WO2009019616A2 - Isoprekinamycin analogs and syntheses thereof - Google Patents

Isoprekinamycin analogs and syntheses thereof Download PDF

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Publication number
WO2009019616A2
WO2009019616A2 PCT/IB2008/003117 IB2008003117W WO2009019616A2 WO 2009019616 A2 WO2009019616 A2 WO 2009019616A2 IB 2008003117 W IB2008003117 W IB 2008003117W WO 2009019616 A2 WO2009019616 A2 WO 2009019616A2
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alkyl
compound
aralkyl
aryl
formula
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PCT/IB2008/003117
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French (fr)
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WO2009019616A3 (en
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Brian B. Hasinoff
Wei Liu
Otunola Adedayo
Gary I. Dmitrienko
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University Of Manitoba
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Publication of WO2009019616A3 publication Critical patent/WO2009019616A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/84—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C225/00—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones
    • C07C225/22—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C245/00—Compounds containing chains of at least two nitrogen atoms with at least one nitrogen-to-nitrogen multiple bond
    • C07C245/12—Diazo compounds, i.e. compounds having the free valencies of >N2 groups attached to the same carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
    • C07C255/41—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by carboxyl groups, other than cyano groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/49—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C255/56—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing cyano groups and doubly-bound oxygen atoms bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/26—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atom of at least one of the carbamate groups bound to a carbon atom of a six-membered aromatic ring
    • C07C271/30—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atom of at least one of the carbamate groups bound to a carbon atom of a six-membered aromatic ring to a carbon atom of a six-membered aromatic ring being part of a condensed ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/02—Systems containing two condensed rings the rings having only two atoms in common
    • C07C2602/04—One of the condensed rings being a six-membered aromatic ring
    • C07C2602/08—One of the condensed rings being a six-membered aromatic ring the other ring being five-membered, e.g. indane
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/56—Ring systems containing bridged rings
    • C07C2603/86—Ring systems containing bridged rings containing four rings

Definitions

  • the present invention generally relates to the fields of synthetic organic chemistry, natural product isolation and study, and cancer treatment. More particularly, it concerns the discoveiy and preparation of compounds comprising a benzo[ ⁇ /]fluorene core, such as isoprekinamycin and derivatives thereof. Methods of use of these compounds for anticancer and antibiotic treatment therapies are also encompassed by the present invention.
  • the kinamcyins first believed to be N-cyanobenzo[b]carbazoles 1 (Omura et a!., 1973) but now known to be diazobenzo[b]fluorenes 2 (Gould et a!., 1994; Mithani et a!., 1994), are of current interest because of antibacterial activity and an in vitro cytotoxicity profile against cancer cells suggestive of a mode of action different than anticancer agents in current clinical use (Hasinoff et a!., 2006).
  • lomaiviticins which are dimeric analogs of the kinamcyins possessing potent cytotoxicity against a range of cancer cell lines, has heightened the interest in these unusual natural products (He et a!., 2001 ).
  • a number of synthetic strategies have been disclosed (Gould, 1997), most notably a total synthesis of the biosynthetic precursor 6 (Hauser and Zhou, 1996) and model studies towards a total synthesis of lomaiviticin B (Nicolaou et a!., 2006).
  • Isoprekinamycin (IPK), first assigned structure 3 (Seaton and Gould, 1989), is now recognized to be the diazobenzo[ ⁇ /]fluorene 4 (Proteau et a!., 2000).
  • the previously rare benzo[ ⁇ /]fluorene class of natural products now boasts several other members (e.g., 7) with various degrees of oxygenation in ring D but lacking a diazo group (Akiyama et al., 1998; Schneider et al., 2006; Baur et al., 2006; Fondja et al., 2006).
  • studies suggest that a substantial diazonium ion character in the diazo group of IPK might play a role in its bioactivity (Laufer and Dmitrienko, 2002).
  • Cancer is a leading cause of death in the United States. Despite significant efforts to find new approaches for treating cancer, the primary treatment options remain surgery, chemotherapy and radiation therapy, either alone or in combination. Surgery and radiation therapy, however, are generally useful only for fairly defined types of cancer, and are of limited use for treating patients with disseminated disease.
  • Chemotherapy is the method that is generally useful in treating patients with metastatic cancer or diffuse cancers such as leukemias. Although chemotherapy can provide a therapeutic benefit, it often fails to result in cure of the disease due to the patient's cancer cells becoming resistant to the chemotherapeutic agent. Similarly, infectious diseases caused by bacteria, for example, are becoming increasingly difficult to treat and cure. For example, more and more bacteria are developing resistance to current antibiotics and chemotherapeutic agents.
  • bacteria examples include both gram positive and gram negative bacteria, including Staphylococcus, Streptococcus, Mycobacterium, Enterococcus, Corynebacterium, Borrelia, Bacillus, Chlamydia, Mycoplasma, and the like.
  • the present invention generally provides novel benzo[ ⁇ /]fluorenes, syntheses of benzo[ ⁇ /]fluorenes, and methods of using these compounds as anticancer and/or antibacterial agents.
  • the present invention is drawn to the preparation of isoprekinamycin (IPK) and derivatives thereof and methods of treating cancer or bacterial infections using these compounds.
  • IPK isoprekinamycin
  • one general aspect of the present invention encompasses a method of synthesizing a compound of formula (A):
  • a 1 -A ⁇ are each independently carbon or nitrogen;
  • X is halo, -OSO2CF3, or any other leaving group as this term is defined herein;
  • Y is alkoxy, aryloxy, aralkyloxy, alkylamino, arylamino, aralkylamino, or -OC(O)R n , wherein R n is alkyl, aryl, or aralkyl;
  • R 1 -R 1 6 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, aiylthio, aralkylthio, alkylamino, arylamino, aralkylamino, nitro, halogen (e.g., fluoro), -S ⁇ 2 (alky
  • the compound of formula (A) is further defined as not the following:
  • any specific compound or genus of compounds described herein may be excluded from this or any other embodiment as described herein.
  • Persons of skill in the art will understand that of A 1 -As in this or any other compound described herein, certain combinations of C and/or N are more chemically feasible than others (e.g., an all-nitrogen aryl group is rarely made).
  • Persons of skill in the art also understand that substituents on aiyl nitrogens may or may not be present in this or any other compound of the present invention. In certain embodiments, when an aiyl ring atom is a nitrogen, the nitrogen displays no substituent.
  • substituents described as being able to form a cyclic group in this or any other compound described herein do not all necessarily form such cyclic groups.
  • Certain pairs of substituents e.g., Ry-R 1(> ) may form a cyclic group while Rn is H.
  • Ris and R 19 may form any boronate ester known to those of skill in the art.
  • Y is fluoro.
  • -OH groups are preferred at certain positions (e.g., R4 and/or R5).
  • a functional group may be protected by a protecting group.
  • Methods may also comprise the addition or removal of a protecting group. Persons of skill in the art are familiar with protecting groups, their installation, and their removal. Certain methods of the present invention comprise reducing conditions.
  • Reducing conditions are well-known to those of skill in the art of synthetic organic chemistry.
  • Non-limiting examples of reducing conditions include conditions comprising an alkyllithium/DIBAL mixture (e.g., n-butyllithium/DIBAL) or borohydride, such as sodium borohydride.
  • Certain methods of the present invention comprise Suzuke reaction conditions. Such conditions are well-known to those of skill in the art of synthetic organic chemistry.
  • the Suzuki reaction is the organic reaction of an aryl- or vinyl- boronic acid with an aryl- or vinyl-halide catalyzed by a palladium(O) complex. It is a widely used reaction to synthesize poly-olefms, styrenes, and substituted biphenyls, for example.
  • the reaction also works with other agents, such as triflates (-OTf), instead of halides, and also with boron-esters instead of boronic acids.
  • -OTf triflates
  • boron-esters instead of boronic acids.
  • the relative reactivity is as shown: -I > -OTf > -Br » -Cl.
  • the reaction relies on a palladium catalyst, such as tetrakis(triphenylphosphine)palladium(0), to effect part of the transformation.
  • the palladium catalyst (more strictly a pre-catalyst) is 4- coordinate, and usually involves phosphine supporting groups.
  • methods comprise anionic reaction conditions.
  • the first intermediate described above may be subjected to anionic reaction conditions.
  • a compound of formula (Ilia), described below (a derivative of (III)) may be subjected to anionic reaction conditions.
  • Persons of skill in synthetic organic chemistry are familiar with such conditions.
  • such conditions may comprise LDA.
  • DBU may be added, in certain embodiments.
  • Other anionic reaction conditions are described by Thebtaranonth and Thebtaranonth, 1994, incorporated herein in its entirety.
  • a compound of formula (IV) may be generated as an intermediate using methods of the present invention: wherein: A 1 -A ⁇ are each independently carbon or nitrogen; Ry-R 16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aiyloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, aiylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO 2 (alkyl), -SO(alkyl), -SO 2 (aryl), -SO(aryl), - S ⁇ 2(aralkyl)
  • the compound of formula (II) is as shown:
  • Rn-Rn are defined as above. Any boronate ester or acid as known to those of skill in the art may be used in methods discussed herein.
  • Compounds of formula (III) may be modified before being subjected to reducing conditions, in certain embodiments. That is, a compound of formula (III) may be generated and then modified to form another compound. This compound may, in certain embodiments, be considered a derivative of a compound of formula (III).
  • a derivative of the compound of formula (III) is a compound of formula (Ilia): wherein: A 1 -As are each independently carbon or nitrogen; Y a is alkoxy, aiyloxy, aralkyloxy, alkylamino, arylamino, aralkylamino, or -OC(O)R e , wherein R e is alkyl, aryl, or aralkyl; Ry n -R 16n are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, aiylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl
  • the compound of formula (A) may, in certain embodiments, be defined as not comprising these compounds.
  • a compound prepared by the methods described herein include these compounds.
  • the following compounds are contemplated as being made:
  • Yi is alkoxy, aryloxy, aralkyloxy, alkylamino, arylamino, aralkylamino, or - OC(O)R n , wherein R n is alkyl, aryl, or aralkyl;
  • R 22 -R 29 arc each independently H, - OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO 2 (alkyl), -SO(alkyl), -SO 2 (aryl), -SO(aryl), -SO 2 (aralkyl), or -SO(aralkyl), or any one or more of R22-R23
  • the compound of formula (V) is further defined as not any of the following:
  • such compounds are specifically included.
  • the following compound is contemplated as a compound of formula
  • R 31 -R 38 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -S ⁇ 2 (alkyl), -SO(alkyl), -
  • R 3 9 is -CN, -CONH 2 , -NH 2 , or -NHCO 2 (alkyl); and
  • R 41 . is alkoxy.
  • R 34 , R 35 and R 52 are each -OH, R 37 is not -CH 3 .
  • Non-limiting examples of compounds of formula (VI) include:
  • a 1 -As arc each independently carbon or nitrogen;
  • R 41 -R. ⁇ are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, aiylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO 2 (alkyl), -SO(alkyl), -SO 2 (aryl), -SO(aryl), - SO ⁇ iaralkyl), or -SO(aralkyl), or any one or more of R41-R42.
  • fluoro e.g., fluoro
  • the compound of formula (VII) is further defined as not any of the following:
  • Non-limiting examples of compounds of formula (VII) include:
  • Also contemplated by the present invention is a method of synthesizing a compound of formula (III): comprising reacting a compound of formula (I): with a compound of formula (II): under Suzuki coupling conditions, wherein: A 1 -A s are each independently carbon or nitrogen; X is halo, -OSO 2 CF;, or any other leaving group as this term is defined herein; Y is alkoxy, aryloxy, aralkyloxy, alkylamino, arylamino, aralkylamino, or - OC(O)R n , wherein R n is alkyl, aryl, or aralkyl; Ry-R 16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino,
  • Another method of the present invention that is contemplated is a method of synthesizing a compound of formula (IV):
  • a 1 -A s are each independently carbon or nitrogen;
  • Y is alkoxy, aryloxy, aralkyloxy, alkylamino, arylamino, aralkylamino, or -OC(O)R n , wherein R a is alkyl, aryl, or aralkyl;
  • Ry-R 16 are each independently H, - OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO 2 (alkyl), -SO(alkyl), -SO 2 (aryl), -SO(aryl), -SO 2 (aralkyl), or
  • Another general aspect of the present invention contemplates the following: In a method of preparing a compound comprising a benzo[ ⁇ /]fluorene, the improvement comprising subjecting a product of a reaction of an aryl boronate ester and an aryl bromide to reducing conditions comprising an alkyllithium/DIBAL (e.g., n-butyllithium/DIBAL) mixture or borohydride, such as sodium borohydride.
  • alkyllithium/DIBAL e.g., n-butyllithium/DIBAL
  • borohydride such as sodium borohydride
  • aiyl boronate esters and aiyl bromides are described herein (e.g., compounds of formulas (I) and (II)).
  • Solvents used in this and other methods discussed herein are well-known to those of skill in the art, and, of course, should be chosen such that that they do not interfere with the desired reaction and are easily removable once the reaction is complete. Solvents that dissolve the reactants completely or nearly completely are also preferred.
  • Another general aspect of the present invention entails a method of obtaining a compound of formula (VIII):
  • the method may further comprise obtaining Streptomvces muruyumuensis.
  • Such methods may be performed by, for example, performing the procedure described in the Examples below. Persons of skill in the art will be familiar with means of obtaining Streptomvces murayamaensis. For example, one may obtain this species from the American Type Tissue Collection, Manassas, Virginia.
  • Any compound of the present invention may be further comprised in a pharmaceutically acceptable exipient, diluent, or vehicle.
  • a compound of formula (A), (IV), (VI), (VII), and/or (VIII) may be comprised in a pharmaceutically acceptable exipient, diluent, or vehicle.
  • Methods of inhibiting the catalytic decatenation activity of topoisomerase Il ⁇ in a cell comprising contacting the cell with an effective amount of a compound of formula (A): or a compound of formula (IV):
  • a 1 -As arc each independently carbon or nitrogen;
  • R 1 -R 1 6 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, aiylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO 2 (alkyl), -SO(alkyl), -SO 2 (aryl), -SO(aryl), - SO 2 (aralkyl), or -SO(aralkyl), or any one or more Of Ri-R 2 , R 2 -R ⁇ , R3-R4, R5-R6, Rr,- R 7 , R 7 -Rs, R9-R10, R 1 O-R 11 , Rn-Ri 2 , R 13
  • the cell in this or any other method of the present invention may be any cell known to those of skill in the art.
  • the cell may be a cancer cell, for example.
  • compounds of the present invention are anticancer agents.
  • the cancer cell may be any type that is known to those of skill in the art.
  • the cancer cell may be a leukemia cell, a breast cancer cell, or a colon cancer cell.
  • Such cells may be of the particular type MCF-7 (breast cancer cells) or HCT-1 16 (colon cancer cells).
  • the cell may be in vivo or in vitro.
  • the compound of formula (A) is selected from the group consisting of:
  • a 1 -As arc each independently carbon or nitrogen;
  • R 1 -R 1 6 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, aiylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO 2 (alkyl), -SO(alkyl), -SO 2 (aryl), -SO(aryl), - SO 2 (aralkyl), or -SO(aralkyl), or any one or more of R r R 2 , R 2 -R 3 , R 3 -R 4 , R 5 -R6, Rr,- R 7 , R 7 -Rs, R9-R10, R 1 O-R 11 , Rn-R
  • Ai-As are each independently carbon or nitrogen;
  • R 1 -R 16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, aiylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO 2 (alkyl), -SO(alkyl), -SO 2 (aryl), -SO(aryl), - SO 2 (aralkyl), or -SO(aralkyl), or any one or more Of Ri-R 2 , R 2 -R 3 , R 3 -R 4 , Rs-R*, Re- R 7 , R 7 -Rs, Ry-Rio, R 1 O-R 11 , Rn-Ri 2 , R 13
  • the subject in this or any other method may be a mammal, for example, such as a human, mouse, rat, or rabbit.
  • the mammal is a human.
  • the subject may have any cancer known to those of skill in the art.
  • the subject may have cancer of the lung, liver, skin, eye, brain, gum, tongue, blood, head, neck, breast, pancreas, prostate, kidney, bone, testicles, ovaiy, cervix, gastrointestinal tract, lymph system, small intestine, colon, or bladder.
  • an "antibacterial agent” or “antibiotic” refers to an agent that prevents the growth of bacteria, and can be bactericidal (kills) or bacteriostatic (inhibits growth).
  • another general aspect of the present invention encompasses a method of inhibiting bacterial growth or killing bacteria in a subject, or treating a bacterial infection in a subject, comprising administering an effective amount of a compound of formula (A):
  • a 1 -As arc each independently carbon or nitrogen;
  • R 1 -R 1 6 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -S ⁇ 2 (alkyl), -SO(alkyl), -S ⁇ 2 (aryl), - SO(aryl), -SO 2 (aralkyl), or -SO(aralkyl), or any one or more Of Ri-R 2 , R2-R3, R3-R4, R 5 -R 6 , R 6 -R 7 , R 7 -Rs, R9-R10, R10-R11, R11-R12, R ⁇
  • the compound of formula (A) is not isoprekinamycin.
  • compounds of the present invention may be used to inhibit the growth of bacteria. Such methods may take place in vivo or in vitro. For example, a bacterial colony can be plated onto agar that incorporates a growth-inhibiting quantity of a compound of the invention. Bacteria that are resistant to the compound of the invention will continue to grow, while bacteria that are not resistant will be inhibited from growing. One of ordinary skill in the art can test for antibacterial effects by growing
  • Methods of the present invention may encompass, for example, treatment of a subject having a bacterial infection in which the infection is caused or exacerbated by any type of bacteria, such as gram-positive bacteria.
  • a compound of the present invention or a pharmaceutical composition thereof is administered to a patient according to the methods of this invention.
  • the compound may be given to a subject, e.g., a human patient or other subject, suffering from a bacterial infection.
  • Administration of the compound can be through any means, e.g., any conventional methods, e.g., oral, intravenous, intradermal, parenteral, transdermal or other methods as described herein.
  • the method of the instant invention may be used for treatment of any bacterial infection of any organ or tissue in the body.
  • the present invention also contemplates administering an effective amount of a compound of the present invention to a subject showing symptoms of bacterial infection, such as fever, headache, sweating, chills, and aches.
  • the bacterial infection may be caused or exacerbated by gram-positive bacteria.
  • gram-positive bacteria include, but are not limited to, methicillin-susceptible and methicillin-resistant staphylococci (including Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. hominis, S. saprophyticus, and coagulase-negative staphylococci), glycopeptide intermediary-susceptible S. aureus (GISA), penicillin-susceptible and penicillin-resistant streptococci (including Streptococcus pneumoniae, S. pyogenes, S. agalactiae, S. avium, S. bovis, S.
  • lactis lactis, S. sanguis and Streptococci Group C, Streptococci Group G and viridans streptococci
  • enterococci including vancomycin susceptible and vancomycin-resistant strains such as Enterococcusfaecalis and E. faecium
  • Clostridium difficile C. clostridiiforme, C. innocuum, C. perfringens, C. ramosum, Haemophilus influenzae, Listeria monocytogenes, Corynebacterium jeikeium, Bifidobacterium spp., Eubacterium aerofaciens, E. lentum, Lactobacillus acidophilus, L. casei, L.
  • Lactococcus spp. Lactococcus spp., Leuconostoc spp., Pediococcus, Peptostreptococcus anaerobius, P. asaccarolyticus, P. magnus, P. micros, P. prevotil, P. productus, Propionibacterium acnes, Actinomyces spp., Moraxella spp. (including M. catarrhalis) and Escherichia spp. (including E. coli).
  • certain compounds of the present invention may, in certain embodiments, be anticancer agents.
  • An "anticancer” agent is capable of negatively affecting cancer in a subject, for example, by killing one or more cancer cells, inducing apoptosis in one or more cancer cells, reducing the growth rate of one or more cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or one or more cancer cells, promoting an immune response against one or more cancer cells or a tumor, preventing or inhibiting the progression of a cancer, or increasing the lifespan of a subject with a cancer.
  • Anticancer agents are well-known in the art and include, for example, chemotherapy agents (chemotherapy), such as DNA intercalators, radiotherapy agents (radiotherapy), a surgical procedure, immune therapy agents (immunotherapy), genetic therapy agents (gene therapy), reoviral therapy, hormonal therapy, other biological agents (biotherapy), and/or alternative therapies.
  • chemotherapy agents such as DNA intercalators
  • radiotherapy agents radiotherapy agents
  • a surgical procedure a surgical procedure
  • immune therapy agents immunotherapy
  • genetic therapy agents gene therapy
  • reoviral therapy hormonal therapy
  • other biological agents biotherapy
  • alternative therapies include, for example, chemotherapy agents (chemotherapy), such as DNA intercalators, radiotherapy agents (radiotherapy), a surgical procedure, immune therapy agents (immunotherapy), genetic therapy agents (gene therapy), reoviral therapy, hormonal therapy, other biological agents (biotherapy), and/or alternative therapies.
  • Compounds of the present invention may, in certain embodiments, be anticancer agents.
  • a compound of the present invention in amount effective to kill the cell.
  • the term "in an amount effective to kill the cell” means that the amount of the compound of the present invention is sufficient so that, when administered to a cell, cell death is induced.
  • a number of in vitro parameters may be used to determine the effect produced by the compositions and methods of the present invention. These parameters include, for example, the observation of net cell numbers before and after exposure to the compositions described herein.
  • the terms "contacted” and “exposed,” when applied to a cell, are used herein to describe the process by which a compound of the present invention is administered or delivered to a target cell or are placed in direct juxtaposition with the target cell.
  • administered and “delivered” are used interchangeably with “contacted” and “exposed.”
  • an “effective” means adequate to accomplish a desired, expected, or intended result.
  • an “effective amount” may be an amount of a compound sufficient to produce a therapeutic benefit (e.g., effective to reproducibly inhibit decrease, reduce, inhibit or otherwise abrogate the growth of a cancer cell).
  • Treatment and “treating” as used herein refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.
  • a subject e.g., a mammal, such as a human
  • a treatment comprising administration of a compound of the present invention.
  • therapeutic benefit refers to anything that promotes or enhances the well- being of the subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.
  • a therapeutically effective amount of a compound of the present invention may be administered to a subject having a cancerous tumor, such that the tumor shrinks.
  • inhibiting or “reducing” or any variation of these terms as used herein includes any measurable decrease or complete inhibition to achieve a desired result. For example, there may be a decrease of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, or any range derivable therein, reduction of tumor size or bacterial infection following administration of a compound of the present invention. In a further example, following administering of a compound of the present invention, a patient suffering from a bacterial infection may experience a reduction the number and/or intensity of symptoms of the infection.
  • Non-limiting examples of typical symptoms associated with a bacterial infection include elevated temperature, sweating, chills, and/or excess white blood cells compared to a normal range. It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention.
  • composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
  • FIG. 1 Inhibition of growth of CHO cells by isoprekinamycin and derivatives thereof.
  • the curved solid lines are non-linear least calculated least squares fits of the MTT absorbance-concentration data to a 3 -parameter logistic equation and yield the IC50 values shown.
  • the left-most data point in each case is the control value in the absence of any added drag..
  • the cells were continuously incubated with the drags for 72 h.
  • the error bars are SEs from 4 replicate wells.
  • IPK isoprekinamycin
  • IPK-diAc isoprekinamycin diacetate
  • IPK-OMe isoprekinamycin O-methyl derivative.
  • FIG. 3 Comparison of the HPLC retention times of the synthetic and natural isoprekinamycin (IPK).
  • HPLC system Waters 600 controller, Waters 996 photodiode array detector, Waters Millennium ⁇ R> software; (2) Column: Nova-Pak ⁇ R> Cl 8 60 A 4 ⁇ m, 3.9 ⁇ 150 mm; (3) linear gradient (20 minutes): 94% H 2 O, 5% CH 3 CN and 0.1% AcOH to 5% H2O, 94% CH 3 CN and 0.1% AcOH at a flow rate of 1.5 mL/minute at room temperature.
  • FIG. 4 Isoprekinamycin-induced growth inhibition of CHO (A) and K562 (B) cells. Attached CHO cells or suspension K562 cells were treated for 72 h with a range of isoprekinamycin concentrations. The solid lines are three-parameter nonlinear least squares calculated logistic fits of the MTT or MTS absorbance- isoprekinamycin concentration data and were used to obtain the IC50 values. The error bars were calculated from replicates.
  • the ability of the compounds to inhibit the topoisomerase Il ⁇ -mediated decatenation of highly networked kDNA was measured in an ATP-containing assay mixture at 37 0 C for 20 minutes.
  • the fluorescence measures the amount of decatenated DNA minicircles in the supernatant of the centrifuged quenched 20 ⁇ L assay mixture.
  • IPK- diacetate inhibited the decatenation activity of topoisomerase Il ⁇ with IC50 values of 9.7 ⁇ 1.9 ⁇ M.
  • the solid line is a non-linear least squares calculated fits of the fluorescence-concentration data to a three-parameter logistic equation.
  • FIG. 6 600 MHz proton NMR spectrum of the compound of formula (VIII), isolated from Streptomyces murayamaensis.
  • the present invention overcomes deficiencies of the prior art by providing methods that enable facile access to benzo[ ⁇ /]fluorene moieties. Since benzo[ ⁇ /]fluorenes are being identified in more and more natural products of therapeutic interest, synthetic access to these natural products is needed, as well as derivatives thereof. The methods of the present invention allow for a variety of these natural products and other benzo[ ⁇ /]fluorene derivatives to be generated. Compounds synthesized by methods of the present invention may be used for therapeutic purposes, such as anticancer and/or antibacterial activity. The compounds described herein may also, in certain embodiments, act as act as electrophilic azo-coupling agents in vitro or in vivo.
  • Isoprekinamycins When IPK was first isolated by Steven Gould and co-workers in 1989, it was assigned an incorrect structure and given the name prekinamycin. Seaton and Gould, 1989. The present inventors later elucidated the correct structure and also revised the name to isoprekinamcyin. Proteau et a!., 2000. A metabolite in S. lmtravamaensis that is believed to be a biosynthetic precursor of the kinamycins is now called prekinamycin, and is an isomer of isoprekinamycin. Hauser and Zhou, 1996; Gould et al., 1996; Birman et .//., 2006.
  • the methods of the present invention provide access to IPK and are generally applicable to the synthesis of a variety of IPK derivatives.
  • amino means -NHT; the term “nitro” means -NCK the term “halo” designates -F, -Cl, -Br or -I and in particular embodiments, halo is
  • halo is Br; the term “mercapto” means -SH; the term “cyano” means -CN; the term “azido” means -N 3 ; the term “silyl” means
  • alkyl includes straight-chain alkyl, branched-chain alkyl, cycloalkyl (alicyclic), cyclic alkyl, heteroatom-unsubstituted alkyl, heteroatom- substituted alkyl, heteroatom-unsubstituted C n -alkyl, and heteroatom-substituted C n -alkyl.
  • “lower alkyls” are contemplated.
  • the term “lower alkyl” refers to alkyls of 1, 2, 3, 4, 5, or 6 carbon atoms.
  • heteroatom- unsubstituted C n -alkyl refers to a radical, having a linear or branched, cyclic or acyclic structure, further having no carbon-carbon double or triple bonds, further having a total of n carbon atoms, all of which are nonaromatic, 3 or more hydrogen atoms, and no heteroatoms.
