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WO1992002522A1 - Syntheses, procedes de preparation et utilisation d'analogues de dynemicines - Google Patents

Syntheses, procedes de preparation et utilisation d'analogues de dynemicines Download PDF

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Publication number
WO1992002522A1
WO1992002522A1 PCT/US1991/005436 US9105436W WO9202522A1 WO 1992002522 A1 WO1992002522 A1 WO 1992002522A1 US 9105436 W US9105436 W US 9105436W WO 9202522 A1 WO9202522 A1 WO 9202522A1
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WO
WIPO (PCT)
Prior art keywords
group
compound
hydroxyl
oxyacetic
fused
Prior art date
Application number
PCT/US1991/005436
Other languages
English (en)
Inventor
Chan-Kou Hwang
Adrian L. Smith
Sebastian V. Wendeborn
Kyriacos C. Nicolaou
Erwin P. Schreiner
Wilhelm Stahl
Wei-Min Dai
Original Assignee
Scripps Clinic And Research Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Scripps Clinic And Research Foundation filed Critical Scripps Clinic And Research Foundation
Priority to FI930380A priority Critical patent/FI930380L/fi
Priority to EP91916729A priority patent/EP0594624A1/fr
Priority to AU86123/91A priority patent/AU661278B2/en
Publication of WO1992002522A1 publication Critical patent/WO1992002522A1/fr
Priority to NO93930314A priority patent/NO930314L/no

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/08—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings

Definitions

  • 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 ring, as well as chimeras that contain such a fused ring system.
  • Me is methyl
  • a potent antibacterial and anticancer agent recently isolated from Micromonospora chersina [(a) Konishi et al, J. Am. Chem. Soc..
  • calicheamicin and esperamicin derivatives are perhaps the best known of the enediyne compounds.
  • the present invention relates to novel fused ring systems that contain an epoxide ring and an
  • the compounds have DNA-cleaving, antibiotic and antitumor activities.
  • a fused ring compound of the invention has a structure that corresponds to the formula
  • A is a double or single bond
  • R 1 is selected from the group consisting of H, C 1 -C 6 alkyl, phenoxycarbonyl, benzyloxycarbonyl, C 1 -C 6 alkoxycarbonyl, substituted C 1 -C 6 alkoxycarbonyl (particularly substituted ethoxycarbonyl), and
  • R 2 is selected from the group consisting of H, carboxyl, hydroxylmethyl and carbonyloxy C 1 -C 6 alkyl;
  • R 3 is selected from the group consisting of H and C 1 -C 6 alkoxy
  • R 4 is selected from the group consisting of H, hydroxyl, C 1 -C 6 alkoxy, oxyacetic acid, oxyacetic C 1 -C 6 hydrocarbyl or benzyl ester, oxyacetic amide, oxyimidazilthiocarbonyl and C 1 -C 6 acyloxy;
  • R 6 and R 7 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;
  • R 8 is hydrogen or methyl, with the proviso that R 8 is hydrogen when W, together with the carbon atoms of the intervening vinylene group is
  • W together with the intervening vinylidene group forms a benzo ring so that a compound has the structural formula shown below.
  • R 5 is selected from the group consisting of hydrogen, C 1 -C 6 alkoxy, hydroxyl, C 1 -C 6 acyloxy, oxyethanol, oxyacetic acid, o-nitrobenzyloxy and halo, and A and the remaining R groups are as before described.
  • R 2 , R 3 , R 5 , R 7 and R 8 are hydrogen so that a compound of the invention corresponds to the structural formula shown below, where R 1 and R 4 are as previously defined.
  • R is C 1 -C 6 alkoxy, hydroxyl
  • a chimeric compound also referred to as a chimer or chimera
  • a chimeric compound 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.
  • the oligosaccharide portion comprises a sugar moiety selected from the group consisting of ribosyl, deoxyribosyl, fucosyl, glucosyl, galactosyl,
  • N-acetylglucosaminyl N-acetylgalactasaminyl, a
  • a monoclonal antibody or binding site portion thereof is bonded to the fused ring compound aglycone portion through an R 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 R 4 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.
  • the DNA to be cleaved, target tumor cells whose growth is to be inhibited or target microbial cells is (are)
  • composition of the invention 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.
  • Figure 1 is a photograph of an ethidium bromide stained 1 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 percent THF at pH 7.4. Lane 1 is the DNA alone as control, lanes 2-6 show the results obtained with 5000, 2000, 1000, 500 and lOO ⁇ M Compound 40, respectively. The designations I, II and III outside the gel indicate 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 0X174 from I DNA by Compounds 40, 47, 42, 54, 55, 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, 47, 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-1).
  • 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-1) .
  • IC 50 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 MB49 murine bladder carcinoma cells by various concentrations of Compound 21 (DY-2).
