HK1060884A - Cytotoxins, prodrugs, linkers and stabilizers useful therefor - Google Patents

Description

Cytotoxins, prodrugs, linkers and stabilizers useful therefor

Cross Reference to Related Applications

This application is a non-provisional filing of U.S. provisional patent application No.60/295,196 filed on day 5/31 2001, No.60/295,259 filed on day 5/31 2001, No.60/295,342 filed on day 5/31 2001, and No.60/304,908 filed on day 11/7/2001. The disclosure of each provisional application is incorporated herein by reference in its entirety.

Background

Many therapeutic agents, particularly those that are particularly effective in cancer chemotherapy, often exhibit acute toxicity in vivo, particularly bone marrow and mucosal toxicity, as well as chronic cardiac and neurological toxicity. Such high toxicity limits their use. The development of more and safer specific therapeutic agents, in particular anti-tumor agents, requires greater effectiveness on tumor cells and a reduction in the number and severity of the side effects (toxicity, destruction of non-tumor cells, etc.) of these products. Another difficulty with some existing therapeutics is their less than ideal stability in plasma. The functional groups added to stabilize these compounds result in a significant reduction in activity. Thus, there is a need to identify methods for stabilizing compounds while maintaining acceptable levels of therapeutic activity.

Research on more selective cytotoxic agents has been extremely active for decades, with dose-limiting toxicity (i.e., undesirable activity of cytotoxins on normal tissues) being one of the major causes of failure of cancer therapy. For example, CC-1065 and duocarmycins are known to be extremely potent cytotoxins.

CC-1065 was first isolated from Streptomyces zelenis in 1981 by Upjohn Company (Hanka et al, J.Antibot.31: 1211 (1978); Martin et al, J.Antibot.33: 902 (1980); Martin et al, J.Antibot.34: 1119(1981)), and was found to have potent antitumor and antimicrobial activity in vitro and in experimental animals (Li et al, Cancer Res.42: 999 (1982)). CC-1065 binds to double-stranded B-DNA in the minor groove (Swenson et al, Cancer Res.42: 2821(1982)), has a sequence preference of 5 ' -d (A/GNTTA) -3 ' and 5 ' -d (AAAAA) -3 ', and alkylates the N3 position of 3 ' -adenine by the CPI left-hand unit in its molecule (Hurley et al, Science 226: 843 (1984)). Despite potent and broad antitumor activity, CC-1065 cannot be used in humans because it results in delayed experimental animal death.

Many analogs and derivatives of CC-1065 and duocarmycins are known in the art. Many studies of the structure, synthesis and properties of compounds have been reviewed. See, e.g., Boger et al, angew.chem.int.ed.engl.35: 1438 (1996); boger et al, chem.Rev.97: 787(1997).

A panel of Kyowa Hakko Kogya co, ltd. has prepared a number of CC-1065 derivatives. See, for example, U.S. Pat. Nos. 5,101,038, 5,641,780, 5,187,186, 5,070,092, 5,703,080, 5,070,092, 5,641,780, 5,101,038, 5,084,468, published PCT application WO 96/10405, and published European application 0537575A 1. None of these patents or applications disclose a strategy to enhance the stability of cytotoxins by generating cleavable prodrugs.

Derivatives of CC-1065 have also been actively prepared by Upjohn Company (Pharmacia Upjohn). See, for example, U.S. patent nos. 5,739,350, 4,978,757, 5,332,837 and 4,912,227. Issued U.S. patents do not disclose or suggest that prodrug strategies would be useful to improve the in vivo stability or reduce toxicity of the compounds disclosed in these patents.

There has also been research focused on developing new therapeutic agents in the form of prodrugs, i.e. compounds that can be converted in vivo into the drug (the active therapeutic compound) by some chemical or enzymatic modification of their structure. For the purpose of reducing toxicity, such transformation is preferably limited to the site of action or target tissue rather than the circulatory system or non-target tissue. However, even prodrugs are problematic because many are characterized by low stability in blood and serum due to the presence of enzymes that degrade or activate the prodrug before it reaches the desired site in the patient.

Thus, despite advances in the art, there remains a need to develop improved therapeutic agents for the treatment of mammals, particularly humans, and more particularly cytotoxins, which exhibit higher specificity of action, reduced toxicity and increased blood stability relative to known compounds of similar structure. The present invention addresses these needs.

Summary of The Invention

The present invention relates to cytotoxins which are analogs of CC-1065 and duocarmycins. The invention also provides linker arms which undergo, for example, enzymatic or reductive cleavage in vivo to release the active drug moiety from the prodrug derivative comprising the linker arm. In addition, the invention includes conjugates between the linker arm of the invention and a cytotoxin, and conjugates between the linker arm, the cytotoxin, and a targeting agent, such as an antibody or a peptide.

The invention also relates to groups useful for stabilizing therapeutic agents and labels. The stabilizing group is selected to limit clearance and metabolism of the therapeutic agent or marker by enzymes that may be present in the blood or non-target tissue, and is further selected to limit transport of the drug or marker into the cell. The stabilizing group acts to retard the degradation of the drug or label and may also provide other physical characteristics of the drug or label. The stabilizing group may also improve the stability of the drug or label during storage, whether in formulated or unformulated form.

In a first aspect, the present invention provides a cytotoxic compound having the structure of formula I:

wherein ring system a is a member selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl. The symbols E and G represent H, substituted or unsubstituted hydrocarbyl, substituted or unsubstituted heterohydrocarbyl, a heteroatom or a single bond. E and G are optionally joined to form a ring system selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl.

In an exemplary embodiment, the ring system a is a substituted or unsubstituted phenyl ring. Ring system a is preferably substituted with one or more aryl substituents as described in the definition section herein. In a preferred embodiment, the phenyl ring is substituted with a CN moiety.