  • a heteroatom-unsubstituted Ci-Cio-alkyl has 1 to 10 carbon atoms.
  • heteroatom- substituted C n -alkyl refers to a radical, having a single saturated carbon atom as the point of attachment, no carbon-carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, all of which are nonaromatic, 0, 1, or more than one hydrogen atom, at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
  • a heteroatom-substituted Ci-Cio-alkyl has 1 to 10 carbon atoms.
  • heteroatom-substituted alkyl groups trifluoromethyl or other perfluoromethyl groups, -CH 2 F, -CH 2 Cl, -CH 2 Br, -CH 2 OH, -CH 2 OCH,, -CH 2 OCH 2 CF,, -CH 2 OC(O)CH,, aminoalkyl groups such as -CH 2 NH 2 , -CH 2 NHCH,, -CH 2 N(CH, ) 2 , and more generally -CH 2 NR 1 R 2 , wherein R 1 and R 2 are each independently alkyl, aryl, or aralkyl, -CH 2 CH 2 Cl, -CH 2 CH 2 OH, CH 2 CH 2 OC(O)CH 3 , -CH 2 CH 2 NHCO 2 C(CH 3 ) 3 , -CH 2 Si(CH 3 ) 3 , and -(CH 2 J n -Y wherein Y is -CO 2 H,
  • alkenyl includes straight-chain alkenyl, branched-chain alkenyl, cycloalkenyl, cyclic alkenyl, heteroatom-unsubstituted alkenyl, heteroatom- s ⁇ bstit ⁇ ted alkenyl, heteroatom- ⁇ ns ⁇ bstit ⁇ ted C n -alkenyl, and heteroatom-substituted C n -alkenyl.
  • heteroatom-unsubstituted C n -alkenyl refers to a radical, having a linear or branched, cyclic or acyclic stiiicture, further having at least one nonaromatic carbon-carbon double bond, but no carbon-carbon triple bonds, a total of n carbon atoms, three or more hydrogen atoms, and no heteroatoms.
  • a heteroatom-unsubstituted C 2 -Cio-alkenyl has 2 to 10 carbon atoms.
  • heteroatom-substituted C n -alkenyl refers to a radical, having a single nonaromatic carbon atom as the point of attachment and at least one nonaromatic carbon-carbon double bond, but no carbon-carbon triple bonds, further having a linear or branched, cyclic or acyclic stiiicture, further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
  • a heteroatom-substituted C 2 -Cio-alkenyl has 2 to 10 carbon atoms.
  • alkynyl includes straight-chain alkynyl, branched-chain alkynyl, cycloalkynyl, cyclic alkynyl, heteroatom-unsubstituted alkynyl, heteroatom- substituted alkynyl, heteroatom-unsubstituted C n -alkynyl, and heteroatom-substituted C n -alkynyl.
  • heteroatom-unsubstituted C n -alkynyl refers to a radical, having a linear or branched, cyclic or acyclic stiiicture, further having at least one carbon-carbon triple bond, a total of n carbon atoms, at least one hydrogen atom, and no heteroatoms.
  • a heteroatom-unsubstituted C 2 -Cio-alkynyl has 2 to 10 carbon atoms.
  • heteroatom- substituted C n -alkynyl refers to a radical, having a single nonaromatic carbon atom as the point of attachment and at least one carbon-carbon triple bond, further having a linear or branched, cyclic or acyclic stiiicture, and having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
  • a heteroatom-substituted C2-Cio-alkynyl has 2 to 10 carbon atoms.
  • aryl includes heteroatom-unsubstituted aryl, heteroatom- substituted aryl, heteroatom-unsubstituted C n -aryl, heteroatom-substituted C n -aryl, heteroaryl, heterocyclic aiyl groups, carbocyclic aiyl groups, biaryl groups, and radicals derived from polycyclic fused hydrocarbons (PAHs).
  • PAHs polycyclic fused hydrocarbons
  • heteroatom- unsubstituted C n -aryl refers to a radical, having a single carbon atom as a point of attachment, wherein the carbon atom is part of an aromatic ring structure containing only carbon atoms, further having a total of n carbon atoms, 5 or more hydrogen atoms, and no heteroatoms.
  • a heteroatom-unsubstituted Cf 1 -C l o-aryl has 6 to 10 carbon atoms.
  • heteroatom- substituted C n -aryl refers to a radical, having either a single aromatic carbon atom or a single aromatic heteroatom as the point of attachment, further having a total of n carbon atoms, at least one hydrogen atom, and at least one heteroatom, further wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
  • a heteroatom-unsubstituted Ci-Cio-heteroaryl has 1 to 10 carbon atoms.
  • Non-limiting examples of heteroatom-substituted aryl groups include the groups: -C 6 H 4 F, -C 6 H 4 Cl, -C 6 H 4 Br, -C 6 H 4 I, -C 6 H 4 OH, -C 6 H 4 OCH 3 , -C 6 H 4 OCH 2 CH 3 , -C 6 H 4 OC(O)CH 3 , -C 6 H 4 NH 2 , -C 6 H 4 NHCH 3 , -C 6 H 4 N(CH 3 ) 2 , -C 6 H 4 CH 2 OH, -C 6 H 4 CH 2 OC(O)CH 3 , -C 6 H 4 CH 2 NH 2 , -C 6 H 4 CF 3 , -C 6 H 4 CN, -C 6 H 4 CHO, -C 6 H 4 CHO, -C 6 H 4 C(O)CH 3 , -C 6 H 4 C(O)C 6 H 5 , -C 6 H 4
  • aralkyl includes heteroatom-unsubstituted aralkyl, heteroatom- substituted aralkyl, heteroatom-unsubstituted C n -aralkyl, heteroatom-substituted C n - aralkyl, heteroaralkyl, and heterocyclic aralkyl groups.
  • heteroatom- unsubstituted C n -aralkyl refers to a radical, having a single saturated carbon atom as the point of attachment, further having a total of n carbon atoms, wherein at least 6 of the carbon atoms form an aromatic ring structure containing only carbon atoms, 7 or more hydrogen atoms, and no heteroatoms.
  • a heteroatom-unsubstituted C ⁇ -Cio-aralkyl has 7 to 10 carbon atoms.
  • Non-limiting examples of heteroatom- unsubstituted aralkyls are: phenylmethyl (benzyl, Bn) and phenylethyl.
  • heteroatom-substituted C n -aralkyl refers to a radical, having a single saturated carbon atom as the point of attachment, further having a total of n carbon atoms, 0, 1 , or more than one hydrogen atom, and at least one heteroatom, wherein at least one of the carbon atoms is incorporated an aromatic ring stiiictures, further wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
  • a heteroatom-substituted C2-Cio-heteroaralkyl has 2 to 10 carbon atoms.
  • acyl includes straight-chain acyl, branched-chain acyl, cycloacyl, cyclic acyl, heteroatom-unsubstituted acyl, heteroatom-substituted acyl, heteroatom- unsubstituted C n -acyl, heteroatom-substituted C n -acyl, alkylcarbonyl, alkoxycarbonyl and aminocarbonyl groups.
  • heteroatom-unsubstituted C n -acyl refers to a radical, having a single carbon atom of a carbonyl group as the point of attachment, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 1 or more hydrogen atoms, a total of one oxygen atom, and no additional heteroatoms.
  • a heteroatom-unsubstituted Ci-Cio-acyl has 1 to 10 carbon atoms.
  • the groups, -CHO, -C(O)CH 3 , -C(O)CH 2 CH 3 , -C(O)CH 2 CH 2 CH 3 , -C(O)CH(CH 3 ) 2 , -C(O)CH(CH 2 ) 2 , -C(O)C 6 H 5 , -C(O)C 6 H 4 CH 3 , -C(O)C 6 H 4 CH 2 CH 3 , and -COC 6 H 3 (CH 3 ) 2 are non-limiting examples of heteroatom- unsubstituted acyl groups.
  • heteroatom-substituted C n -acyl refers to a radical, having a single carbon atom as the point of attachment, the carbon atom being part of a carbonyl group, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 0, 1 , or more than one hydrogen atom, at least one additional heteroatom, in addition to the oxygen of the carbonyl group, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
  • a heteroatom-substituted Ci-Cio-acyl has 1 to 10 carbon atoms..
  • the groups, -C(O)CH 2 CF 3 , -CO 2 H, -CO 2 CH 3 , -CO 2 CH 2 CH 3 , -CO 2 CH 2 CH 2 CH 3 , -CO 2 CH(CH 3 ) 2 , -CO 2 CH(CH 2 ) 2 , -C(O)NH 2 (carbamoyl), -C(O)NHCH 3 , -C(O)NHCH 2 CH 3 , -CONHCH(CH 3 ) 2 , -CONHCH(CH 2 ) 2 , -CON(CHj) 2 , and -CONHCH 2 CF, are non-limiting examples of heteroatom-substituted acyl groups.
  • alkoxy includes straight-chain alkoxy, branched-chain alkoxy, cycloalkoxy, cyclic alkoxy, heteroatom-unsubstituted alkoxy, heteroatom-substituted alkoxy, heteroatom-unsubstituted C n -alkoxy, and heteroatom-substituted C n -alkoxy.
  • heteroatom-unsubstituted C n -alkoxy refers to a group, having the structure -OR, in which R is a heteroatom-unsubstituted C n -alkyl, as that term is defined above.
  • Heteroatom-unsubstituted alkoxy groups include: -OCH 3 , -OCH 2 CH;*, -OCH 2 CH 2 CH 3 , -OCH(CH 3 ) 2 , and -OCH(CH 2 ) 2 .
  • the term "heteroatom-substituted C n -alkoxy” refers to a group, having the structure -OR, in which R is a heteroatom- substituted Cn-alkyl, as that term is defined above.
  • -OCH 2 CF 3 is a heteroatom-substituted alkoxy group.
  • alkenyloxy includes straight-chain alkenyloxy, branched-chain alkenyloxy, cycloalkenyloxy, cyclic alkenyloxy, heteroatom-unsubstituted alkenyloxy, heteroatom-substituted alkenyloxy, heteroatom-unsubstituted C n - alkenyloxy, and heteroatom-substituted C n -alkenyloxy.
  • heteroatom- unsubstituted C n -alkenyloxy refers to a group, having the structure -OR, in which R is a heteroatom-unsubstituted C n -alkenyl, as that term is defined above.
  • heteroatom-substituted C n -alkenyloxy refers to a group, having the structure -OR, in which R is a heteroatom-substituted C n -alkenyl, as that term is defined above.
  • alkynyloxy includes straight-chain alkynyloxy, branched-chain alkynyloxy, cycloalkynyloxy, cyclic alkynyloxy, heteroatom-unsubstituted alkynyloxy, heteroatom-substituted alkynyloxy, heteroatom-unsubstituted C n - alkynyloxy, and heteroatom-substituted C n -alkynyloxy.
  • heteroatom- unsubstituted C n -alkynyloxy refers to a group, having the structure -OR, in which R is a heteroatom-unsubstituted C n -alkynyl, as that term is defined above.
  • heteroatom-substituted C n -alkynyloxy refers to a group, having the structure -OR, in which R is a heteroatom-substituted C n -alkynyl, as that term is defined above.
  • aryloxy includes heteroatom-unsubstituted aryloxy, heteroatom- substituted aryloxy, heteroatom-unsubstituted C n -aryloxy, heteroatom-substituted C n - aryloxy, heteroaryloxy, and heterocyclic aryloxy groups.
  • heteroatom- unsubstituted C n -aryloxy refers to a group, having the structure -OAr, in which Ar is a heteroatom-unsubstituted C n -aryl, as that term is defined above.
  • a non-limiting example of a heteroatom-unsubstituted aryloxy group is -OCf 1 H 5 .
  • heteroatom-substituted C n -aiyloxy refers to a group, having the structure -OAr, in which Ar is a heteroatom-substituted C n -aryl, as that term is defined above.
  • aralkyloxy includes heteroatom-unsubstituted aralkyloxy, heteroatom-substituted aralkyloxy, heteroatom-unsubstituted C n -aralkyloxy, heteroatom-substituted C n -aralkyloxy, heteroaralkyloxy, and heterocyclic aralkyloxy groups.
  • heteroatom-unsubstituted C n -aralkyloxy refers to a group, having the structure -OAr, in which Ar is a heteroatom-unsubstituted C n -aralkyl, as that term is defined above.
  • heteroatom-substituted C n -aralkyloxy refers to a group, having the structure -OAr, in which Ar is a heteroatom-substituted C n -aralkyl, as that term is defined above.
  • acyloxy includes straight-chain acyloxy, branched-chain acyloxy, cycloacyloxy, cyclic acyloxy, heteroatom-unsubstituted acyloxy, heteroatom- substituted acyloxy, heteroatom-unsubstituted C n -acyloxy, heteroatom-substituted C n - acyloxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylate groups.
  • heteroatom-unsubstituted C n -acyloxy refers to a group, having the stiiicture -OAc, in which Ac is a heteroatom-unsubstituted C n -acyl, as that term is defined above.
  • -OC(O)CH? is a non-limiting example of a heteroatom-unsubstituted acyloxy group.
  • heteroatom-substituted C n -acyloxy refers to a group, having the structure -OAc, in which Ac is a heteroatom-substituted C n -acyl, as that term is defined above.
  • -OC(O)OCH? and -OC(O)NHCH? are non-limiting examples of heteroatom-unsubstituted acyloxy groups.
  • alkylamino includes straight-chain alkylamino, branched-chain alkylamino, cycloalkylamino, cyclic alkylamino, heteroatom-unsubstituted alkylamino, heteroatom-substituted alkylamino, heteroatom-unsubstituted C n -alkylamino, and heteroatom-substituted C n -alkylamino.
  • heteroatom- unsubstituted C n -alkylamino refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two saturated carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, containing a total of n carbon atoms, all of which are nonaromatic, 4 or more hydrogen atoms, a total of 1 nitrogen atom, and no additional heteroatoms.
  • a heteroatom-unsubstituted Ci-Cio-alkylamino has 1 to 10 carbon atoms.
  • heteroatom-unsubstituted C n -alkylamino includes groups, having the structure -NHR, in which R is a heteroatom-unsubstituted C n -alkyl, as that term is defined above.
  • a heteroatom-unsubstituted alkylamino group would include -NHCH 3 , -NHCH 2 CH 3 , -NHCH 2 CH 2 CH 3 , -NHCH(CH 3 ) 2 , -NHCH(CH 2 ) 2 , -NHCH 2 CH 2 CH 2 CH 3 , -NHCH(CH 3 )CH 2 CH 3 , -NHCH 2 CH(CH 3 ) 2 , -NHC(CH 3 ) 3 , -N(CH 3 ) 2 , -N(CH 3 )CH 2 CH 3 , -N(CH 2 CH 3 ) 2 , JV-pyrrolidinyl, and JV-piperidinyl.
  • heteroatom-substituted C n -alkylamino refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two saturated carbon atoms attached to the nitrogen atom, no carbon-carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, all of which are nonaromatic, 0, 1 , or more than one hydrogen atom, and at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
  • a heteroatom-substituted Ci-Cio-alkylamino has 1 to 10 carbon atoms.
  • the term "heteroatom-substituted C n -alkylamino" includes groups, having the stiiicture -NHR, in which R is a heteroatom-substituted C n -alkyl, as that term is defined above.
  • alkenylamino includes straight-chain alkenylamino, branched- chain alkenylamino, cycloalkenylamino, cyclic alkenylamino, heteroatom- unsubstituted alkenylamino, heteroatom-substituted alkenylamino, heteroatom- unsubstituted C n -alkenylamino, heteroatom-substituted C n -alkenylamino, dialkenylamino, and alkyl(alkenyl)amino groups.
  • heteroatom- unsubstituted C n -alkenylamino refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, containing at least one nonaromatic carbon-carbon double bond, a total of n carbon atoms, 4 or more hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms.
  • a heteroatom-unsubstituted C 2 -Cio-alkenylamino has 2 to 10 carbon atoms.
  • heteroatom-unsubstituted C n -alkenylamino includes groups, having the stiiicture -NHR, in which R is a heteroatom-unsubstituted C n - alkenyl, as that term is defined above.
  • heteroatom-substituted C n - alkenylamino refers to a radical, having a single nitrogen atom as the point of attachment and at least one nonaromatic carbon-carbon double bond, but no carbon- carbon triple bonds, further having one or two carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
  • heteroatom- substituted C n -alkenylamino includes groups, having the stiiicture -NHR, in which R is a heteroatom-substituted C n -alkenyl, as that term is defined above.
  • alkynylamino includes straight-chain alkynylamino, branched- chain alkynylamino, cycloalkynylamino, cyclic alkynylamino, heteroatom- unsubstituted alkynylamino, heteroatom-substituted alkynylamino, heteroatom- unsubstituted C n -alkynylamino, heteroatom-substituted C n -alkynylamino, dialkynylamino, alkyl(alkynyl)amino, and alkenyl(alkynyl)amino groups.
  • heteroatom-unsubstituted C n -alkynylamino refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, containing at least one carbon-carbon triple bond, a total of n carbon atoms, at least one hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms.
  • a heteroatom-unsubstituted C ⁇ -Cio-alkynylamino has 2 to 10 carbon atoms.
  • heteroatom-unsubstituted C n -alkynylamino includes groups, having the stiiicture -NHR, in which R is a heteroatom-unsubstituted C n - alkynyl, as that term is defined above.
  • heteroatom-substituted Cn- alkynylamino refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two carbon atoms attached to the nitrogen atom, further having at least one nonaromatic carbon-carbon triple bond, further having a linear or branched, cyclic or acyclic stiiicture, and further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
  • heteroatom-substituted C2-Cio-alkynylamino has 2 to 10 carbon atoms.
  • heteroatom-substituted C n -alkynylamino includes groups, having the stiiicture -NHR, in which R is a heteroatom-substituted C n -alkynyl, as that term is defined above.
  • arylamino includes heteroatom-unsubstituted arylamino, heteroatom-substituted arylamino, heteroatom-unsubstituted C n -arylamino, heteroatom-substituted C n -arylamino, heteroarylamino, heterocyclic arylamino, and alkyl(aryl)amino groups.
  • heteroatom-unsubstituted C n -aiylamino refers to a radical, having a single nitrogen atom as the point of attachment, further having at least one aromatic ring structure attached to the nitrogen atom, wherein the aromatic ring structure contains only carbon atoms, further having a total of n carbon atoms, 6 or more hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms.
  • a heteroatom-unsubstituted Cf 1 -C io-aiylamino has 6 to 10 carbon atoms.
  • heteroatom-unsubstituted C n -arylamino includes groups, having the structure -NHR, in which R is a heteroatom-unsubstituted C n -aryl, as that term is defined above.
  • heteroatom-substituted C n -arylamino refers to a radical, having a single nitrogen atom as the point of attachment, further having a total of n carbon atoms, at least one hydrogen atom, at least one additional heteroatoms, that is, in addition to the nitrogen atom at the point of attachment, wherein at least one of the carbon atoms is incorporated into one or more aromatic ring structures, further wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
  • a heteroatom-substituted Cf-Cio-arylamino has 6 to 10 carbon atoms.
  • heteroatom-substituted Cn- arylamino includes groups, having the structure -NHR, in which R is a heteroatom- substituted C n -aryl, as that term is defined above.
  • aralkylamino includes heteroatom-unsubstituted aralkylamino, heteroatom-substituted aralkylamino, heteroatom-unsubstituted C n -aralkylamino, heteroatom-substituted C n -aralkylamino, heterocyclic aralkylamino groups, and diaralkylamino groups.
  • heteroatom- unsubstituted Cn-aralkylamino refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two saturated carbon atoms attached to the nitrogen atom, further having a total of n carbon atoms, wherein at least 6 of the carbon atoms form an aromatic ring structure containing only carbon atoms, 8 or more hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms.
  • a heteroatom-unsubstituted C ⁇ -Cio-aralkylamino has 7 to 10 carbon atoms.
  • heteroatom-unsubstituted C n -aralkylamino includes groups, having the structure -NHR, in which R is a heteroatom-unsubstituted C n -aralkyl, as that term is defined above.
  • heteroatom-substituted C n -aralkylamino refers to a radical, having a single nitrogen atom as the point of attachment, further having at least one or two saturated carbon atoms attached to the nitrogen atom, further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein at least one of the carbon atom incorporated into an aromatic ring, further wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
  • heteroatom-substituted C ⁇ -Cio-aralkylamino has 7 to 10 carbon atoms.
  • heteroatom-substituted C n -aralkylamino includes groups, having the structure -NHR, in which R is a heteroatom-substituted C n -aralkyl, as that term is defined above.
  • amido includes straight-chain amido, branched-chain amido, cycloamido, cyclic amido, heteroatom-unsubstituted amido, heteroatom-substituted amido, heteroatom-unsubstituted C n -amido, heteroatom-substituted C n -amido, alkylcarbonylamino, arylcarbonylamino, alkoxycarbonylamino, aryloxycarbonylamino, acylamino, alkylaminocarbonylamino, arylaminocarbonylamino, and ureido groups.
  • heteroatom-unsubstituted C n - amido refers to a radical, having a single nitrogen atom as the point of attachment, further having a carbonyl group attached via its carbon atom to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 1 or more hydrogen atoms, a total of one oxygen atom, a total of one nitrogen atom, and no additional heteroatoms.
  • a heteroatom- unsubstituted Ci-Cio-amido has 1 to 10 carbon atoms.
  • heteroatom- unsubstituted C n -amido includes groups, having the stiiicture -NHR, in which R is a heteroatom-unsubstituted C n -acyl, as that term is defined above.
  • the group, -NHC(O)CH?, is a non-limiting example of a heteroatom-unsubstituted amido group.
  • heteroatom-substituted C n -amido refers to a radical, having a single nitrogen atom as the point of attachment, further having a carbonyl group attached via its carbon atom to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, further having a total of n aromatic or nonaromatic carbon atoms, 0, 1, or more than one hydrogen atom, at least one additional heteroatom in addition to the oxygen of the carbonyl group, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.
  • a heteroatom-substituted Ci-Cio-amido has 1 to 10 carbon atoms.
  • the term "heteroatom-substituted C n -amido" includes groups, having the stiiicture -NHR, in which R is a heteroatom-unsubstituted C n -acyl, as that term is defined above.
  • the group, -NHCO 2 CH 3 is a non-limiting example of a heteroatom-substituted amido group.
  • alkylthio includes straight-chain alkylthio, branched-chain alkylthio, cycloalkylthio, cyclic alkylthio, heteroatom- ⁇ ns ⁇ bstit ⁇ ted alkylthio, heteroatom-substituted alkylthio, heteroatom- ⁇ ns ⁇ bstit ⁇ ted C n -alkylthio, and heteroatom-substituted C n -alkylthio.
  • heteroatom-unsubstituted C n - alkylthio refers to a group, having the structure -SR, in which R is a heteroatom- unsubstituted C n -alkyl, as that term is defined above.
  • the group, -SCH ⁇ is an example of a heteroatom-unsubstituted alkylthio group.
  • heteroatom- substituted C n -alkylthio refers to a group, having the stiiicture -SR, in which R is a heteroatom-substituted C n -alkyl, as that term is defined above.
  • alkenylthio includes straight-chain alkenylthio, branched-chain alkenylthio, cycloalkenylthio, cyclic alkenylthio, heteroatom-unsubstituted alkenylthio, heteroatom-substituted alkenylthio, heteroatom-unsubstituted C n - alkenylthio, and heteroatom-substituted C n -alkenylthio.
  • heteroatom- unsubstituted C n -alkenylthio refers to a group, having the structure -SR, in which R is a heteroatom-unsubstituted C n -alkenyl, as that term is defined above.
  • heteroatom-substituted C n -alkenylthio refers to a group, having the structure -SR, in which R is a heteroatom-substituted C n -alkenyl, as that term is defined above.
  • alkynylthio includes straight-chain alkynylthio, branched-chain alkynylthio, cycloalkynylthio, cyclic alkynylthio, heteroatom-unsubstituted alkynylthio, heteroatom-substituted alkynylthio, heteroatom-unsubstituted C n - alkynylthio, and heteroatom-substituted C n -alkynylthio.
  • heteroatom- unsubstituted C n -alkynylthio refers to a group, having the structure -SR, in which R is a heteroatom-unsubstituted C n -alkynyl, as that term is defined above.
  • heteroatom-substituted C n -alkynylthio refers to a group, having the stiiicture -SR, in which R is a heteroatom-substituted C n -alkynyl, as that term is defined above.
  • arylthio includes heteroatom-unsubstituted arylthio, heteroatom- substituted arylthio, heteroatom-unsubstituted C n -arylthio, heteroatom-substituted C n - arylthio, heteroarylthio, and heterocyclic arylthio groups.
  • heteroatom- unsubstituted C n -arylthio refers to a group, having the structure -SAr, in which Ar is a heteroatom-unsubstituted C n -aryl, as that term is defined above.
  • the group, -SCr 1 Hf is an example of a heteroatom-unsubstituted arylthio group.
  • heteroatom-substituted C n -arylthio refers to a group, having the stiiicture -SAr, in which Ar is a heteroatom-substituted C n -aryl, as that term is defined above.
  • aralkylthio includes heteroatom-unsubstituted aralkylthio, heteroatom-substituted aralkylthio, heteroatom-unsubstituted C n -aralkylthio, heteroatom-substituted C n -aralkylthio, heteroaralkylthio, and heterocyclic aralkylthio groups.
  • heteroatom-unsubstituted C n -aralkylthio refers to a group, having the structure -SAr, in which Ar is a heteroatom-unsubstituted C n -aralkyl, as that term is defined above.
  • the group, -SCHoCf 1 H 5 is an example of a heteroatom- unsubstituted aralkyl group.
  • heteroatom-substituted C n -aralkylthio refers to a group, having the structure -SAr, in which Ar is a heteroatom-substituted Cn- aralkyl, as that term is defined above.
  • acylthio includes straight-chain acylthio, branched-chain acylthio, cycloacylthio, cyclic acylthio, heteroatom-unsubstituted acylthio, heteroatom- substituted acylthio, heteroatom-unsubstituted C n -acylthio, heteroatom-substituted C n - acylthio, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylate groups.
  • heteroatom-unsubstituted Cn-acylthio refers to a group, having the structure -SAc, in which Ac is a heteroatom-unsubstituted C n -acyl, as that term is defined above.
  • the group, -SCOCH? is an example of a heteroatom-unsubstituted acylthio group.
  • heteroatom-substituted C n -acylthio refers to a group, having the structure -SAc, in which Ac is a heteroatom-substituted C n -acyl, as that term is defined above.
  • a substituted substituent as described above, may be employed.
  • an unsubstituted substituent, as described above may be employed.
  • the claimed invention is also intended to encompass salts of any of the compounds of the present invention.
  • salt(s) as used herein, is understood as being acidic and/or basic salts formed with inorganic and/or organic acids and bases.
  • Zwitterions are understood as being included within the term “salt(s)” as used herein, as are quaternary ammonium salts such as alkylammonium salts.
  • Nontoxic, pharmaceutically acceptable salts are preferred as described below, although other salts may be useful, as for example in isolation or purification steps.
  • pharmaceutically acceptable salts refers to salts of compounds of this invention that are substantially non-toxic to living organisms.
  • Typical pharmaceutically acceptable salts include those salts prepared by reaction of a compound of this invention with an inorganic or organic acid, or an organic base, depending on the s ⁇ bstit ⁇ ents present on the compounds of the invention.