  • a compound of the invention contains an enediyne macrocycle linked to a fused ring that
  • A is a double or single bond
  • R 1 is selected from the group consisting of H, C 1 -C 6 alkyl, phenoxycarbonyl, benzyloxycarbonyl, C 1 -C 6 alkoxycarbonyl, substituted C 1 -C 6 alkoxycarbonyl
  • R 2 is selected from the group consisting of H, carboxyl, hydroxylmethyl and carbonyloxy-C 1 -C 6 alkyl;
  • R 3 is selected from the group consisting of H and C 1 -C 6 alkoxy
  • R 4 is selected from the group consisting of H, hydroxyl, C 1 -C 6 alkoxy, oxyacetic acid (-OCH 2 CO 2 H) , C 1 -C 6 hydrocarbyl or benzyl oxyacetic acid ester, oxyacetic amide, oxyethanol (-OCH 2 CH 2 OH) , oxyimidazylthiocarbonyl and C 1 -C 6 acyloxy;
  • R 6 and R 7 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 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 6-membered rings all but two of which rings are aromatic, and in which that aromatic hydrocarbyl ring system, W, is joined [a, b] to the structure shown; and
  • R 8 is hydrogen or methyl with the proviso that R 8 is hydrogen when W together with the intervening vinylidene group is 9,10-dioxoanthra.
  • R 6 and R 7 groups are shown in Scheme III and are discussed in relation thereto, and
  • the bond, A, between the R 2 and R 3 substituents can be a double or single bond.
  • the bond A is preferably a single bond.
  • a C 1 -C 6 alkyl group, as can be present in R 1 is exemplified by methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, pentyl, 2-methylpentyl, hexyl, cyclohexyl, cyclopentyl and the like.
  • a substituted C 1 -C 6 alkyl group is also contemplated as an R 1 group.
  • substituted alkyl groups include hydroxyalkyl groups such as 2-hydroxyethyl, 4-hydroxyhexyl and
  • C 1 -C 6 alkyl and substituted C 1 -C 6 alkyl groups are further contemplated as the C 1 -C 6 alkyl portion of a carbonyloxy C 1 -C 6 alkyl group of R 2 ; i.e., a C 1 -C 6 alkyl ester of a R 2 carboxyl group, and of a R 1 urethane group.
  • Those same alkyl groups can constitute the alkyl portion of a C 1 -C 6 alkoxy group of R 3 or R 4 .
  • a C 1 -C 6 acyloxy group as is present in R 4 or R 5 is a carboxylic acid derivative of an appropriate alkyl group, above, except for, for example, cyclohexyl and iso-propyl, and is limited to a
  • C 1 -C 6 acyloxy groups include formyloxy, acetoxy, propionoxy, butyryloxy, iso-butyryloxy, pentanoyloxy, 2-methylbutyryloxy,
  • the alcohol-carbonyl portion of a urethane R 1 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 acetyleni ⁇ 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.
  • a halo formate derivative such as a chloroformate like phenylchloroformate
  • Such groups can also be prepared by base-catalyzed exchange from a formed carbamate using the substituted ethyl alcohol as is illustrated hereinafter.
  • Exemplary C 1 -C 6 alkoxycarbonyl groups and substituted C 1 -C 6 alkoxycarbonyl groups contain a before-described C 1 -C 6 alkoxy group or substituted C 1 -C 6 alkoxy group linked to the carbonyl group and can be formed by reaction of a C 1 -C 6 alkylchloroformate.
  • Exemplary substituted ethoxycarbonyl groups that are a particularly preferred group of substituted C 1 -C 6 alkoxycarbonyl group have a substituent other than hydrogen at the 2-position of the ethoxy group, and include 2-trimethylsilylethoxycarbonyl,
  • 2-triphenylphosphoniumethoxycarbonyl halide e.g., chloride, bromide or iodide
  • R 1 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 R 1 contains a 2-substituted-ethoxycarbonyl group such as a 2-phenylsulfonyl-, 2-naphthylsulfonyl- and
  • Phenylsulfonylethoxycarbonyl, ⁇ -naphthyl- and ⁇ -naphthylsulfonylethoxycarbonyl are particularly preferred R 1 groups, with phenoxycarboxyl being a preferred R 1 group.
  • R 8 group can be methyl or hydrogen with the proviso that R 8 is hydrogen when W along with the intervening vinylene group carbon atoms forms a 9,10-dioxoanthra ring. It is particularly preferred that R 8 be methyl when W forms a benzo ring.
  • oxyethanol (-OCH 2 CH 2 OH) , oxyacetic acid (-OCH 2 CO 2 H) , oxyacetic C 1 -C 6 hydrocarbyl esters such as the before-discussed C 1 -C 6 alkyl groups such as ethyl oxyacetate (-OCH 2 CO 2 CH 2 CH 3 ) , as well as C 1 -C 6 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 C 1 -C 6 and benzyl esters that have been
  • a pharmaceutically acceptable non-toxic salt of the oxyacetic acid such as sodium, potassium, ammonium, calcium and magnesium is also contemplated.
  • An oxyacetic acid amide corresponds to the chemical formula -OCH 2 C0NR 13 R 14 wherein R 13 is hydrogen (H) or C 1 -C 6 alkyl (as before) and R 14 is independently hydrogen, C 1 -C 6 alkyl, phenyl, 1- or 2-napthyl, 1- or 2-anthryl, or a peptide having 1 to about six amino acid residues; or R 13 and R 14 together with the amido nitrogen atom form a 5- or 6-membered ring as is present in pyrrolidine, piperidine or morpholine.
