 0 simllar reactions to those disclosed in WO 92/02522 with dif ~erent intermediate s~eps that permit a stereoc:ontrolled synthesis. These differant steps are outlined hereinafter.
Racemat~s are useful as the data of W0 92/02522 and the literature indicate. However, separate enantiomers have also been prepared and a compound having the same absolute st~reochemistry as that of dynemicin A, the (+) isomer, has ~een fsund to be more potent against some cancer cell lines, ~. g . Molt4 T
2 0 cell leu3cemia and Capan-l pancreatic carcinoma, than the other, (~ enantiomer-. Separatad (~) and (-) isomers al50 e~ibit similar potencies against other cancer cell lines suc:h as SK-MEL-28 melanom~. Thus, a ~u~ed ring enediyne disclosed herein is contemplated as either or both of the separated (~) and (-j, single, enantiomeric m~lecules (enantiomers).
E:x~mplary R~ and R7 groups other than hydragen, ~rhich is~: preferred for both, are discussed hereina~t~r .
As :n~ted above, t~e bond, A, between the R2 and }~ substituents can ke a double or single bond . The ~: bond A is prefera3~1y a single bond.

:

~, W093J23~6 PCT/U~93/04708 ~, ~ 3 6~ !

A C1-C6 alkyl group, as can be present in R1 is exemplified by methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, p~ntyl, 2~ethylpentyl, hexyl, cyc~ohexyl, cyclopentyl and the l~ke. A ~ubstitut~d C1-C6 alkyl group i~ also contemplated as an R1 groupO Such substituted alkyl groups include hydroxyalkyl groups such as 2-hydroxyetbyl, 4-hydroxyhexyl and 3-hydroxypropyl, haloalXyl groups such as 2-chlorobutyl, 3-halopentyl such as 3-~luoropentyl, and the likeO The above C~-C6 alkyl and s~bstituted Ct-C6 alkyl groups are further contemplated as the C1-C6 aIkyl portion of a carbonyloxy C1-C~ alkyl group o~ R2; i.e., a Cl-C6 alkyl ester of a R2 carboxyl group, and of a R1 urethane group. Those same alkyl groups can constitute the alkyl portion of a C1 C~ alkoxy group of R3 or R4. A C~
acylo~y group as i~ present in * or R5 (discussed hereinafter~ is a rarboxylic acid derivative of an appropriate alkyl group, above, except for, for example, cyclohe~yI and iso-propyl t and is limited to a cyclopentylcarboxyl group for the cyclopentane derivatives. Examples Qf such C1 C~ BCylOXy groups ~ : include formyloxy, acetoxy, propionoxy, butyryloxy, iso-: ~ buty ~ loxy, pentanoyloxy,~2-methylbutyryloxy, pivaloyloxy, hexanoyloxy, and the like.
The alcohol-carbonyl portion of a urethane i typicall:y formed by the reaction o~ a correspon~ing halo formate deri~ative, such as a chl~roformate like phenylc~loroformate, with the ~econdary amine nitrogen : atom that is:formed by addition of an acètyl~nic group-::co~taining~moiety to the 6-position or a correspondingly numbered:position of a ~u~sed ring system such as that shown in Scheme II hereinafter. Such groups can also be : prepared~by ~ase-catalyzed exch~ange from a formed :carbamate using the~ubstituted ethyl alcohol a~ is illustr~ted~he~eina~ter.

W093/23~6 2 ~ 3 ~ ~ 3 ~ ~CT/US93/~4708 Exemplary Cl-C~ alkoxycarbonyl groups and substi~uted Ct-C6 alkoxycarbonyl groups contain a before-described Cl-C6 alko~y group or substituted C1-C~
al~oxy group linked to th~ carbonyl group and can be formed by rea~tion o~ a Cl C~ alXylchloroforma~e~
Exempla~y substituted ethoxycarbonyl groups that are a particularly prefexred group of substituted Cl-C~
alkoxycarbQnyl group ha~e a substituent o~her than hydrogen at the 2-position o~ the ethoxy group, and in,_lude 2~(trimethylsilyl)ethoxycarbonyl, 2-(phenylsulfonyl)ethoxycarbonyl, ~- or 2-(naphthylsulfonyl)~thoxycar~onyl, ~- or ~-2-(anthracylsulfonyl)ethoxycar~onyl, 2-propenoxycaxbonyl, ~-hydroxyethoxycar~onyl, 2-(triphenylphosphoniume)thoxycar~onyl halide (e.g.
chloride, bromide or iodide3 and 2-(trimethylammonium)ethoxycarbonyl halide (as before).
It is particularly preferred that R~ be a group that can be enz ~ atically or otherwise removed - 20 : intracellularly ~o pro~id~ ~he resul~ing secondary amine free of a substituent group. A compound wh~re Rl contains a 2 substituted~ethoxycarbonyl group such as a 2-(phenylsulfonyl)-, 2-(naphthylsulfonyl)- and 2-~anthracylsulfonyl)- as ar0 shown in Scheme III (shown 2~ as Rl therein) can ~orm the free secondary amine compound ~ia a ~-elimination under relatively mild conditions. An e~hoxy carbo~yl group can also be named i an ethyl,ene oxycarbonyl:group. ! i Phenylsulfonylethoxycarbonyl, ~-naphthyl- and ~-napht~ylsu1fonylathoxycarbonyl (collectiYely referred to as n~phthylsulfonylethoxycarbonyl~ are particularly pre~erred R~ groups,: wit~ phenoxycar~oxyl being a preferred R1 group. When an Rl group is o-nitrobenzyloxycarbonyl, W light-irradiation (about : ~ :
`:

W093/23~6 PCT/US93/04708 `~3 6~3 ~ -14-~90-400 nm) provides cleavage of that group from a fused xing system, t~ereby pro~iding a free am-ne group.
The ab~olute stexeochemistry of 2-(phenyl)- or 2-(naphthylsulfonyl)etho~ycarbonyl R1 ~roup can al50 lead differences in potency when the ethoxy portion of that g~oup is also su~stituted at its 2-position by one or two Cl-C6 alkyl groups discussed before such as methyl. Tha presence of a chiral, enantiomeric 2-(phenylsulfonyl) 2-(mon- or di-C1=C6 alkyl)ethoxycarbonyl R1 grsup in an otherwise racemic fused ring ~nediyne compound provides a pair of diastereomers, whereas a single enantiomer is formed : when bo~h parts of the molecule are themselves chiral enantiomers.
Thus,~ the 2-(S~ methyl deri~ati~e was more potent against seYeral cancer cells lines tharl was the : 2-(R)-(+~-me~hyl derivati~e. Both were more potent than ; was th~ 2,2 dim~thyl derivatives.
Each of the 2-(phenylsulfonyl)-2-(mono- or di-Cl-C6 alkyl)-ethoxy~carbonyl-contai~ing ~used ring enediyne compounds w~s le~s potent than the Yery potent un~allylated deri~ativ~s. ~ These differences in potency can be used to a~just the potency and selectivity of a ' conte~plated compound. : : '~
Exemplary R6 and R7 that together with the :~ ~ intarvening vinylene group form a on¢, two or three fused aro~atic~six-membered:ring sys~em that includes enzo,:naph~ho and an hra rings, as well as 1 ,.
6p8-dimethoxynaphtho and 6,8-diazanaphtho. As no~ed : before, it is~preferred that both ~b and R7 be:~ydr~gen.
~n R8 group can~be methyl or hydrogen with ~he : pro~iso that R8 is hydrogen whe~ W along with the intervening viny}ene group carbon atoms forms a 9,10-dioxoanthra ~ing.~ It is particuIarly preferred tha~ ~8 35 ~ be methy~l~when W forms:a benzo ring.

:: : : ~ :`

: W093/23~6 ~ 1 3 ~ t~ PCT/US93/04708 R4 groups that ~r~ hydrogen, hydroxyl, 1, oxy~thanol ~QC~2CH2OH), oxyacetic acid (-OCH2C02H), oxyacetic C~-C~ hydrocarbyl ~sters such as the before-discussed Cl-~6 alkyl groups such a~ ethyl oxyacetate S
5(-OC~2CO2C~2~3), as well as Cl-C6 unsaturated es~ers such as the allyl, propargyl, ~-butenyl and the like, as well as the benzyl ester and o~yacet~c amides cons~itute particularly preferred embod~me~ts ~f the in~ention.
A pharmaceutically acceptable non-toxic salt 10of the oxyacetic ac~d such as sodium, potassium, ammonium, calcium and magnesium is ~lso contemplated.
An oxyaeetic acid amide corresponds ~o the chemical formula ~ocH2coN~13Rl4 wherein R13 is hydrogen (H) or Cl-C6 alkyl (as before) and ~1~ is independently hydrogen, C1-C6 alXyl, phenyl, l~ or 2-n~pthyl, 1- or 2~anth~yl, or a peptide~haYing 1 to a~out six amino acid residues;
or R13 and ~4 together with the nitrogen atom fo~m a S
or 6-memb~red ring as is pr~sent in pyrrolidine, piperidine,~orpholine, imidazole or pyrrole.
A particu~larly con~emplated peptide is distamycin, or~a deri~ative thereof as discussed in Taylor et al., Tetra~ 3z~, 40O457 (1~84) and ~aker et al., J. A~ Chem So~ 2700 (1989). Distamycin : derivatives are themselves kno~n DNA-cleaving agents.
25~ Another particularly preferred peptide is ; ` -Ala-Ala-Ala-, C(-Ala )3] which sequence is re ogniz2d and cleaved by: a~ i~tracelIular ly~osomal enz~me~
Furtherjsuitable peptide linkers that are cl~av~d : enzymatically ~n viVo ar~ well known to skilled workers~
0 See, for ex~mple, Reisfeld ~t al.l H_m3~L5~u~e}
2L:341 (1991) and the citations therein. So called acid-cleavable linkers such as ~-aconitate~and the li~e as are also well known can also be used alone or in conjunction with a cleavable peptlde linker. See for example, Reisf~ld e~ al., ~Y~U-~Ll~