  • Non-limiting examples of inorganic acids which may be used to prepare pharmaceutically acceptable salts include: hydrochloric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid and the like.
  • organic acids which may be used to prepare pharmaceutically acceptable salts include: aliphatic mono- and dicarboxylic acids, such as oxalic acid, carbonic acid, citric acid, succinic acid, phenyl-heteroatom-substituted alkanoic acids, aliphatic and aromatic sulfuric acids and the like.
  • Pharmaceutically acceptable salts prepared from inorganic or organic acids thus include hydrochloride, hydrobromide, nitrate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydroiodide, hydrofluoride, acetate, propionate, formate, oxalate, citrate, lactate, p-toluenesulfonate, methanesulfonate, maleate, and the like.
  • Suitable pharmaceutically acceptable salts may also be formed by reacting the agents of the invention with an organic base such as methylamine, ethylamine, ethanolamine, lysine, ornithine and the like.
  • Pharmaceutically acceptable salts include the salts formed between carboxylate or sulfonate groups found on some of the compounds of this invention and inorganic cations, such as sodium, potassium, ammonium, or calcium, or such organic cations as isopropylammonium, trimethylammonium, tetramethylammonium, and imidazolium.
  • any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Stahl and. Wermuth,
  • cyclic group refers to a carbocycle group (e.g., cyclohexyl), a heterocycle group (e.g., pyrrolidinyl), an aryl group, or any combination thereof.
  • nucleophile or “nucleophilic” generally refers to atoms bearing lone pairs of electrons. Such terms are well known in the art and include -NHT, thiolate, carbanion and hydroxyl.
  • leaving group generally refers to a group readily displaceable by a nucleophile, such as an amine, an alcohol, or a thiol nucleophile.
  • Such leaving groups are well known and include carboxylates, N- hydroxysuccinimide, N-hydroxybenzotriazole, triflates, tosylates, mesylates, alkoxy, thioalkoxy and the like.
  • the term "functional group” generally refers to how persons of skill in the art classify chemically reactive groups. Examples of functional groups include hydroxyl, amine, sulfhydryl, amide, carboxyls, carbonyls, etc.
  • protecting group refers to a moiety attached to a functional group to prevent an otherwise unwanted reaction of that functional group.
  • Protecting groups are well-known to those of skill in the art. Non-limiting exemplary protecting groups fall into categories such as hydroxy protecting groups, amino protecting groups, sulfhydryl protecting groups and carbonyl protecting groups. Such protecting groups may be found in Greene and Wuts, 1999, which is incorporated herein by reference in its entirety.
  • Compounds of the present invention may contain one or more asymmetric centers and thus can occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In certain embodiments, a single diastereomer is present. All possible stereoisomers of the compounds of the present invention are contemplated as being within the scope of the present invention.
  • derivative such as an IPK derivative or a derivative of any of the compounds discussed herein (e.g., a derivative of a compound of formula (III)) refers to a chemically modified compound that still retains the desired effects of the compound prior to the chemical modification. Such derivatives may have the addition, removal, or substitution of one or more chemical moieties on the parent molecule.
  • Non-limiting examples of the types modifications that can be made to the compounds and structures disclosed herein include the addition or removal of lower alkanes such as methyl, ethyl, propyl, or substituted lower alkanes such as hydroxymethyl or aminomethyl groups; carboxyl groups and carbonyl groups; hydroxyls; nitro, amino, amide, and azo groups; sulfate, sulfonate, sulfono, sulfhydryl, sulfonyl, sulfoxido, phosphate, phosphono, phosphoiyl groups, and halide substituents.
  • lower alkanes such as methyl, ethyl, propyl, or substituted lower alkanes
  • carboxyl groups and carbonyl groups hydroxyls; nitro, amino, amide, and azo groups
  • Additional modifications can include an addition or a deletion of one or more atoms of the atomic framework, for example, substitution of an ethyl by a propyl; substitution of a phenyl by a larger or smaller aromatic group.
  • heteroatoms such as N, S, or O can be substituted into the structure instead of a carbon atom to generate, for example, a heterocycloalkyl structure.
  • Prodrugs and solvates of compounds of the present invention are also contemplated herein.
  • the term "prodiiig” as used herein, is understood as being a compound which, upon administration to a subject, such as a mammal, undergoes chemical conversion by metabolic or chemical processes to yield a compound any of the formulas herein, or a salt and/or solvate thereof (Bundgaard, 1991 ; Bundgaard, 1985).
  • Solvates of compounds of the present invention are preferably hydrates.
  • a benzo[ ⁇ /]fluorene of the present invention may be converted to a benzo[/?]fluorene in vivo or in vitro.
  • Solvent choices for the methods of the present invention will be known to one of ordinary skill in the art. Solvent choices may depend, for example, on which one(s) will facilitate the solubilizing of all the reagents or, for example, which one(s) will best facilitate the desired reaction (particularly when the mechanism of the reaction is known). Solvents may include, for example, polar solvents or non-polar solvents. Solvents choices include, but are not limited to, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, dioxane, methanol, ethanol, hexane, methylene chloride and acetonitrile. In some preferred embodiments, the solvent is dimethylformamide.
  • More than one solvent may be chosen for any particular reaction or purification procedure. Water may also be admixed into any solvent choice. Further, water, such as distilled water, may constitute the reaction medium instead of a solvent. Persons of ordinary skill in the art will be familiar with methods of purifying compounds of the present invention. One of ordinary skill in the art will understand that compounds of the present invention can generally be purified at any step, including the purification of intermediates as well as purification of the final products. In certain embodiments, purification is performed via silica gel column chromatography or HPLC.
  • a compound of the present invention may be administered to kill tumor cells by any method that allows contact of the active ingredient with the agent's site of action in the tumor.
  • a compound of the present invention may be administered by any conventional methods available for use in conjunction with pharmaceuticals, either as an individual therapeutically active ingredient or in a combination of therapeutically active ingredients.
  • a compound of the present invention may be administered alone, but will generally be administered with a pharmaceutically acceptable earner selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • a compound of the present invention may be extensively purified and/or dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. Such methods are well-known in the art.
  • the active compounds will then generally be formulated for administration by any known route, such as parenteral administration. Methods of administration are discussed in greater detail below.
  • a compound of the present invention can be provided in prodrug form, meaning that an environment to which a compound of the present invention is exposed alters the prodrug into an active, or more active, form.
  • the term "precursor” covers compounds that are considered “prodrugs.”
  • the benzo[ ⁇ /]fluorenes described herein may be converted to benzo[/?]fluorenes in vivo, which then, in turn, exhibit biological and/or therapeutic effects.
  • compositions of the present invention may comprise an effective amount of one or more candidate substances (e.g., a compound of the present invention) or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier.
  • candidate substances e.g., a compound of the present invention
  • additional agents dissolved or dispersed in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of a pharmaceutical composition that contains at least one candidate substance or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drag stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, pp 1289-1329, 1990). Except insofar as any conventional earner is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
  • preservatives e.g., antibacterial agents, antifungal agents
  • isotonic agents e.g., absorption delaying agents, salts, preservatives, drugs, drag stabilizers, gels, binders, excipients, disintegration agents, lubricants
  • the candidate substance may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, locally, via inhalation (e.g., aerosol inhalation), via injection, via infusion, via continuous infusion, via localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination
  • the composition is administered to a subject using a drug delivery device.
  • a drug delivery device Any drug delivery device is contemplated for use in delivering a pharmaceutically effective amount of a compound of the present invention.
  • the actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • the dose can be repeated as needed as determined by those of ordinary skill in the art.
  • a single dose is contemplated.
  • two or more doses are contemplated.
  • the time interval between doses can be any time interval as determined by those of ordinary skill in the art.
  • the time interval between doses may be about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 6 hours to about 10 hours, about 10 hours to about 24 hours, about 1 day to about 2 days, about 1 week to about 2 weeks, or longer, or any time interval derivable within any of these recited ranges.
  • compositions may comprise, for example, at least about 0.1% of a compound of the present invention.
  • a compound of the present invention may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
  • the composition may comprise various antioxidants to retard oxidation of one or more component.
  • the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.
  • parabens e.g., methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal, or combinations thereof.
  • the candidate substance may be formulated into a composition in a free base, neutral, or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine, or procaine. Other salts are described herein.
  • a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc. ), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods.
  • isotonic agents such as, for example, sugars, sodium chloride, or combinations thereof.
  • nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays.
  • Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained.
  • the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5.
  • antimicrobial preservatives similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation.
  • various commercial nasal preparations are known and include drags such as antibiotics or antihistamines.
  • the candidate substance is prepared for administration by such routes as oral ingestion.
  • the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof.
  • Oral compositions may be incorporated directly with the food of the diet.
  • carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof.
  • the oral composition may be prepared as a syrup or elixir.
  • a syrap or elixir may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.
  • an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, or combinations thereof.
  • a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.: or combinations thereof the foregoing.
  • a binder such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof
  • an excipient such as
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients.
  • certain methods of preparation may include vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered liquid medium thereof.
  • the liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose.
  • the preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
  • composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
  • prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin, or combinations thereof.
  • a compound of the present invention may be combined with traditional drugs. It is contemplated that this type of combination therapy may be used in vitro or in vivo.
  • an anticancer agent may be used in combination with a compound of the present invention.
  • an antibiotic may be used in combination with a compound of the present invention.
  • a compound of the present invention may be provided in a combined amount with an effective amount of an anticancer agent to reduce or block DNA replication in cancerous cells (e.g., tissues, tumors).
  • This process may involve administering the agents at the same time or within a period of time wherein separate administration of the substances produces a desired therapeutic benefit.
  • This may be achieved by contacting the cell, tissue, or organism with a single composition or pharmacological formulation that includes two or more agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition includes one agent and the other includes another.
  • Anti-bacterial classes and agents are well-known in the art, and include, for example, the classes of aminoglycoside antibiotics, cephalosporins, penicillins, quinolones, sulfonamides, tetracyclines, beta-lactams and macrolides.
  • Non-limiting specific examples of antibacterial agents include linezolid, tigecycline, tetracycline, oxytetracycline, doxycycline, minocycline, vancomycin, enrofloxacin, erythromycin, tyrocidine, griseofulvin, streptomycin, polymyxin, cephalosporin, ampicillin, cephalothin, lincomycin, gentamicin, carbenicillin, cephalexin and clindamycin.
  • These lists of antibiotics are not exhaustive and one skilled in the art can readily determine other antibiotics which may be employed.
  • the compounds of the present invention may precede, be co-current with and/or follow the other agents by intervals ranging from minutes to weeks.
  • the agents are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell, tissue or organism.
  • one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) as the candidate substance.
  • one or more agents may be administered within of from substantially simultaneously, about 1 minute, 5 minutes, 10 minutes, 20 minutes 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 1, 2, 3, 4, 5, 6, 7 or 8 hours, 9
  • High/low resolution electron impact (EI) mass spectra were measured by the WATSPEC Mass Spectrometry Facility (Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada) and the McMaster Regional Center for Mass Spectrometry (Department of Chemistry, McMaster University, Hamilton, Ontario, Canada).
  • Gas chromatography-mass spectrometry was performed on a HP GCD 1800 with a column (HP5) length of 30.0 cm and diameter of 0.25 mm. The following temperature program was applied: initial temperature 70 0 C, rising rate 10 °C/minute, final temperature 265 0 C held for 20.0 minutes. Elemental analyses were performed by the M-H-W Laboratories (Phoenix, Arizona, USA).
  • Anhydrous THF and EtoO were freshly distilled from sodium/benzophenone under nitrogen prior to use.
  • Anhydrous CH2CI2 was freshly distilled from CaH2 under nitrogen prior to use. All commercial reagents were purchased from Aldrich Chemical Co., Strem Chemicals Inc., Alfa Aesar, Lancaster Synthesis Ltd. or BDH Inc. and were used as received unless otherwise indicated. Deionized water was supplied by a Biolab vertical series reverse osmosis system.
  • the -78 0 C and 0 0 C designations refer to dry ice/acetone and ice/water slush respectively.
  • Flash column chromatography was carried out using the Merck silica gel (70-230 mesh) and SiliCycle silica gel (60 A). Reactions were magnetically stirred and monitored by thin layer chromatography (TLC) with Merck pre-coated silica gel plates (silica gel 60 F 254 011 aluminum sheet). All reported yields are isolated yields.
  • Scheme 4 depicts non-limiting examples of syntheses of the present invention.
  • Scheme 1 shows a preparation of isoprekinamycin (IPK) derivative 16, accompanied by syntheses of various benzo[ ⁇ /]fluorenes as intermediates.
  • IPK isoprekinamycin
  • Scheme 5 represents another set of non-limiting synthetic examples of the present invention. Adaptation of the approach of Scheme 4 to the preparation of IPK is presented in Scheme 5, accompanied by the synthesis of various benzo[ ⁇ /]fluorenes as intermediates. Synthesis of 4-Hydroxy-2, 3-dihydroindan-l-one (see Loudon and Razdan, 1954)
  • Spectroscopic comparison of the synthetic and natural IPK was carried out in the following manner: ( 1 ) for 1 H NMR spectral comparison, saturated CD2CI2 solutions of the synthetic and natural IPK were examined on a 500 MHz NMR spectrometer; (2) for IR spectral comparison, saturated CH 2 CI 2 solutions of the synthetic and natural IPK were examined on a FT-IR spectrometer using a demountable liquid-cell IR kit (Aldrich Zl 1200-3) with two CaF2 windows (32 mm ⁇ 3 mm) and a light path of 0.1 mm, and the solvent and air backgrounds were deducted from the recorded spectra; (3 ) for HPLC retention time and UV-vis spectra comparison, the synthetic and natural IPK and also a mixture of both compounds were analyzed with a Waters HPLC system equipped with a photodiode array detector. See FIG. 3 for HPLC data.
  • CHO cells type AA8; ATCC CRL-1859
  • HEPES alpha minimum essential medium
  • Human leukemia K562 cells obtained from the American Type Culture Collection, were maintained as suspension cultures in DMEM (Invitrogen, Burlington, Canada) containing 10% fetal calf seiiim (FCS) and 2 mM L-glutamine.
  • IC50 values the concentration of isoprekinamycin that reduces the absorbance of MTT or MTS by one-half, for growth inhibition in both assays were measured by fitting the absorbance-drag concentration data to a three-parameter logistic equation as described. Hasinoff et al, 1997.
  • Topoisomerase Il ⁇ kDNA decatenation inhibition assay A spectrofluorometric decatenation assay was used to determine the inhibition of topoisomerase Il ⁇ by IPK-diacetate as was done for kinamycin A and kinamycin C (Hasinoff et al., 2006).
  • kDNA consists of highly catenated networks of circular DNA.
  • the 20 ⁇ L reaction mixture contained 0.5 mM ATP, 50 mM Tris-HCl (pH 8.0), 120 mM KCl, 10 mM MgCl 2 , 30 ⁇ g/mL bovine serum albumin, 40 ng kDNA, test compound (0.5 ⁇ L in dimethyl sulfoxide) and 10 ng of topoisomerase Il ⁇ protein (the amount that gave approximately 80% decatenation).
  • test compound 0.5 ⁇ L in dimethyl sulfoxide
  • 10 ng of topoisomerase Il ⁇ protein the amount that gave approximately 80% decatenation.
  • the assay incubation was carried out at 37 0 C for 20 minutes and was terminated by the addition of 12 ⁇ L of 250 mM Na 2 EDTA. Samples were centrifuged at 8000 g at 25 0 C for 15 minutes and 20 ⁇ L of the supernatant was added to 180 ⁇ L of 600-fold diluted PicoGreen ⁇ R> dye (Molecular Probes, Eugene, OR) in a 96-well plate. The fluorescence, which was proportional to the amount of kDNA, was measured in a Fluostar Galaxy (BMG, Durham North Carolina, USA) fluorescence plate reader using an excitation wavelength of 485 nm and an emission wavelength of 520 nm.
  • Topoisomerase Il-cleaved DNA complexes produced by anticancer drugs may be trapped by rapidly denaturing the complexed enzyme with sodium dodecyl sulfate (SDS) (Burden et a!., 2001 ).
  • SDS sodium dodecyl sulfate
  • the cleavage of double-stranded closed circular pBR322 DNA to form linear DNA was followed by separating the SDS-treated reaction products using ethidium bromide gel electrophoresis as described.12
  • the 20 ⁇ L cleavage assay reaction mixture contained 100 ng of topoisomerase Ik/.
  • pBR322 plasmid DNA 80 ng pBR322 plasmid DNA (MBI Fermentas, Burlington, Canada), 0.5 mM ATP in assay buffer ( 10 mM Tris-HCl, 50 mM KCl, 50 mM NaCl, 0.1 mM EDTA, 5 mM MgCl 2 , 2.5% (v/v) glycerol, pH 8.0, and drag (0.5 ⁇ L in dimethyl sulfoxide). The order of addition was assay buffer, DNA, drag, and then topoisomerase Il ⁇ .
  • the reaction mixture was incubated at 37 0 C for 10 minutes and quenched with 1% (v/v) SDS /25 mM Na 2 EDTA.
  • the reaction mixture was treated with 0.25 mg/ml proteinase K (Sigma) at 55 0 C for 30 minutes to digest the protein.
  • the linear pBR322 DNA cleaved by topoisomerase Il ⁇ . was separated by electrophoresis (2 hours at 8 V/cm) on a TAE (Tris base (4 mM)/glacial acetic acid (0.1 1% (v/v))/Na 2 EDTA (2 mM) buffer) ethidium bromide (0.5 ⁇ g/mL) agarose gel ( 1.2%, wt/v).
  • TAE Tris base (4 mM)/glacial acetic acid (0.1 1% (v/v))/Na 2 EDTA (2 mM) buffer
  • ethidium bromide 0.5 ⁇ g/mL)
  • isoprekinamycin strongly inhibited growth of CHO cells and K562 cells with IC50 values of 5.8 and 6.4 ⁇ M, respectively. These values compare to IC50 values of 1.4 and 3.4 ⁇ M, respectively for the widely used anticancer drug etoposide.
  • IPK-diacetate inhibits the decatenation activity of topoisomerase Il ⁇
  • IPK-diacetate strongly inhibited (IC50 value of 9.7 ⁇ M) the decatenation activity of human topoisomerase Il ⁇ .
  • This assay is a measure of the ability of these compounds to inhibit the catalytic activity only and is not a measure of whether these compounds act as topoisomerase II poisons as do some widely used anticancer drags. Fortune and Osheroff, 2000; Li and Liu, 2001.
  • IPK up to a concentration of 120 ⁇ M did not inhibit topoisomerase II.
  • Inhibition of topoisomerase Il ⁇ activity by IPK or IPK-diacetate was not accompanied by stabilization of the covalent topoisomerase Il ⁇ -DNA cleavable complex
  • topoisomerase II alters DNA topology by catalyzing the passing of an intact DNA double helix through a transient double-stranded break made in a second helix and is critical for relieving torsional stress that occurs during replication and transcription and for daughter strand separation during mitosis (Fortune and Osheroff, 2000; Li and Liu, 2001 ).
  • the IC50 value for 16 with respect to K562 cells was determined to be 78 iiM, and the IC50 value for the C ⁇ O assay was determined to be 1 ⁇ M.
  • the IC50 value for 16 in the topoisomerase Il ⁇ assay described in Example 19 was determined to be 51 ⁇ M.
  • Streptomyces murayamaensis (ATCC 21414) was purchased from the American Type Culture Collection. Slants of Krainsky's agar were inoculated with Streptomyces sps, incubated at 27° C for 15 days and then stored at 4° C. Media composition :
  • Krainsky's medium Glucose 10 g; K ⁇ PO 4 0.5 g; asparagine 0.5 g; agar 10 g per L.
  • Seed medium Glucose 20 g; soybean meal 20 g; NaCl 3 g in 1 L distilled water. The medium was adjusted to pH 8 (using NaOH) before sterilization.
  • Oatmeal medium Trace metals medium containing rolled oats (90 g cooked in 1 L of distilled water for 10 minutes, then added to 1 L of cold distilled water). The boiled mixture was filtered to remove oatmeal shaft, leaving a fine starch filtrate solution. Trace metal (6 mL) was added to the filtrate before making up the total volume to 3 L using more distilled water (medium pH was adjusted to 8 with NaOH before sterilization).
  • Trace metal salts solution contained: ZnCl 2 (40 mg), FeSO 4 WH 2 O (200 mg), CuCl 2 WH 2 O ( 10 mg), MnCMH 2 O ( 10 mg), H 3 BO 3 (5 mg), (NH 4 V 1 Mo 7 O 24 ⁇ H 2 O ( 10 mg), concentrated HCl (2 mL) in 1 L of distilled water. Seed preparation and Shake Flask Fermentation: Seed medium (200 mL in a 1 L flask) was inoculated with 5-10 colonies
  • the isolation of compound VIII was based on a modification of the procedure employed by Cone et al. in the isolation of kinamycins and isoprekinamycin ( 1989).
  • the acidified fermented oatmeal-broth was centrifuged at 10,000 rpm for ten minutes to remove the mycelia pellet, resuspended in 50 mL distilled water, sonicated ( 1-4° C for 5 minutes) and recombined with the supernatant.
  • the whole suspension was stirred with toluene (400 mL to 1 L broth) for 90 minutes and the resulting emulsion was filtered through Celite and the Celite was washed with acetone.
  • the aqueous layer was separated and washed with ethyl acetate (2 x 250 mL for eveiy L of broth) and the total organic layer was separated and dried over anhydrous NaTSO 4 .
  • the dried organic layer was concentrated in vacuo to give a dark brown residue.
  • the concentrate was dissolved in a minimal amount of CHCh and applied to a Silicar CC- 4 column packed in CHCh. Elution with CHCh first gave a dark oily fraction but further elution with 1% ethyl acetate in CHCh gave a brown-purple fraction that was confirmed (HPLC) to contain kinamycins.
  • the stiiicture of VIII was assigned on the basis of proton NMR analysis, mass spectrometry analysis and HMQC and HMBC NMR experiments at 600 MHz.

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Abstract

The present invention relates to the preparation of various benzo[α]fluorenes. The benzo[α]fluorene moiety appears in a variety of natural products, some of which have been shown to exhibit anticancer and antibacterial effects. Methods of the present invention provide facile access to some of these natural products as well as derivatives thereof.

Description

DESCRIPTION
ISOPREKINAMYCIN ANALOGS AND SYNTHESES THEREOF
BACKGROUND OF THE INVENTION
The present application claims benefit of priority to U.S. Provisional Application Serial No. 60/954,806, filed August 8, 2007, and U.S. Provisional Application Serial No. 60/955,187, filed August 10, 2007, the entire contents of both of which are hereby incorporated by reference.
1. Field of the Invention The present invention generally relates to the fields of synthetic organic chemistry, natural product isolation and study, and cancer treatment. More particularly, it concerns the discoveiy and preparation of compounds comprising a benzo[</]fluorene core, such as isoprekinamycin and derivatives thereof. Methods of use of these compounds for anticancer and antibiotic treatment therapies are also encompassed by the present invention.
2. Description of Related Art
The kinamcyins, first believed to be N-cyanobenzo[b]carbazoles 1 (Omura et a!., 1973) but now known to be diazobenzo[b]fluorenes 2 (Gould et a!., 1994; Mithani et a!., 1994), are of current interest because of antibacterial activity and an in vitro cytotoxicity profile against cancer cells suggestive of a mode of action different than anticancer agents in current clinical use (Hasinoff et a!., 2006). The discoveiy of the lomaiviticins, which are dimeric analogs of the kinamcyins possessing potent cytotoxicity against a range of cancer cell lines, has heightened the interest in these unusual natural products (He et a!., 2001 ). A number of synthetic strategies have been disclosed (Gould, 1997), most notably a total synthesis of the biosynthetic precursor 6 (Hauser and Zhou, 1996) and model studies towards a total synthesis of lomaiviticin B (Nicolaou et a!., 2006). Two recent total syntheses of kinamycin C (Lei and Porco, 2006; Nicolaou et a!., 2007) and a synthesis of the racemic form of the O-methyl ether of kinamycin C have also been disclosed. Kumamoto et a!., 2007.
Figure imgf000003_0001
Isoprekinamycin (IPK), first assigned structure 3 (Seaton and Gould, 1989), is now recognized to be the diazobenzo[</]fluorene 4 (Proteau et a!., 2000). The previously rare benzo[</]fluorene class of natural products now boasts several other members (e.g., 7) with various degrees of oxygenation in ring D but lacking a diazo group (Akiyama et al., 1998; Schneider et al., 2006; Baur et al., 2006; Fondja et al., 2006). In this group, studies suggest that a substantial diazonium ion character in the diazo group of IPK might play a role in its bioactivity (Laufer and Dmitrienko, 2002).
Cancer is a leading cause of death in the United States. Despite significant efforts to find new approaches for treating cancer, the primary treatment options remain surgery, chemotherapy and radiation therapy, either alone or in combination. Surgery and radiation therapy, however, are generally useful only for fairly defined types of cancer, and are of limited use for treating patients with disseminated disease. Chemotherapy is the method that is generally useful in treating patients with metastatic cancer or diffuse cancers such as leukemias. Although chemotherapy can provide a therapeutic benefit, it often fails to result in cure of the disease due to the patient's cancer cells becoming resistant to the chemotherapeutic agent. Similarly, infectious diseases caused by bacteria, for example, are becoming increasingly difficult to treat and cure. For example, more and more bacteria are developing resistance to current antibiotics and chemotherapeutic agents. Examples of such bacteria include both gram positive and gram negative bacteria, including Staphylococcus, Streptococcus, Mycobacterium, Enterococcus, Corynebacterium, Borrelia, Bacillus, Chlamydia, Mycoplasma, and the like.
Therefore, a need exists for additional chemotherapeutics and antimicrobial agents to treat cancer and infectious diseases. A continuing effort is being made by individual investigators, academia and companies to identify new and potentially useful chemotherapeutic and antimicrobial agents.
To date, there have been no reports of the total synthesis of any of the benzo[</]fluorene natural products. Accordingly, a need exists for facile synthetic routes to be developed such that these natural products and their derivatives may be prepared and studied for their anticancer, antibacterial and other effects.
SUMMARY OF THE INVENTION
The present invention generally provides novel benzo[</]fluorenes, syntheses of benzo[</]fluorenes, and methods of using these compounds as anticancer and/or antibacterial agents. In certain aspects, the present invention is drawn to the preparation of isoprekinamycin (IPK) and derivatives thereof and methods of treating cancer or bacterial infections using these compounds.