  • a particularly contemplated peptide is distamycin, or a derivative thereof as discussed in Taylor et al., Tetrahedron. 40:457 (1984) 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 prepared.
  • Another particularly preferred peptide is -Ala-Ala-Ala-, [(-Ala-) 3 ].
  • R 4 group that contains a derivatized oxyacetic acid amide or ester can also include a
  • a Mab 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 R 3 ).
  • 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.
  • 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.
  • 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
  • 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
  • Exemplary antibody binding site portions include F(ab), F(ab'), F(ab') 2 and F v portions of an intact antibody 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.
  • anti-tumor Mabs are noted in the table below, listed by the name utilized in a
  • a fused ring enediyne compound of the invention can also be glycosidically linked to a sugar moiety to form a second type chimeric molecule.
  • the fused ring enediyne compound takes the place of the aglycone as in an antibi ic molecule such as doxorubicin, calicheamicin or esperamicin, with the sugar moiety taking the place of the oligosaccharide portion.
  • Bonding between the used 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 hydroxyl group.
  • a preferred spacer group is an oxyethanol group that can be an R 4 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 is complete, except for any blocking groups on otherwise reactive
  • a sugar moiety is 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,
  • 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.
  • the 1-position hydroxyl group of an otherwise protected sugar as with t BuMe 2 Si or Et 3 Si groups is reacted with
  • DAST diethylaminosulfur trifluoride
  • the 1, 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 groups (are derivatives of anthraquinone) and are therefore referred to as 9, 10-dioxoanthra rings.
  • a benzo, naphtho or 9,10-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 (a, 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
  • one or two substituents can be present at one or two of the remaining positions of the radical. Symmetrical substitution by the same substituent is preferred because of the lessened
  • R 5 substituent is present on a benzo ring, that substituent is referred to as R 5 , which designation for convenience includes hydrogen.
  • R 5 is thus selected from the group consisting of hydrogen (no substituent), C 1 -C 6 alkoxy, benzyloxy, o-nitrobenzyloxy, hydroxyl, C 1 -C 6 acyloxy, oxyethanol, oxyacetic acid, oxyacetic acid C 1 -C 6
  • R 10 and R 11 are selected from the group consisting of
  • W is more preferably a benzo group that contains a single substituent R 5 .
  • R 5 is situated in the benzo ring meta or para to the nitrogen atom bonded to R 1 . That R 5 group is more preferably selected from the group consisting of hydroxyl, C 1 -C 6 alkoxy, benzyloxy,
  • R 5 When R 5 is meta to the above nitrogen atom, it is preferred that the R 5 group be an electron releasing group such as hydroxyl or a C 1 -C 6 acyloxy group that can provide a hydroxyl group intracellularly.
  • a C 1 -C 6 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.
  • R 5 is para to the above nitrogen atom, it is preferred that the R 5 group be an o-nitrobenzyloxy group, oxyethanol, oxyacetic acid or oxyacetic acid
  • a particularly preferred compound has a structure corresponding to Formula Xlb, hereinafter.
  • a naphtho ring can have three substituents.
  • This ring can have a 4-position radical, R 5 , selected from the group consisting of hydroxyl, C 1 -C 6 alkoxy, benzyloxy, C 1 -C 6 acyloxy and halo, and substituents at the 5- (R 10 ) and 8-positions (R 11 ) that are selected from the group consisting of hydroxyl, C 1 -C 6 alkoxy,
  • a 9 , 10-dioxoanthra ring can have three substituents at the 4- (R 5 ) , 5- (R 9 ) and 8-positions (R 12 ) that are
  • R 5 , R 9 and R 12 can define the same groups, and all three groups can be written as either R 5 , R 9 or R 12 , but they are shown separately herein.
  • R 2 and R 3 be hydrogen, and that R 6 and R 7 be hydrogen.
  • the fused ring system W together with the depicted vinylene group be substituted benzo, or an unsubstituted benzo, naphtho or 9,10-dioxoanthra ring. 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 forms a benzo ring.
  • One particularly preferred group of compounds of the invention in which W is an R 5 -substituted benzo ring corresponds to structural Formula X.
  • R 1 is selected from the group consisting of H,
  • R 2 is selected from the group consisting of H, carboxyl, hydroxylmethyl and carbonyloxy C 1 -C 6 alkyl
  • R 3 is selected from the group consisting of H and C 1 -C 6 alkoxy
  • R 4 is selected from the group consisting of H, hydroxyl, oxyacetic acid (-OCH 2 CO 2 H) , oxyacetic C 1 -C 6 hydrocarbyl or benzyl ester, oxyacetic amide,
  • R 5 is selected from the group consisting of hydrogen, C 1 -C 6 alkoxy, benzyloxy, o-nitrobenzyloxy, hydroxyl, C 1 -C 6 acyloxy, oxyethanol, oxyacetic acid, oxyacetic acid C 1 -C 6 hydrocarbyl ester and halo;
  • R 6 and R 7 are each H or together form with the intervening vinylidine group form a one, two or three fused aromatic ring system, and R 8 is methyl or
  • a still more preferred group of compounds of the invention correspond to structural Formulas XI, XIa and Xlb.