,:

W093/23~6 PCT/US93/04708 2~36~3~ ` , la_C~ aD/~II, 11(21: 341 (1991) and Mue}ler et al., ~ioconl~u~ate Chem., 2:32S (1990), and the citations therein.
Appropriate diamine and dicarboxylic acid S groups can be added at the carboxy- and amino-terminl of the peptides or acid labile linker~, respectively, to join the fus~d ring e~ediyne t~ the Mab, as is discussed below. Exemplary diamines are the ~ C2-C6 alkylene diamines such as ethylene diamine, 1, 30propylene diamines and 1,6-hexylene diamine. Exemplary ~ C4-C6 dicarboxylic acids include succinic, maleic, glutaric and adipic acids.
An R4 group that contains a derivatized oxyacetic acid amide or ester can also include a peptidyl spacer containing z~ro to a~out 6 residues such as (-Ala-j 3 that links the campound to a monoclonal antibody or an~antibody binding site portion thereof, coll~ctively r~ferred to herein as a "Mab~'. An Rs group as d~iscussed in detail hereina~ter as a substituent of W
as in a compound of Formula XIb can also constitute a useful spacer ~for bonding to a Mab.
: The Mab utilized immunor~ac~s substantially :~ ; only with target tumcr cells; i. eO ~ is tumor cell : specific, and thereby proYides ~urther specificity to the drug mol~cules. Such a Mab-linked fused ring enediyne i. one type of chimeric molecule of the invention. ~ .
The;:spacer portion of the enantiomeric l ;
: :compound-Mab ~pnstruct serves to link the two portions : 30 ~ the mol~:cule together~. :~hen th~ra are zero peptide :res~idues present,:~a lysine ep~ilon-amino group of the ~:: Mab fo~s the amido~bond with an R~ group as spacer.
: The spacer peptide chain, when present, is typically comprised of~mino acid residues having small side :ch~ains such as glycine~or alanine, or relatively -~ WC~93/2304~ 21~ J~ PCI/US93/0470 hydrophilic side chains such as serine, glutamine and aspartic aeid. A peptide spacer is typically free of cysteine residues, but can contain c:ystine residues and oth~ ise can have substantial~y any structure that does not inter~ere with bonding ~etween the two portions of ~he chimeric compound. A peptide can ~e prepared by an one of se~eral synthetic m~thods as are w~ll knowrl. A
particularly preferred peptide spaGer includes an amine acid residue sequenc:Q that is recognized and cleaYed by an enzyme such as a lysosomal or other proteolytic enzye prPsent within a target neoplastic cell so tnat the fused rlng enediyne can be ~reed from the Mab after endocytosis, as is well known.
The Mab portion of the above ohimeric construc~ can constitute an intact antibody molec:ule oî
IgG or IgM isotype, in which case, a pluxality of compounds can be present per antibody molecule. The ~inding site portions of an arlti~ody can 21so be utilized, in which case, at least one c:ompound i5 linked to t~e proteinaceous antibody bindin~ 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 ~;ite portions inc}ude F(ab);, F(ab'), F~ab')2 and Fv portions of an intact antibody molecule, and ::an be prepared by well known methods. An intact monoclonal antibody and a porS:ion that includes its antibody co~ining site ' portion can be collec~ively rsferred to as a para~ope-containirlg m~lecul~.
Exe~plary anti-tumor Ma3: s are noted in the :: ~ tabl~ below, listed by the name utilized in a pu~lication~ along with its deposit acce~sion nu~er at ~: ~the American Type ~Culture Collection (ATCC~, 12301 :35 ~ ~ Parklawn Drive, Rockville, M ryland 20852 U~S~Ao ~ and WO 93~23~46 PCr/US93/04708 f.

2 ~. 3 6 ~

the tumor antigen with whis:h the ~ab paratop~ is reported to react. A citation to a discussion of each Elab and its immunoreacti~Jity is pro~rided by the footnote under the antigen listing.
s : . :

:

:

~: :

` : :~ : : :: : :

: : : : : : : -._.~ ,.. , . ~._ . _._. ~_~,~_ ~. ~, _ ~ .. _ ,._ .,~ ___._ .__ _._ _.. _ ,. _. _ ___ _.. _ __:.. _ .. _ .___.. , _ _ ,,,. _, ,_ .~_ _ _ __ ._ _ _ .. : . ~ , .

;. W093/23~6 ~ ~ 35~?,~ PCT/US~3/04708 ~` 19~;

Mab A~CC No. ~a~igça B 3.6 HB 8890 ~D31 14.8 HB 9~18 GD22 llC6~ -- GD33 9.2.27 - Condritin sul~a~e proteoglycan4 R24 ~ - GD35 H~29/26 HB 82q7 colon cancer glycoprotein gp 296 HT29/36 HB 8248 colon cancer glycoprotein gp296 CLT85 H~ 8240 colon cancer~
: F64~.S -- mammary carcinoma7 R~8.1 - pan carcinoma . : 7OKd protein7 :
F36/22 H~ ~215 human breast : . ~ carcinoma8 ;~ : T16 HB 827g human bladder tumor,:
~ ~ ~ glycoprotein gp489 T43 HB 827S human bladder:tumor9 T101 ~B 8273 human hladder ~umor9 116-NS-l9~:HB 8059: rolorectal carcinoma`
mon~sialoganglioside~
:25 ~126XB;85~8 GD211 ~ C~H 6:~ HB 8532 colon cancer12 :~: CLG 479HB~241 colon cancer12 9.9~ CRL 8019 CEA13 ~: CLN~S -- lung carcinoma14 30~ 8B ~ colon carcinoma15 ~ J
Sl/4~ lung adenocarcinoma LM~O9 -- vitrQnectin recep~or17 .

:-WO 93/23~46 PCr/US93/04708 Cheresch et al ., Proc ~a 1:1 Acad~i~USA , ~:5155--~15g (1985) ~ 5767 5771 (1~84) 2 Cheresch et al. 5~5~ ~:5112-5118 (19~6) Cheresch et al.,, ~1~ 68P~ (1986) Bumol ~t al. ~ Proc~ Na~l .~ad. Sci . . USA, 79 :1245 ~982~; Harp~2~ ek al., ~, ~:2096 (1984) 5 U. S . Patent No . 4, 507, 3gl 6 U. S . Patent No. 4 ,, S79, 827 7 U . S . Patent No . 4, 522, 918 8 European Patent Application No. 8440~420. 0, pu~lication No. 0 118 365, published September 12, 9 European Patent Application No. 84102517 . 4, publication No. 0 11~ 891, published September 19, 25~ ~ 10 U.S. Patent No. 4;,471,057 ~ , : - , ~
Cheresch ~ al- I ~I., 102:~88 (1986):
: IJ . S . Patent No . 4, 67$, 287 ; 30 ~12 U.S. Patent No. 4,579,827 13 U . S . Patent No ., 4, 3 4 9~, 5 2 $
14 Patent Apl?lication PCT/USa3/007~1, WO 83/04313 15 European Patent ~pplication No. 85300~10. 4, publication :No.~ 0~151 030, publish~d August 7, 1~85 :
16 ~Varki et~al., an::er Res., 44:6Bl ~1984)~;~:Bumol e~
al., H~bridoma~ 7:407 (I988) 7 ~ Cheresch~et alO, J Biol:._ hem., ~ 17~703 tl9Bl7); , ! ' : :Sm~th ~t al~ ~51 h~D-, 265:2168 (199~

45~ An~nantiomeric;fused ring~enediyne compound : :o~the invention can: also be glycosidical~ly linked~to a : suga~r:m~i~ty~ o~form a:secQnd type c~imeric molecule.~
In~such a chimer,~: the enantiomeric fus~d ring 2nediyne compound~tak~s~the place of the aqlycone as in an W093/23~6 2~ ~23~ P~T/US93J04708 antibiotic ~ol~cule such as doxorubicin, calicheamicin or esperamicin, with the sugar moiety taking the place o~ ~he oligosaccharlde porkion. Bonding betwesn the enantiomeric fus~d ring ~nediyne compound aglycone and oligosaccharide is ~ypical}y via a hydroxyl group of a spacer group that is itself linked to the fused ring enediyne through a reacted hydroxyl group. A preferred spacer group is an oxye hanol group that can be an R4 group ar can be an R5 s~bstituent of W as is discussed and illustrated hereinafter. The glycosidically bonded saccharid~ thus forms an ether bond via the hydroxyl group o~ the oxyethanol group.
The oligosaccharide por~ion of the molecule is typically added after ~he synthesis o~ the fused ring enediyne compound (aglycone) portion is complete, except for any blockin~ groups on otherwise reactive functionalities of the aglycons t~at are typically remo~ed a~ter additisn of the:oli~osaccharide portion.
A sugar moie~y i5 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-acety}glucosaminyl, N-acetylgalactosaminyl moiety or t~e m~re preferred : ~ 25 ~ saccharides whos~structurés are shown bel~w, wherein a waYy line adjacent a bond indicate~ the position of linkag .

:: :

:

: ~ : : :

~ . .. ...... . .. ....... ... . . .