Accordingly, one general aspect of the present invention encompasses a method of synthesizing a compound of formula (A):
Figure imgf000004_0001
comprising: (a) reacting a compound of formula (I):
Figure imgf000005_0001
with a compound of formula (II):
Figure imgf000005_0002
to generate a first intermediate of formula (III):
Figure imgf000005_0003
and (b) subjecting the first intermediate or a derivative thereof to reducing conditions, wherein: A1-A^ are each independently carbon or nitrogen; X is halo, -OSO2CF3, or any other leaving group as this term is defined herein; Y is alkoxy, aryloxy, aralkyloxy, alkylamino, arylamino, aralkylamino, or -OC(O)Rn, wherein Rn is alkyl, aryl, or aralkyl; R1-R16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, aiylthio, aralkylthio, alkylamino, arylamino, aralkylamino, nitro, halogen (e.g., fluoro), -Sθ2(alkyl), -SO(alkyl), - Sθ2(aiyl), -SO(aryl), -Sθ2(aralkyl), or -SO(aralkyl), or any one or more of R1-R2, R2- R*, R3-R4, R5-R6, R6-R7, Ry-Rs, R9-R10, R10-R11, R11-R12, R13-R14, R14-R15, or R15-R16 taken together form a cyclic group; Rn is H, -CH2CN or -CH2CO2Rt>, wherein Rb is alkyl, aryl, or aralkyl; and Ris and R19 are each independently H, alkyl, aryl, or aralkyl, or Ris and R19 taken together with the boron atom to which they are attached form the following substituent:
Figure imgf000006_0001
In certain embodiments, the compound of formula (A) is further defined as not the following:
Figure imgf000006_0002
Indeed, any specific compound or genus of compounds described herein may be excluded from this or any other embodiment as described herein. Persons of skill in the art will understand that of A1-As in this or any other compound described herein, certain combinations of C and/or N are more chemically feasible than others (e.g., an all-nitrogen aryl group is rarely made). Persons of skill in the art also understand that substituents on aiyl nitrogens may or may not be present in this or any other compound of the present invention. In certain embodiments, when an aiyl ring atom is a nitrogen, the nitrogen displays no substituent. It is intended that the substituents described as being able to form a cyclic group in this or any other compound described herein do not all necessarily form such cyclic groups. Certain pairs of substituents (e.g., Ry-R1(>) may form a cyclic group while Rn is H. Ris and R19 may form any boronate ester known to those of skill in the art. In certain embodiments in this or any other formula, Y is fluoro. In certain embodiments, -OH groups are preferred at certain positions (e.g., R4 and/or R5).
In any compound described herein, a functional group may be protected by a protecting group. Methods may also comprise the addition or removal of a protecting group. Persons of skill in the art are familiar with protecting groups, their installation, and their removal. Certain methods of the present invention comprise reducing conditions.
Reducing conditions are well-known to those of skill in the art of synthetic organic chemistry. Non-limiting examples of reducing conditions include conditions comprising an alkyllithium/DIBAL mixture (e.g., n-butyllithium/DIBAL) or borohydride, such as sodium borohydride.
Certain methods of the present invention comprise Suzuke reaction conditions. Such conditions are well-known to those of skill in the art of synthetic organic chemistry. Typically, the Suzuki reaction is the organic reaction of an aryl- or vinyl- boronic acid with an aryl- or vinyl-halide catalyzed by a palladium(O) complex. It is a widely used reaction to synthesize poly-olefms, styrenes, and substituted biphenyls, for example. Miyaura, et <//., 1979; Miyaura and Suzuki, 1979; Suzuki, 1991 ; Miyaura and Suzuki, 1995; Suzuki, 1999 (each of which is incorporated herein by reference in its entirety). The reaction also works with other agents, such as triflates (-OTf), instead of halides, and also with boron-esters instead of boronic acids. The relative reactivity is as shown: -I > -OTf > -Br » -Cl. The reaction relies on a palladium catalyst, such as tetrakis(triphenylphosphine)palladium(0), to effect part of the transformation. The palladium catalyst (more strictly a pre-catalyst) is 4- coordinate, and usually involves phosphine supporting groups.
In certain embodiments, methods comprise anionic reaction conditions. For example, the first intermediate described above may be subjected to anionic reaction conditions. A compound of formula (Ilia), described below (a derivative of (III)), may be subjected to anionic reaction conditions. Persons of skill in synthetic organic chemistry are familiar with such conditions. For example, such conditions may comprise LDA. DBU may be added, in certain embodiments. Other anionic reaction conditions are described by Thebtaranonth and Thebtaranonth, 1994, incorporated herein in its entirety.
Various intermediates may be prepared using methods of the present invention, such as the intermediate of formula (III). Such intermediates may be purified, or may be used without purification in a subsequent step. All such intermediates are contemplated by the present invention. For example, a compound of formula (IV) may be generated as an intermediate using methods of the present invention:
Figure imgf000008_0001
wherein: A1-A^ are each independently carbon or nitrogen; Ry-R16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aiyloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, aiylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO2(alkyl), -SO(alkyl), -SO2(aryl), -SO(aryl), - Sθ2(aralkyl), or -SO(aralkyl), or any one or more Of Ry-R1O, R1O-R11, R11-R12, R13-R14, R14-R15, or Ri S-R16 taken together form a cyclic group; R20 is -CN, -CONH2, - NH2, or -NHCθ2(alkyl); and R21 is alkoxy. Non-limiting examples of compounds of formula (IV) include the following:
Figure imgf000008_0002
Figure imgf000009_0001
In certain embodiments, the compound of formula (II) is as shown:
Figure imgf000009_0002
wherein Rn-Rn are defined as above. Any boronate ester or acid as known to those of skill in the art may be used in methods discussed herein.
Compounds of formula (III) may be modified before being subjected to reducing conditions, in certain embodiments. That is, a compound of formula (III) may be generated and then modified to form another compound. This compound may, in certain embodiments, be considered a derivative of a compound of formula (III). (This is not to imply that any compound that is a derivative of another must be made from the parent compound, although this is certainly optional.) In certain embodiments, a derivative of the compound of formula (III) is a compound of formula (Ilia):
Figure imgf000010_0001
wherein: A1-As are each independently carbon or nitrogen; Ya is alkoxy, aiyloxy, aralkyloxy, alkylamino, arylamino, aralkylamino, or -OC(O)Re, wherein Re is alkyl, aryl, or aralkyl; Ryn-R16n are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, aiylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., flυoro), -SO2(alkyl), - SO(alkyl), -SO2(aryl), -SO(aryl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of Ryn-RiOa, Rioa-Ri ia, Ri ia"Ri2a, Ri3a-Ri4a, Ri4a-Ri5a, or R1 Sn-R16n taken together form a cyclic group; and Rnn is H, -CH2CN or -CH2CO2R0, wherein R0 is alkyl, aryl, or aralkyl, wherein the compound of formula (III) is not the same as the compound of formula (Ilia), and the compound of formula (Ilia) is then subjected to the reducing conditions.
Also contemplated by the present invention are compounds made by any of the methods described herein. In certain embodiments, the following compounds are excluded in such methods:
Figure imgf000010_0002
For example, the compound of formula (A) may, in certain embodiments, be defined as not comprising these compounds. In certain embodiments, a compound prepared by the methods described herein include these compounds. In certain embodiments, the following compounds are contemplated as being made:
Figure imgf000011_0001
Also contemplated by methods of the present invention are compounds of formula (V):
Figure imgf000011_0002
wherein: Yi is alkoxy, aryloxy, aralkyloxy, alkylamino, arylamino, aralkylamino, or - OC(O)Rn, wherein Rn is alkyl, aryl, or aralkyl; R22-R29 arc each independently H, - OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO2(alkyl), -SO(alkyl), -SO2(aryl), -SO(aryl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of R22-R23. R23-R24. R24-R25. R26-R27. R27-R2^ or R2^-R29 taken together form a cyclic group; and R30 is H, -CH2CN or -CH2CO2Rb, wherein Rb is alkyl, aryl, or aralkyl. In certain embodiments, the compound of formula (V) is further defined as not any of the following:
Figure imgf000011_0003
Figure imgf000012_0001
In certain embodiments, such compounds are specifically included. In certain embodiments, the following compound is contemplated as a compound of formula
(V):
Figure imgf000012_0002
Other general aspects of the present invention contemplate a compound of formula (VI):
Figure imgf000012_0003
wherein: R31-R38 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -Sθ2(alkyl), -SO(alkyl), -
SO2(aryl), -SO(aryl), -Sθ2( aralkyl), or -SO(aralkyl), or any one or more of R31-R32,
R32-R33, R33-R34, R35-R36, R36-R37, or R37-R38 taken together form a cyclic group; R39 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and R41. is alkoxy. In certain embodiments, when R34, R35 and R52 are each -OH, R37 is not -CH3. Non-limiting examples of compounds of formula (VI) include:
Figure imgf000013_0001
Other aspects of the present invention encompass a compound of formula
(VII):
Figure imgf000013_0002
wherein: A1-As arc each independently carbon or nitrogen; R41-R.^ are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, aiylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO2(alkyl), -SO(alkyl), -SO2(aryl), -SO(aryl), - SO^iaralkyl), or -SO(aralkyl), or any one or more of R41-R42. R42-R43. R43-R44. R45- R46, R46-R47, or R47-R4S taken together form a cyclic group. In certain embodiments, the compound of formula (VII) is further defined as not any of the following:
Figure imgf000014_0001
Non-limiting examples of compounds of formula (VII) include:
Figure imgf000014_0002
Also contemplated by the present invention is a method of synthesizing a compound of formula (III):
Figure imgf000015_0001
comprising reacting a compound of formula (I):
Figure imgf000015_0002
with a compound of formula (II):
Figure imgf000015_0003
under Suzuki coupling conditions, wherein: A1-As are each independently carbon or nitrogen; X is halo, -OSO2CF;, or any other leaving group as this term is defined herein; Y is alkoxy, aryloxy, aralkyloxy, alkylamino, arylamino, aralkylamino, or - OC(O)Rn, wherein Rn is alkyl, aryl, or aralkyl; Ry-R16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO2(alkyl), -SO(alkyl), -SO2(aryl), -SO(aryl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of Ry-R1(), Ri0-Rn, Rn-Ri2, R13-R14, R14-R15, or R15-R16 taken together form a cyclic group; Rn is H, -CH2CN or -CH2CO2R11, wherein R11 is alkyl, aryl, or aralkyl; and Ris and Riy are each independently H, alkyl, aryl, or aralkyl, or Ris and Riy taken together with the boron atom to which they are attached form the following substituent:
Figure imgf000016_0001
Another method of the present invention that is contemplated is a method of synthesizing a compound of formula (IV):
Figure imgf000016_0002
comprising cyclizing a compound of formula (III):
Figure imgf000016_0003
under anionic cyclization conditions, wherein: A1-As are each independently carbon or nitrogen; Y is alkoxy, aryloxy, aralkyloxy, alkylamino, arylamino, aralkylamino, or -OC(O)Rn, wherein Ra is alkyl, aryl, or aralkyl; Ry-R16 are each independently H, - OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO2(alkyl), -SO(alkyl), -SO2(aryl), -SO(aryl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of Ry-R1O, R1O-R11, Rn-Ri2, R13-Ru, Ru-Ri5, or R15-R16 taken together form a cyclic group; Rn is -CH2CN or -CH2CO2R11, wherein R1, is alkyl, aryl, or aralkyl; R20 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and R21 is alkoxy.
Another general aspect of the present invention contemplates the following: In a method of preparing a compound comprising a benzo[</]fluorene, the improvement comprising subjecting a product of a reaction of an aryl boronate ester and an aryl bromide to reducing conditions comprising an alkyllithium/DIBAL (e.g., n-butyllithium/DIBAL) mixture or borohydride, such as sodium borohydride. Other reducing conditions may be employed, as recognized by a skilled artisan. Persons of skill in the art will be familiar with aiyl boronate esters and aryl bromides that are compatible in this method. Certain aiyl boronate esters and aiyl bromides are described herein (e.g., compounds of formulas (I) and (II)). Solvents used in this and other methods discussed herein are well-known to those of skill in the art, and, of course, should be chosen such that that they do not interfere with the desired reaction and are easily removable once the reaction is complete. Solvents that dissolve the reactants completely or nearly completely are also preferred.
Another general aspect of the present invention entails a method of obtaining a compound of formula (VIII):
Figure imgf000017_0001
comprising isolating the compound from Streptomvces munnwnaensis . In further embodiments, the method may further comprise obtaining Streptomvces muruyumuensis. Such methods may be performed by, for example, performing the procedure described in the Examples below. Persons of skill in the art will be familiar with means of obtaining Streptomvces murayamaensis. For example, one may obtain this species from the American Type Tissue Collection, Manassas, Virginia. Any compound of the present invention may be further comprised in a pharmaceutically acceptable exipient, diluent, or vehicle. For example, a compound of formula (A), (IV), (VI), (VII), and/or (VIII) may be comprised in a pharmaceutically acceptable exipient, diluent, or vehicle.
Methods of inhibiting the catalytic decatenation activity of topoisomerase Ilα in a cell are also contemplated by the present invention, comprising contacting the cell with an effective amount of a compound of formula (A):
Figure imgf000018_0001
or a compound of formula (IV):
Figure imgf000018_0002
wherein: A1-As arc each independently carbon or nitrogen; R1-R16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, aiylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO2(alkyl), -SO(alkyl), -SO2(aryl), -SO(aryl), - SO2(aralkyl), or -SO(aralkyl), or any one or more Of Ri-R2, R2-R^, R3-R4, R5-R6, Rr,- R7, R7-Rs, R9-R10, R1O-R11, Rn-Ri2, R13-Ru, Ru-Ri5, or R15-R16 taken together form a cyclic group; R20 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and R21 is alkoxy. The cell in this or any other method of the present invention may be any cell known to those of skill in the art. The cell may be a cancer cell, for example. Thus, in certain embodiments, compounds of the present invention are anticancer agents. The cancer cell may be any type that is known to those of skill in the art. For example, the cancer cell may be a leukemia cell, a breast cancer cell, or a colon cancer cell. Such cells may be of the particular type MCF-7 (breast cancer cells) or HCT-1 16 (colon cancer cells). The cell may be in vivo or in vitro. In particular embodiments, the compound of formula (A) is selected from the group consisting of:
Figure imgf000019_0001
Other methods of the present invention contemplate methods of inhibiting cell growth, comprising contacting the cell with an effective amount of a compound of formula (A):
Figure imgf000019_0002
or a compound of formula (IV):
Figure imgf000019_0003
wherein: A1-As arc each independently carbon or nitrogen; R1-R16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, aiylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO2(alkyl), -SO(alkyl), -SO2(aryl), -SO(aryl), - SO2(aralkyl), or -SO(aralkyl), or any one or more of RrR2, R2-R3, R3-R4, R5-R6, Rr,- R7, R7-Rs, R9-R10, R1O-R11, Rn-Ri2, R13-Ru, Ru-Ri5, or Ri5-Ri6 taken together form a cyclic group; R20 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and R21 is alkoxy. The cell may be a cancer cell, as described above. The cell may be in vivo or in vitro.
Also contemplated are methods of treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of a compound of formula (A):
Figure imgf000020_0001
or a compound of formula (IV):
Figure imgf000020_0002
wherein: Ai-As are each independently carbon or nitrogen; R1-R16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, aiylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -SO2(alkyl), -SO(alkyl), -SO2(aryl), -SO(aryl), - SO2(aralkyl), or -SO(aralkyl), or any one or more Of Ri-R2, R2-R3, R3-R4, Rs-R*, Re- R7, R7-Rs, Ry-Rio, R1O-R11, Rn-Ri2, R13-Ru, Ru-Ri5, or R1S-R16 taken together form a cyclic group; R20 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and R21 is alkoxy. The subject in this or any other method may be a mammal, for example, such as a human, mouse, rat, or rabbit. In particular embodiments, the mammal is a human. The subject may have any cancer known to those of skill in the art. For example, the subject may have cancer of the lung, liver, skin, eye, brain, gum, tongue, blood, head, neck, breast, pancreas, prostate, kidney, bone, testicles, ovaiy, cervix, gastrointestinal tract, lymph system, small intestine, colon, or bladder.
Compounds of the present invention, such as IPK or derivatives thereof, may also behave as antibiotics. An "antibacterial agent" or "antibiotic" refers to an agent that prevents the growth of bacteria, and can be bactericidal (kills) or bacteriostatic (inhibits growth). Thus, another general aspect of the present invention encompasses a method of inhibiting bacterial growth or killing bacteria in a subject, or treating a bacterial infection in a subject, comprising administering an effective amount of a compound of formula (A):
Figure imgf000021_0001
or a compound of formula (IV):
Figure imgf000021_0002
to the subject, wherein: A1-As arc each independently carbon or nitrogen; R1-R16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen (e.g., fluoro), -Sθ2(alkyl), -SO(alkyl), -Sθ2(aryl), - SO(aryl), -SO2(aralkyl), or -SO(aralkyl), or any one or more Of Ri-R2, R2-R3, R3-R4, R5-R6, R6-R7, R7-Rs, R9-R10, R10-R11, R11-R12, R^-Ri-t, R14-R15, or Ri5-Ri6 taken together form a cyclic group; R20 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and R2i is alkoxy.
In certain embodiments, the compound of formula (A) is not isoprekinamycin. In particular aspects, compounds of the present invention may be used to inhibit the growth of bacteria. Such methods may take place in vivo or in vitro. For example, a bacterial colony can be plated onto agar that incorporates a growth-inhibiting quantity of a compound of the invention. Bacteria that are resistant to the compound of the invention will continue to grow, while bacteria that are not resistant will be inhibited from growing. One of ordinary skill in the art can test for antibacterial effects by growing
(e.g., culturing) bacteria in the presence and absence of a compound of the invention. Those compounds that inhibit the growth of the bacteria are selected for further tests, while those compounds that do not inhibit bacterial growth are not selected for further tests. See also, e.g.. Clinical and Laboratory Standards Institute, 2006. Methods of the present invention may encompass, for example, treatment of a subject having a bacterial infection in which the infection is caused or exacerbated by any type of bacteria, such as gram-positive bacteria. In certain embodiments, a compound of the present invention or a pharmaceutical composition thereof is administered to a patient according to the methods of this invention. For example, the compound may be given to a subject, e.g., a human patient or other subject, suffering from a bacterial infection. Administration of the compound can be through any means, e.g., any conventional methods, e.g., oral, intravenous, intradermal, parenteral, transdermal or other methods as described herein. The method of the instant invention may be used for treatment of any bacterial infection of any organ or tissue in the body. The present invention also contemplates administering an effective amount of a compound of the present invention to a subject showing symptoms of bacterial infection, such as fever, headache, sweating, chills, and aches.
In certain embodiments, the bacterial infection may be caused or exacerbated by gram-positive bacteria. These gram-positive bacteria include, but are not limited to, methicillin-susceptible and methicillin-resistant staphylococci (including Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. hominis, S. saprophyticus, and coagulase-negative staphylococci), glycopeptide intermediary-susceptible S. aureus (GISA), penicillin-susceptible and penicillin-resistant streptococci (including Streptococcus pneumoniae, S. pyogenes, S. agalactiae, S. avium, S. bovis, S. lactis, S. sanguis and Streptococci Group C, Streptococci Group G and viridans streptococci), enterococci (including vancomycin susceptible and vancomycin-resistant strains such as Enterococcusfaecalis and E. faecium), Clostridium difficile, C. clostridiiforme, C. innocuum, C. perfringens, C. ramosum, Haemophilus influenzae, Listeria monocytogenes, Corynebacterium jeikeium, Bifidobacterium spp., Eubacterium aerofaciens, E. lentum, Lactobacillus acidophilus, L. casei, L. plantarum, Lactococcus spp., Leuconostoc spp., Pediococcus, Peptostreptococcus anaerobius, P. asaccarolyticus, P. magnus, P. micros, P. prevotil, P. productus, Propionibacterium acnes, Actinomyces spp., Moraxella spp. (including M. catarrhalis) and Escherichia spp. (including E. coli).
As discussed herein, certain compounds of the present invention may, in certain embodiments, be anticancer agents. An "anticancer" agent is capable of negatively affecting cancer in a subject, for example, by killing one or more cancer cells, inducing apoptosis in one or more cancer cells, reducing the growth rate of one or more cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or one or more cancer cells, promoting an immune response against one or more cancer cells or a tumor, preventing or inhibiting the progression of a cancer, or increasing the lifespan of a subject with a cancer. Anticancer agents are well-known in the art and include, for example, chemotherapy agents (chemotherapy), such as DNA intercalators, radiotherapy agents (radiotherapy), a surgical procedure, immune therapy agents (immunotherapy), genetic therapy agents (gene therapy), reoviral therapy, hormonal therapy, other biological agents (biotherapy), and/or alternative therapies. Compounds of the present invention may, in certain embodiments, be anticancer agents.
To kill a cell in accordance with the present invention, one would generally contact the cell with a compound of the present invention in amount effective to kill the cell. The term "in an amount effective to kill the cell" means that the amount of the compound of the present invention is sufficient so that, when administered to a cell, cell death is induced. A number of in vitro parameters may be used to determine the effect produced by the compositions and methods of the present invention. These parameters include, for example, the observation of net cell numbers before and after exposure to the compositions described herein. The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a compound of the present invention is administered or delivered to a target cell or are placed in direct juxtaposition with the target cell.
The terms "administered" and "delivered" are used interchangeably with "contacted" and "exposed."
As used herein, the term "effective" (e.g., "an effective amount") means adequate to accomplish a desired, expected, or intended result. For example, an "effective amount" may be an amount of a compound sufficient to produce a therapeutic benefit (e.g., effective to reproducibly inhibit decrease, reduce, inhibit or otherwise abrogate the growth of a cancer cell).
"Treatment" and "treating" as used herein refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, a subject (e.g., a mammal, such as a human) having cancer may be subjected to a treatment comprising administration of a compound of the present invention.
The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well- being of the subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, a therapeutically effective amount of a compound of the present invention may be administered to a subject having a cancerous tumor, such that the tumor shrinks.
The terms "inhibiting" or "reducing" or any variation of these terms as used herein includes any measurable decrease or complete inhibition to achieve a desired result. For example, there may be a decrease of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, or any range derivable therein, reduction of tumor size or bacterial infection following administration of a compound of the present invention. In a further example, following administering of a compound of the present invention, a patient suffering from a bacterial infection may experience a reduction the number and/or intensity of symptoms of the infection. Non-limiting examples of typical symptoms associated with a bacterial infection include elevated temperature, sweating, chills, and/or excess white blood cells compared to a normal range. It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention.
Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device and/or method being employed to determine the value.
As used herein the specification, "a" or "an" may mean one or more, unless clearly indicated otherwise. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Inhibition of growth of CHO cells by isoprekinamycin and derivatives thereof. The curved solid lines are non-linear least calculated least squares fits of the MTT absorbance-concentration data to a 3 -parameter logistic equation and yield the IC50 values shown. The left-most data point in each case is the control value in the absence of any added drag.. The cells were continuously incubated with the drags for 72 h. The error bars are SEs from 4 replicate wells. IPK = isoprekinamycin; IPK-diAc = isoprekinamycin diacetate; IPK-OMe = isoprekinamycin O-methyl derivative. FIG. 2. Inhibition of topoisomerase Ilα-catalyzed decatenation of kDNA by isoprekinamycin derivatives. The curved solid lines are non-linear least calculated least squares fits of the kDNA-associated fluorescence-concentration data to a 4- parameter logistic equation and yield the IC50 values shown. The left-most data point in each case is the control value in the absence of any added drug. IPK itself did not inhibit topoisomerase Ilα up to it solubility limit of -10 μM. IPK-diAc = isoprekinamycin diacetate; IPK-OMe = isoprekinamycin O-methyl derivative.
FIG. 3. Comparison of the HPLC retention times of the synthetic and natural isoprekinamycin (IPK). HPLC system: Waters 600 controller, Waters 996 photodiode array detector, Waters Millennium<R> software; (2) Column: Nova-Pak<R> Cl 8 60 A 4 μm, 3.9 \ 150 mm; (3) linear gradient (20 minutes): 94% H2O, 5% CH3CN and 0.1% AcOH to 5% H2O, 94% CH3CN and 0.1% AcOH at a flow rate of 1.5 mL/minute at room temperature.
FIG. 4. Isoprekinamycin-induced growth inhibition of CHO (A) and K562 (B) cells. Attached CHO cells or suspension K562 cells were treated for 72 h with a range of isoprekinamycin concentrations. The solid lines are three-parameter nonlinear least squares calculated logistic fits of the MTT or MTS absorbance- isoprekinamycin concentration data and were used to obtain the IC50 values. The error bars were calculated from replicates. FIG. 5. IPK-diacetate inhibits the decatenation activity of topoisomerase Ilα.
The ability of the compounds to inhibit the topoisomerase Ilα-mediated decatenation of highly networked kDNA was measured in an ATP-containing assay mixture at 37 0C for 20 minutes. The fluorescence measures the amount of decatenated DNA minicircles in the supernatant of the centrifuged quenched 20 μL assay mixture. IPK- diacetate inhibited the decatenation activity of topoisomerase Ilα with IC50 values of 9.7 ± 1.9 μM. The solid line is a non-linear least squares calculated fits of the fluorescence-concentration data to a three-parameter logistic equation.
FIG. 6. 600 MHz proton NMR spectrum of the compound of formula (VIII), isolated from Streptomyces murayamaensis.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention overcomes deficiencies of the prior art by providing methods that enable facile access to benzo[</]fluorene moieties. Since benzo[</]fluorenes are being identified in more and more natural products of therapeutic interest, synthetic access to these natural products is needed, as well as derivatives thereof. The methods of the present invention allow for a variety of these natural products and other benzo[</]fluorene derivatives to be generated. Compounds synthesized by methods of the present invention may be used for therapeutic purposes, such as anticancer and/or antibacterial activity. The compounds described herein may also, in certain embodiments, act as act as electrophilic azo-coupling agents in vitro or in vivo.
A. Isoprekinamycins When IPK was first isolated by Steven Gould and co-workers in 1989, it was assigned an incorrect structure and given the name prekinamycin. Seaton and Gould, 1989. The present inventors later elucidated the correct structure and also revised the name to isoprekinamcyin. Proteau et a!., 2000. A metabolite in S. lmtravamaensis that is believed to be a biosynthetic precursor of the kinamycins is now called prekinamycin, and is an isomer of isoprekinamycin. Hauser and Zhou, 1996; Gould et al., 1996; Birman et .//., 2006.
In 2002, Dmitrienko and co-workers prepared a derivative of IPK, 4, using the synthetic methodology shown in Scheme 1 below (Laufer and Dmitrienko, 2002) (incorporated herein by reference in its entirety):
Figure imgf000027_0001
Figure imgf000028_0001
(The numbering of the compounds in this section does not necessarily correspond to the numbering in the Examples section below).
A subsequent attempt to apply the synthetic methodology outlined above to the preparation of IPK at the University of Waterloo failed since there was an unexpectedly strong tendency for a competing lactone forming process that prevented the formation of the five-membered B ring. This result is outlined in the scheme below and is described in more detail in the Ph.D. Thesis of Dr. Radoslaw S. Laufer at the University of Waterloo, 2002, incorporated herein by reference in its entirety. This was assumed to be a consequence of the steric hindrance suggested in Scheme 2 below:
Figure imgf000029_0001
A later study by Matthew Buck at the University of Waterloo, which is described in his M. Sc. Thesis (2003) (incorporated herein by reference in its entirety), showed that the problem of lactone formation (instead of Friedel-Crafts cyclization) unexpectedly persisted even if the steric hindrance suggested in the work by Laufer was eliminated (see Scheme 3). As a result, the approach to the diazobenzo[</]fluorene system originally examined by Laufer was abandoned as a possible route to IPK and useful analogues.
Figure imgf000029_0002
These negative results prompted the present inventors to pursue an alternate synthetic route towards compounds of the present invention. One route that was pursued is shown in Scheme 4:
Figure imgf000030_0001
However, attempts to apply the synthetic method which yielded compound 16 as indicated in Scheme 4 (and described in more detail in the Examples below) to the preparation of IPK also failed and prompted a major modification in the synthetic route. This route is shown below in Scheme 5, shown below and described in the Examples below. See also Liu et a!., 2007, incorporated herein by reference in its entirety.