  • R 1 , R 4 , R 5 and R 8 are as previously defined.
  • R 1 is most preferably phenoxycarbonyl phenylsulfonylethoxycarbonyl
  • R 8 is most preferably hydrogen (H) to provide a compound of
  • R 4 is most preferably H, hydroxyl, imidazylthiocarbonyloxy, benzyl oxyacetate and C 1 -C 6 hydrocarbyl oxyacetate such as ethyl oxyacetate.
  • R 5 in Formulas XI and XIa is H, but is more preferably
  • a compound or chimera of the invention is useful as a DNA cleaving agent, and also as an
  • a cytoxic (antitumor) agent as are dynemicin A, calicheamicin, esperamicin and
  • 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 described hereinafter as well as those described by et al., J. Pro. Chem.. 54:2781 (1989); 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.
  • a compound or chimer of the invention is useful against Gram-positive bacteria such as S. aureus and epidermis. Micrococcus luteus and Bacillus subtillis as is dynemicin A. Such a compound or chimer also exhibits antimicrobial activity against E. coli.
  • Antimicrobial and antitumor assays can also be carried out by techniques described in U.S. Patent No.
  • a before-described compound can also be shown to undergo a Bergman 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.
  • compositions are 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.
  • a compound or chimer of the present invention 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.
  • the agent is typically dissolved or dispersed in a physiologically tolerable carrier.
  • a carrier or diluent is a material useful for administering the active compound and must be
  • physiologically tolerable in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
  • pharmaceutically tolerable and “pharmaceutically acceptable” are used interchangeably and refer to molecular entities and compositions 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.
  • a compound or chimer of the invention can be utilized, dissolved or dispersed in a liquid
  • composition such as a sterile suspension or solution, or as isotonic preparation containing suitable
  • 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 Remmington's Pharmaceutical Sciences. Mack Publishing Co., Easton, PA (1980).
  • 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,
  • compositions in liposome form can contain 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.
  • an active agent can also be used in compositions such as tablets or pills, preferably containing a unit dose of the compound or chimer.
  • the agent active ingredient
  • 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
  • the tablets or pills can be any suitable tolerable carriers.
  • the tablets or pills can be any suitable tolerable carriers.
  • the tablets or pills can be any suitable tolerable carriers.
  • the tablets or pills can be any suitable tolerable carriers.
  • composition 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 formulation isotonic with the blood of the intended recipient.
  • the tablets or pills can also be provided with an enteric layer in the form of an envelope that serves to resist disintegration in the stomach and permits the active ingredient to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings including polymeric acids or mixtures of such acids with such materials a& shellac, shellac and cetyl alcohol, cellulose acetate phthalate, and the like.
  • 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.
  • unit dose 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
  • suitable unit dosage forms in accord with this invention are tablets, capsules, pills, powder packets, granules, wafers, cachets, teaspoonfuls, dropperfuls, ampules, vials, segregated multiples of any of the foregoing, and the like.
  • a compound or chimer of the invention is present in such a pharmaceutical composition in an amount effective to achieve the desired result.
  • a compound or chimer of the invention can be utilized in an amount sufficient to provide a
  • an effective amount of a compound or chimer of the invention is about 0.1 to about 15 mg per kilogram of body weight or an amount sufficient to provide a concentration of about 0.01 to about 50 ⁇ g/mL to the bloodstream.
  • a compound or chimer of the invention exhibits antimicrobial activity in a concentration range of about 0.01 mg to about 50 ⁇ g/mL.
  • a compound or chimer of the invention is useful in cleaving DNA, as a cytotoxic agent and 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
  • 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 the invention (active
  • 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.
  • the desired result is carried out in vitro. contact is maintained by simply admixing the DNA or target cells with the composition and maintaining them together under the appropriate conditions of temperature and for cell growth to occur, as for
  • 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.
  • a mammal such as a rodent like a rat, mouse, or rabbit
  • a farm animal like a horse, cow or goat
  • a primate like a monkey, ape or human
  • compositions administration of the composition to the mammal by oral, nasal or anal administration or by introduction
  • contact in vivo is achieved via the blood or lymph systems.
  • Exemplary methods oi die 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 ten other neoplastic cell lines are discussed.
  • 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 for these syntheses is illustrated below in Scheme I, with the general forward synthesis shown in Scheme II, thereafter.
  • the basic fused 3-, 4- or 5-fused six-membered ring system is first formed.
  • an oxygen-containing substituent R 4 having an oxygen atom of that group bonding to the 10-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 10-position (steps a and b) of Compound 5.
  • mCPBA m-chloroperbenzoic acid
  • the acetyl group is removed to form the alcohol (Compound 6, step c), which is then blocked with a t-butyldimethylsilyl group (Si t BuMe 2 ; 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
  • the epoxide ring is added next between the 6- and 10-positions by oxidation as with (mCPBA) as in
  • the formed epoxide ring is on the opposite side of the ring plane from the 6-position acetylenic group, and preferably, the epoxide is in an ⁇ -configuration, whereas the acetylenic group is ⁇ .