WO 93/231~6 PCI`~US93/047~ ~
p~3~Q~34 D ;2 ;2 --~ 'V ~ ~Oh llN~ ~O OH E9 O ~ A ~ H
~~/ ~N~

~ ~f ~N~ oJ
08 I~J Ol~o, ~~ ~N
~o o ~J~ol~
~S~) D

~~ ~~ ~ oa~ H~

s~ ~ : E

~` ;W093/23~6 ~.~ L 3 6 ?~ 3 ~ PCT/US93/04708 ~23~

The position of the glycosyl bond to be formed in the sugar mo'~ty us~d for ~orming a chimeric compound is typically activated prior to linkage to the fused ring ~n~diyne compound. For example, the l-position S hydroxyl group o~ an otherwise pro~ected sugar (as with ~BuMe2Si or E~Si groups~ i5 reacted with diethylami~osulfur ~r~fluoride (DAST) in THF and in the pres~nc~ of ~A molecular sie~es at -78C ~o form the 1-fluorod~ri~ative. The ena~tiomeric enediyne ha~ing a free hydroxyl group is:th~n react~d with the ~-fluro-protected saccharide in the presence of silYer perGhlorate and s~annous chloride to provide a protected desired, typically blocked, enantiomeric chimer molecule. I
Similarly, ~reatmen~ of l-position hydroxyl of : an otherwise protected saccharide with sodium hydride and trichloracetonitrile t~randler et al.~, Ca~hYdr.
135:203 ~19853; Sc ~ idt, ~En~l., 25:212 tl986)] in methylene chloride at about room temp~rature pro~ides a l-~-trichloroacetimidate roup to acti~ate the saccharide ~or coupling with the fused ring enediyne t aglycon~ hydroxyl ~. Coupling is then carried out~in boron trifluoride etherate in : methylene chlorid~ to pro~ide th~ protected desired ; ` 25 : ~chimer compoundc : once the enantiomeric a~lyco~e and oligosaccharide::are~coup~ed, the protecting groups that are present are removed to provide the desired compound, which is then reco~ered using andar~ technilques! '`
: Exemplarx~:syntheses are:discu~sed hereinaft~rO
The~, 2 or 3 six-membered rlng fused rings that alon~ with the depic~ed ~inylene group cons~itu~e the structure:W are aromatic hydrocar~yl ring~. Such rings can thus be benzo, naphtho a~d anthra rings, using ~ ~ ~ 35 ~used ring nomenclatur~. T~e anthra (ant~racene) : :~ ~ : : :

WO 93J23046 PCr/lJS93/û4708 ,q,,3 ~

derivati~e rings contemplated here contain 9, }0-dioxo groups (are derivatives of anthraquinone) and are ~herefore xe~erred to as 9 ~10-dioxoanthra rings .
Whexe a benzo, naphtho or 9, 10-dioxoanthra ring forms par~ of the fused ring syste~, those fused rings are bonded ~o the remaining fused ring system through the car~on atoms of the 1 arld 2-positions or are (a, b~. A ~enzo, napht~o or 9,lO~dioxoanthra fused ring portion can also contain one or more substituents a~ the ring posit~ ns remaining for substitution. Thos2 - substituent groups are selec~ed from the grc~up c:onsisting of hydroxyl, C1-C6 alkoxy, Xt C~-C6 acyloxy and halo (chlorsl bro1lao or iodo).
For a benzo ring,. one or two substituents ::an be present at one or two c: f the remaining positions of the radical O Symmetr? cal substitution by ~he same substitue;lt is preferred 3:~ecau e of the les~;ened possibility for isomer formation. ~hen a sirlgle ;~ s~s~ituent is pr~sent on a ~nzo ring, that substituent is referred to as RS, which designation for convenience ihcludes hydro~en. Rs is thus selected from the group eonsisting of hydrogen (no substituent)~, C1-C6 alkoxy , car~oxyl, Cl-C6 hydrocarbyl or benæyl carboxylate, : benzyloxy, o-nitrobenzyloxy, hydroxy, Cl-C6 acyloxy,o~yethanol, oxyethanol teirtiary amino or quater~ary : ammoniu~ C2~C6 alkyl carboxylic acid~ester, oxya~etic acid, oxyacetic a id C1 C6 hydrocarbyl ester, oxyacetic " j j acid amide,~3-hydroxyprop l-ynyl and halo.
: It is preferred t~at a hydroxyl group or agroup that can~form a hydroxyl group intracellularly be ~ : present, sucn~ that:a hydroxyl gxoup b~ present : :
: : ~ intracellularly:a~ a~;position ~ to the nitrogen in the adjacent ring. When two substituents are present on : ~ ~ a benzo ring, they~are referred to as R10 and R11 and are : :

WO 93/23046 7 ~ ~ 6 2 `~` ~ PCI/US93/047û8 ~25--~el~cted from ~he group consi t ng of C1-C6 alkoxy, benzyloxy, oxo, C1-C~5 acyloxy, hydroxyl and halo.
W i5 more preferably a benzo ~roup hat contains a sîngle sub~ti~uent ~. In one particularly S preferred embodiment, R5 is situated ln the benzo ring or pax a to the nitrogan ato~n bonded to R1 . That R5 group is mor~ praferably selected from the group con isting of hydroxyl, C1-C~, allcoxy, benzyloxy, o-nitrobenzy}oxy, C1-C6 acyloxy, carboxyl, C1-C~s hydrocarbyl or benzyl carboxylate, cxyethanol, oxyacetic:
acid, oxycacetic Cl~C6 hydrocar~yl ester, oxyacetic acid amide, oxyethanol tertiary amino or q~aters~ary ammonium-substitllted C~-C6 alkyl carboxylate or 3 hydroxyprop ~ l-ynyl . An ~5 o~cyacetic acid or 1~ oxyethanol or 3-hydroxyp~op-l-ynyl g~oup is ~seful for linking the aglyconQ to an oligosaccharide or antib~dy co ~ îning si~e portion via an ether or ester group, as dis:cussed previously for R~.
When Rs is: ~ to the above nitroqen atom, it is preferred that the~ R5 group be an electron releasing group such as hydroxyl or a C1-C~ acyloxy group that can - : p~o~ide a hydroxyl group in~racellularly. A C1-C6 acyloxy group is believed to be a pro-drug form of the : hydro~yl group that i~ cleaved intracellularly by an : 25 ~ endogenus esterase or the li~e to provide the hydroxyl :: ~ group. The~presence of such an electron releasing ~roup appears to assist:in enhancing the potency o~ the compound against target tum~r cells. It is believed that the ~nhanced potency i5 due to enhanced triggering : ~f khe epoxide opening a~d~cyclization r~actions.
When R5 i~ ~LE~ ~o the abov~ ~itrogen a~om, it is~pre~er~ed t~a~ the R5 gr~p be an:o nitrobenzyloxy group, o~yethanol, car~oxyl, C~-C6 hydrocarbyl or ~enzyl carboxylate~, oxyacetic acid or oxyaeetic acid Cl-C6 ::; :

:~

~: `

WO 93/23046 PCr/US93/0470~

~,~3~ 'J~ -26-hydrocarbyl e~ter. Thosa ~roups are particularly useful for the preparation of chimeras.
The pr~sence of an R5 substituent E~E~ to the nitrogen that is an c~xyethanol, oxyacetic acid or S oxyac:etic aci d amide as discussed for an R4 group before, is also u eful for providing e~anced water solubility to a fused ring ~nediyne ;:ompound discussed herein. On~ particularly preferred compound contains an oxyethanol R5 group ~ to ~he nitrc)gen atom.
Another part:icularly prefered ~5 s~stituent that is ~a to the nitrogen atom is an oxyethanol ester of a tertiary or ~uaternary amine substituted C2-C6 alkyl carboxylic acid (carboxyla~e). These substituents provide still further enhancements to water solubility because of the formal charge of a quaternary ammonium group or the protonation of the ~ertiary amine at physiological pH:Yalues, e.g. pH 7.~-7.4.
Ex~mplary~C2-C6 alky} carboxylio acids are those discussed in con~unction with a Ct-C6 acyloxy group. The amine substituent is preferably ~onded to the carbon atom farthest down the alkyl chain from the : carboxyl group and is ~herefore an ~-(omega) substituent. Thus, tertiary amine and quaternary a~monium de~ivatives of ~-amino acids such as glycine, ~-alanine, y-aminobutyric acid and 6-aminocaproic acid are preferred.~
The amine portion of an oxyethanol tertiary amin~ or qua,ternary am~onium-substituted C2-C6 alkyl carboxylic acid ester ha~ the ~tructure -NR2~R2~ or 3 0 -~NR20R2lR22 wherein R20,: R21 and R~ are e ch ind pendently -C6 alkyl,~r RZ~ and R2l together with th~ nitrogen atom form~a 5 or 6 membered ring, or R20, R21 and Rz2 .
~R20 2Z) together with the nitrogen atom form a pyridinium or pyrazinium~group. Exemplary Cl C6 alkyl groups have :~ 35 already been diseussed, and methyl is preferred for each W~93~3~6 2 ~ 3 ~ 2 3 ~ PCT/VS93/04708 -~7- i o~ R2022.. Exemplary 5 and 6-membered ring compounds form~d by R20, R21 and the nitrogen atom include plperidine, morpholine, pyrrolidine, imidazole, pyrrole and pip~razine. Thus~ exe~plary _NR2~R21 portion~ of tertiary amine groups includ~ dimethylamino, diethylamino, h~xyliso-propylamino, di-sec butylamino, N-morpholinyl, N-piperidyl and N-imidazyl. Exemplary quaternary ammonium groups include trime~hylammonium, ethyldimethylammonium, ethyli~o-propylhexylammonium, N-mathylmorpholinium, N-butylpiperidinium, pyridinium and pyrazinium. A sui~able anion for the ~uaternary group is of course contemplated and inc}u~es halide ions such as chloride and bromid~, sulfate, acetate or another Cl-C6 acylo~ ~roup anion. An N,N,N-1~ trimethylgly~ine chloride ester of an o ~ ethanol :~ su~stituent is particularly preferred.
A particularly:preferred compound has astructure corresponding to Formula XIb, hereinafter~
A naphtho ring can have three subs~ituents.
20 ~ This ring c~n hav~ a 4-position radical, R5, selected : from the group consistin~ of hydroxyl, C1-C6 alkoxy, benzyloxy, C1-~C6 acyloxy, carboxyl, C1 C6 hydrocarbyl or : ~enzyl car~oxylate, and halo, and substituents at the 5-(R10) and 8-positions ~R11~ that are selected from the group consisting of hydroxyl, Cl-C6 alkoxy, benzy1Oxy, C1-C~ acyloxy, oxo and halo radicals. A ~,lO-dioxoanthra ring can have three substituents at the 4 (R5), 5-j~ (R9)! and 8-positi~ns (R12) that are ; independ~ntly~selected from the group consisting of 3Q hydroxyl,: C1-C~ alkoxy/ b~nzyloxy, Cl-C6 acyIoxy and halo. T~us, R5, R9 and R1~ can define the sam groups, and all three ~roups can ~e written as ~ither R5l R9 or : : R12, ~u~ they are shown separately herein.
, :: :

PCI /US93/047~8 ~VO 93/2304~
,3C~ ?~3 ~ 2~ 1 Exemplary structural formulas for a contemplated enan~iomeric fus~d ring compound are illustrated 1: elaw by s~ructural Formulas II-IX, wherein r each of the R groups is as discussed before.