Figure imgf000031_0001
The methods of the present invention provide access to IPK and are generally applicable to the synthesis of a variety of IPK derivatives. B. Chemical Definitions
As used herein, the term "amino" means -NHT; the term "nitro" means -NCK the term "halo" designates -F, -Cl, -Br or -I and in particular embodiments, halo is
F while in other particular embodiments, halo is Br; the term "mercapto" means -SH; the term "cyano" means -CN; the term "azido" means -N3; the term "silyl" means
-SiH,, and the term "hydroxy" means -OH.
The term "alkyl" includes straight-chain alkyl, branched-chain alkyl, cycloalkyl (alicyclic), cyclic alkyl, heteroatom-unsubstituted alkyl, heteroatom- substituted alkyl, heteroatom-unsubstituted Cn-alkyl, and heteroatom-substituted Cn-alkyl. In certain embodiments, "lower alkyls" are contemplated. The term "lower alkyl" refers to alkyls of 1, 2, 3, 4, 5, or 6 carbon atoms. Indeed, with any use of the word "alkyl" or "alk" (such as "aralkyl" or "alkoxy") as used herein, "lower alkyl" groups are also contemplated (e.g., "lower aralkyl"). The term "heteroatom- unsubstituted Cn-alkyl" refers to a radical, having a linear or branched, cyclic or acyclic structure, further having no carbon-carbon double or triple bonds, further having a total of n carbon atoms, all of which are nonaromatic, 3 or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted Ci-Cio-alkyl has 1 to 10 carbon atoms. The groups, -CH, (Me), -CH2CH, (Et), -CH2CH2CH, (w-Pr), -CH(CH,)2 (/.W-Pr), -CH(CH2)2 (cyclopropyl), -CH2CH2CH2CH3 («-Bu), -CH(CH3)CH2CH, (Λ?c-butyl), -CH2CH(CH,)2 (/.w-butyl), -C(CH,), (tert-butyi), -CH2C(CH,), («eO-pentyl), cyclobutyl, cyclopentyl, and cyclohexyl, are all non- limiting examples of heteroatom-unsubstituted alkyl groups. The term "heteroatom- substituted Cn-alkyl" refers to a radical, having a single saturated carbon atom as the point of attachment, no carbon-carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, all of which are nonaromatic, 0, 1, or more than one hydrogen atom, at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted Ci-Cio-alkyl has 1 to 10 carbon atoms. The following groups are all non-limiting examples of heteroatom-substituted alkyl groups: trifluoromethyl or other perfluoromethyl groups, -CH2F, -CH2Cl, -CH2Br, -CH2OH, -CH2OCH,, -CH2OCH2CF,, -CH2OC(O)CH,, aminoalkyl groups such as -CH2NH2, -CH2NHCH,, -CH2N(CH, )2, and more generally -CH2NR1R2, wherein R1 and R2 are each independently alkyl, aryl, or aralkyl, -CH2CH2Cl, -CH2CH2OH, CH2CH2OC(O)CH3, -CH2CH2NHCO2C(CH3 )3, -CH2Si(CH3 )3, and -(CH2Jn-Y wherein Y is -CO2H, -SO3H, -CONH2, -SONH2, 1-tetrazolyl, 1-imidizolyl, - N+(RiR2R3) where R1, R2, R3 are each independently alkyl, aryl, or arylalkyl.
The term "alkenyl" includes straight-chain alkenyl, branched-chain alkenyl, cycloalkenyl, cyclic alkenyl, heteroatom-unsubstituted alkenyl, heteroatom- sυbstitυted alkenyl, heteroatom-υnsυbstitυted Cn-alkenyl, and heteroatom-substituted Cn-alkenyl. The term "heteroatom-unsubstituted Cn-alkenyl" refers to a radical, having a linear or branched, cyclic or acyclic stiiicture, further having at least one nonaromatic carbon-carbon double bond, but no carbon-carbon triple bonds, a total of n carbon atoms, three or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted C2-Cio-alkenyl has 2 to 10 carbon atoms. Heteroatom- unsubstituted alkenyl groups include: -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CH-C6H5. The term "heteroatom-substituted Cn-alkenyl" refers to a radical, having a single nonaromatic carbon atom as the point of attachment and at least one nonaromatic carbon-carbon double bond, but no carbon-carbon triple bonds, further having a linear or branched, cyclic or acyclic stiiicture, further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted C2-Cio-alkenyl has 2 to 10 carbon atoms. The groups, -CH=CHF, -CH=CHCl and -CH=CHBr, are non-limiting examples of heteroatom-substituted alkenyl groups.
The term "alkynyl" includes straight-chain alkynyl, branched-chain alkynyl, cycloalkynyl, cyclic alkynyl, heteroatom-unsubstituted alkynyl, heteroatom- substituted alkynyl, heteroatom-unsubstituted Cn-alkynyl, and heteroatom-substituted Cn-alkynyl. The term "heteroatom-unsubstituted Cn-alkynyl" refers to a radical, having a linear or branched, cyclic or acyclic stiiicture, further having at least one carbon-carbon triple bond, a total of n carbon atoms, at least one hydrogen atom, and no heteroatoms. For example, a heteroatom-unsubstituted C2-Cio-alkynyl has 2 to 10 carbon atoms. The groups, -C≡CH, -C=CCH3, and -C=CC6H5 are non-limiting examples of heteroatom-unsubstituted alkynyl groups. The term "heteroatom- substituted Cn-alkynyl" refers to a radical, having a single nonaromatic carbon atom as the point of attachment and at least one carbon-carbon triple bond, further having a linear or branched, cyclic or acyclic stiiicture, and having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one heteroatom, wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted C2-Cio-alkynyl has 2 to 10 carbon atoms. The group, -C=CSi(CH3 )3, is a non-limiting example of a heteroatom- substituted alkynyl group.
The term "aryl" includes heteroatom-unsubstituted aryl, heteroatom- substituted aryl, heteroatom-unsubstituted Cn-aryl, heteroatom-substituted Cn-aryl, heteroaryl, heterocyclic aiyl groups, carbocyclic aiyl groups, biaryl groups, and radicals derived from polycyclic fused hydrocarbons (PAHs). The term "heteroatom- unsubstituted Cn-aryl" refers to a radical, having a single carbon atom as a point of attachment, wherein the carbon atom is part of an aromatic ring structure containing only carbon atoms, further having a total of n carbon atoms, 5 or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted Cf1-C lo-aryl has 6 to 10 carbon atoms. Non-limiting examples of heteroatom-unsubstituted aiyl groups include phenyl (Ph), methylphenyl, ( dimethyl )phenyl, -C6H4CH2CH3, -C6H4CH2CH2CH3, -C6H4CH(CH3 )2, -C6H4CH(CH2 )2, -C6H3(CH3)CH2CH3, -C6H4CH=CH2, -C6H4CH=CHCH3, -C6H4C=CH, -C6H4C=CCH3, naphthyl, quinolyl, indolyl, and the radical derived from biphenyl. The term "heteroatom- substituted Cn-aryl" refers to a radical, having either a single aromatic carbon atom or a single aromatic heteroatom as the point of attachment, further having a total of n carbon atoms, at least one hydrogen atom, and at least one heteroatom, further wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-unsubstituted Ci-Cio-heteroaryl has 1 to 10 carbon atoms. Non-limiting examples of heteroatom-substituted aryl groups include the groups: -C6H4F, -C6H4Cl, -C6H4Br, -C6H4I, -C6H4OH, -C6H4OCH3, -C6H4OCH2CH3, -C6H4OC(O)CH3, -C6H4NH2, -C6H4NHCH3, -C6H4N(CH3 )2, -C6H4CH2OH, -C6H4CH2OC(O)CH3, -C6H4CH2NH2, -C6H4CF3, -C6H4CN, -C6H4CHO, -C6H4CHO, -C6H4C(O)CH3, -C6H4C(O)C6H5, -C6H4CO2H, -C6H4CO2CH3, -C6H4CONH2, -C6H4CONHCH3, -C6H4CON(CH3 )2, furanyl, thienyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, and imidazoyl.
The term "aralkyl" includes heteroatom-unsubstituted aralkyl, heteroatom- substituted aralkyl, heteroatom-unsubstituted Cn-aralkyl, heteroatom-substituted Cn- aralkyl, heteroaralkyl, and heterocyclic aralkyl groups. The term "heteroatom- unsubstituted Cn-aralkyl" refers to a radical, having a single saturated carbon atom as the point of attachment, further having a total of n carbon atoms, wherein at least 6 of the carbon atoms form an aromatic ring structure containing only carbon atoms, 7 or more hydrogen atoms, and no heteroatoms. For example, a heteroatom-unsubstituted Cγ-Cio-aralkyl has 7 to 10 carbon atoms. Non-limiting examples of heteroatom- unsubstituted aralkyls are: phenylmethyl (benzyl, Bn) and phenylethyl. The term "heteroatom-substituted Cn-aralkyl" refers to a radical, having a single saturated carbon atom as the point of attachment, further having a total of n carbon atoms, 0, 1 , or more than one hydrogen atom, and at least one heteroatom, wherein at least one of the carbon atoms is incorporated an aromatic ring stiiictures, further wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted C2-Cio-heteroaralkyl has 2 to 10 carbon atoms.
The term "acyl" includes straight-chain acyl, branched-chain acyl, cycloacyl, cyclic acyl, heteroatom-unsubstituted acyl, heteroatom-substituted acyl, heteroatom- unsubstituted Cn-acyl, heteroatom-substituted Cn-acyl, alkylcarbonyl, alkoxycarbonyl and aminocarbonyl groups. The term "heteroatom-unsubstituted Cn-acyl" refers to a radical, having a single carbon atom of a carbonyl group as the point of attachment, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 1 or more hydrogen atoms, a total of one oxygen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted Ci-Cio-acyl has 1 to 10 carbon atoms. The groups, -CHO, -C(O)CH3, -C(O)CH2CH3, -C(O)CH2CH2CH3, -C(O)CH(CH3)2, -C(O)CH(CH2)2, -C(O)C6H5, -C(O)C6H4CH3, -C(O)C6H4CH2CH3, and -COC6H3(CH3 )2, are non-limiting examples of heteroatom- unsubstituted acyl groups. The term "heteroatom-substituted Cn-acyl" refers to a radical, having a single carbon atom as the point of attachment, the carbon atom being part of a carbonyl group, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 0, 1 , or more than one hydrogen atom, at least one additional heteroatom, in addition to the oxygen of the carbonyl group, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted Ci-Cio-acyl has 1 to 10 carbon atoms.. The groups, -C(O)CH2CF3, -CO2H, -CO2CH3, -CO2CH2CH3, -CO2CH2CH2CH3, -CO2CH(CH3 )2, -CO2CH(CH2 )2, -C(O)NH2 (carbamoyl), -C(O)NHCH3, -C(O)NHCH2CH3, -CONHCH(CH3 )2, -CONHCH(CH2 )2, -CON(CHj)2, and -CONHCH2CF,, are non-limiting examples of heteroatom-substituted acyl groups.
The term "alkoxy" includes straight-chain alkoxy, branched-chain alkoxy, cycloalkoxy, cyclic alkoxy, heteroatom-unsubstituted alkoxy, heteroatom-substituted alkoxy, heteroatom-unsubstituted Cn-alkoxy, and heteroatom-substituted Cn-alkoxy. The term "heteroatom-unsubstituted Cn-alkoxy" refers to a group, having the structure -OR, in which R is a heteroatom-unsubstituted Cn-alkyl, as that term is defined above. Heteroatom-unsubstituted alkoxy groups include: -OCH3, -OCH2CH;*, -OCH2CH2CH3, -OCH(CH3 )2, and -OCH(CH2 )2. The term "heteroatom-substituted Cn-alkoxy" refers to a group, having the structure -OR, in which R is a heteroatom- substituted Cn-alkyl, as that term is defined above. For example, -OCH2CF3 is a heteroatom-substituted alkoxy group.
The term "alkenyloxy" includes straight-chain alkenyloxy, branched-chain alkenyloxy, cycloalkenyloxy, cyclic alkenyloxy, heteroatom-unsubstituted alkenyloxy, heteroatom-substituted alkenyloxy, heteroatom-unsubstituted Cn- alkenyloxy, and heteroatom-substituted Cn-alkenyloxy. The term "heteroatom- unsubstituted Cn-alkenyloxy" refers to a group, having the structure -OR, in which R is a heteroatom-unsubstituted Cn-alkenyl, as that term is defined above. The term "heteroatom-substituted Cn-alkenyloxy" refers to a group, having the structure -OR, in which R is a heteroatom-substituted Cn-alkenyl, as that term is defined above.
The term "alkynyloxy" includes straight-chain alkynyloxy, branched-chain alkynyloxy, cycloalkynyloxy, cyclic alkynyloxy, heteroatom-unsubstituted alkynyloxy, heteroatom-substituted alkynyloxy, heteroatom-unsubstituted Cn- alkynyloxy, and heteroatom-substituted Cn-alkynyloxy. The term "heteroatom- unsubstituted Cn-alkynyloxy" refers to a group, having the structure -OR, in which R is a heteroatom-unsubstituted Cn-alkynyl, as that term is defined above. The term "heteroatom-substituted Cn-alkynyloxy" refers to a group, having the structure -OR, in which R is a heteroatom-substituted Cn-alkynyl, as that term is defined above.
The term "aryloxy" includes heteroatom-unsubstituted aryloxy, heteroatom- substituted aryloxy, heteroatom-unsubstituted Cn-aryloxy, heteroatom-substituted Cn- aryloxy, heteroaryloxy, and heterocyclic aryloxy groups. The term "heteroatom- unsubstituted Cn-aryloxy" refers to a group, having the structure -OAr, in which Ar is a heteroatom-unsubstituted Cn-aryl, as that term is defined above. A non-limiting example of a heteroatom-unsubstituted aryloxy group is -OCf1H5. The term "heteroatom-substituted Cn-aiyloxy" refers to a group, having the structure -OAr, in which Ar is a heteroatom-substituted Cn-aryl, as that term is defined above.
The term "aralkyloxy" includes heteroatom-unsubstituted aralkyloxy, heteroatom-substituted aralkyloxy, heteroatom-unsubstituted Cn-aralkyloxy, heteroatom-substituted Cn-aralkyloxy, heteroaralkyloxy, and heterocyclic aralkyloxy groups. The term "heteroatom-unsubstituted Cn-aralkyloxy" refers to a group, having the structure -OAr, in which Ar is a heteroatom-unsubstituted Cn-aralkyl, as that term is defined above. The term "heteroatom-substituted Cn-aralkyloxy" refers to a group, having the structure -OAr, in which Ar is a heteroatom-substituted Cn-aralkyl, as that term is defined above.
The term "acyloxy" includes straight-chain acyloxy, branched-chain acyloxy, cycloacyloxy, cyclic acyloxy, heteroatom-unsubstituted acyloxy, heteroatom- substituted acyloxy, heteroatom-unsubstituted Cn-acyloxy, heteroatom-substituted Cn- acyloxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylate groups. The term "heteroatom-unsubstituted Cn-acyloxy" refers to a group, having the stiiicture -OAc, in which Ac is a heteroatom-unsubstituted Cn-acyl, as that term is defined above. For example, -OC(O)CH? is a non-limiting example of a heteroatom-unsubstituted acyloxy group. The term "heteroatom-substituted Cn-acyloxy" refers to a group, having the structure -OAc, in which Ac is a heteroatom-substituted Cn-acyl, as that term is defined above. For example, -OC(O)OCH? and -OC(O)NHCH? are non-limiting examples of heteroatom-unsubstituted acyloxy groups.
The term "alkylamino" includes straight-chain alkylamino, branched-chain alkylamino, cycloalkylamino, cyclic alkylamino, heteroatom-unsubstituted alkylamino, heteroatom-substituted alkylamino, heteroatom-unsubstituted Cn-alkylamino, and heteroatom-substituted Cn-alkylamino. The term "heteroatom- unsubstituted Cn-alkylamino" refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two saturated carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, containing a total of n carbon atoms, all of which are nonaromatic, 4 or more hydrogen atoms, a total of 1 nitrogen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted Ci-Cio-alkylamino has 1 to 10 carbon atoms. The term "heteroatom-unsubstituted Cn-alkylamino" includes groups, having the structure -NHR, in which R is a heteroatom-unsubstituted Cn-alkyl, as that term is defined above. A heteroatom-unsubstituted alkylamino group would include -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -NHCH(CH3 )2, -NHCH(CH2 )2, -NHCH2CH2CH2CH3, -NHCH(CH3)CH2CH3, -NHCH2CH(CH3 )2, -NHC(CH3 )3, -N(CH3 )2, -N(CH3)CH2CH3, -N(CH2CH3 )2, JV-pyrrolidinyl, and JV-piperidinyl. The term "heteroatom-substituted Cn-alkylamino" refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two saturated carbon atoms attached to the nitrogen atom, no carbon-carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, all of which are nonaromatic, 0, 1 , or more than one hydrogen atom, and at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted Ci-Cio-alkylamino has 1 to 10 carbon atoms. The term "heteroatom-substituted Cn-alkylamino" includes groups, having the stiiicture -NHR, in which R is a heteroatom-substituted Cn-alkyl, as that term is defined above.
The term "alkenylamino" includes straight-chain alkenylamino, branched- chain alkenylamino, cycloalkenylamino, cyclic alkenylamino, heteroatom- unsubstituted alkenylamino, heteroatom-substituted alkenylamino, heteroatom- unsubstituted Cn-alkenylamino, heteroatom-substituted Cn-alkenylamino, dialkenylamino, and alkyl(alkenyl)amino groups. The term "heteroatom- unsubstituted Cn-alkenylamino" refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, containing at least one nonaromatic carbon-carbon double bond, a total of n carbon atoms, 4 or more hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted C2-Cio-alkenylamino has 2 to 10 carbon atoms. The term "heteroatom-unsubstituted Cn-alkenylamino" includes groups, having the stiiicture -NHR, in which R is a heteroatom-unsubstituted Cn- alkenyl, as that term is defined above. The term "heteroatom-substituted Cn- alkenylamino" refers to a radical, having a single nitrogen atom as the point of attachment and at least one nonaromatic carbon-carbon double bond, but no carbon- carbon triple bonds, further having one or two carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom- sυbstitυted C2-Cio-alkenylamino has 2 to 10 carbon atoms. The term "heteroatom- substituted Cn-alkenylamino" includes groups, having the stiiicture -NHR, in which R is a heteroatom-substituted Cn-alkenyl, as that term is defined above.
The term "alkynylamino" includes straight-chain alkynylamino, branched- chain alkynylamino, cycloalkynylamino, cyclic alkynylamino, heteroatom- unsubstituted alkynylamino, heteroatom-substituted alkynylamino, heteroatom- unsubstituted Cn-alkynylamino, heteroatom-substituted Cn-alkynylamino, dialkynylamino, alkyl(alkynyl)amino, and alkenyl(alkynyl)amino groups. The term "heteroatom-unsubstituted Cn-alkynylamino" refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, containing at least one carbon-carbon triple bond, a total of n carbon atoms, at least one hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted C^-Cio-alkynylamino has 2 to 10 carbon atoms. The term "heteroatom-unsubstituted Cn-alkynylamino" includes groups, having the stiiicture -NHR, in which R is a heteroatom-unsubstituted Cn- alkynyl, as that term is defined above. The term "heteroatom-substituted Cn- alkynylamino" refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two carbon atoms attached to the nitrogen atom, further having at least one nonaromatic carbon-carbon triple bond, further having a linear or branched, cyclic or acyclic stiiicture, and further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, and at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted C2-Cio-alkynylamino has 2 to 10 carbon atoms. The term "heteroatom-substituted Cn-alkynylamino" includes groups, having the stiiicture -NHR, in which R is a heteroatom-substituted Cn-alkynyl, as that term is defined above.
The term "arylamino" includes heteroatom-unsubstituted arylamino, heteroatom-substituted arylamino, heteroatom-unsubstituted Cn-arylamino, heteroatom-substituted Cn-arylamino, heteroarylamino, heterocyclic arylamino, and alkyl(aryl)amino groups. The term "heteroatom-unsubstituted Cn-aiylamino" refers to a radical, having a single nitrogen atom as the point of attachment, further having at least one aromatic ring structure attached to the nitrogen atom, wherein the aromatic ring structure contains only carbon atoms, further having a total of n carbon atoms, 6 or more hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted Cf1-C io-aiylamino has 6 to 10 carbon atoms. The term "heteroatom-unsubstituted Cn-arylamino" includes groups, having the structure -NHR, in which R is a heteroatom-unsubstituted Cn-aryl, as that term is defined above. The term "heteroatom-substituted Cn-arylamino" refers to a radical, having a single nitrogen atom as the point of attachment, further having a total of n carbon atoms, at least one hydrogen atom, at least one additional heteroatoms, that is, in addition to the nitrogen atom at the point of attachment, wherein at least one of the carbon atoms is incorporated into one or more aromatic ring structures, further wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted Cf-Cio-arylamino has 6 to 10 carbon atoms. The term "heteroatom-substituted Cn- arylamino" includes groups, having the structure -NHR, in which R is a heteroatom- substituted Cn-aryl, as that term is defined above.
The term "aralkylamino" includes heteroatom-unsubstituted aralkylamino, heteroatom-substituted aralkylamino, heteroatom-unsubstituted Cn-aralkylamino, heteroatom-substituted Cn-aralkylamino, heteroaralkylamino, heterocyclic aralkylamino groups, and diaralkylamino groups. The term "heteroatom- unsubstituted Cn-aralkylamino" refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two saturated carbon atoms attached to the nitrogen atom, further having a total of n carbon atoms, wherein at least 6 of the carbon atoms form an aromatic ring structure containing only carbon atoms, 8 or more hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms. For example, a heteroatom-unsubstituted Cγ-Cio-aralkylamino has 7 to 10 carbon atoms. The term "heteroatom-unsubstituted Cn-aralkylamino" includes groups, having the structure -NHR, in which R is a heteroatom-unsubstituted Cn-aralkyl, as that term is defined above. The term "heteroatom-substituted Cn-aralkylamino" refers to a radical, having a single nitrogen atom as the point of attachment, further having at least one or two saturated carbon atoms attached to the nitrogen atom, further having a total of n carbon atoms, 0, 1, or more than one hydrogen atom, at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein at least one of the carbon atom incorporated into an aromatic ring, further wherein each heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted Cγ-Cio-aralkylamino has 7 to 10 carbon atoms. The term "heteroatom-substituted Cn-aralkylamino" includes groups, having the structure -NHR, in which R is a heteroatom-substituted Cn-aralkyl, as that term is defined above.
The term "amido" includes straight-chain amido, branched-chain amido, cycloamido, cyclic amido, heteroatom-unsubstituted amido, heteroatom-substituted amido, heteroatom-unsubstituted Cn-amido, heteroatom-substituted Cn-amido, alkylcarbonylamino, arylcarbonylamino, alkoxycarbonylamino, aryloxycarbonylamino, acylamino, alkylaminocarbonylamino, arylaminocarbonylamino, and ureido groups. The term "heteroatom-unsubstituted Cn- amido" refers to a radical, having a single nitrogen atom as the point of attachment, further having a carbonyl group attached via its carbon atom to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 1 or more hydrogen atoms, a total of one oxygen atom, a total of one nitrogen atom, and no additional heteroatoms. For example, a heteroatom- unsubstituted Ci-Cio-amido has 1 to 10 carbon atoms. The term "heteroatom- unsubstituted Cn-amido" includes groups, having the stiiicture -NHR, in which R is a heteroatom-unsubstituted Cn-acyl, as that term is defined above. The group, -NHC(O)CH?, is a non-limiting example of a heteroatom-unsubstituted amido group. The term "heteroatom-substituted Cn-amido" refers to a radical, having a single nitrogen atom as the point of attachment, further having a carbonyl group attached via its carbon atom to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, further having a total of n aromatic or nonaromatic carbon atoms, 0, 1, or more than one hydrogen atom, at least one additional heteroatom in addition to the oxygen of the carbonyl group, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substituted Ci-Cio-amido has 1 to 10 carbon atoms. The term "heteroatom-substituted Cn-amido" includes groups, having the stiiicture -NHR, in which R is a heteroatom-unsubstituted Cn-acyl, as that term is defined above. The group, -NHCO2CH3, is a non-limiting example of a heteroatom-substituted amido group. The term "alkylthio" includes straight-chain alkylthio, branched-chain alkylthio, cycloalkylthio, cyclic alkylthio, heteroatom-υnsυbstitυted alkylthio, heteroatom-substituted alkylthio, heteroatom-υnsυbstitυted Cn-alkylthio, and heteroatom-substituted Cn-alkylthio. The term "heteroatom-unsubstituted Cn- alkylthio" refers to a group, having the structure -SR, in which R is a heteroatom- unsubstituted Cn-alkyl, as that term is defined above. The group, -SCH^, is an example of a heteroatom-unsubstituted alkylthio group. The term "heteroatom- substituted Cn-alkylthio" refers to a group, having the stiiicture -SR, in which R is a heteroatom-substituted Cn-alkyl, as that term is defined above. The term "alkenylthio" includes straight-chain alkenylthio, branched-chain alkenylthio, cycloalkenylthio, cyclic alkenylthio, heteroatom-unsubstituted alkenylthio, heteroatom-substituted alkenylthio, heteroatom-unsubstituted Cn- alkenylthio, and heteroatom-substituted Cn-alkenylthio. The term "heteroatom- unsubstituted Cn-alkenylthio" refers to a group, having the structure -SR, in which R is a heteroatom-unsubstituted Cn-alkenyl, as that term is defined above. The term "heteroatom-substituted Cn-alkenylthio" refers to a group, having the structure -SR, in which R is a heteroatom-substituted Cn-alkenyl, as that term is defined above.
The term "alkynylthio" includes straight-chain alkynylthio, branched-chain alkynylthio, cycloalkynylthio, cyclic alkynylthio, heteroatom-unsubstituted alkynylthio, heteroatom-substituted alkynylthio, heteroatom-unsubstituted Cn- alkynylthio, and heteroatom-substituted Cn-alkynylthio. The term "heteroatom- unsubstituted Cn-alkynylthio" refers to a group, having the structure -SR, in which R is a heteroatom-unsubstituted Cn-alkynyl, as that term is defined above. The term "heteroatom-substituted Cn-alkynylthio" refers to a group, having the stiiicture -SR, in which R is a heteroatom-substituted Cn-alkynyl, as that term is defined above.