  • step g The oxygen-linked R 4 group Si t BuMe 2 is replaced with a hydrogen (step g) and the alcohol so formed is next converted to a ketone.
  • Compound 11 (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 reacting (Z) 1-chloro-4-trimethylsilyl-but-1-en-3-yne (Compound 12) with the ketone Compound 11 in the
  • step i palladium 11 acetate
  • Scheme III also illustrates formation of a benzo ring W by reaction of an aniline compound with ethyl
  • a derivative of compound such as Compounds 2 or 21 having a hydroxyl group (R 5 , or R 4 of Scheme III) can be prepared following the synthetic route
  • 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 ring compound of the invention where R 4 is hydroxyl (OH) (step k).
  • a base such as lithium diisopropylamide (LDA)
  • LDA lithium diisopropylamide
  • R 4 group can later be replaced with a hydrogen or derivatized as discussed hereinafter.
  • R 6 and R 7 are preferably hydrogen (H) . However, R 6 and R 7 along with the
  • intervening vinylidene group can together form an aromatic mono-, di- or tri-cyclic ring system that can be hydrocarbyl or heterocyclic.
  • 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, R 2 ) as a single unit, as is shown in Scheme XI.
  • step e The product of step e is reacted first with a chloroformate whose R 1 group is shown hereinafter, and then with diacetylide or an aromatic diacetylide ring system compound (R 2 as shown hereinafter) blocked with a trimethylsilyl group (TMS) and containing a mono-Grignard reagent in step f to form the partially linked macrocyclic ring precursor.
  • a chloroformate whose R 1 group is shown hereinafter
  • R 2 aromatic diacetylide ring system compound
  • TMS trimethylsilyl group
  • the epoxide ring is formed in step g by reaction with mCPBA.
  • the Si t BuMe 2 group is removed in step h by reaction with n BuNF, and the resulting alcohol is oxidized with pyridinium chlorochromate in the presence of molecular sieves in step i to form the ketone.
  • step j The macrocyclic ring is closed in step j by reaction with lithium diisopropylamide (LDA).
  • LDA lithium diisopropylamide
  • step k The hydroxyl group formed in step j is reacted in step k with an appropriate 2-haloacetic acid derivative (R 3 as shown hereinbelow) to form the final product.
  • R 3 CH 2 COOH
  • Subscripted R groups are used in this scheme to distinguish R groups therein from the superscripted R groups defined elsewhere herein.
  • R 3 is a peptide that typically contains zero to about six amino acid residues that links a monoclonal antibody, "Mab”, to the
  • R 3 "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.
  • a vicinal diyne aromatic compound suitable for introduction at the 6-position (based on Compound 4) of a 3-, 4- or 5-fused six-membered ring system can be prepared by alkylation of a vicinal dihalide with trimethylsilylacetylene in the presence of
  • Exemplary vicinal dihalo aromatic compounds commercially available from Aldrich Chemical Co. of Milwaukee, WI include 1,2-diiodobenzene,
  • TMS trimethylsilyl groups
  • An appropriate vicinal diacetylide can also be prepared via a vicinal, dihydroxymethyl compound.
  • 1,4-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
  • the above method of synthesis can also be applied to unsubstituted aromatic compounds, such as vicinal dicarboxylic acids, anhydrides or esters.
  • aromatic compounds such as vicinal dicarboxylic acids, anhydrides or esters.
  • 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).
  • DIBAL diisobutylaluminum hydride
  • the resulting dihydroxymethyl compounds are thereafter reacted as described above to form a desired compound.
  • an aromatic diacetylide contain its two vicinal acetylenic groups symmetrically bonded to the ring system so as to minimize isomer formation.
  • 2,3-disubstituted-naphthalene, anthracene or quinoxaline compounds are utilized, or a ⁇ ,7-disubstituted-quinoxaline, or the like.
  • step iii provides the O-blocked
  • Scheme III provides Compounds 29 (steps v-viii) and 30 (steps ix and xii).
  • Compound 30 is then reacted with lithium iodide in pyridine, oxidized with 2,3-dichloro-5,6-dicyanobenzoguinone (DDQ) , and reacted with pivaloyl chloride (Piv) to form Compound 31 (steps xiii-xv).
  • step xxiii thiocarbonyldiimidazole
  • step xxiv tri-n-butylstannane and AIBN
  • step xxiv hydroxide ion
  • 1-aminoanthraquinone (Aldrich Chemical Co.) can be used as a starting material.
  • the amino group is blocked as with a t-Boc group
  • the quinone function is reduced as with DIBAL
  • the resulting phenolic hydroxyl groups are blocked with pivaloyl chloride.
  • t-Boc group is then removed as by reaction with trifluoroacetic acid (TFA) and the aromatic ring is annelated with ethyl cyclohexanone-2-carboxylate, followed by 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.
  • TFA trifluoroacetic acid
  • the TES group is removed and the resulting hydroxyl group is oxidized to a ketone as by Swern oxidation. That ketone is then reacted with LDA and methyl chloroformate to form a carboxy enol whose hydroxyl group can be methylated with diazomethane.