:

.~ ~

;
, :; ~ : : : ; :

-` WC)93/2304~6 2 ~ 3~2? IP~/US93/04708 ~9 ,R7 R7 R~N~R ; ~ ~ ~R3 3 ~ ~J :
;~" R10 ~J

1 ~ \

Rl R5 :YII~ ~ b~ IX

:: ~: :~ ::: ` :

W093/23046 PCT/US~3/04708 f ~3G~ -30-In addition to the b~fore-stated preference regardtng ~8 and tha. bond A be a .ingle bo~d, several other structural features and ~ubstituen~s are preferred.
~hus, it is pr~ferred that R2 and R3 be hydrogen, and tha~ R6 and R7 be hydrogen. It is al~o preferred th~t the ~u~ed xing sys~em ~ tog2.her with the depicted Yinylene g~oup b~ su~stituted b2nzs, or an unsubstituted benzo, naphtho or 9,10-dioxoanthra ring.
It is furth~r preferred that ~he fused ring compound - contain a total of 3-fused sixomembered rings so that W
together with th~ depicted vinylene group forms a be~zo ring.
one particularly preferr~d group of en~ntiomeric compounds of the invention in which ~ is an Rs-substituted benzo ring corresponds to structural Formula X.
p/?

ZS r~X

l ~ ~ Rs wherein A i5 a d~ubl~ or single ~ond;
R1 is selected from the group ~onsisting of H, Cl- ~6 alkyl, phenoxycarbonyl, benzcxvc2rbonyl, Ct-C~
lko~ycarbonyl, substituted C1 C~ alXoxycarbonyl ~particularly substitu~ed ethoxycar~onyl where the substituent is phenylsulfonyl or naphthylSulfQnyl, with W093/~3~6 ~ 1 3 ~ ~ 3 ~ PCT/US93/04708 ~31-phenylsulfonyl most part~cularly preferred), o~nitrobenzyloxycarbonyl, and 9-fluorenylmethylo~ycarbonyl;
R2 is selected from the group consisting of H, carboxyl, hydroxylm~thyl and carbonyloxy Cl-C6 alkyl;
R3 is selected fro~ the group consisting of H
and Cl-C6 alkoxy;
R~ is selected from th~ ~roup consistin~ of H, hydraxyl, oxyacetic acid (-OCH~CO2H~, oxyacetic Cl-C6 10 hydrocarbyl or benzyl ester, o~yacetic amide, oxye~hanol, oxyimidazylthiocarbonyl and C1-C6 a yloxy:
R5 is selected from the group consisting of hydrogen, Cl-C6 alkoxy, benzy~oxy, o-nitrobenzyloxy, hydro~yl, Cl-C6 acy~oxy, carbo~yl, Cl-C6 hydrocar~yl or benzyl carboxylate, oxyethanol, oxyacetic acid, oxyacetic ac~d C1-C6 hydrocarbyl es~er, halo, oxyacetic acid amide, oxyethanol ~e ~ iary amino- or quaternary ammonium-eubstituted C2-C6 alkyl carboxylate and 3-hydroxyprop-1-ynyl; and R6 and ~7 are each ~ cr ~ogether form with ~he : intervening ~inylidine group form a one, two or three fused aromatic;:ring system, and R8 is methyl or hydrogen.
A still more preferred group of enanti~meric compounds o~ the invention correspond to structural ; : Formulas XI, XIa and XIb.:

;'~ 30 R~N~ a~N~--l ~N~t~
` ` ~ ~ ~5 ~ 35 ~ A4 ~
X~: ~ : Xla Xlb :

W093l23~6 PCT~USg3/047~ t ~,3 ~

wherein R9, ~4, Rs and R8 are as pre~iously defined.
Of the i~di~idual enantiomeriG compounds corresponding to ~truc~ural ~ormulas XI, XIa and XIb, there ar~ further pr2ferenc~s for Rl, R~ a~d *. These prefer~nces also relate ~o ~he previously discussed compounds.
Thus; Rl i5 most preferably phenoxycar~onyl 2-~phenylsulo~yl)ethoxycarbonyl, 2-(naphthylsulfonyl)ethoxycarbonyl or hydrogen. R8 is most preferab}y hydroge~ ~) to provide a compound of Formulas XIa or XIb. R4 i5 most preferably H, hydroxyl, lmidazylthiocarbonyloxy, ben~yl oxyacetate and C1-C6 hydrocarbyl oxyacetate such as ethyl oxyacetate. R in Formulas XI a~d XIa is ~, but is mor~ preferably hydroxyl, C1-C~ a}koxy, benzyloxy, Cl-C6 acyloxy, oxyethanol, oxyace~ic acid, oxyacetic acid C1 C6 hydrocarbyl or benzy} ester and oonitrobenzyloxy, oxyacetic acid amide, oxyethanol t~rtiary amino- or quaternary ammonium-substituted C2-C6 alkyl carboxylate ~ or 3--hydroxyprop-l~ynyl as in Formula XIb. It is noted ; that an P~5 o-nitrobenzylo ~ group is not usually used in : ~ a pharmaceutical composition discussed hereinafter.
~h~ struG~ural formulas of particu1arly preferr@d enatiomeric ~ompounds ar~ shown bel.ow, generally:as the preferred (~) stereoi~omers, along wit~
compound ~umbe~s as utilized in WO 92~02522. Syntheses for~the:~racemates of those compounds ar~ disclosed in ~O 92/02522, as wel} as in Nicolaou e~ al., SCi nce, 256:1172-1178 tl992);, and th~ citations t~erein, as are analytica1:d~ta. In the ~srmulas below and e~sewhere ;~ her~ein~ Ph = ph~nyl, Me ~ methyl, NBnO =
: o~nitrobenzyloxy and tBuCO2 a pi~aloyl~

P~/US93/0~70 . ~ 931~3~6 ,~
:

~,~ PhO ~

OCH2CO2CH2CH, 2t (+j-24c PhO~

(~)40 ~ 41 PhO~ PhS~0)2(C~z)20 (+)-41 a : : MeQ
OMe (C~ hO'~

MeO (~)-42 C~ ~o NBn :

WO 93/23046 PCI'~US93/0470B
3 6 ~ 3 _ 3 ~

(~)45 ~ PhS2--PhO~ PhO~

'BuCO2 OH IBL~O2 ~ 59a (+)-59b PhS(0)2(CH2)2~ ~ Phs(o)2(cH2)2 (+) 120 O~ 153 ~~

1 60 (~1 61 : OMe 0 .,0 ~H~, Ph'S--O N' _ (~) 250 ~ H
O

O

` W0~3/23~6 ~136~3~1 PC~/US93/04708 A beforeodiscussed compound is chiral, and as such can exist in two enantlomeric forms ~+) and t~)-The compounds are generally shown in ~he absolute ster~oconfiguration of d~nemicin A ~Landley ~t al., ~ 5l~o4_J~ ~ 4395 (1991), and Wender et al., P~oc. ~atl. ~cad. s~i~ u~a, ~:883S (1991)].
A contemplated enan~iomeric fused ring enediyne compound can ~e synthesized a~ a racemic .
mixture of the enantiomers as discussed in W0 92/02522 :~
and resolved into single:~enantiomers for use herein, or can be synthesized as an optically~pure single .
~ enantiomer, which i5 pre~err~d. The synthesis of si~gle ;:
enantiomeric Compounds (+)- and (~ 5 are discussad hereinafter, and analogous syntheses can be applied'to the preparation of an enantiome~ of any of the other compounds~disclosed herein. ~s is also: discussed : hereina~ter, the ~nantiomeric Compounds ~5 exhibited similar DNA cleaYing activities to eaoh other and to the racemate, but exhibited some:startling differences between themsel~es~and the known;racemate in ;.
: cy~toxicity :when~assayed against cancer cell lines.
~;
II. F~3~l~aceutical Co 05~ ~ -: An enantiomeric compound or chimera o f the 2~ invention :is:useful as a DNA cleaving agent, and~also as an:antimi~robial~nd a:cytoxic (antitumor) ag2nt, as are ;`
dynemicin ~,~ calicheamiein, e~peramicin and ., .
neocarzinosta~in. :A compound of the in~ent~ion can also ~ ' there~ore~be;referred ~o as~an '~a~tive agen~" or "alctiv~
;~ 30 ~ ingredient~ : J ,''' DNA;clea~age~can~b~;assay~d using~th~ ' :techniqu~s:describ~d hereinaft~r as well as those described by ~antlo et:~al:~ . or~. Ch_ , 54: 2781 (1989)~;~Micolaou~et al.;,:~ D~ bA~ 2:7147 3~ 89~: Nicol~aou ~ l.:, J~ 5l ~ 50~ 0:7247 W093/23~6 ~ PCT/VS93/04708 ~ .
(1988) or Z~in et al., S~ien~ç, 2~:1198 (1988) and the citations therein~
~ n enantiom2ri compound or chimer of the inventlon is use~ul against Gram-positive bacteria such S as ~. aureus and ~iderm~s, Mi~rococcus luteus and }-clJJ~}~c}5iL~ as is dyne~icin A. Such a compound or chimer also exhibits antimicrobial activity against ~
._coli, Pseudomonas 3~}~Lh3Q~, C-~ndida albucans and ~:
~sPe~qill-is fumi~atus. Activity o~ an enantiomeric compound of the inven~ion againct the above microorganisms can be de~ermined using various well known techniques. See, for example, Konishi et al., J~ Antibiotics, ~ 14~9 (1989). Antimicrobial and antitumor assays can also be arried out by ~echni ~ es ~.
described in U.S~ Patent No. ~,837,206, whose disclosures are incorporated by reference, as well as by ...
the procedures described hereinafter. ~:
A ~efore-de~cribed enantiomeric ~ompound can also be shown to undergo a Bergman cycloaromatization reaction in the pre3ence of benzyl mercaptan, -~-triethylamine and 1~4~cyclohexadiene as discussed in -:
Haseltine et al., J. Am.:Ch~m. Soc., 111:7638 (1989~
This reaction ~orms a tetracyclic reaction as is formed duxing 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 enantiomeric compound or chim~r of the invention as . :
active agent. A pharmaceutical composition isipreplared by any of the methods well known in the art of pharmacy all of which:involve bringing into asso lation the I~
: active compound and the c rrier therefor. For .~.
ther~peutic use, a compound or chimer of the presant invention can be administered~in the f~rm of conventional pharmaceutical co=positi~ns. Such '.