The term "arylthio" includes heteroatom-unsubstituted arylthio, heteroatom- substituted arylthio, heteroatom-unsubstituted Cn-arylthio, heteroatom-substituted Cn- arylthio, heteroarylthio, and heterocyclic arylthio groups. The term "heteroatom- unsubstituted Cn-arylthio" refers to a group, having the structure -SAr, in which Ar is a heteroatom-unsubstituted Cn-aryl, as that term is defined above. The group, -SCr1Hf, is an example of a heteroatom-unsubstituted arylthio group. The term "heteroatom-substituted Cn-arylthio" refers to a group, having the stiiicture -SAr, in which Ar is a heteroatom-substituted Cn-aryl, as that term is defined above. The term "aralkylthio" includes heteroatom-unsubstituted aralkylthio, heteroatom-substituted aralkylthio, heteroatom-unsubstituted Cn-aralkylthio, heteroatom-substituted Cn-aralkylthio, heteroaralkylthio, and heterocyclic aralkylthio groups. The term "heteroatom-unsubstituted Cn-aralkylthio" refers to a group, having the structure -SAr, in which Ar is a heteroatom-unsubstituted Cn-aralkyl, as that term is defined above. The group, -SCHoCf1H5, is an example of a heteroatom- unsubstituted aralkyl group. The term "heteroatom-substituted Cn-aralkylthio" refers to a group, having the structure -SAr, in which Ar is a heteroatom-substituted Cn- aralkyl, as that term is defined above. The term "acylthio" includes straight-chain acylthio, branched-chain acylthio, cycloacylthio, cyclic acylthio, heteroatom-unsubstituted acylthio, heteroatom- substituted acylthio, heteroatom-unsubstituted Cn-acylthio, heteroatom-substituted Cn- acylthio, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylate groups. The term "heteroatom-unsubstituted Cn-acylthio" refers to a group, having the structure -SAc, in which Ac is a heteroatom-unsubstituted Cn-acyl, as that term is defined above. The group, -SCOCH?, is an example of a heteroatom-unsubstituted acylthio group. The term "heteroatom-substituted Cn-acylthio" refers to a group, having the structure -SAc, in which Ac is a heteroatom-substituted Cn-acyl, as that term is defined above. In certain embodiments, a substituted substituent, as described above, may be employed. In certain embodiments, an unsubstituted substituent, as described above, may be employed.
The claimed invention is also intended to encompass salts of any of the compounds of the present invention. The term "salt(s)" as used herein, is understood as being acidic and/or basic salts formed with inorganic and/or organic acids and bases. Zwitterions (internal or inner salts) are understood as being included within the term "salt(s)" as used herein, as are quaternary ammonium salts such as alkylammonium salts. Nontoxic, pharmaceutically acceptable salts are preferred as described below, although other salts may be useful, as for example in isolation or purification steps.
The term "pharmaceutically acceptable salts," as used herein, refers to salts of compounds of this invention that are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of a compound of this invention with an inorganic or organic acid, or an organic base, depending on the sυbstitυents present on the compounds of the invention.
Non-limiting examples of inorganic acids which may be used to prepare pharmaceutically acceptable salts include: hydrochloric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid and the like. Examples of organic acids which may be used to prepare pharmaceutically acceptable salts include: aliphatic mono- and dicarboxylic acids, such as oxalic acid, carbonic acid, citric acid, succinic acid, phenyl-heteroatom-substituted alkanoic acids, aliphatic and aromatic sulfuric acids and the like. Pharmaceutically acceptable salts prepared from inorganic or organic acids thus include hydrochloride, hydrobromide, nitrate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydroiodide, hydrofluoride, acetate, propionate, formate, oxalate, citrate, lactate, p-toluenesulfonate, methanesulfonate, maleate, and the like. Suitable pharmaceutically acceptable salts may also be formed by reacting the agents of the invention with an organic base such as methylamine, ethylamine, ethanolamine, lysine, ornithine and the like.
Pharmaceutically acceptable salts include the salts formed between carboxylate or sulfonate groups found on some of the compounds of this invention and inorganic cations, such as sodium, potassium, ammonium, or calcium, or such organic cations as isopropylammonium, trimethylammonium, tetramethylammonium, and imidazolium.
It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Stahl and. Wermuth,
2002, which is incorporated herein by reference.
As used herein, the term "cyclic group" refers to a carbocycle group (e.g., cyclohexyl), a heterocycle group (e.g., pyrrolidinyl), an aryl group, or any combination thereof.
As used herein, the term "nucleophile" or "nucleophilic" generally refers to atoms bearing lone pairs of electrons. Such terms are well known in the art and include -NHT, thiolate, carbanion and hydroxyl. As used herein, the term "leaving group" generally refers to a group readily displaceable by a nucleophile, such as an amine, an alcohol, or a thiol nucleophile.
Such leaving groups are well known and include carboxylates, N- hydroxysuccinimide, N-hydroxybenzotriazole, triflates, tosylates, mesylates, alkoxy, thioalkoxy and the like.
The term "functional group" generally refers to how persons of skill in the art classify chemically reactive groups. Examples of functional groups include hydroxyl, amine, sulfhydryl, amide, carboxyls, carbonyls, etc.
As used herein, "protecting group" refers to a moiety attached to a functional group to prevent an otherwise unwanted reaction of that functional group. Protecting groups are well-known to those of skill in the art. Non-limiting exemplary protecting groups fall into categories such as hydroxy protecting groups, amino protecting groups, sulfhydryl protecting groups and carbonyl protecting groups. Such protecting groups may be found in Greene and Wuts, 1999, which is incorporated herein by reference in its entirety.
Compounds of the present invention may contain one or more asymmetric centers and thus can occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In certain embodiments, a single diastereomer is present. All possible stereoisomers of the compounds of the present invention are contemplated as being within the scope of the present invention.
However, in certain aspects, particular diastereomers are contemplated. The chiral centers of the compounds of the present invention can have the S- or the R- configuration, as defined by the IUPAC 1974 Recommendations. The present invention is meant to comprehend all such isomeric forms of the compounds of the invention.
Modifications or derivatives of the compounds disclosed throughout this specification are contemplated as being useful with the methods and compositions of the present invention. Derivatives may be prepared and the properties of such derivatives may be assayed for their desired properties by any method known to those of skill in the art.
In certain aspects, "derivative," such as an IPK derivative or a derivative of any of the compounds discussed herein (e.g., a derivative of a compound of formula (III)) refers to a chemically modified compound that still retains the desired effects of the compound prior to the chemical modification. Such derivatives may have the addition, removal, or substitution of one or more chemical moieties on the parent molecule. Non-limiting examples of the types modifications that can be made to the compounds and structures disclosed herein include the addition or removal of lower alkanes such as methyl, ethyl, propyl, or substituted lower alkanes such as hydroxymethyl or aminomethyl groups; carboxyl groups and carbonyl groups; hydroxyls; nitro, amino, amide, and azo groups; sulfate, sulfonate, sulfono, sulfhydryl, sulfonyl, sulfoxido, phosphate, phosphono, phosphoiyl groups, and halide substituents. Additional modifications can include an addition or a deletion of one or more atoms of the atomic framework, for example, substitution of an ethyl by a propyl; substitution of a phenyl by a larger or smaller aromatic group. Alternatively, in a cyclic or bicyclic structure, heteroatoms such as N, S, or O can be substituted into the structure instead of a carbon atom to generate, for example, a heterocycloalkyl structure.
Prodrugs and solvates of compounds of the present invention are also contemplated herein. The term "prodiiig" as used herein, is understood as being a compound which, upon administration to a subject, such as a mammal, undergoes chemical conversion by metabolic or chemical processes to yield a compound any of the formulas herein, or a salt and/or solvate thereof (Bundgaard, 1991 ; Bundgaard, 1985). Solvates of compounds of the present invention are preferably hydrates. For example, a benzo[</]fluorene of the present invention may be converted to a benzo[/?]fluorene in vivo or in vitro.
Solvent choices for the methods of the present invention will be known to one of ordinary skill in the art. Solvent choices may depend, for example, on which one(s) will facilitate the solubilizing of all the reagents or, for example, which one(s) will best facilitate the desired reaction (particularly when the mechanism of the reaction is known). Solvents may include, for example, polar solvents or non-polar solvents. Solvents choices include, but are not limited to, tetrahydrofuran, dimethylformamide, dimethylsulfoxide, dioxane, methanol, ethanol, hexane, methylene chloride and acetonitrile. In some preferred embodiments, the solvent is dimethylformamide. More than one solvent may be chosen for any particular reaction or purification procedure. Water may also be admixed into any solvent choice. Further, water, such as distilled water, may constitute the reaction medium instead of a solvent. Persons of ordinary skill in the art will be familiar with methods of purifying compounds of the present invention. One of ordinary skill in the art will understand that compounds of the present invention can generally be purified at any step, including the purification of intermediates as well as purification of the final products. In certain embodiments, purification is performed via silica gel column chromatography or HPLC.
In view of the above definitions, other chemical terms used throughout this application can be easily understood by those of skill in the art. Terms may be used alone or in any combination thereof. C. Pharmaceutical Preparations
Certain of the methods set forth herein pertain to methods involving the administration of a pharmaceutically and/or therapeutically effective amount of a compound of the present invention for chemotherapeutic and/or antibacterial purposes, or other purposes. For example, in certain embodiments, a compound of the present invention may be administered to kill tumor cells by any method that allows contact of the active ingredient with the agent's site of action in the tumor.
A compound of the present invention may be administered by any conventional methods available for use in conjunction with pharmaceuticals, either as an individual therapeutically active ingredient or in a combination of therapeutically active ingredients. A compound of the present invention may be administered alone, but will generally be administered with a pharmaceutically acceptable earner selected on the basis of the chosen route of administration and standard pharmaceutical practice.
A compound of the present invention may be extensively purified and/or dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle, where appropriate. Such methods are well-known in the art. The active compounds will then generally be formulated for administration by any known route, such as parenteral administration. Methods of administration are discussed in greater detail below. Moreover, it will be generally understood that a compound of the present invention can be provided in prodrug form, meaning that an environment to which a compound of the present invention is exposed alters the prodrug into an active, or more active, form. It is contemplated that the term "precursor" covers compounds that are considered "prodrugs." For example, the benzo[</]fluorenes described herein may be converted to benzo[/?]fluorenes in vivo, which then, in turn, exhibit biological and/or therapeutic effects.
1. Pharmaceutical Formulations and Routes for Administration to Subjects Pharmaceutical compositions of the present invention may comprise an effective amount of one or more candidate substances (e.g., a compound of the present invention) or additional agents dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one candidate substance or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drag stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, pp 1289-1329, 1990). Except insofar as any conventional earner is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
The candidate substance may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, locally, via inhalation (e.g., aerosol inhalation), via injection, via infusion, via continuous infusion, via localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 1990).
In particular embodiments, the composition is administered to a subject using a drug delivery device. Any drug delivery device is contemplated for use in delivering a pharmaceutically effective amount of a compound of the present invention.
The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
The dose can be repeated as needed as determined by those of ordinary skill in the art. Thus, in some embodiments of the methods set forth herein, a single dose is contemplated. In other embodiments, two or more doses are contemplated. Where more than one dose is administered to a subject, the time interval between doses can be any time interval as determined by those of ordinary skill in the art. For example, the time interval between doses may be about 1 hour to about 2 hours, about 2 hours to about 6 hours, about 6 hours to about 10 hours, about 10 hours to about 24 hours, about 1 day to about 2 days, about 1 week to about 2 weeks, or longer, or any time interval derivable within any of these recited ranges.
In certain embodiments, it may be desirable to provide a continuous supply of a pharmaceutical composition to the patient. This could be accomplished by catheterization, followed by continuous administration of the therapeutic agent. The administration could be intra-operative or post-operative.
In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of a compound of the present invention. In other embodiments, a compound of the present invention may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.
The candidate substance may be formulated into a composition in a free base, neutral, or salt form. Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine, or procaine. Other salts are described herein.
In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc. ), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. It may be preferable to include isotonic agents, such as, for example, sugars, sodium chloride, or combinations thereof.
In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants in the present invention. Such compositions are generally designed to be compatible with the target tissue type. In a non-limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in certain embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drags such as antibiotics or antihistamines.
In certain embodiments the candidate substance is prepared for administration by such routes as oral ingestion. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. In certain embodiments, carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof. In other aspects of the invention, the oral composition may be prepared as a syrup or elixir. A syrap or elixir, and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof. In certain embodiments an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, or combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.: or combinations thereof the foregoing. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, certain methods of preparation may include vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin, or combinations thereof. 2. Combination Therapy
In order to increase the effectiveness of a compound of the present invention, a compound of the present invention may be combined with traditional drugs. It is contemplated that this type of combination therapy may be used in vitro or in vivo. In a non-limiting example, an anticancer agent may be used in combination with a compound of the present invention. In another non-limiting example, an antibiotic may be used in combination with a compound of the present invention.
For example, a compound of the present invention may be provided in a combined amount with an effective amount of an anticancer agent to reduce or block DNA replication in cancerous cells (e.g., tissues, tumors). This process may involve administering the agents at the same time or within a period of time wherein separate administration of the substances produces a desired therapeutic benefit. This may be achieved by contacting the cell, tissue, or organism with a single composition or pharmacological formulation that includes two or more agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition includes one agent and the other includes another.
Compounds of the present invention may also be combined with one or more known antibacterial agents. Anti-bacterial classes and agents are well-known in the art, and include, for example, the classes of aminoglycoside antibiotics, cephalosporins, penicillins, quinolones, sulfonamides, tetracyclines, beta-lactams and macrolides. Non-limiting specific examples of antibacterial agents include linezolid, tigecycline, tetracycline, oxytetracycline, doxycycline, minocycline, vancomycin, enrofloxacin, erythromycin, tyrocidine, griseofulvin, streptomycin, polymyxin, cephalosporin, ampicillin, cephalothin, lincomycin, gentamicin, carbenicillin, cephalexin and clindamycin. These lists of antibiotics are not exhaustive and one skilled in the art can readily determine other antibiotics which may be employed.
The compounds of the present invention may precede, be co-current with and/or follow the other agents by intervals ranging from minutes to weeks. In embodiments where the agents are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agents would still be able to exert an advantageously combined effect on the cell, tissue or organism. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) as the candidate substance. In other aspects, one or more agents may be administered within of from substantially simultaneously, about 1 minute, 5 minutes, 10 minutes, 20 minutes 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 1, 2, 3, 4, 5, 6, 7 or 8 weeks or more, and any range derivable therein, prior to and/or after administering the candidate substance. Various combination regimens of the agents may be employed. Non-limiting examples of such combinations are shown below, wherein a compound of the present invention is "A" and a second agent, such as an anticancer agent or an antibiotic, is "B":
Figure imgf000054_0001
D. Examples
The following examples are included to demonstrate certain preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
1H NMR spectra were recorded on a Brϋker AVANCE500 (500 MHz), Brϋker AC300 (300 MHz) and Brϋker AVANCE300 (300 MHz) NMR spectrometer. Chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane (TMS). The following abbreviations are used for NMR peak multiplicities: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; dt, doublet of triplet; m, multiplet; b, broad; w, weak. L"C NMR spectra were broad band decoupled and recorded on a Bruker AVANCE500 ( 125.8 MHz), Biϊiker AC300 (75.5 MHz) and Bruker AVANCE300 (75.5 MHz) NMR spectrometer using the carbon signal of the deuterated solvent as the internal standard. HMQC and HMBC experiments were performed on a Bruker AVANCE500 spectrometer. IR spectra were determined on a Perkin-Elmer RX I FT-IR spectrometer as KBr discs unless otherwise indicated. High/low resolution electron impact (EI) mass spectra (MS) were measured by the WATSPEC Mass Spectrometry Facility (Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada) and the McMaster Regional Center for Mass Spectrometry (Department of Chemistry, McMaster University, Hamilton, Ontario, Canada). Gas chromatography-mass spectrometry (GC-MS) was performed on a HP GCD 1800 with a column (HP5) length of 30.0 cm and diameter of 0.25 mm. The following temperature program was applied: initial temperature 70 0C, rising rate 10 °C/minute, final temperature 265 0C held for 20.0 minutes. Elemental analyses were performed by the M-H-W Laboratories (Phoenix, Arizona, USA).
Anhydrous THF and EtoO were freshly distilled from sodium/benzophenone under nitrogen prior to use. Anhydrous CH2CI2 was freshly distilled from CaH2 under nitrogen prior to use. All commercial reagents were purchased from Aldrich Chemical Co., Strem Chemicals Inc., Alfa Aesar, Lancaster Synthesis Ltd. or BDH Inc. and were used as received unless otherwise indicated. Deionized water was supplied by a Biolab vertical series reverse osmosis system.
The -78 0C and 0 0C designations refer to dry ice/acetone and ice/water slush respectively. Flash column chromatography was carried out using the Merck silica gel (70-230 mesh) and SiliCycle silica gel (60 A). Reactions were magnetically stirred and monitored by thin layer chromatography (TLC) with Merck pre-coated silica gel plates (silica gel 60 F254011 aluminum sheet). All reported yields are isolated yields.
The following non-limiting examples pertain to compounds prepared as shown in Schemes 4 and 5, shown below. Scheme 4 depicts non-limiting examples of syntheses of the present invention. In particular, Scheme 1 shows a preparation of isoprekinamycin (IPK) derivative 16, accompanied by syntheses of various benzo[</]fluorenes as intermediates.
Figure imgf000056_0001
Scheme 5 represents another set of non-limiting synthetic examples of the present invention. Adaptation of the approach of Scheme 4 to the preparation of IPK is presented in Scheme 5, accompanied by the synthesis of various benzo[</]fluorenes as intermediates.
Figure imgf000057_0001
Figure imgf000058_0001
Synthesis of 4-Hydroxy-2, 3-dihydroindan-l-one (see Loudon and Razdan, 1954)
AlCh (58.19 g, 0.436 mol) and NaCl ( 12.03 g, 0.256 mol) were mixed and heated in an oil bath. When the bath temperature was about 150 0C, dihydrocoumarin
( 10 mL, 0.079 mol) was added slowly while the bath temperature was maintained between 150 0C and 180 0C. The bath temperature was then raised to 200 0C and the mixture was stirred for 1 hour. The mixture was cooled to room temperature and quenched with 100 g crushed ice and 50 ml cone. HCl at 0 0C. The suspension was stirred at room temperature for 30 minutes and the crude product (9.688 g) was obtained as a gray solid upon filtration.
Figure imgf000058_0002
Synthesis of 4-Methoxy-2,3-dihydroindan-l-one To a solution of the crude indanone (9.688 g, 0.065 mol) and K2CO3 (56.94 g,
0.41 1 mol) in acetone (667 mL) was added Me2SO4 (45.36 g, 0.360 mol) dropwise at room temperature and the reaction mixture was then refluxed for 2.5 hours. The solution was cooled to room temperature, filtrated and concentrated. The residue was mixed with water ( 100 mL) and triethylamine ( 100 ml), followed by stirring at room temperature for 1 hour. The resulting solution was extracted with EtOAc (3 x 200 mL) and the organic phase was dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (EtOAc:Hexane = 1 :9, v/v) to obtain the title compound as a white solid (8.317 g, 65%). 1H NMR (300 MHz, CDCh): δ 7.30-7.34 (m, 2 H), 6.96-7.01 (m, 1 H), 2.97-3.01 (m, 2 H), 2.62-2.66 (m, 2 H); 1T NMR (75.5 MHz, CDCh): δ 207.2, 156.9, 143.9, 138.5, 128.7, 1 15.1, 1 14.6, 55.3, 36.0. 22.4; MS (GC-MS): 13.34 minutes, m/z 162 (M+).
Figure imgf000059_0001
Synthesis of 7-Methoxy-3-oxo-2, S-dihydro-lH-indene-l-carboxylic acid
A modified literature procedure (Pang and Kozikowski, 1991 ) was used to prepare this compound. To a solution of 4-methoxy-l-indanone (2.000 g, 12.33 mmol) in anhydrous THF (25 mL) at -78 0C was added freshly prepared LDA in THF ( 12.95 mmol in 65 mL) dropwise, and the reaction mixture was stirred for 1 hour. TMSCl ( 1.64 mL, 12.95 mmol) was then added slowly at -78 0C and the reaction mixture was further stirred for 1 hour. The second batch of freshly prepared LDA in THF ( 18.50 mmol in 65 mL) was added at -78 0C and the reaction mixture was stirred for one more hour. Diy ice ( 100 g) was added and the reaction mixture was stirred at room temperature for 1.5 hours. The reaction was quenched with 2 M HCl in an ice bath and then extracted with ether (3 x 150 mL). The ether phase was concentrated and the residue was redissolved in enough 2 M aqueous NaOH solution until the pH was ca. 12. The basic aqueous solution was washed with ether (3 x 20 mL) and then acidified with cone. HCl until the pH reached ca. 1. The now acidic aqueous solution was then extracted with a mixture of ether and THF (9: 1, v/v, 4 x 100 mL). The resulting organic phase was dried over Na^SO4 and concentrated to give the crude product as a yellow solid (2.270 g).
Figure imgf000060_0001
Synthesis of Methyl 7-methoxy-3-oxo-2,3-tlihydro-lH-indene-l-carboxylate (9)
To a solution of the crude carboxylic acid (2.270 g) in methanol (50 mL) was added cone. Η2SO4 ( 10 drops) and the reaction mixture was refluxed for 48 hours. The solution was concentrated to ca. 5 mL, followed by addition of saturated aqueous NaHCCh solution to adjust the pH to ca. 8. The solution was extracted with EtOAc (3 x 80 mL) and the organic phase was dried over NaTSO4 and concentrated. The residue was purified by flash chromatography (EtOAc:Hexane = 1 :4, v/v) to obtain the ester as a yellow solid ( 1.845 g, 68% for two steps). 1H NMR (300 MHz, CDCh): δ 7.40 (t, 7.7 Hz, 1 H), 7.33 (d, 8.1 Hz, 1 H), 7.04 (d, 7.8 Hz, 1 H), 4.19 (dd, 3.5 Hz, 8.2 Hz, 1 H), 3.85 (s, 3 H), 3.69 (s, 3 H), 2.95 (dd, 8.2 Hz, 1 1.7 Hz, 1 H), 2.73 (dd, 3.5 Hz, 1 1.7 Hz,l H); 13C NMR (75.5 MHz, CDCh): δ 204.0, 173.4, 157.0, 140.5, 138.4, 130.6, 1 15.8, 1 15.5, 55.8, 52.4, 41.1, 41.0; Elemental analysis: calculated for CnH12O4: C, 65.45, H, 5.49; found: C, 65.22, H, 5.49.
Figure imgf000060_0002
Synthesis of Methyl 2-bromo-7-methoxy-3-oxo-3H-indene-l-carboxylate (10)
To a solution of compound 9 (2.330 g, 10.59 mmol) in freshly distilled CH2Ch (46 mL) was added Br2 in CH2Cl2 ( 1 M, 26.5 mL) at room temperature and the solution was stirred for 24 hours. The solution was diluted with CH2Cl2 (300 mL) and washed with water (3 x 25 mL). The CH2Cl2 phase was dried over Na2SO4 and concentrated. The ciiide product was obtained as a red solid. To a solution of the above ciiide product in CH2CI2 ( 100 mL) at 0 0C was added DBU ( 1.59 mL, 10.59 mmol) slowly and the reaction mixture was stirred at this temperature for 1 hour. The reaction solution was washed with water (3 x 15 mL), dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (EtOAc:Hexane = 1 :5, v/v) to obtain the title compound as a red solid (2.365 g, 75% for two steps). 1H NMR (300 MHz, CDCh): δ 7.28 (t, 4.5 Hz, 1 H), 7.18 (d, 7.2 Hz, 1 H), 7.01 (d, 8.4 Hz, 1 H), 3.98 (s, 3 H), 3.85(s, 3 H); 1T NMR (75.5 MHz, CDCh): δ 188.9, 164.9, 152.1, 148.4, 131.7, 129.7, 128.0, 119.6, 1 17.8, 1 16.8, 56.4, 52.9; IR (KBr): 1737, 1723 cm"1; MS (GC-MS): 18.52 minutes, m/z 296 (M+ containing 79Br) 298 (M+ containing MBr) ( 1 : 1 ); Elemental analysis: calculated for Ci2H9BrO4: C, 48.51, H, 3.05; found: C, 48.70, H, 3.02; HRMS: calculated for C12H9 79BrO4: 295.9684, found: 295.9689.
EXAMPLE 6
Figure imgf000061_0001
Synthesis of Methyl 2-(2-(cyanomethyl)phenyl)-7-methoxy-3-oxo-3H-indene-l- carboxylate (ild) (see Littke et «/., 2000 and Netherton and Fu, 2001)
A mixture of compound 10 (273 mg, 0.916 mmol), KF ( 160 mg, 2.49 mmol), Pd2(dbab'CHCh (51 mg, 0.049 mmol) and [(t-BubPH]BF4 (28 mg, 0.099 mmol) was deoxygenated five times with an argon balloon and a vacuum pump. Pinacolboronate (200 mg, 0.824 mmol) was dissolved in a mixture of THF and water ( 19:1, v/v, 8 mL) and the solution was deoxygenated three times by the thaw-freeze process. The deoxygenated solution was then added to the solid mixture and stirred for 24 hours at room temperature under argon atmosphere. The solution was diluted with ether ( 100 mL) and washed with water (3 x 15 mL). The organic phase was dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (Et2O:Hexane = 1 : 1, v/v) to obtain the product as a red orange solid (255 mg, 93%). 1H NMR (500 MHz, CDCh): δ 7.54 (d, 7.7 Hz, 1 H), 7.4 (dt, 1.1 Hz, 7.5 Hz, 1 H), 7.28 (m, 2 H), 7.24 (d, 7.6 Hz, 1 H), 7.18 (d, 6.80 Hz, 1 H), 7.03 (d, 8.4 Hz, 1 H), 3.85 (s, 3 H), 3.76 (s, 3 H), 3.75 (s, 2 H); 1 T NMR ( 125.8 MHz, CDCh): δ 194.76, 165.76, 152.95, 148.06, 132.24, 132.10, 130.46, 129.63, 129.49, 128.96, 128.56, 127.93, 127.61, 1 19.12, 1 17.58, 1 17.1 1, 56.15, 52.40, 22.49; IR (KBr): 2246, 1735, 171 1 cm"1; Elemental analysis: calculated for C2OHi5NO4: C, 72.06, H, 4.54; found: C, 71.88, H, 4.72; HRMS: calculated for C20H15NO4: 333.1001, found: 333.0985.
Figure imgf000062_0001
Synthesis of 6,7-Dimethoxy-ll-oxo-llH-benzo[«]fluorene-5-carbonitrile (12) To a solution of compound 11 (255 mg, 0.766 mmol) in anhydrous THF ( 15 mL) at room temperature was added LDA in THF (0.781 mmol in 6 mL) slowly and the solution was stirred for 1 hour. The reaction was quenched with saturated aqueous NH4Cl solution ( 15 mL) leading to the formation of some red orange precipitate. The precipitate was dissolved in EtOAc (600 mL) and then washed with H2O (3 x 30 mL ). The organic phase was dried over Na2SO4 and concentrated to obtain the crude product as a red orange solid.
To a solution of the above crude product in DMF ( 100 mL) was added K2CO^ (0.529 g, 3.83 mmol) at room temperature and the mixture was stirred for 15 minutes, followed by addition of CHJ (0.48 mL, 7.66 mmol). The solution was heated at 80 0C for 45 minutes then filtrated and concentrated. The solution residue was diluted with CHCh (300 mL) and washed with H2O (3 x 30 mL). The organic phase was dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (CHCh:Hexane = 3: 1, v/v) to give the product as an orange solid ( 140 mg, 58% for two steps). 1H NMR (300MHz, CDCh): δ 9.05 (m, 1 H), 8.03 (m, 1 H), 7.58 (m, 2 H), 7.32 (m, 2 H), 7.15 (d, 8.4 Hz, 1 H), 4.04 (s, 3 H), 4.00 (s, 3 H); 1 T NMR (75.5 MHz, CDCh): δ 194.1, 157.8, 154.8, 139.2, 135.6, 135.0, 132.0, 129.2, 129.1, 127.5, 126.8, 124.7, 124.6, 120.2, 1 17.0, 1 15.1, 1 10.8, 63.9, 56.3 (one peak missing or overlap). HRMS: calculated for C211HuNO.,: 315.0895, found: 315.0896.