  • carbamate derivative of a dynemicin analog can be prepared from a phenoxycarbonyl derivative such as
  • Scheme II outlined the construction of Compound 2 starting from the quinoline derivative Compound 4 [(a) Masamune et al., J. Org. Chem.. 29:681-685 (1964); (b) Curran et al., J. Org. Chem.. 49:2063-2065 (1984); (c) Hollingsworth et al., J. Org. Chem.. 1537-1541 (1948)].
  • step a treatment of Compound 4 with m-chloroperbenzoic acid (mCPBA) in dichloromethane gave the corresponding N-oxide (step a) which underwent regiospecific rearrangement [Boekelheide et al., J. Am. Chem. Soc.. 76:1286-1291 (1954)] upon heating in acetic anhydride (step b) to give the acetoxy derivative
  • step f Treatment of Compound 8 with mCPBA led to epoxide Compound 9 (85 percent) (step f), which was converted to ketone Compound 11 via alcohol Compound 10 by desilylation (step g) followed by oxidation (step h).
  • step i Coupling Compound 11 with the vinyl chloride derivative Compound 12 via Pd(OAc 2 )-CuI catalysis (step i) followed by AgNO 3 /KCN treatment (step j) resulted in the
  • step k treatment of Compound 3 with LDA in toluene at -78°C gave the targeted dynemicin A model
  • Compound 2 (80 percent based on 25 percent recovery of starting material). Compound 2 is also referred to as DY-l in Figures 10 and 11.
  • the acetylenic moieties are bent from linearity with the following angles: C14, 160.4°; C15, 170.8°; C18, 171.6° and C19, 162.0°.
  • the distance between carbons C14 and C19 (cd distance) [Nicolaou et al., J. Am. Chem. Soc.. 110:4866-4868 (1988)] was found to be 3.63A, a value that agrees well with the calculated one for the MMX minimized structure of
  • TMSOTf trimethylsilyl trifluoromethylsulfonate
  • reaction of Compound 2 with Co 2 (CO) 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 Co 2 (CO) 8 resulted in the formation of a monocobalt complex in addition to dicobalt derivative Compound 18 and starting material Compound 2.
  • CD 2 Cl 2 resulted in the incorporation of two deuterium atoms in Compound 17 confirming methylene chloride as an effective hydrogen atom donor in these aromatization studies.
  • step b When step b is carried out with one equivalent of mCBPA, the corresponding sulfoxides, Compounds 21b and 46b are prepared.
  • Freshly prepared Compound 40 was reacted with either phenol (i, PhOH) or thiophenol (ii, PhSH) using two equivalents of either nucleophile at 25°C for two hours to provide Compounds 49 (25 percent yield) or 50 (33 percent yield, respectively, as step d.
  • phenol i, PhOH
  • thiophenol ii, PhSH
  • Compound 48 proved to be quite stable under basic or neutral conditions.
  • Enediyne model system Compound 43 (Scheme IX) was designed for its potential to generate species 42 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.
  • the methoxy Compound 41 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-H 2 O in benzene: 1,4-cyclohexadiene (1:1) at 25°C for one hour, Compound 41 afforded the aromatized product Compound 61 (32 percent yield).
  • Treatment of Compound 41 as per steps a and b of Scheme V yields the corresponding methoxyphenyl derivative Compound 41a having a hydrogen in place of the hydroxyl of Compound 41.
  • Step h utilized acetonitrile as a solvent and a one hour reaction time as compared with step a of 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 equivalents of trimethylsilylacetylene, 0.05 equivalents of Pd(PPh 3 ) 4 , 0.2 equivalents of Cul and two equivalents of triethylamine in acetonitrile (as were present in steps a and b) with a reaction time of 20 hours at 25°C to provide Compound 82 in 76 percent yield.
  • Steps c-g of both reactions were identical. Reaction of Compounds 77 or 88 with one equivalent each of mCPBA leads to preparation of the corresponding sulfoxides. Compounds 77a and 88a.
  • Chimeric compounds that include both a fused ring enediyne as the aglycone and a before-discussed mono- or oligosaccharide as the oligosaccharide portion is also contemplated, as noted earlier.
  • the previously depicted saccharides are related to the calicheamicin oligosaccharide.
  • the before-depicted saccharides correspond to the calicheamicin oligosaccharide (Structures F and G), the oxime precursor thereto (Structures D and E), and fragments thereof (Structures A-C). More specifically, the disaccharide Structure A corresponds to the
  • the calicheamicin A and E rings with the hydroxylamine link to a B ring analog corresponds to the monosaccharide Structure B corresponds to the A ring alone with the hydroxylamine-linked B ring analog.
  • the trisaccharide thiobenzoate 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
  • calicheamicin oligosaccharide whereas 5-ring Structure E is the FMOC-deblocked 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
  • the disaccharide-linked hydroxylamine compound is prepared in a manner analogous to that of Compound 12 of Nicolaou et al., J. Am. Chem. Soc.. 112:8193-8195 (1990), except that an o-nitrobenzyl (shown as NBnO or ONBn in the schemes) glycoside is utilized instead of the methyl glycoside precursor.
  • NBnO or ONBn in the schemes an o-nitrobenzyl glycoside
  • 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).