.-- ~
~ PCr~US93/04708 : W093/23iff~6 ~ ~ 3 ~ 2 ~ ~ ! ".

co~positions can be formulat2d ~o as to be suitahflQ for oral or parenteral administr~tion, or as suppositories.
In these composqftions, the enantiomeric agent is , ~., typfically diss~lved or dispersed in a physiologically ;~
S tolerable car~ier. ,~
A carrier or diluent :ls a material use.ul for a~ministering the active compou~d and must be .;-"pharmaceutically ac~eptable" in:the sense of being ~ ~
cofmpatible with the o~her ingredients of ~he composition ,;
and not deleterious to the recipi~ent thereqf. As used : herein, the phràses "physiologically ~oleràble" and ,".
"pharmaceutically acceptablel'~are used interc~angeably and ~efer to mol~cular entities and compositions that do not produce an:~alIergic or similar untoward reaction, 15:: such:;as gast~ic Up52t,~ dizziness and the like,~ when admlnistered~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 o;f administration. ~ , ; '20~ ~: : As~an~example of a:useful composition, an ,.
anantiomeric:~compound or chimer of the~invention (active ,:
agent): can~be~util:ized, dissolvfed or:dispersed~'in a ~ ,~
uid~ao~position~such as ~a sterile suspension or ,:, solutiofn,~'~or~as is~tonic preparation:f_ontain~lng sultable~ ;.2:5 ~ preservatives.~Particular~ well-suited~for ,he present~ .
purposes:~;arfe~ injec~ ~ le media constitute~d by aqueous~
injectable buf~ered or unbufferfPd isotbnifc`and sterile -,, aline'~or'glùcose''solution ,~as wel~ asiwatær~allon~ or , hi h:~th ~ co~pounds or ch;rers~can be incorporated for~
admin~:st~ion~:in~Iude~12~ored~emulsions with edlble oils~such~as~cott~nseed~oil, s~same oil, coconut oil, peanut~oil~,-and;~the~:~like,~:as well~as elixirs~ and similar pharm ceutic~1 v-hicles. ~ Ex~mplary further liqu-d W093/23~6 ~ ~ PCTIU~93/0470~ ~
~ ~36 r~ ~

diluents can b~ ~ound in ~inaton's ~ ceutical Sciences, ~ack Publishing CoO, Easton, PA (1980).
An active agent can al~o ~e administered in th~ form of lipo~omes. As ~s known in the art, lipo~Qmes 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 ~edium. Any non-toxic, physiologically acceptable and m~tabolizable lipid ~0 capable of ~orming liposomes can be used. The present composi~ions in liposome form can contain s~abilizers, preservatives, excipients, and the like in addition to the agent. The preferre~ lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural : 15 and synthetic.
Methods of forming Iiposomes are known in the art. S~e, for example, Presco~t, Ed., ~ethods_in cell gy, Vo}. XIV, ~cademic press, New York, N.Y.
(1976), p.33 et seq.
~n active agent can also be used in compositions such as tablets or pills, prefera~ly containing a unit dose of the enantiomer~c compound or chimer. To this end, the agent (active ingredient) is mixed wit~ Gonventional ta~leting ingredients such as corn ~tarch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, ~ums, or similar materials as non~toxic, physiologically i tolerable carriers.: The tablets or pill~ can be laminated or otherwi~e compounded to pro~ide unit dosage forms affording prolonged or delayed ac~ion.
;It should b~ understood that in addition to the aforemention~d ca~rier ingredients the pharmaceutical formulation described herein can include, as appropriate,: one or mor~ additional carri~r ingredients such as diluents, buffers, f1avoring agents, .

::

:`:

` W093/23~6 PCT/US93~047Q8 213~i2?4 binders, surface active agents, thic~eners, lubricants, preserYati~es (including antioxidants) and the like, and substances included fo~ ~he pu~pose of rendering the formulati~n isotonic wi~h the ~lood of the intended recipient.
T~e tablets or pill~ can also be provided with an enteric layer in th~ form of an env~lope that serves to r~sist disintegration in the stomach and permits the actiYe ingredient to pa~s 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 a~ids with such materials as~shellac, shellac and cetyl alcohol, cellulose acetate pht~alate, a~d ~he 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~ Method~ for producing enteric coated tablets are des~ribed in U.S. Patent 4,~79,125 to Sipos, ~20~ which is herein incorporat~d~by reference.
The term "unit dosel', as used herein,- refers ~ to p~ysically discrete units suitable as unitary dosage ; for administration to wanm blooded anima}s, each such un~it containing a predetermined quantity of kh~:agent : 25 ~calculated to produce the desired therapeutic e~fect in : a~sociation with:~he pharmaceutically accep~ablQ
diluent~ Examples of suitable uni~ dosage forms in ac~ord~wi~h this:im ention are tablets, ca~sules, pills, powder packets, granules:, waf~rs, cach~ts, ~easpoonfuls, 30~ ~ droppe~uls~, ampule , vial:s, segregated multiple~ of any of;the ~o~egoing~, and t~e like.
A~pr~vious~y noted pr~ferred or particularly preferred~compouffd or c~imer is;preferred or : particularly preferred ~or~use in a pharmaceutical ; 35:~ : compo~itlon~

:: : :
~ ~ .

WO9~/23~6 P~T/US93~0~708 ~, 3, 3 6 ~ !

An enantiomeric co~pound or chimer of the invention is present in ~uch a pharmaceutical composition in an amoun~ effective to achiev~ the desired result. For example, where in vitro DNA
cleavage i5 th2 desired result, a compound or chimer of the inven~ion can be utiliz~d in an amount sufficient to pro~ide a concentr~tion of about l.0 to about 5000 micromolar (~M) wit~ a ~N~ concentration of about 0.02 ~g/~L. As a cytotoxic ~anti~umor) agent, an effective amount of an enantiomeric compQund or chimer of the - invention ~s about 0.05 to about 50 mg per kilogram, and more preferably about Or ~ to about 15 mg per kilogram of body weight or an amount sufficient to provide a !
concentration of about 0.01 to about ~a ~g/mL ~o the bloodstream. A compound or chimer of the invention exhi~its antimicrobial activity in a concentration range `
of about 0.0~ mg to about 50 ~g/mL. The above concentrations and dosages vary with the pa~ticular compound of the inven~ion utilized as well as with the target, e.g., DNA, tumor, microbe, as is well known.
Lower dosages are preferred when multiple administration utilized.
:
III. Methods An enantiomeric compound or chimer o~ the inv~ntion is useful i~ cleaYing DNA, as a cytotoxic agent and also in i~hi~iti~g the growth of neoplastic cells, and ~isiuti}ized in a method for effe~king such a ~: : rei~ult. An en~ntiomeric compound or chimer of th~
invention is typically utilized in a before-descri~ed compo~ition. ~ ;
: ~ In accordance with such a method, DNA to be clea~ed Dr target cells to be kill~d or whose grow~h is ko be inhibited are contacted with a compoun~ or chimer of the invenkion (a:ctive ingredient), typic~lly in a -``~ W093/23~ ~ 1 3 6 2 3 ~} P~T/US93/0470$

composition as before, in ~n amount effective or sufficien~ ~or such a purpose, as discussed before, dissolved or dispersed in a physiologically tolerable (pharmaceuti~ally acceptable) diluent. That con~act is maintained for a time sufficient for the desir~d result to be o~tain~d; i.e., ~NA cleaved, cells killed or neoplastic call growth i~hibi~ed.
As is discussed hereina~ter, preliminary Rtudies indlcate that the principal mechanism of cytotoxicity ~xhibited by an enantiomeric fused ring ene~iyne disclosed herein is DNA cleavage within the contacted calls. ~ithout being bound by th~o~y, it is nevertheless believed that both DNA cleavage ~IL~La~e and cytotoxi~y or cell growth inhibition by contact with a disclosed fused ring enediyne operate substantially : similarly.
Whare th~ desired result is carried out in vitE~, contact is maintained by ~imply admi~ing the DNA
or target cells with the composi~ion and maintaining them together under the appropriate conditions of : temperature and for cell growth to occur, as for control, untreated cells~ Thus, a single admixing and ccntacting is t ~ ically sufficient for in ~itro~
purposes~ ~
~: 25 The above method is also u~eful in ~ivo, as where a mammal such as~a rodent ~ike a rat, mouse, or rabbi~, a farm~ani~al:like a horse, cow or goat, or a primat~ like a monkey, ap~ or human is tre t~d . Here, contact o~ a composition and the cells to be killed or 30 ~ whose growt~ :i5 to be i~hibited is achieved by a~mini~tration of the co~position to the mammal by oral, nasal or anal administra~ion~or by introduction intraYenously, subcutaneously or intraperitoneally.
Thus, contact in Vi YO is achie~ d via the blood or lymph systems.