Figure imgf000063_0001
Synthesis of 6,7-Dimethoxy-ll-oxo-llH-benzo[«]fluorene-5-carboxamide (13)
(see Katritzky et «/., 1989)
To a solution of compound 12 ( 140 mg, 0.443 mmol) and K2CO^ (95 mg, 0.687 mmol) in DMSO (55 mL) at 0 0C was added 30% H2O2 ( 10 mL) slowly, then the mixture was stirred at room temperature for 21 hours. The reaction was quenched with water (60 mL), followed by extraction with EtOAc (300 mL). The organic phase was washed with water (3 x 30 mL), dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (EtOAc:Hexane = 3: 1, v/v) to give the product as an orange solid ( 151 mg, 100%). 1H NMR (300 MHz, DMSO-d6): δ 8.88 (d, 8.3 Hz, 1 H), 8.12 (s, br, 1 H), 7.88 (s, br, 1 H), 7.67 (d, 8.3 Hz, 1 H), 7.52 (m, 2 H), 7.34 (m, 2 H), 7.20 (d, 6.6 Hz, 1 H), 3.93 (s, 3 H), 3.75 (s, 3 H); 1 T NMR (75.5 MHz, DMSO-d6): 5 194.4, 168.2, 154.8, 149.2, 140.7, 138.7, 135.9, 132.4, 131.7, 129.1, 127.7, 127.6, 127.4, 127.3, 125.5, 123.7, 121.2, 1 16.7, 63.9, 56.5; IR (CH2Cl2 film): 3389.8, 1692.6, 1654.5, 1272.1, 1046.8 cm"1; MS (EI): m/z 333.1 ( 100, M+), 316.1 (44).
Figure imgf000063_0002
Synthesis of Methyl 6,7-dimethoxy-ll-oxo-llH-benzo[«]fluoren-5-ylcarbamate
(14) (see Moriarty et «/., 1993)
To a solution of compound 13 ( 151 mg, 0.453 mmol) in methanol ( 160 mL) was added KOH (64 mg, 1.13 mmol) at room temperature. The solution was cooled to 0 0C and stirred for 10 minutes. PhI(OAc)2 ( 146 mg, 0.453 mmol) was added and the mixture was stirred at O0C for additional 15 minutes. The solution was then warmed to room temperature and further stirred for 7 hours. The solution was concentrated and the residue was mixed with CH2Cl2 (200 mL) and water ( 100 mL). The aqueous phase was extracted with CH2Cl2 (2 x 100 mL). The combined organic phase was dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (EtOAc:Hexane = 3: 1, v/v) to give the title compound as an orange solid ( 1 15 mg, 70%). 1H NMR (300 MHz, CDCh): 5 9.05 (d, 8.4 Hz, 1 H), 7.77 (d, 8.5 Hz, 1 H), 7.49 (t, 7.5 Hz, 1 H), 7.40 (t, 7.5 Hz, 1 H), 7.29 (m. 2 H), 7.07 (m, 1 H), 6.81 (s, br, 1 H), 3.98 (s, 3 H), 3.81 (s, 3 H), 3.80 (s, 3 H); 13C NMR (75.5 MHz, CDCh): 5 194.2, 155.6, 154,3, 147.5, 139.8, 136.7, 133.6, 131.3, 130.8, 128.6, 128.3, 128.0, 126.6, 124.4, 123.9, 1 19.5, 1 16.8, 62.6, 56.3, 53.1; IR (CH2Cl2 film): 3275.8, 1730.0, 1690.0, 1283.6, 1261.9, 1238.0 cm 1; MS (EI): m/z 363.1 (80, M+), 331.1 ( 100), 316.1 (60), 288.1 ( 19).
EXAMPLE 10
Figure imgf000064_0001
Synthesis of 5-Amino-6, 7-dimethoxy-llH-benzo[α]fluoren-ll-one (15)
To a solution of compound 14 ( 1 15 mg, 0.317 mmol) in ethanol ( 125 mL) was added aqueous LiOH solution (2 M, 0.79 mL) slowly at room temperature and the reaction mixture was refluxed for 21 hours. The solution was concentrated and the residue was dissolved in EtOAc (300 mL) and washed with water (3 x 30 mL). The organic phase was dried over Na2SO4 and concentrated. The residue was purified by flash chromatography ( EtOAc :Hexane = 2:3, v/v) to give the product as a red solid (99 mg, 100%). 1H NMR (300 MHz, DMSO-(I6): δ 8.85 (d, 8.4 Hz, 1 H), 8.12 (d, 8.6 Hz, 1 H), 7.44 (t, 7.6 Hz, 1 H), 7.27 (m, 2 H), 7.18 (s, 1 H), 7.15 (m, 2 H), 7.06 (d, 6.9 Hz, 1 H), 3.91 (s, 3 H), 3.66 (s, 3H); 1 T NMR (75.5 MHz, DMSO-(I6): δ 190.8, 154.7, 148.0, 140.5, 139.1, 136.7, 131.9, 130.2, 129.4, 126.6, 124.2, 123.9, 123.6, 121.3, 1 19.3, 1 15.2, 1 12.9, 61.7, 56.5; IR (CH2Cl2 film): 3315.8, 3216.1, 1654.0, 1613.2, 1546.9, 1519.9, 1285.9, 1212.8, 1097.7, 1055.4 cm"1; MS (EI): m/z 305.1 ( 100, M+), 290.1 (81 ).
EXAMPLE 11
Figure imgf000065_0001
Synthesis of 5-Diazo-5H-7-methoxy-llH-benzo[«]fluoren-6, 11-dione (16)
To a solution of compound 15 (99 mg, 0.33 mmol) in ethanol ( 150 ml) was added cone. HCl (2.5 mL) at room temperature. The solution was cooled to 0 0C, and an aqueous NaNO2 solution (28 mg/0.41 mmol in 5 mL) was added slowly. The solution was stirred at 0 0C for 4 hours, followed by addition of NaΗCO? powder (2.5 g), and the mixture was stirred for additional 20 minutes. The remained NaΗCO? was filtered and the filtrate was concentrated. The residue was dissolved in EtOAc (300 mL) and washed with water (3 x 30 mL). The organic phase was dried over Na2SO4 and concentrated. The residue was purified by flash chromatograph (EtOAc:Ηexane = 1 : 1, v/v) to obtain the title compound as a purple solid (58 mg, 59%). 1H NMR (500 MHz, CD2Cl2): δ 8.86 (d, 8.1 Hz, 1 H), 7.51 (t, 7.4 Hz, 1 H), 7.40 (d, 8.0 Hz, 1 H), 7.33 (m, 2 H), 7.25 (d, 6.7 Hz, 1 H), 7.13 (d, 8.1 Hz, IH), 3.96 (s, 3 H); 1T NMR ( 125.8 MHz, CD2Cl2)*: 5 196.2, 170.5, 154.8, 133.7, 133.6, 131.4, 129.4, 128.6, 127.5, 126.5, 125.7, 122.2, 121.2, 1 19.5, 1 17.0, 56.5 (missing two peaks); IR (CH2Cl2 solution): 3061.0, 2945.0, 2838.1, 2095.7, 1710.8, 1620.1, 1480.1, 1336.0 cm-i; MS (EI): m/z 302.1 (30, M+), 274.1 (96), 259.1 ( 100), 231.1 (25), 187.1 (24), 175.1 (23); HRMS (EI): calculated for
Figure imgf000065_0002
302.0691, found: 302.0691. *As a result of low solubility of the title compound in CD2Cl2, no 1 C NMR signal was observed for the quaternary carbon attached to the diazo group. A cross peak at 90.0 ppm, assignable to the carbon atom attached to the diazo group, is clear evidence, however, in the HMBC experiment.
EXAMPLE 12
Figure imgf000066_0001
Synthesis of (3-methoxy-5-methylphenyl)methanol (see Ishii and Sakaguchi,
1999)
To a solution of 3,5-dimethylanisole (4.752 g, 34.90 mmol) in HOAc (50 mL) were added AMiydroxyphthalimide (0.569 g, 3.490 mmol) and Co(OAc)2 »4H2O (0.174 g, 0.698 mmol). The solution was stirred at 100 0C for 72 hours. The solution was concentrated, and 1 M aqueous NaOH solution was added to the residue to adjust the pH to ca. 12. The resulting solution was extracted with CH2Cl2 (3 x 100 mL), and the organic phase was dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (EtOAc:Hexane = 1 :20, v/v) to recover the starting material 3,5-dimethylanisole ( 1.712 g, 36%) and meanwhile to obtain the ciiide aldehyde. The pH of the basic aqueous phase was adjusted to ca. 1 by adding cone. HCl and the resulting solution was extracted with EtOAc ( 100 mL \ 4). The EtOAc phase was dried over Na2SO4 and concentrated to obtain the crude carboxylic acid. The crude aldehyde and carboxylic acid were combined (3.425 g) and subjected to the next reduction step.
To a mixture of LiAlH4 ( 1.299 g, 34.25 mmol) suspended in anhydrous THF (75 mL) at 0 0C was added slowly the anhydrous THF solution of the above ciiide products (3.425 g in 9 mL), and the reaction mixture was further stirred at 0 0C for 15 minutes. The solution was then wanned to room temperature and stirred for 20 hours. Saturated aqueous NH4Cl solution was added at 0 0C to quench the excess LiAlH4 until no more gas bubbles were generated, followed by filtration. The filtrate was dried over Na2SO4 and concentrated. The residue was purified by flash chromatograph (EtOAc:Hexane = 1 :5, v/v) to obtain the title compound as a white solid ( 1.487 g, 28% for two steps). 1H NMR (300 MHz, CDCh): δ 6.71 (s, 1 H), 6.68 (s, 1 H), 6.62 (s, 1 H), 4.53 (s, 2 H), 3.74 (s, 3 H), 3.08 (s, 1 H), 2.30 (s, 3 H); 1 T NMR (75.5 MHz, CDCh): 5 159.7, 142.4, 139.5, 120.0, 1 13.9, 109.2, 64.9, 55.1, 21.4; IR (CH2Cl2 film): 3366.1, 2999.8, 2940.0, 2871.3, 2839.1, 1613.5, 1462.7, 1325.3, 1295.3, 1 193.6, 1 152.5, 1068.2 cm"1; MS (EI): m/z 152.09 ( 100, M+), 137.07 (23), 123.09 (39), 109.07 (22), 91.05 ( 18), 77.02 ( 1 1 ).
Figure imgf000067_0001
Synthesis of (2-iodo-3-methoxy-5-methylphenyl)ethanol (17)
To a solution of compound 16 (3.743 g, 24.62 mmol) in anhydrous diethyl ether ( 160 mL) was added «-BuLi in hexane ( 1.6 M, 33.86 mL) slowly, and the solution was warmed to room temperature and stirred for 4 hours, after which the solution was cooled to 0 0C again. Anhydrous THF (80 ml) was then added to the solution and the reaction mixture was stirred for 1 hour, followed by slow addition of I2 (7.687 g, 30.29 mmol) dissolved in minimum amount of THF and the mixture was further stirred for 30 minutes at 0 0C. The reaction mixture was washed with 10% aqueous Na2S2O? solution and then mixed with saturated aqueous NH-tCl solution (50 mL), followed by extraction with Et2O ( 100 mL \ 3). The organic phase was dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (EtOAc:Hexane = 1 :5, v/v) to obtain the title compound as a white solid (5.068 g, 74%). 1H NMR (300 MHz, CDCh): 5 6.86 (s, 1 H), 6.54 (s, 1 H), 4.59 (s, 2 H), 3.83 (s, 3 H), 2.60 (s, 1 H), 2.30 (s, 3 H); 1 T NMR (75.5 MHz, CDCh): 5 157.7, 144.0, 139.7, 121.7, 1 1 1.1, 85.2, 69.4, 56.5, 21.4; IR (CH2Cl2 FiIm): 3262.1, 2902.6, 1651.8, 1575.5, 1457.7, 1433.9, 1409.8, 1356.2, 1307.6, 1 168.2, 1099.7, 1041.8, 1009.6, 839.7, 582.0 cm-i; MS (EI): m/z 278.0 ( 100, M+), 263.0 (5). EXAMPLE 14
Figure imgf000068_0001
Synthesis of 2-(2-iodo-3-methoxy-5-methylphenyl)acetonitrile (18)
To a solution of compound 17 (5.068 g, 18.23 mmol) in anhydrous CH2CI2 ( 100 mL) was added CBr4 (6.348 g, 19.14 mmol) at room temperature. The solution was cooled to 0 0C and PPI13 (5.021 g, 19.14 mmol) was added slowly. The solution was then warmed to room temperature and stirred for 6 hours. The solution was concentrated and the residue was purified by flash chromatography ( EtOAc :Hexane = 1 :50, v/v) to obtain the title compound as a white solid. To a solution of the above white solid in anhydrous DMSO (30 mL) was added NaCN ( 1.340 g, 27.35 mmol) and the mixture was stirred for 36 hours. The reaction mixture was diluted with water ( 150 ml) and extracted with CH2CI2 (2 x 100 mL). The organic phase was dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (EtOAc:Hexane = 1 :20, v/v) to obtain the title compound as a white solid (3.606 g, 69% for two steps). 1H NMR (300 MHz, CDCh): δ 6.91 (s, 1 H), 6.55 (s, 1 H), 3.82 (s, 3 H), 3.73 (s, 2 H), 2.30 (s, 3 H); 13C NMR (75.5 MHz, CDCh): δ 158.4, 140.3, 134.5, 122.2, 1 17.6, 1 1 1.4, 87.4, 56.6, 30.2, 21.4; IR (CH2Cl2 film): 3020.3, 2968.7, 2942.4, 2920.1, 2250.9, 1572.7, 1458.9, 1408.0, 1391.9, 1319.7, 1298.3, 1243.0, 1 184.4, 1081.5, 1015.7 cm"1; MS (EI): m/z 287.0 ( 100, M+), 272.0 (9); HRMS (EI): calculated for C10H10ONI: 286.9807, found: 286.9816.
EXAMPLE 15
Figure imgf000068_0002
Synthesis of 2-(3-methoxy-5-methyl-2-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)phenyl) acetonitrile (19) (see Baudoin et «/., 2000)
To a solution of compound 18 ( 1.500 g, 5.226 mmol) in anhydrous dioxane ( 18 mL) were added pinacolborane (2.28 mL, 15.98 mmol) and EhN (2.91 mL, 20.91 mmol), and the mixture was degassed three times by the thaw-freeze process. Pd(OAc)2 (59 mg, 0.2613 mmol) and (2-biphenyl)dicyclohexyl-phosphine (366 mg, 1.045 mmol) were added to the mixture then the reaction mixture was degassed again by the thaw-freeze process. The mixture was stirred at 100 0C for 4 hours. Saturated aqueous NH4CI solution (20 mL) was added to the solution at 0 0C followed by extraction with CH2CI2 (2 x 100 mL). The organic phase was dried over Na2SO4 and concentrated, and the residue was purified by flash chromatography (EtOAc:Hexane = 1 : 10, v/v) to obtain the title compound as a white solid ( 1.229 g, 82%). 1H NMR (300 MHz, CDCh): δ 6.77 (s, 1 H), 6.56 (s, 1 H), 3.76 (s, 2 H), 3.71 (s, 3 H), 2.28 (s, 3 H), 1.31 (s, 12 H); 1 T NMR (75.5 MHz, CDCb): δ 164.2, 142.3, 135.7, 121.6, 1 18.5, 1 10.8, 83.8, 55.8, 24.7, 23.4, 21.7; IR (CH2Cl2 film): 3458.1, 2978.7, 2935.6, 2838.5, 2249.0, 1612.1, 1567.3, 1 144.2, 1084.9, 1063.8 cm"1; MS (EI): m/z 287.2 ( 100, M+), 286.2 (27), 272.2 (26), 228.2 (38), 214.2 (25), 200.2 (37), 187.1 (99), 186.1 (32), 161.1 ( 18), 131.1 (26), 130.1 ( 12); HRMS (EI): calculated for C16H22O3N10B: 286.1729, found: 286.1722.
EXAMPLE 16
Figure imgf000069_0001
Synthesis of Methyl 2-(2-(cyanomethyl)-6-methoxy-4-methylphenyl)-7-methoxy-
3-oxo-3H-indene-l-carboxylate (20)
Compound 10 ( 132 mg, 0.443 mmol) was mixed with KF (77 mg, 1.329 mmol), Pd2(dbab»CHCh (23 mg, 0.022 mmol) and [(f-BubPH]BF4 ( 13 mg, 0.044 mmol) and the mixture was deoxygenated five times with an argon balloon and vacuum pump. Compound 19 ( 1 14 mg, 0.339 mmol) was dissolved in a mixture of
THF and water ( 19: 1, v/v, 4 mL) and the solution was deoxygenated three times using the thaw-freeze process. The deoxygenated solution was then added to the mixture of solids and the reaction mixture was stirred at room temperature for 24 hours under argon atmosphere. The solution was then diluted with Et2O ( 100 mL) and washed with H2O (3 x 10 mL). The aqueous phase was extracted with Et2O (2 x 50 mL) and all Et2θ phases were combined. The organic phase was dried over NaTSO4 and concentrated. The residue was purified by flash chromatograph (EtOAc:Hexane = 1 :3, v/v) to obtain the title compound as an orange solid ( 127 mg, 85%). 1H NMR (300 MHz, CDCh): δ 7.27 (m, 1 H), 7.16 (d, 6.8 Hz, IH), 7.01 (d, 8.3 Hz, IH), 6.93 (s, 1 H), 6.67 (s, 1 H), 3.83 (s, 3 H), 3.75 (s, 3 H), 3.73 (d, 19.0 Hz, 1 H), 3.69 (s, 3 H), 3.64 (d, 19.0 Hz, 1 H), 2.35 (s, 3 H); 1 T NMR (75.5 MHz, CDCh): δ 195.1, 166.0, 158.1, 152.8, 148.3, 141.0, 131.8, 131.1, 130.8, 130.4, 128.0, 121.2, 1 19.0, 1 18.0, 1 17.0, 1 15.1, 1 1 1.4, 56.2, 55.8, 52.4, 22.0, 21.8; IR (CH2CI2 film): 2950.0, 2842.3, 2251.6, 1733.1, 1717.1, 1610.0, 1575.8, 1479.4, 1464.0, 1337.9, 1274.2, 1242.6, 1202.1, 1 173.8, 1092.5, 1055.1 cm"1; MS (EI): m/z 377.2 ( 100, M+), 345.1 (25), 318.1 (40), 286.1 (65), 59.2 ( 12); HRMS (EI): calculated for C22Hi9NO5: 377.1263, found: 377.1263.
Figure imgf000070_0001
Synthesis of 1, 6, 7-trimethoxy-3-methyl-ll-oxo-llH-benzo[«]fluorene-5- carboxamide (27)
Preparation of lithium complex solution (see Kim and Aim, 1984): To a solution of /-Bu2AlH ( 1.75 mL, 1.75 mmol) in anhydrous THF (2.05 mL) at -78 0C was added «-BuLi in hexane ( 1.20 mL, 1.75 mmol) slowly, the mixture was further stirred for 1 hour. Preparation of LDA: To a solution of /-Pr2NH (0.26 mL, 1.834 mmol) in anhydrous THF (8.54 mL) at 0 0C was added W-BuLi in hexane ( 1.20 mL, 1.75 mmol) slowly, and the mixture was further stirred for 30 minutes. To a solution of compound 20 (66 mg, 0.175 mmol) in anhydrous THF (4 mL) at -78 0C was added the above lithium complex solution (0.5 mL) slowly, and the reaction mixture was further stirred for 1 hour, followed by slow addition of the freshly prepared LDA solution ( 1.25 mL). The solution was stirred for 1 hour at -78 0C and then wanned to room temperature to be further stirred for 5 hours. Saturated aqueous NH4CI solution ( 10 mL) was added to quench the reaction and the mixture was stirred for 20 minutes at 0 0C. The resulting solution was extracted with CH2CI2 (3 x 50 mL). The organic phase was dried over Na2SO4 and concentrated to obtain the crude product as a red orange solid (75 mg). To a solution of the above crude product (75 mg) in anhydrous DMF ( 10 mL) was added K2CO? ( 121 mg) and the reaction mixture was stirred at room temperature for 10 minutes, followed by addition of CHJ (0.1 1 mL). The reaction mixture was heated and stirred at 80 0C for 45 minutes. The solution was cooled to room temperature and diluted with H2O ( 100 mL), followed by extraction with Et2θ (3 x 100 mL). The organic phase was washed with H2O (3 x 30 mL), dried over Na2SO4 and concentrated to obtain the ciiide product as a red orange solid (63 mg).
To a solution of the above ciiide product (63 mg) and K2CO? (37 mg) in DMSO (25 ml) at 0 0C was added slowly 30% H2O2 (5 mL). The solution was warmed to room temperature and stirred for 24 hours. The reaction mixture was diluted with H2O ( 100 mL) and then extracted with EtOAc (3 x 100 mL). The EtOAc phase was washed with H2O (30 mL \ 3 ), dried over Na2SO4 and concentrated. The residue was purified by flash chromatograph (EtOAc:Hexane = 2: 1, v/v) to obtain the title compound as a red orange solid (34 mg, 51% for three steps). 1H NMR (300 MHz, DMSO-(I6): δ 8.04 (s, 1 H), 7.78 (s, 1 H), 7.35 (m, 1 H), 7.26 (d, 8.2 Hz, 1 H), 7.15 (d, 6.8 Hz, 1 H), 7.00 (s, 1 H), 6.83 (s, 1 H), 3.91 (s, 3 H), 3.87 (s, 3 H), 3.70 (s, 3 H), 2.38 (s, 3 H); 1 T NMR (75.5 MHz, DMSO-d6): δ 190.4, 168.5, 156.2, 154.4, 149.5, 140.1, 138.0, 137.3, 135.8, 133.6, 131.8, 131.6, 126.9, 120.2, 1 18.4, 1 16.5, 1 16.4, 1 10.1, 63.5, 56.1, 55.6, 22.2; IR (CH2Cl2 FiIm): 3425.0, 3337.5, 2937.5, 2837.5, 1706.3, 1670.9, 1605.8, 1554.6, 1483.8, 1355.5, 1266.3, 1047.6 cm"1; MS (EI): m/z 377.2 ( 100, M+); HRMS (EI): CaIc for C22Hi9O5N: 377.1263, found: 377.1268. EXAMPLE 18
Figure imgf000072_0001
Synthesis of Methyl 1, 6, 7-trimethoxy-3-methyl-ll-oxo-llH-benzo[α]fluoren-5- ylcarbamate (28) To a solution of compound 27 (24 mg, 0.065 mmol) and KOΗ (9 mg, 0.16 mmol) in anhydrous methanol ( 14 mL) at 0 0C was added PhI(OAc)2 (21 mg, 0.065 mmol). The reaction mixture was stirred for 30 minutes at 0 0C, then wanned to room temperature and stirred for 3 hours. The solution was diluted with EtOAc ( 150 mL) and washed with brine (3 x 15 mL). The organic phase was dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (EtOAc:Ηexane = 1 : 1, v/v) to obtain the title compound as a red orange solid (26 mg, 99%). 1H NMR (300 MHz, CD2Cl2): δ 7.24 (m, 2 H), 7.12 (s, 1 H), 7.06 (d, 7.9 Hz, 1 H), 6.82 (s, 1 H), 6.66 (s, 1 H), 3.94 (s, 6 H), 3.78 (s, 3 H), 3.73 (s, 3 H), 2.37 (s, 3 H); 13C NMR (75.5 MHz, CD2Cl2): δ 190.3, 156.3, 155.6, 153.9, 149.0, 139.8, 137.6, 136.4, 133.7, 132.1, 130.7, 130.5, 127. 3, 1 18.9, 1 18.6, 1 16.1, 1 14.8, 109.6, 61.8, 56.0, 55.6, 52.7, 21.8; IR (CH2Cl2 film): 3304.4, 2939.3, 1706.2, 1605.5, 1559.8, 1483.9, 1273.5, 1045.8 cm"1; MS (EI): 407.1 ( 100, M+), 375.1 ( 15); HRMS (EI): calculated for C23H21O6N: 407.1369, found: 407.1377.
EXAMPLE 19
Figure imgf000072_0002
5-amino-l,6,7-trimethoxy-3-methyl-llH-benzo[«]fluoren-ll-one (29)
To a solution of compound 28 (26 mg, 0.065 mmol) in ethanol ( 16 mL) was added aqueous LiOH solution (4 M, 0.16 mL, 0.65 mmol) and the mixture was refluxed for 24 hours. The solution was concentrated to ca. 5 mL and then diluted with CΗ2CI2 ( 100 mL). The resulting solution was washed with LLO (3 x 10 mL), and the aqueous phase was extracted with CH2Cl2 (2 x 50 mL). The combined CH2Cl2 phase was dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (EtOAc:Hexane = 1 : 1, v/v) to obtain the title compound as a dark red solid (23 mg, 100%). 1H NMR (300 MHz, DMSO-d6): δ 7.46 (s, 1 H), 7.26 (m, 1 H), 7.13 (d, 8.3 Hz, 1 H), 7.02 (d, 6.9 Hz, 1 H), 6.75 (m, br, 3 H), 3.88 (s, 3 H), 3.82 (s, 3 H), 3.62 (s, 3 H), 2.36 (s, 3 H); 1 T NMR (75.5 MHz, DMSO-d6): δ 187.5, 156.6, 154.2, 146.1, 140.8, 138.9, 136.8, 134.6, 131.3, 126.6, 123.8, 120.7, 1 18.7, 1 15.9, 1 15.1, 1 14.6, 1 10.7, 61.1, 56.6, 55.8, 22.0; IR (CH2Cl2 FiIm): 3394.6, 1627.8, 1513.1, 1457.0, 1269.9 cm"1; MS (EI): m/z 349.1 ( 100, M+), 334.1 (27), 319.1 ( 18); HRMS (EI): calculated for C21H19O4N: 349.1314, found: 349.1323.
EXAMPLE 20
Figure imgf000073_0001
Synthesis of Dimethylisoprekinamycin (30)
To a solution of compound 29 ( 15 mg, 0.044 mmol) in ethanol and water (5: 1, v/v, 12 mL at 0 0C was added dilute HCl ( 1.2 M, 0.91 mL) slowly and the mixture was stirred for 10 minutes. An aqueous solution Of NaNO2 (4 mg/0.044 mmol in 0.5 mL) was added and the reaction was stirred at 0 0C for 1 hour, followed by addition of aqueous NaHCO? solution ( 150 mg in 2.0 mL), and the mixture was stirred at 0 0C for additional 20 minutes. The reaction mixture was diluted with EtOAc ( 150 mL) and washed with H2O (3 x 20 mL). The organic phase was dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (EtOAc:Hexane =
1 :2, v/v) to obtain the title compound as a dark purple solid ( 10 mg, 63%). 1H NMR (500 MHz, CD2Cl2): δ 7.33 (m, 1 H), 7.27 (d, 6.5 Hz, 1 H), 7.16 (d, 8.2 Hz, 1 H), 6.86 (s, 1 H), 6.69 (s, 1 H), 3.99 (s, 3 H), 3.98 (s, 3 H), 2.48 (s, 3 H); 1 T NMR ( 125.8 MHz, CD2Cl2)*: 5 192.2, 171.2, 158.3, 154.4, 142.1, 140.9, 139.2, 133.9, 130.9, 130.4, 127.4, 120.5, 1 16.7, 1 12.5, 1 10.7, 109.1, 56.7, 55.8, 21.7; IR (CH2Cl2 solution): 2996.6, 2926.9, 2852.8, 21 12.6, 1722.3, 1608.6, 1563.4, 1483.9, 1468.0, 1361.4 cm 1; MS (EI): l-n/z 346.1 (78, M+), 318.1 ( 100), 303.1 (97), 275.1 (27), 247.1 (22), 219.1 ( 15), 189.1 (23), 187.1 ( 12); HRMS (EI): calculated for C20Hi4O4N2: 346.0954, found: 346.0953. *As a result of low solubility of the title compound in CD2Cl2, no L"C NMR signal was observed for the quaternary carbon attached to the diazo group. A cross peak at 88.0 ppm, assignable to the carbon atom attached diazo group, is clear evidence, however, in the HMBC experiment.