  • the hydroxylamine linked A ring derivative (Structure B) can be prepared starting with Compound 9 of Nicolaou et al., J. Am. Chem. Soc. 112:8193-8195 (1990). There, the 2-hydroxyl is blocked with a
  • t-butyldimethylsilyl (t BuMe 2 Si) group as before, and the carbonate group removed by reaction of sodium hydride in ethylene glycol-THF at room temperature.
  • the keto group can be prepared by oxidation with dibutylstannic oxide ( n Bu 2 SnO) in methanol at 65°. The 3-position hydroxyl is similarly blocked with a l BuMe 2 Si group, and the oxime formed as above.
  • Structures 112a-ll5a and 112b-115b are interchangeable, since the absolute stereochemistry of the aglycons has not been determined. Physical data for Compounds 115a and 115b are provided hereinafter. 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.
  • Racemic compound 120 was reacted with Compound 123 as shown in Scheme XV.
  • Compound 123 was prepared 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 itself prepared in a manner analogous to that discussed in Nicolaou et al., J. Am. Chem. Soc., 112:8193-8195
  • diastereomeric anomers 124a and 124b present as a ⁇ - to ⁇ -anomer ratio of about 5:1, the ⁇ -anomer being shown as Compound 124a, and the ⁇ -anomer, Compound 124b, not being shown.
  • Scheme XVI shows only the oxime-containing ring of Compound 126a, with the remaining portions indicated by the wavy lines.
  • reduction of Compound 126a with sodium cyanoborohydride in BF 3 ⁇ Et 2 O at -50°C provided a 65 percent yield of epimeric Compounds 127a and 127b, that were present in about equal amounts.
  • Those compounds were epimeric at the 4-position of the A ring as indicated by the number 4 and the arrow.
  • an R 5 group such as that shown in Formula X is preferably a hydroxyl group or a group convertible thereto intracellualarly.
  • 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 25°C for 14 hours to form Compound 134 in 96 percent yield.
  • Compound 134 was reacted with m-aminophenol in THF 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
  • Compound 136 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 137b 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 LiAlH 4 in THF at reflux for three hours and then with oxygen and SiO 2 at 25°C for 24 hours to form Compound 138 in 50 percent yield.
  • Compounds 142a, 142b, 143a or 143b can be used to form a fused-ring enediyne compound of the invention using the steps outlined in the prior reaction schemes, such as Compounds 59a and 59b.
  • 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
  • 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. Oxidation of the free acid as in step b provides Compound 154.
  • the carboxylic acid group of Compound 154 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.
  • Compound 41 was utilized at a 2 mM concentration at pH 5.0.
  • Compounds 40, 42 and 54 were further found to cause significant DNA cleavage when incubated at 5mM with supercoiled ⁇ X174 DNA at pH 8.0 ( Figure 2).
  • 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
  • IC 50 Values of IC 50 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-1 African green monkey kidney cells (ATCC CCL 70) or WI-38 human lung cells (ATCC CCL 75).
  • Each of the dynemicin A analog Compounds 24a-g exhibited anti-tumor activity against MIA PaCa-2 cells, with the esters (Compounds 24b-g) being more potent than the free acid (Compound 24a).
  • Compounds 24a-g were also active against MB-49 cells and inactive against CV-1 and WI-38 cells.
  • the described chemistry supports the viability of two paths as triggering mechanisms for the dynemicin A-type cascade by showing that a lone pair of electrons on a heteroatom (N or O) strategically positioned on the aromatic ring in relation to the epoxide moiety serves to initiate the cycloaromatization reaction.
  • a heteroatom N or O
  • Such reactive species can be generated within the cell by enzymatic reactions, or as shown above, be released from suitable precursors under mild conditions in the
  • a vial containing a 50 micromolar per base pair solution of 0X174 Form I double stranded DNA in 2.0 microliters of pH 7.4 phosphate (50 mM) buffers were added 6.0 microliters of the same buffer solution and 2.0 microliters of a 5.0 millimolar ethanol solution of Compound 40.
  • MIA PaCa-2 cells, MB-49, CV-1 or WI-38 cells 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 incubated for 4 hours (37°C, 7 percent CO 2 ).
  • tissue culture (T.C.) medium RPMI 1640 plus 10 percent fetal calf serum and 2 mM L-glutathione
  • T.C. media to give a density of 1 X 10 5 cells/mL.
  • T.C. medium A minimum volume of 300 ⁇ L is required per 96-well plate.
  • TCA trichloroacetic acid
  • SRB Sulforhodamine B dissolved in 1 percent acetic acid to each well of 96- well plates using multichannel pipettor. b. Incubate plates at room temperature for 30 minutes.
  • IC 50 values i.e., the concentration of
  • Molt-4 (All are human cancer cell lines as
  • M-14 and M-21 are human melanoma cell lines, whereas P-3 is a human non-small cell lung carcinoma cell line.
  • Table 3 contains data for two fused-ring enediyne compounds (Compounds 47 and 120) as well as data for six chimers.
  • naphthylsulfonylethoxy group were about 10 to 100 times more active than similar compounds having a phenoxy group as part of the carbamate.