:~ :

W~ 93/~3046 PC~r/US93/04708 Although a ~ingle admini tration (admixture) and its resulting contact i~; usually suf f i~ient to maintain the required contac:t and o3: tain a desired result i~ ~itro, multiple a~ministrations ar~ typically utilized in ViYo. Thus, because of a body's brea3~down and excreting pathways, c:ontact betw~en an active ingredienk of a composition and the target ce}ls is typically maintained by repeated administration of a compound of the invention over a period o~ time such as days, weeks or monl:hs, or more, depending upon the ~arget cells.
Exemplary methods of the invention for DNA
cleavage and: inhibition of MIA PaCa-2 human pancreatic carcin~ma (ATCC CRL 1420) and MB~9 murine bladder carcinoma target cells (obtained from Dr. Lan ~o Chen of the Dana Farber Cancer ~nstitute, :E~o ton t MA) as well as several other neoplastic cell lines are discussed in WO 9~/025~, and in Nicolaou et al., Science, 256:1172-1178 tl992), and in the citations therein~
2 0 Exemplary concentrations f or ~
cytoxscity studies vary with the cells to be killed, and can range from about lO 5M to about 10-l5M, as is seen from the data in Tables 1~ hereinafter. Exemplary concentrations and dosage~; for in v ivo use can be those used ~or dynemic:in A or calicheamicin rll~ Typical ~,~
~ri~ do~ages are about 1 to abc~u~ 100 mg/kg body weight o~ the recipi~nk mammal. Exemplary concentrations useful ~or in: vitro: ::leavage o~ ~DNA range ~r~m about 0 .1 to about 5 mM.

~v. c~b~;
An enantiomeric contemplated compound of the invention can be~ prepared by a number of routes, several of which: are illustrated in W0 92/02522, as well as in Nicolaou et al., Sclence, 2S6:1172-1178 (19~2) and ~he ~.

~ ~: WO 93J2304S 2 ~ 3 6 ~ 3 ~ P~r/US93/04708 --q 3 cikations therein. The retrosyn~hetic plan ~or those syntheses is illustrated below in Scheme I.

5 Schem~ I

PhO~N~'~ PhOJ~N~

~ 3 In the reactions shown in Wo 92/02522, the compounds prepared such as Compourld 3 were racemates, even thou~h only one enantic: mer was shown . As is shown in the schemes that follow, a pair of diastereomeric precursor mol~cules to Compound 3 were prepared, separa~ed and then ::onverted to a single enantiomer from which enantiomeric Compound 3 were prepar~d. After preparation of a Compound 3 enantiomer or an analosue thereof having one or more substituents discussed be~ore, that ~nantiomer is con~re:rted to a desired enantiomeric: ~used ring compound that ~f Formulas I-XI, XIa or XIb.
I , , ! :. j : , Brief ly, the ~a i :: hydroxyl-substituted 3 -, 4 ~
or 5-~used six-m~mbered rirlg system i~; first formed such as Compound 6 discussed in Wo 92/0:~522, or Compound 4ao thzlt is hown in Scheme II, below.

:

WC) ~3/230~ PCIJU~93/04708 2~ ~ .
~.~.1 ~ ..

Scherre 11 ¢~ b ¢~OSI~uMe2 aC400x O~r--- 402 4~1:X=O
c H

H

PhO~ ~ d ~ H~

Me~s;
e 1~ \~

c~

~ : o 11~ o 1~
PhO~ ~ PhO~

405: R _ StMe3 : 406: R = SiMe3 407: ~ _ H ~ C 4a~: R = H

: ` : :: ~
.

, ~ ~

W093l~3W6 ~3~1 PCT/US93/04708 Thus, hydroxy qui~oline Compound ~00 was oxidized to ketone Campound 401 using Jones reagent tl.3 equiv~lents of Jones' rea~ent, ~.0 e~uivalents of H2SO~, AcOH-acetone (1:1), ze~o - 2SC, 30 minutes, 98 percentJ, a~ step a, and then converted to enol silyl ~ther Co~pound 402 in high yi~ld in step ~ by trea ment with 1.2 equivale~ts of ~Bu~eSiOTf, 1.5 equivalents of ET~N in CH2Cl~ at 25C for three hours 99 percent.
Sequential treatment of Co~pound 402 with ethyny1magnesium ~romide (1.1 equiv~lents) and phenyl chloroformate (1.1 equivalents) and in THF at -~8 - 25C, for one hour, and then 10 per~ent ~Cl at 25C for ten minutes as step c afforded acetylenic Compound 403 in 92 percent overall yield. Ketalization of Compound ~03 with ~2R,3R)-2,3-butanediol (l.S
equivalents, plus O.2 equivalents of TsVH-H~O in r~fluxing benz~ne for 20 hours) gave an inseparable mixture of diastereomers Compound ~04 (about 1:1 by 1H
NMR) in 95 percent yield as step ~. That mixture was coupled with ~inyl chloride Co~pound ~2 under the influence of Pd(O)-Cu(I) catalysis ~1.5 equi~alents of Compound 1~, 0.05 e~ui~alents of Pd(PPh3), 0.2 equi~alents of CuI, and 1.5 equivalents Of nBuNH2 in ~ benzæne at 25C for two hours} aforded a 1:1 mixture of : 25 enediyne Compounds ~OS and 406 t63 percent yield) in step ~. ~lash column chromatograp~y (silica ge~, 0~2 percent ethyl acetate in benzene) led to pure ` i diastereoisomeric Compounds 405 ~Rf=0.22 (silica gel, 0.2S percent ethyl acetate in benzene); t~DZS~427 ~C
~: 30 0.88, benzen~)~ and 406 ~R~z0.20 ~silica gel, 0.2 percent ethyl acetate in benzene); [~D~-397 ( 0 - 9 benzene3~ in 45 and 42 perce~t yield, respectively.
Separate remo~al of t~e trimethylsilyl group from Compounds ~05 and ~06 ~4~0 equivalents of AgNO3 in E~H:THF:~20 (1:1:13 at 25C for two hours and then 7.0 ! -WO 93i~46 . PCr/USg3/04708 2 i~ 3 6 r~ --4 ~--equivalent~ of NaCN at 25 for 30 minutes) led to enediyne Compound~ 407 and 408 in hi~h yi~ ds as step f .
Assignment of absolute st~rel~che~nistry in this series was based on X-ray crystallographic analysis of Compound 7 4~.
Transformation of the diastereomeric Compounds 4~7 and ~0~ ~o the target~d Compounds ~+) 45 and ~
was carried out as illustrated in Schem~ III, below, f or the synthesis of Compound (+)-~50 Scheme 111 ' 1~ ~

1~X~'O
4C~
407 b ~: ~

PhO~---- PhO~

. ` 3 t) - : .
:

:

~ ~ 3 5 , ~ :

` ~093J23~6 PCT/US93104708 2 ~ 4 -~7-Thus, acid hydrolysis o~ ketal Compound 407 ~0.2 e~uivalents of TsOH-H~O in benzene:acetone: H2O
(100:1:1) at refl~x for 5iX hour ) a~forded enone Compound ~O9 (85 perc2nt~ 'n ~ep a, which was converted to epoxyketone Compound 3 in step b using ~CPBA under basic condition~ ~.0 ~qui~alents of ~CPBA in aqueous NaHCO3:~H~Cl2 gl:1~ at 256 for 1.5 hours; 43 percent yi21d based on 87 per ent conversion]. Steps for transforming Compound 3 into Co~pound ~5 ~ollowed t~e pathway for synth~sis of the racemic Compound 45 ` dîscussed in relation to Scheme ~I and steps a and b of Scheme YIII of WO 92jo2522. Enantiomer t-~5 was prepared similarly.
It is noteworthy that enantiomer Compound (~)-45 had the same absolute stereochemistry tthe absolute star20chemistry of dynemicin A was suggested based on a wo~king model of its interaction wit~ DNA, see: Landl~y et al., J. A~. ~hem Soc~ 4395 (1991) and Wender, ~ , ~:8835 (1991)]
and sign of optical rotation as dynemicin A
t(~)-45:~a~D25~586 (c 0.5~, ~enzene), dynemicin A
a~3D24~270 (C 0.01, D~F IKonishi et al., J~
Anti~ot., ~:1449 (1989); ~onishi et al., ~QÇ~ 3715 (1990)]. Enantiomer (-~-45 ~]D25-562 ~c 0.50, ben~ene~
The above-discussed enantioselective syntheses of Compounas (+~-45 ~hd (-)-4S are general ~or any of the ~nantio~eric dynemicin analogs discussed herein.
Thus, e ther enantiomer of any desired fused ring dy~emicin analog can be readily prepared via diastereome~Ic Xe~aliz~tion o~ a compound such as Compound ~03, or more generally, a co~po~nd of stsuctural Formulas XII and XIIa, b210w, wherein R1,R2,*,Rs,~8,:A and W are as be~ore des~ribed, to form a compound of stru~tural Formulas XIII and XIIIa, below, PCI`/US93/0470~ ' ' ~,~36~3 ~ -48 wherein R24 and R2s are ind~pendently C1 C3 alkyl or phenyl, q is zero or 1 such ~hat the parenthesized CH2 group i a~sent or pre~;ent, respecti~rely, and ketalization forms at least two diastereom~rs.
H H
R1~ ~ ~2 R~ ~ R2 (~R3 ~;~R3 XII R5 XIIz R ~d~ R~ ~ R2 ~R2 ~R~

R2~ ~2s R5 R24 (~ H23q p~2s XII:I: X~:IIa f 3 0 ~ should ~ ~o course, b~ understoc)d that ( 2R, 3R) -2, 3-butanediol as used herein is not the only use~ul dic ny chiral diol that contains unre~ctive ~ub~tituents in ~he above reactions and can form a 5- or 6-membered~ring:ketal can be used. For example, 35 ~ ~2S,3S)-2,3-butanediol, (~R,4R~-2,4-pen~anediol, : : :

.