EXAMPLE 21
Figure imgf000074_0001
Synthesis of Isoprekinamycin 4
To a solution of compound 30 (8 mg, 0.022 mmol) in anhydrous CH2Cl2 (2 mL) at -78 0C was added slowly a CH2Cl2 solution of BCl5 ( 1 M, 0.09 mL, 0.09 mmol), and the reaction mixture was stirred for 30 minutes. The reaction mixture was warmed to room temperature and further stirred for 90 minutes, followed by addition of ice ( 1 g) and CH2Cl2 ( 100 mL). The CH2Cl2 solution was washed with water (3 x 10 mL), dried over Na2SO4 and concentrated. The residue was purified by flash chromatography (Et2O:CH2Cl2 = 1 :200, v/v) to obtain the title compound as a dark purple solid (5 mg, 64%). 1H NMR (500 MHz, CD2Cl2): 5 12.32 (s, 1 H), 1 1.61 (s, 1 H), 7.26 (d, 6.7 Hz, 1 H), 7.18 (t, 7.6 Hz, 1 H), 7.06 (d, 8.3 Hz, 1 H), 6.72 (s, 1 H), 6.69 (s, 1 H), 2.41 (s, 1 H); IR (CH2Cl2 solution): 3686.1, 3600.7, 2926.6, 2855.0, 2126.1, 1733.9, 1686.8, 161 1.5, 1560.5, 1466.5 cm"1; UV-vis (HPLC): 21 1.0, 247.4, 284.0, 561.3 nm. EXAMPLE 22 Comparison of Synthetic and Natural Isoprekinamycin
Spectroscopic comparison of the synthetic and natural IPK was carried out in the following manner: ( 1 ) for 1H NMR spectral comparison, saturated CD2CI2 solutions of the synthetic and natural IPK were examined on a 500 MHz NMR spectrometer; (2) for IR spectral comparison, saturated CH2CI2 solutions of the synthetic and natural IPK were examined on a FT-IR spectrometer using a demountable liquid-cell IR kit (Aldrich Zl 1200-3) with two CaF2 windows (32 mm \ 3 mm) and a light path of 0.1 mm, and the solvent and air backgrounds were deducted from the recorded spectra; (3 ) for HPLC retention time and UV-vis spectra comparison, the synthetic and natural IPK and also a mixture of both compounds were analyzed with a Waters HPLC system equipped with a photodiode array detector. See FIG. 3 for HPLC data.
Synthetic and natural isoprekinamycin exhibited the same Rf values in the following three different solvent systems: Rf = 0.8 (EtOAc:Hexane = 3:2), Rf = 0.38 (Et2O:Hexane = 1 : 1 ), Rf= 0.14 (CH2Cl2:Hexane = 2: 1 ).
1H NMR (500 MHz in CD2Cl2)
Figure imgf000075_0001
IR Frequency (saturated CH2Cl2 solution)
Figure imgf000076_0002
Absorption of the diazo group
UV-vis (HPLC)
Figure imgf000076_0001
EXAMPLE 23
Biological and Biochemical Assays Studying IPK, IPK-diacetate and IPK-O- methyl
Methods ami Materials
Growth Inhibition Assays:
Unless indicated, all chemicals were from Sigma (Oakville, Canada). The 3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sυlfophenyl)-2H- tetrazolium (MTS) CellTiter 96 (R) AQυeoυs One Solution Cell Proliferation Assay kit was obtained from Promega (Madison, WI, U.S.A.). The errors quoted are standard errors from non-linear least squares analysis (SigmaPlot, Systat, Point Richmond, CA).
Cell culture and growth inhibition assays Chinese hamster ovaiy (CHO) cells (type AA8; ATCC CRL-1859), obtained from the American Type Culture Collection, were grown in alpha minimum essential medium (α-MEM; Invitrogen, Burlington, Canada) containing 20 mM HEPES (4-(2- hydroxyethyl)piperazine-l-ethanesulfonic acid) as described (Hasinoff et al., 1997). Human leukemia K562 cells, obtained from the American Type Culture Collection, were maintained as suspension cultures in DMEM (Invitrogen, Burlington, Canada) containing 10% fetal calf seiiim (FCS) and 2 mM L-glutamine. The spectrophotometric 96-well plate cell growth inhibition 3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyl tetrazolium bromide (MTT) (for CHO cells) and MTS (for K562 cells) (Promega, San Luis Obispo, CA) assays, which measures the ability of the cells to enzymatically reduce MTT or MTS after treatment with various concentrations of drugs, have been described (Hasinoff et al., 2006). The drags were dissolved in DMSO. The final concentration of DMSO did not exceed 0.5% (v/v) and was an amount that had no significant effect on cell growth. The cells were incubated with the drags for 72 hours and then assayed with either MTT or MTS. IC50 values, the concentration of isoprekinamycin that reduces the absorbance of MTT or MTS by one-half, for growth inhibition in both assays were measured by fitting the absorbance-drag concentration data to a three-parameter logistic equation as described. Hasinoff et al, 1997.
Topoisomerase Ilα kDNA decatenation inhibition assay A spectrofluorometric decatenation assay was used to determine the inhibition of topoisomerase Ilα by IPK-diacetate as was done for kinamycin A and kinamycin C (Hasinoff et al., 2006). kDNA consists of highly catenated networks of circular DNA. Topoisomerase Ilα decatenates kDNA in an ATP-dependent reaction to yield individual minicircles of DNA. The 20 μL reaction mixture contained 0.5 mM ATP, 50 mM Tris-HCl (pH 8.0), 120 mM KCl, 10 mM MgCl2, 30 μg/mL bovine serum albumin, 40 ng kDNA, test compound (0.5 μL in dimethyl sulfoxide) and 10 ng of topoisomerase Ilα protein (the amount that gave approximately 80% decatenation). Using a high copy yeast expression vector, full-length human topoisomerase Ilα was expressed, extracted and purified as described previously (Hasinoff et al., 2005). The final dimethyl sulfoxide concentration of 2.5% (v/v) was shown in controls not to affect the activity of topoisomerase Ilα. The assay incubation was carried out at 37 0C for 20 minutes and was terminated by the addition of 12 μL of 250 mM Na2EDTA. Samples were centrifuged at 8000 g at 25 0C for 15 minutes and 20 μL of the supernatant was added to 180 μL of 600-fold diluted PicoGreen<R> dye (Molecular Probes, Eugene, OR) in a 96-well plate. The fluorescence, which was proportional to the amount of kDNA, was measured in a Fluostar Galaxy (BMG, Durham North Carolina, USA) fluorescence plate reader using an excitation wavelength of 485 nm and an emission wavelength of 520 nm. pBR322 DNA topoisomerase II-mediated cleavage assays
Topoisomerase Il-cleaved DNA complexes produced by anticancer drugs may be trapped by rapidly denaturing the complexed enzyme with sodium dodecyl sulfate (SDS) (Burden et a!., 2001 ). The cleavage of double-stranded closed circular pBR322 DNA to form linear DNA was followed by separating the SDS-treated reaction products using ethidium bromide gel electrophoresis as described.12 The 20 μL cleavage assay reaction mixture contained 100 ng of topoisomerase Ik/. protein or topoisomerase II nuclear extract, 80 ng pBR322 plasmid DNA (MBI Fermentas, Burlington, Canada), 0.5 mM ATP in assay buffer ( 10 mM Tris-HCl, 50 mM KCl, 50 mM NaCl, 0.1 mM EDTA, 5 mM MgCl2, 2.5% (v/v) glycerol, pH 8.0, and drag (0.5 μL in dimethyl sulfoxide). The order of addition was assay buffer, DNA, drag, and then topoisomerase Ilα. The reaction mixture was incubated at 37 0C for 10 minutes and quenched with 1% (v/v) SDS /25 mM Na2EDTA. The reaction mixture was treated with 0.25 mg/ml proteinase K (Sigma) at 55 0C for 30 minutes to digest the protein. The linear pBR322 DNA cleaved by topoisomerase Ilα. was separated by electrophoresis (2 hours at 8 V/cm) on a TAE (Tris base (4 mM)/glacial acetic acid (0.1 1% (v/v))/Na2EDTA (2 mM) buffer) ethidium bromide (0.5 μg/mL) agarose gel ( 1.2%, wt/v). The DNA in the gel was imaged by its fluorescence on an Alpha Innotech Fluorochem 8900 imaging system. Results
Isoprekinamycin-induced cell growth inhibition
As shown in FIG. 4, isoprekinamycin strongly inhibited growth of CHO cells and K562 cells with IC50 values of 5.8 and 6.4 μM, respectively. These values compare to IC50 values of 1.4 and 3.4 μM, respectively for the widely used anticancer drug etoposide.
IPK-diacetate inhibits the decatenation activity of topoisomerase Ilα
As shown in FIG. 5, IPK-diacetate strongly inhibited (IC50 value of 9.7 μM) the decatenation activity of human topoisomerase Ilα. This assay is a measure of the ability of these compounds to inhibit the catalytic activity only and is not a measure of whether these compounds act as topoisomerase II poisons as do some widely used anticancer drags. Fortune and Osheroff, 2000; Li and Liu, 2001. IPK up to a concentration of 120 μM did not inhibit topoisomerase II. Inhibition of topoisomerase Ilα activity by IPK or IPK-diacetate was not accompanied by stabilization of the covalent topoisomerase Ilα-DNA cleavable complex
Several widely used anticancer agents, including etoposide, are thought to be cytotoxic by virtue of their ability to stabilize a covalent topoisomerase II-DNA intermediate (the cleavable complex) (Fortune and Osheroff, 2000; Li and Liu, 2001 ). Topoisomerase II alters DNA topology by catalyzing the passing of an intact DNA double helix through a transient double-stranded break made in a second helix and is critical for relieving torsional stress that occurs during replication and transcription and for daughter strand separation during mitosis (Fortune and Osheroff, 2000; Li and Liu, 2001 ). Thus, DNA cleavage assay experiments as described (Katritzky et a!., 1989) were carried out using etoposide as a control to see whether the test compounds stabilized the cleavable complex. The addition of etoposide to the experimental mixture containing topoisomerase Ilα and supercoiled pBR322 DNA induced formation of linear pBR322 DNA. Linear DNA was identified by comparison with linear pBR322 DNA produced by action of the restriction enzyme HindIII acting on a single site on pBR322 DNA. The addition of up to 250 μM IPK or 125 μM of IPK- diacetate to the reaction mixture induced little or no detectable formation of cleaved linear pBR322 DNA. EXAMPLE 24
Biological and Biochemical Assays Studying 5-Diazo-5H-7-methoxy-llH- benzo[α]fluoren-6, 11-dione (16)
Using the cell culture and growth inhibition assays described above, the IC50 value for 16 with respect to K562 cells was determined to be 78 iiM, and the IC50 value for the CΗO assay was determined to be 1 μM. The IC50 value for 16 in the topoisomerase Ilα assay described in Example 19 was determined to be 51 μM.
EXAMPLE 25 Isolation of a Compound of the Present Invention from Streptomyces murayamaensis
Stock culture and medium:
Streptomyces murayamaensis (ATCC 21414) was purchased from the American Type Culture Collection. Slants of Krainsky's agar were inoculated with Streptomyces sps, incubated at 27° C for 15 days and then stored at 4° C. Media composition :
Krainsky's medium: Glucose 10 g; KΗPO4 0.5 g; asparagine 0.5 g; agar 10 g per L.
Seed medium: Glucose 20 g; soybean meal 20 g; NaCl 3 g in 1 L distilled water. The medium was adjusted to pH 8 (using NaOH) before sterilization.
Oatmeal medium: Trace metals medium containing rolled oats (90 g cooked in 1 L of distilled water for 10 minutes, then added to 1 L of cold distilled water). The boiled mixture was filtered to remove oatmeal shaft, leaving a fine starch filtrate solution. Trace metal (6 mL) was added to the filtrate before making up the total volume to 3 L using more distilled water (medium pH was adjusted to 8 with NaOH before sterilization). Trace metal salts solution contained: ZnCl2 (40 mg), FeSO4WH2O (200 mg), CuCl2WH2O ( 10 mg), MnCMH2O ( 10 mg), H3BO3 (5 mg), (NH4V1Mo7O24^H2O ( 10 mg), concentrated HCl (2 mL) in 1 L of distilled water. Seed preparation and Shake Flask Fermentation: Seed medium (200 mL in a 1 L flask) was inoculated with 5-10 colonies
(depending on the size of the colonies) and incubated for 48 h at 240 rpm and 27° C. Oatmeal medium was inoculated ( 10 %) with 48 h-old seed culture and fermentation was carried out at 240 rpm and 27° C for 96 h. The fermentation broth was acidified with 6 N HCl to pH 3 and kept in a freezer (cold and dark) for future use. Isolation of metabolites:
The isolation of compound VIII was based on a modification of the procedure employed by Cone et al. in the isolation of kinamycins and isoprekinamycin ( 1989). The acidified fermented oatmeal-broth was centrifuged at 10,000 rpm for ten minutes to remove the mycelia pellet, resuspended in 50 mL distilled water, sonicated ( 1-4° C for 5 minutes) and recombined with the supernatant. The whole suspension was stirred with toluene (400 mL to 1 L broth) for 90 minutes and the resulting emulsion was filtered through Celite and the Celite was washed with acetone. The aqueous layer was separated and washed with ethyl acetate (2 x 250 mL for eveiy L of broth) and the total organic layer was separated and dried over anhydrous NaTSO4. The dried organic layer was concentrated in vacuo to give a dark brown residue. The concentrate was dissolved in a minimal amount of CHCh and applied to a Silicar CC- 4 column packed in CHCh. Elution with CHCh first gave a dark oily fraction but further elution with 1% ethyl acetate in CHCh gave a brown-purple fraction that was confirmed (HPLC) to contain kinamycins. Further column chromatography of this fraction using ethyl acetate in hexanes ( 1 :5) gave isoprekinamcyins (Rf = 0.32) and O- methylisoprekinamycin (VIII) (Rf = 0.2) as well as kinamycins A, C and D.
The stiiicture of VIII was assigned on the basis of proton NMR analysis, mass spectrometry analysis and HMQC and HMBC NMR experiments at 600 MHz.
************************
All of the methods and apparatuses disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and apparatuses and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
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Florida, 1994, Chapter 4.

Claims

1. A method of synthesizing a compound of formula (A):
Figure imgf000084_0001
comprising:
(a) reacting a compound of formula (I):
Figure imgf000084_0002
with a compound of formula (II):
Figure imgf000084_0003
to generate a first intermediate of formula (III):
Figure imgf000084_0004
and (b) subjecting the first intermediate or a derivative thereof to reducing conditions, wherein:
Ai-A^ are each independently carbon or nitrogen;
X is halo or -OSO2CF3;
Y is alkoxy, aryloxy, aralkyloxy, alkylamino, arylamino, aralkylamino, or - OC(O)Rn, wherein Ra is alkyl, aryl, or aralkyl;
R1-R16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, nitro, halogen, -SO2(alkyl), -SO(alkyl), - Sθ2(aiyl), -SO(aryl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of Ri-R2, R2-R^, R3-R4, R5-R6, R6-R7, Ry-Rs, R9-R10, R10-R11, Rn-Ri2, R13-R14, R14-R15, or R15-R16 taken together form a cyclic group;
Rn is H, -CH2CN or -CH2CO2Rb, wherein Rb is alkyl, aryl, or aralkyl; and
Ris and R19 are each independently H, alkyl, aryl, or aralkyl, or Ris and R19 taken together with the boron atom to which they are attached form the following substituent:
Figure imgf000085_0001
and wherein the compound of formula (A) is further defined as not the following:
Figure imgf000085_0002
2. The method of claim 1, wherein the reducing conditions are conditions comprising an alkyllithium/DIBAL mixture or borohydride.
3. The method of claim 1, wherein the reaction comprises Suzuki reaction conditions.
4. The method of claim 1, further comprising anionic cyclization of the first intermediate.
5. The method of claim 4, wherein the anionic cyclization step comprises reaction conditions comprising LDA or DBU.
6. The method of claim 1, further comprising the generation of a second intermediate of formula (IV):
Figure imgf000086_0001
wherein:
Ai-A^ are each independently carbon or nitrogen;
Ry-R16 arc each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen, -Sθ2(alkyl), - SO(alkyl), -SO2(aryl), -SO(aiyl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of R9-Ri0, Ri0-Rn, Rn-Rn, R^-Ru, Ru-Ri5, or R15-R16 taken together form a cyclic group;
R20 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and
R2I is alkoxy.
7. The method of claim 5, wherein the second intermediate is selected from the group consisting of:
Figure imgf000087_0001
8. The method of claim 1, wherein the compound of formula (II) is further defined as the following compound:
Figure imgf000088_0001
wherein Rn-Ri7 are defined as in claim 1.
9. A compound made by the method of claim 1, provided that the compound is further defined as not any of the following:
Figure imgf000088_0002
10. The compound of claim 9, further defined as a compound selected from the group consisting of:
Figure imgf000088_0003
Figure imgf000089_0001
1 1. A compound of formula (V):
Figure imgf000089_0002
wherein:
Yi is alkoxy, aryloxy, aralkyloxy, alkylamino, arylamino, aralkylamino, or - OC(O)Rn, wherein Ra is alkyl, aiyl, or aralkyl;
R22-R29 arc each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen, -Sθ2(alkyl), - SO(alkyl), -SO2(aryl), -SO(aiyl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of R22-R2^, R23-R24, R24-R25, R26-R27, R27-R2S, or R2^-R29 taken together forai a cyclic group; and
R30 is H, -CH2CN or -CH2CO2Rb, wherein Rb is alkyl, aiyl, or aralkyl;
wherein the compound of formula (V) is further defined as not any of the following:
Figure imgf000090_0001
Figure imgf000090_0002
12. The compound of formula (V), further defined as
Figure imgf000090_0003
13. A compound of formula (VI):
Figure imgf000090_0004
wherein:
R31-R.^ are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen, -Sθ2(alkyl), - SO(alkyl), -SO2(aryl), -SO(aryl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of R31-R32, R32-R33, R33-R34, R35-R36, R36-R37, or R37-R38 taken together forai a cyclic group;
R39 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and
R4 0 is alkoxy; provided that when R34 and R35 are each -OH, R37 is not -CH3.
14. The compound of claim 13, further defined as a compound selected from the group consisting of:
Figure imgf000091_0001
Figure imgf000092_0001
15. A compound of formula (VII):
Figure imgf000092_0002
wherein:
Ai-A^ are each independently carbon or nitrogen;
R-ti-R-ts are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, aiylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen, -Sθ2(alkyl), - SO(alkyl), -SO2(aryl), -SO(aiyl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of R41-R42, R42-R43, R43-R44, R45-R46, R46-R47, or R47-R_ts taken together form a cyclic group; and wherein the compound of formula (VII) is further defined as not any of the following:
Figure imgf000093_0001
16. The compound of claim 15, further defined as
Figure imgf000093_0002
17. A method of synthesizing a compound of formula (III):
Figure imgf000094_0001
comprising reacting a compound of formula (I):
Figure imgf000094_0002
with a compound of formula (II):
Figure imgf000094_0003
under Suzuki coupling conditions, wherein:
Ai-A^ are each independently carbon or nitrogen;
X is halo or -OSO2CF3;
Y is alkoxy, aryloxy, aralkyloxy, alkylamino, arylamino, aralkylamino, or - OC(O)Rn, wherein Ra is alkyl, aryl, or aralkyl;
R9-R16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen, -Sθ2(alkyl), - SO(alkyl), -SO2(aryl), -SO(aiyl), -SO2(aralkyl), or -SO(aralkyl), or any one or more Of R9-Ri0, Ri0-Rn, R11-R12, R13-R14, R14-R15, or R15-R16 taken together form a cyclic group;
Rn is H, -CH2CN or -CH^CO^Rb, wherein R11 is alkyl, aiyl, or aralkyl; and Ris and R19 are each independently H, alkyl, aryl, or aralkyl, or Ris and R19 taken together with the boron atom to which they are attached form the following substituent:
Figure imgf000095_0001
18. A method of synthesizing a compound of formula (IV):
Figure imgf000095_0002
comprising cyclizing a compound of formula (III):
Figure imgf000095_0003
under anionic cyclization conditions, wherein:
Ai-A^ are each independently carbon or nitrogen;
Y is alkoxy, aryloxy, aralkyloxy, alkylamino, aiylamino, aralkylamino, or OC(O)Rn, wherein Rn is alkyl, aryl, or aralkyl; Ry-R16 arc each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, aiylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen, -SO2(alkyl), - SO(alkyl), -SO2(aryl), -SO(aryl), -SO2( aralkyl), or -SO(aralkyl), or any one or more of R9-R10, Rio-Rn, Rn-Rn, R^-Ru, R14-R15, or R15-R16 taken together form a cyclic group;
Rn is -CH2CN or -CH2CO2Rb, wherein Rb is alkyl, aryl, or aralkyl;
R20 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and
R2I is alkoxy.
19. In a method of preparing a compound comprising a benzo[</]fluorene, the improvement comprising subjecting a product of a reaction of an aryl boronate ester and an aiyl bromide to reducing conditions comprising an alkyllithium/DIBAL mixture or borohydride.
20. A method of obtaining a compound of formula (VIII):
Figure imgf000096_0001
comprising isolating the compound from Streptomvces munnwnaensis .
21. The method of claim 20, further comprising obtaining Streptomvces muruyumuensis.
22. The compound of claim 9, further comprised in a pharmaceutically acceptable exipient, diluent, or vehicle.
23. The compound of claim 1 1, further comprised in a pharmaceutically acceptable exipient, diluent, or vehicle.
24. The compound of claim 13, further comprised in a pharmaceutically acceptable exipient, diluent, or vehicle.
25. The compound of claim 15, further comprised in a pharmaceutically acceptable exipient, diluent, or vehicle.
26. The compound of formula (VIII) of the method of claim 20, further comprised in a pharmaceutically acceptable exipient, diluent, or vehicle.
27. A method of inhibiting the catalytic decatenation activity of topoisomerase Ilα in a cell, comprising contacting the cell with an effective amount of a compound of formula (A):
Figure imgf000097_0001
or a compound of formula (IV):
Figure imgf000097_0002
wherein:
Ai-A^ are each independently carbon or nitrogen;
Ri-R-16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen, -Sθ2(alkyl), - SO(alkyl), -SO2(aryl), -SO(aiyl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of Ri-R2, R2-R?, R3-R4, R5-R6, R6-R7, Ry-Rs, R9-R10, R10- Rn, Rn-Ri2, R13-R14, R14-R15, or R15-R16 taken together form a cyclic group; R20 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and R2I is alkoxy.
28. The method of claim 27, wherein the cell is a cancer cell.
29. The method of claim 28, wherein the cancer cell is a leukemia cell, a breast cancer cell, or a colon cancer cell.
30. The method of claim 27, wherein the cell is in vivo.
31. The method of claim 27, wherein the cell is in vitro.
32. The method of claim 27, wherein the compound of formula (A) is selected from the group consisting of:
Figure imgf000098_0001
33. A method of inhibiting cell growth, comprising contacting the cell with an effective amount of a compound of formula (A):
Figure imgf000099_0001
or a compound of formula (IV):
Figure imgf000099_0002
wherein:
Ai-A^ are each independently carbon or nitrogen;
R1-R16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen, -Sθ2(alkyl), - SO(alkyl), -SO2(aryl), -SO(aryl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of Ri-R2, R2-R;, R3-R4, R5-R6, R6-R7, R7-Rs, R9-R10, R10- Rn, Rn-Ri2, R13-R14, R14-R15, or R15-R16 taken together form a cyclic group;
R20 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and
R2I is alkoxy.
34. The method of claim 33, wherein the cell is a cancer cell.
35. A method of treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of a compound of formula (A):
Figure imgf000100_0001
or a compound of formula (IV):
Figure imgf000100_0002
wherein:
Ai-A^ are each independently carbon or nitrogen;
R1-R16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen, -Sθ2(alkyl), - SO(alkyl), -SO2(aryl), -SO(aiyl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of Ri-R2, R2-R;, R3-R4, R5-R6, R6-R7, R7-Rs, R9-R10, R10- Rn, Rn-Ri2, R13-R14, R14-R15, or R15-R16 taken together forai a cyclic group;
R20 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and
R2I is alkoxy.
36. The method of claim 35, wherein the subject is a mammal.
37. The method of claim 36, wherein the mammal is a human.
38. The method of claim 35, wherein the subject has cancer of the lung, liver, skin, eye, brain, gum, tongue, blood, head, neck, breast, pancreas, prostate, kidney, bone, testicles, ovaiy, cervix, gastrointestinal tract, lymph system, small intestine, colon, or bladder.
39. A method of inhibiting bacterial growth or killing bacteria in a subject, comprising administering an effective amount of a compound of formula (A)
Figure imgf000101_0001
or a compound of formula (IV):
Figure imgf000101_0002
to the subject, wherein:
Ai-A^ are each independently carbon or nitrogen;
Ri-R-16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen, -Sθ2(alkyl), - SO(alkyl), -SO2(aryl), -SO(aryl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of Ri-R2, R2-R , R3-R4, R5-R6, R6-R7, Ry-Rs, R9-R10, R1O- Rn, Rn-Ri2, R13-R14, R14-R15, or R15-R16 taken together form a cyclic group;
R20 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and
R2I is alkoxy; provided the compound is not isoprekinamycin.
40. A method of treating bacterial infection in a subject comprising administering an effective amount of a compound of formula (A)
or a compound of formula (IV):
Figure imgf000102_0002
to the subject, wherein:
A1-A^ arc each independently carbon or nitrogen;
Ri-R-16 are each independently H, -OH, alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, acyl, acyloxy, alkylthio, arylthio, aralkylthio, alkylamino, arylamino, aralkylamino, aminoalkyl, nitro, halogen, -Sθ2(alkyl), - SO(alkyl), -SO2(aryl), -SO(aiyl), -SO2(aralkyl), or -SO(aralkyl), or any one or more of Ri-R2, R2-R;, R3-R4, R5-R6, R6-R7, R7-Rs, R9-R10, R10- Rn, Rn-Ri2, R13-R14, R14-R15, or R15-R16 taken together form a cyclic group;
R20 is -CN, -CONH2, -NH2, or -NHCO2(alkyl); and
R2I is alkoxy; provided the compound is not isoprekinamycin.
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