  • Compounds having an electron releasing group relative to hydrogen para to the carbamate nitrogen atom tended to be equal to less active than those with hydrogen, whereas compounds with an intracellular-formed electron releasing group (e.g. Compounds 59a and 59b) relative to hydrogen meta to that nitrogen atom tended to be more active than compounds having hydrogen at that position.
  • the data Table 3 indicate that the chimeras are effective. Those data also indicate that the presence of the FMOC group 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
  • 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 59b, 41b, 4lc and 41d exhibited IC 50 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 IC 50 values against Molt-4 cells were also 10,000- 100,000 times smaller than the IC 50 values for those compounds against NHDF cells.
  • Example 3 N-Phenyloxycarbonyl-6-(3(Z)-hexene-1,5- diynyl)-6a:10a-epoxy-10-oxo- 5,6,6a,7,8,9,10,10a- octahydrophenanthridine (Compound 3.
  • Example 8 N-Phenyloxycarbonyl-10-tert- butyldimethylsilyloxy-6a:1 Oa-epoxy- 6-ethyl-5,6,6a,7,8, 9,10,10a- octahydrophenanthridine (Compound 9.
  • Alcohol Compound 10 (6.009, 17.4 mmol) was dissolved in dry dichlorometh ne (180 mL) and treated with powdered, activated 4A molecule... sieves (l g) and pyridinium chlorochromate (6.25 g, 29.0 mmol).
  • Compound 17 has been prepared by several methods as indicated below.
  • keton Compound 17 (53.7 mg, 92 percent).
  • thionoimidazolide 20 160 mg, 95 percent. 20: white crystalline solid, mp 178-179°C dec. (from
  • TMSOTf Trimethylsilyl trifluoromethylsulfonate

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Abstract

L'invention se rapporte à un composé avec système d'anneaux soudés, qui contient un groupe époxide sur l'un des côtés des anneaux soudés et un anneau macrocyclique d'énédiyne sur l'autre côté des anneaux soudés. De tels composés ont une propriété de clivage de l'ADN, un pouvoir antimicrobien et une propriété d'inhibition de la croissance tumorale. Sont également décrits des composés chimériques contenant comme aglycone le composé avec système d'anneaux soudés, liés à (i) une fraction de sucre faisant office de partie oligosaccharide ou (ii) un anticorps monoclonal ou une partie de site de combinaison anticorpale de ce dernier qui entre en immunoréaction avec des cellules tumorales cibles. Des compositions contenant un tel composé ou une chimère ainsi que des procédés de préparation d'un tel composé sont également décrits.
PCT/US1991/005436 1990-08-01 1991-07-31 Syntheses, procedes de preparation et utilisation d'analogues de dynemicines WO1992002522A1 (fr)

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FI930380A FI930380L (fi) 1990-08-01 1991-07-31 Dynemicinanaloger: syntes, foerfarande foer framstaellning och anvaendning
EP91916729A EP0594624A1 (fr) 1990-08-01 1991-07-31 Syntheses, procedes de preparation et utilisation d'analogues de dynemicines
AU86123/91A AU661278B2 (en) 1990-08-01 1991-07-31 Dynemicin analogs: syntheses, methods of preparation and use
NO93930314A NO930314L (no) 1990-08-01 1993-01-29 Dynemicinanaloger samt fremgangsmaater for fremstilling avslike

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EP0641207A4 (fr) * 1992-05-21 1996-08-14 Scripps Research Inst Analogues de cynemicine enantiomeres, leur preparation et utilisation.
US5763451A (en) * 1994-07-27 1998-06-09 California Institute Of Technology Dynemicin analogs

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Title
J.A.C.S. Vol. 109, pages 3461-3462 (1987) JERJY GOLIK et al., "Esperamicins, a Novel Class of potent antitumor antibiotics. 2. Structure of Esperamicin X", see entire document. *
J.A.C.S. Vol. 109, pages 3462-3464 (1987) JERZY GOLIK et al., "Esperamicins, a novel class of potent antitumor antibiotics. 3. Structures of Esperamicins A1, A2, and A1b". *
J.A.C.S. Vol. 109, pages 3464-3466 (1987) MAY D. LEE et al., "Calicheamicins, a novel family of antitumor Antibiotics, 1. Chemistry and partial structure of Calicheamicin gamma", see entire document. *
J.A.C.S. Vol. 109, pages 3466-3468 (1987) MAY D. LEE et al., "Calicheamicins, a novel family of antitumor Antibiotics, 2. Chemistry and structure of Calicheamicin gamma", see entire document. *
J.A.C.S. Vol. 110, pages 6921-6932 (1988) PHILIP MAGNUS et al., "Synthesis of a remarkably stable bicycle (7.3.1.) diynen Esperamicin A./Calicheamicin gamma system. Structural requirements for facile formation of a 1,4-Diyn". *
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See also references of EP0594624A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0641207A4 (fr) * 1992-05-21 1996-08-14 Scripps Research Inst Analogues de cynemicine enantiomeres, leur preparation et utilisation.
US5763451A (en) * 1994-07-27 1998-06-09 California Institute Of Technology Dynemicin analogs
US5849750A (en) * 1994-07-27 1998-12-15 California Institute Of Technology Dynemicin analogs

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