W093/23~6 2 ~ 3 f3 2 ~ 4 PCT/U593/04708 4g-~2S,4S) pentanediol, ~S~- or (R)-1,2-propanediol, (S)-or (R)-2-phenyl-1,2-prapanediol~ (S)- or (R)-1-phenyl-1,2-ethanediol and the l~ke can be us~d. Symmetrical diols such as the chiral 2,3-butanedi~ls and 2,4-pentanediols are pre~erred. Diastereomeric compounds cf Formulas XIII and XIIa are also contemplated as are separàted enantiom~rs of Formulas XII and XIIa.
As was discussed earlîer, th~ stereochemistry of a 2-phenylsulfony}-2-mono- or di-Cl-C6 alkyl - ethoxycarbonyl R1 substituent can also have an effect on the pote~cy of a eontemplated enantiom~r. The discussion below illustrates an exemplary stereoselectl~e~synkhesis for exemplary chiral R
group~.
Racemic Compound 21 was u~ed as the basis for the synthe~is of racemic Compound 45 as discussed in regard to Scheme VIII of WO 92/025~2, and was also u~ed as a starting material for synthesis of 2-~Ct-C6 alkyl) : 20 2-(phenylsulf:onyl3ethoxyc~rbonyl derivatives. The required chiral 2 phenylthio-l-propanols were prepared as illustrated in Sche~e IV, below, by an asymmetric : raduction method based on the chemistry of 4(R)-: : isopropyl-1,3 thiazolidine 2-thione), ~o~pound ~21 ~ujita et al., in "Advances in Heterocyclic ~hemistry'l, .45~ 6 (1989):; Nagao et al., Or Chem., 51:2391 (1986)], that was coupled with racemic 2-m~thylphenylthioacetic a~id (Compound ~20~. Compou~d ~O wa itself pr~pa~ed by reackion of phenylthioacetic ~ acid methyl~e~ter and m~thyl iodide in the presence o~
lithium diisopropylamide ~LDAj at -7ac~

::
:
: ~ ~35:~

:
`: :
: ~ : ~ : :

WO 93~23~ PCI/US93tO470~ l ~50--I

S~herne IV

~OH ~_~ h>~ Mc~
HN S ¦ 422 \ 423 421 ~
~ H J ¦b ¦b PhS>~OH Me>~OH

~ 4 : (~ 425 Thus, the racemic acid (Compound ~20) and Compound ~21 were coupled in step ~ ~ in the presence of 1:~2; equivalents o~ DCC and 0.2 equivalents of DP~P in 20 ~ C~2C:12 at 25~ r one hour to provid.e the diasl:ereomeric :imides,~ t:ompounds 422~ and ~23. Those compound: were separated by flash col~ chromatography to provide 42 and; 22 percerlt ;yields, respective}y~. ~Compound 42~
C~D25-56~i.0O (~C 0.1,~ ETO~), R~ = 0.41 (silie:a, 10 percent :~ ~TzO in petrolaum ethe~r); Compound ~23 [~]D225-268.0 0.1, ETO~), Rt~- 0.27;~(silica, lO percent ET20 in:
: petrol~eum èther~.]~ ~
Separate:raductio~s of Co~pound ~22;and ~23 i with LiA1~4 (one::~quivalent in THF~at zero deg~ees ~ for~
30 ~ two m~nutes~ provided~Compounds 42~and 425 as t~e (5)~ a~d~(R)~ (+~ isomers in; 73 and 79 percen~
yields,~`respécti~ely-,~in st~p~b.~ Compound ~24 ~[~3D
lO~ 3~ ~R~0.~6~ ETOH);;~ ~ompound 425~ D25 = 9.~o (~ 0.87, ETOH).~ The~assignment:~of absolute stereochemistry was ~ :~
35~ ma~e by an independent~synthe~i~ of Compound 425.:

WO 93~23046 2 ~ 3 5 ?1 I?j ~ Pcr/usg3/04708 ~, Compound 426, 2, 2- (di~ethyl) -2-phenylthioethanol was prepared by reactic: n of ethyl isobutyrate with ~iphenyld~sulf ~de in the presenc:e of ~DA, followed by redu~tion with LiAlH~,.
Compounds ~24 l 42~ and 42~ were then reacted with racemic Compound 2 ~L a~; an e~xemplary compound to demonstrate the reaction and ~o form ~he fused ring enediyne~; ~2?, 428 and 429, a~ter oxida~ion. These reactions are illustrated below in Scheme ~1.

- Scheme V

PhS~OH + ~hO ~) a "s' J~

424: R1 _ tl, R2 _ Me (i)-21 ~,27: R1 = H, F~2 _ Me 425: ~1 = Me, R2 = ~1 428: R1 = Me, R2 = H
426~ = R2 _ Me 429: Rl = R~ = Me Thus, 1, 2 equivalents of eac~ of Compounds 424-426 was separately reacted with one equi~alent of WO 92/02522 Compound 21 and 1. 2 e~ivalents of NaH in T~IF at 25t: for 0 . 5 hours in step ~. The c:ompounds ~o prepared wexe then ~eE~arat~ly reacted in step b with 2 . 5 val~nts of mCPBA in CH2Cl~ at zero degrees C for 0 . 5 . hours to provide Compounds 4a7, 428 and 429 in 79, 79 ;: a~d 6~ percent yields , respectively. Each of Compounds ~27 and 428 was an inse~arable pair of diast~reomers ~single enantiomers at Rl linlced to a rac:emate).
: ~ DNA cleaving prop~rties- of Compounds 427-~29 at 5.0 mM each wer~ assayed and ::ompared to Compsund 21 at l. û mPX using ~X1?4 DNA (50 ~M per base pair~ at p~
~; ~ valu~s of 8.5 and 9.0 at 37:: for 48 hours. see Figs.
: 35 : la and 1b.
:: :
.

:

W093/23~6 ~36~.~3~ PCT/US93/04708 As seen in Figs. la and lb, Compounds 427 and 428 (lanes 3 and 4) exhibited greatly reduced in ~itro DNA cleaving ability a~ basic pH values relative to racemic Compound 21 (lane 5), whereas Compound ~29 (lane 6) exhi~i~ed no DN~ cleaving ability. Phenyl isopropenyl sulfon~ (lane 6) and 2 (phenylsulfonyl)propanol (lane 7~ us~d as controls confirmed that Compounds 427 and ~28 cleavGd DN~ by benzenoid diradicals generated from ~he enediyne core (the fu-~ed ring enediyne freed of the R1 group).
Because phenyl ~in~l sulfone (lane 8) is an alkylating - a~ent, it was not surprising to see Form IT DNA at the concentration used for these studies. Separate studies at 1.0 mM showed no DNA cleavage. The increased DNA
d mage from Compounds ~27 and ~28 at~the higher pH value supports th~ concep~ of a bas~-catalyzed ~-~limination ; of~ the R1 group leading to formation of the DNA-cleaving m~aterial.
: : Further biological e~aluation data for :20 ~ Comp;ound ~27~2~and 429 are provided hereinafter in .
Tables 1 and 2.

Best ~ode ~o~ Sarryin~_out the Invention ~:

:
Mekho~
DNA~cleavage~studi~s, and cytotoxicity studies were~carried out~as dis~ussed in Wo 92/02522, Nicolaou ~t al., Science, ~ 1172-1178 (1992) and the citations therein. Compound~dat~ for a:contemplated fused ring 30~ ~ dynemicin A analogue racema~es are provided:in : WO~g2/02527~or in the a~ove published liter~ture.
The ce~ll;lines~assayed were obtained from the s American Type~:Culture~Col1ec~ion (ATC~) o~ Rock~ille M~ryland, except~ for norfflal :human dermal fibroblasts :W0~3/23~46 ~ PCr/US93/04708 ~NHDF) that were obtained from Clonetics Corporation, San Di~go, t:alifo~nia.
Controi studies wer~ also carried out using the ~Eollowing well known ant.ic:~ncer ~nlgs with the S ~ollowing IC50 Yalues for ~DF and can::er cells. The results of these studues are ~hown below, ~5~g~0 Values fMolarit~L
~uq . N~IDF Cancer Cells Doxorubicirl ~l.l;X10 10 - 9.8X10-8 Dynemicin A lO~8 1.6XlO 8 _ 9.8Xl0~1 Calicheamicin 2.5Xl0 9 5XlO 5 1o 12~
Marpholinodoxorubicin -- 1.6X10 7 - 9~8xlq-9 Taxol lo~8 10 7 - 10 9 Methotrexate 5X10 5 ~10 4 l~-a ~-Platin 5X10 5 10 4 ~ lo 6 Melphelan 10 ~ 10 4 - lo 6 * UCLP~-P3 ce}ls were susceptible at l0 12M~ All other cells were susceptible at 1.5~X10 10 M or higher concentxat ions .
** Molt-4 cells were susceptible at 10~12M~ All other c~lls were susceptible at 3~X10 9~ or higher 2 5 conc:entrations .

Compourlds (+)~S, ( ~ )-45, and ~ 5 clea~ed ~X174 supercoiled DNA under basic conditions ~pH 8 . 5) with comparable potencies (at 1000 and lOO ~LM
c!oncentrations)-~ ~These r~sults may arise ~rom the lack of an extended aromatic ring skeleton in these compo~nds as c:ompared to dynemicin A, which was proposed to .
intercalate into D~;IA prior to drug activation, see:
Sugiura et al., ~ ~, Q7 : 3831 (1~90) ]-The data i n Table 1~ below, show that the enantiomer utiliz d t:an result in dramatically differing .

: ~

-WO93/23046 t h ~'36~ ~ PCT/US93/~4708 cytotoxicite~. Thus, against some cancer cell lines, there was no di~ference in cyto~oxicity among the rac~mate and the two enant~o~ers~ e.g~ SK-Mel-28 c211s, wh~reas with other cells such as Molt-4 T cell leukemia cells, the (+~-enantiomer was about one million times more potent than was the (-)-enan~iomer.

~ytotoxicites of EnedL~es (~-45~ t+) 45 and f-)-45 3 t M 2 Cell ~ype CelL line (~)-45 _t+)-q5 ~ 5 Melanoma ~ SX-Mel 28 6.3Xl0 6 6.~X10 6 6.3X10 6 Pancreatic carcinoma Capan-1 l.6Xl0 6 3.9Xl0 7 1.6X10 6 13reast carcinoma 8CF-~/AD~* 1.6X10-~7.8X10'7 l.~Xl0 6 Promyeocytlc~
laukemia HI.-60: 3~9X10'6 ~9.8XlO 8 7.8X10-7 ~: : T-cell : `
leukemia Molt-4 : l.OXl011 l.OXl013 l.OX10-7 ~
:
: ~ *Adriamycin resistant cell lin~
~ .
, : Cytotoxic~ty studies uslng Compounds 2:1 and ;;;30 :~27-~:29 were contuc~ed as~discussed~b-fore. The reduced otency~in c~11 killing by Compounds 427-~2~ again ~` ~refl-c~ed that ~he C2 methyl grou~ts) attachad next to he.~ulfon- residue hinder~d th~ actiYatlldn ~f these agents Yia ~ a ,~-elimination process. ~s shown in Table 35 ~ ;~ 2,:~bel~w,~ s~ignifioant~dif~er~noes~were obtained with the most~sensit1v-~Mo~lt-4~leukemia cell:line ~103 to 106-S:old~less~actlv~ by attaching~a methyl~group at the C2 pos~i~i:on~;~108-~ol~d~ ss a`c~ive by attachin~two me~hyl groups at ~he:same:pos~itionl.~ The diferential in 40 : ~ cyto~toxici~ies~for ~ompounds~;4~7 and 428 was intriguing WO 93~23046 ~ 3 ~ ~ ~ I P~r/US93/04708 in that it suggests the invov}ement of chiral molecules in th~ activation of these agents in li~ing cellsO The reduced cytotoxicity o~ Compound ~27 against normal cell lines while maintaining considerable ac:ti~rity against cancer cell lines i noteworthy in the ~ontext of selective th~reapeutic agents.

Table 2 Cyto~oxi city tIC5~ of Designed Enediynes Containing ~-Sulfone Triç ~ers _ _ _ , _ _ _ Com~ound Cell Line Z1 427 42~ ~29 NHDF 6 3Xlo~6 <10~4 <10 ~ Non-Toxic CHO 6.3X10 6 ~10 4 <10 4 Non-Toxic Molt-4 lo~l2 10 9 1o-6 10-4 HL-60 9.8X10 87.8X10-7 l.~Xl0 6 ~.5X~ 0~5 C~pan~-l 7.8X10 83.1X10 ~ 6.~3X10 ~ 5.OXl0 5 Po388 9~ 8XlO a1~6Xl0 6 1.~,X10 6 1.3X10-5 Ov~car 3 7.8Xl0 73. lXl0 6 1~ 3X10-s 5~OXlO S
RT-29 3.9X10 77.8X10-7 1.3Xl0 5 2.5Xl0-5 ~ ~C~-P3 7 . 8X10 73 . 1X10 6 1. 3Xl0 5 5. 0~10 5 M~:F-7 3.1X~0~62 5X1~ 5 <10 4 2.5Xl0 5 H-322 3. lXl0'61. 3XlO S Non-Toxic 5~OX10 5 5K-Mel~28 6.3X10 5 ~.~Xl0 5 <10'4 <lO-4 - "
:; 35 Although the pres~nt invention has now been described in terms of c~rtain pref~rred embodiments, and ~: 4 0 exempli~ied with respect thereto , one skilled in the art : ~ : will readily appraciate that various modifi ations, changes, omissions and sub titutions may be made without departing from: ~the sp ' ri~ thereof .

.

Claims (31)

CLAIMS:

1. An enantiomer of 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-C4 alkoxycarbonyl, substituted C1-C6 alkoxycarbonyl, o-nitrobanzyloxycarbonyl, and 1-9-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 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 an 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 enantiomer 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 enantiomer according to claim 1 wherein said aromatic hydrocarbyl ring system W is selected from the group consisting of a benzo ring, a naphtho ring and a 9,10-dioxoanthra ring.

4. The enantiomer according to claim 3 wherein the formed aromatic hydrocarbyl ring system is a benzo ring.

5. The enantiomer according to Claim 4 wherein the benzo ring is 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, carboxyl, C1-C6 hydrocarbyl or benzyl carboxylate, oxyethanol, oxyacetic acid, oxyacetic acid amide, oxyethanol tertiary amino or quaternary ammonium C2-C6 alkyl carboxylate, 3-hydroxyprop-1-ynyl, an oxyacetic C1-C6 hydrocarbyl or benzyloxy ester and halo.

6. The enantiomer according to claim 1 wherein A is a single bond.

7. An enantiomer of 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, o-nitrobenzyloxycarbonyl, 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, carboxyl, C1-C6 hydrocarbyl or benzyl carboxylate, oxyethanol, oxyacetic acid, oxyacetic acid, oxyacetic acid amide, oxyethanol tertiary amino or quaternary ammonium C2-C6 alkyl carboxylate, 3-hydroxyprop-1-ynyl, 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.

8. The enantiomer according to claim 7 wherein R2, R3, R5, R6 and R7 are H.

9. The enantiomer according to claim 8 wherein R1 is phenoxycarbonyl, 2-(phenylsulfonyl)ethoxycarbonyl, 2-(phenylsulfonyl)-2(C1-C6 alkyl)ethoxycarbonyl or 2-(naphthylsulfonyl)ethoxycarbonyl.

10. The enantiomer according to claim 9 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.

11. An enantiomer of a fused-ring compound corresponding to the formula wherein R1 is selected from the group consisting of H, phenoxycarbonyl, benzyloxycarbonyl, 2-(phenylsulfonyl)ethoxycarbonyl, 2-(phenylsulfonyl)-2-(C1-C6 alkyl)ethoxycarbonyl, 2-(naphthylsulfonyl)ethoxycarbonyl and o-nitrobenzyloxycaxbonyl;
R4 is selected from the group consisting of H, hydroxyl, oxyacetic acidt oxyacetic amide, oxyacetic C1-C6 hydrocarbyl or benzyl ester and oxyethanol; and R5 is situated meta or para to the nitrogen atom bonded to R1 and is selected from the group consisting of hydroxyl, C1-C6 alkoxy, benzyloxy, C1-C6 acyloxy, carboxyl, C1-C6 hydrocarbyl or benzyl carboxylate, oxyethanol, oxyacetic acid, oxyacetic acid amide, oxyethanol tertiary amino or quaternary ammonium C2-C6 alkyl carboxylate, 3-hydroxyprop-1-ynyl, an oxyacetic C1-C6 hydrocarbyl or benzyloxy ester and o-nitrobenzyloxy.

12. The fused ring compound according to claim 11 wherein R1 is 2-(phenylsulfonyl)ethoxycarbonyl, 2-(phenylsulfonyl)-2-(C1-C6 alkyl)ethoxycarbonyl, phenoxycarbonyl or 2-(naphthylsulfonyl)ethoxycarbonyl.

13. The fused ring compound according to claim 12 wherein R4 is H.

14. The fused ring compound according to claim 24 wherein R5 is hydroxyl, oxyethanol or C1-C6 acyloxy.

15. A pharmaceutical composition that comprises a DNA-cleaving or cytotoxic amount of an enantiomer 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, o-nitrobenzyloxycarbonyl, 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 an 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.

16. The composition according to claim 15 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.

17. The composition according to claim 16 wherein said aromatic hydrocarbonyl ring system W is selected from the group consisting of a benzo ring, a naphtho ring and a 9,10-dioxoanthra ring.

18. The composition according to claim 16 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, carboxyl, C1-C6, hydrocarbyl or benzyl carboxylate, oxyethanol, oxyacetic acid, oxyacetic acid amide, oxyethanol tertiary amino or quaternary ammonium C2-C6 alkyl carboxylate, 3-hydroxyprop-1-ynyl; oxyacetic C1-C6 hydrocarbyl or benzyloxy ester and halo.

19. The composition according to claim 16 wherein A is a single bond.

20. The composition according to claim 17 wherein R1 is phenoxycarbonyl, 2-(phenylsulfonyl)ethoxycarbonyl, 2-(phenylsulfonyl)-2-(C1-C6 alkyl) ethoxycarbonyl, or 2-(naphthylsulfonyl)ethoxycarbonyl.

21. The composition according to claim 20 wherein W is benzo.

22. The composition according to claim 17 wherein R6 and R7 are both H.

23. The composition according to claim 17 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, benyzloxy, C1-C6 acyloxy, oxyethanol, oxyacetic acid, oxyacetic C1-C6 hydrocarbyl ester, oxyacetic acid amide, oxyethanol tertiary amino or quaternary ammonium C2-C6 alkyl carboxylate and 3-hydroxyprop-1-ynyl.

24. 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 portion is an enantiomer of 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, o-nitrobenzyloxycarbonyl, 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 an 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 R6 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.

25. The chimeric compound according to claim 24 wherein A is a single bond, R2, R3, R6, R7 and R8 are hydrogen, and W is benzo.

26. The chimeric compound according to claim 25 wherein said aglycone portion is bonded to an oligosaccharide portion.

27. The chimeric compound according to claim 25 wherein R1 is phenoxycarbonyl, 2-(phenylsulfonyl)ethoxycarbonyl, 2-(phenylsulfonyl)-2 (C1-C6)ethoxycarbonyl or 2-(naphthylsulfonyl)ethoxycarbonyl.

28. A compound of the 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, o-nitrobenzyloxycarbonyl, 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;
R8 is hydrogen or methyl;
W together with the bonded vinylene group forms an 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;
R24 and R25 are independently C1-C3 alkyl or phenyl; and q is zero or 1.

29. The compound according to claim 28 wherein A is a sing1e bond and R2, R3 amd R8 are H.

30. The compound according to claim 29 wherein W together with the bonded vinylene group forms a benzo ring.

31. The compound according to claim 30 wherein R1 is phenoxycarbonyl.