And R³The curves within the six-membered rings that are linked indicate that the ring system may have one or more unsaturations at any position within the ring, and may represent aromaticity.

The symbol X represents a radical selected from O, S and NR²³Is a member of (1). R²³Is a member selected from the group consisting of H, substituted or unsubstituted hydrocarbyl, substituted or unsubstituted heterohydrocarbyl, and acyl.

Symbol R³Represents selected from (═ O), SR¹¹、NHR¹¹And OR¹¹Wherein R is¹¹Is H, substituted or unsubstituted hydrocarbyl, substituted or unsubstituted heterohydrocarbyl, acyl, C (O) R¹²、C(O)OR¹²、C(O)NR¹²R¹³、C(O)OR¹²、P(O)(OR¹²)₂、C(O)CHR¹²R¹³、C(O)OR¹²、SR¹²Or SiR¹²R¹³R¹⁴. Symbol R¹²、R¹³And R¹⁴Independently represent H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted aryl, wherein R¹²And R¹³Optionally joined together with the nitrogen atom to which they are attached to form a 4-to 6-membered substituted or unsubstituted heterocycloalkyl ring system, optionally containing two or more heteroatoms.

R⁴And R⁵Is independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstitutedArylalkyl, halogen, NO₂、NR¹⁵R¹⁶、NC(O)R¹⁵、OC(O)NR¹⁵R¹⁶、OC(O)OR¹⁵、C(O)R¹⁵、OP(O)OR¹⁵OR¹⁶And OR¹⁵Is a member of (1). R¹⁵And R¹⁶Independently represent H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, arylalkyl, and substituted or unsubstituted peptidyl, wherein R is¹⁵And R¹⁶Optionally joined together with the nitrogen atom to which they are attached to form a 4-to 6-membered substituted or unsubstituted heterocycloalkyl ring system, optionally containing two or more heteroatoms.

R⁴、R⁵、R¹¹、R¹²、R¹³、R¹⁵And R¹⁶Optionally containing one or more cleavable groups within their structure. Exemplary cleavable groups include, but are not limited to, peptides, amino acids, and disulfides.

R⁶Is a single bond, which may or may not be present. When R is⁶When present, R⁶And R⁷The linkage forms a cyclopropyl ring. R⁷Is CH₂-X¹or-CH₂-. When R is⁷is-CH₂When it is a component of a cyclopropane ring. Symbol X¹Represents a leaving group. The skilled person will understand R⁶And R⁷In a manner that does not violate the principle of valence.

In another aspect, the invention provides a cleavable linker arm comprising a group cleavable by an enzyme. Cleavable linkers generally confer in vivo cleavability to the construct. Thus, the linking group may comprise one or more groups that will cleave in vivo, e.g., in the bloodstream, at a higher rate than a construct lacking such groups. Conjugates of the linker arm with therapeutic and diagnostic agents are also provided. The linking group may be used to create prodrug analogs of the therapeutic agent, reversibly linking the therapeutic or diagnostic agent to a targeting agent, detectable label or solid support. Connection ofThe groups may be combined into a complex comprising a cytotoxin of the invention. The linking group has the general formula described in formula II:

in the above formula, the symbol E represents an enzymatically cleavable moiety (e.g., peptide, ester, etc.). Symbol R, R^I、R^IIAnd R^IIIRepresentative, for example, include H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, poly (ethylene glycol), acyl, targeting agent, detectable label member. In a presently preferred embodiment, the oxygen of the carboxyl moiety is attached to a detectable label, a therapeutic moiety, or a solid support.

In a further aspect, the invention provides a cleavable linker arm, which is based on a disulfide moiety. Thus, there is provided a compound having the structure of formula III:

by the symbol R, R^I、R^II、R^III、R^IV、R^VAnd R^VIThe atomic group represented is as above R, R^I、R^IIAnd R^IIIThe method is as follows.

Other aspects, advantages and objects of the present invention will become apparent from the following detailed description.

Brief description of the drawings

Figure 1 illustrates an exemplary cleavable urethane linking group of the present invention conjugated to a cytotoxin.

FIG. 2 illustrates exemplary cytotoxins of the present invention.

Figure 3 illustrates an exemplary cleavable disulfide linker of the present invention conjugated to a cytotoxin.

Detailed description of the invention and preferred embodiments

Abbreviations

As used herein, "Ala" means alanine. "Boc" represents t-butyloxycarbonyl. "DDQ" means 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone. The symbol "E" as used herein represents an enzymatically cleavable group. "EDCI" is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide. As used herein, "FMOC" means 9-fluorenylmethoxycarbonyl. "Leu" is leucine. The symbol "PMB" represents p-methoxybenzyl. "TBAF" means tetrabutylammonium fluoride. The abbreviation "TBSO" means t-butyldimethylsilyl ether. "TFA" refers to trifluoroacetic acid. The symbol "Q" represents a therapeutic agent, a diagnostic agent, or a detectable label.

Definition of

Unless defined to the contrary, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Nucleic acid and peptide synthesis was performed using standard procedures. Generally, the enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The procedures and operations are generally performed according to conventional methods in the art and various general references (see generally Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference), which methods are incorporated herein by reference. The nomenclature used herein and the laboratory procedures in analytical chemistry and organic synthesis described below are those well known and commonly employed in the art. Chemical synthesis and chemical analysis were performed using standard procedures or modifications thereof. The term "therapeutic agent" is intended to mean a compound that, when present in a therapeutically effective amount, produces a desired therapeutic effect in a mammal. With respect to the treatment of cancer, it is desirable that the therapeutic agent also be able to enter the target cell.

The term "cytotoxin" is intended to mean a therapeutic agent that has a desired effect of being cytotoxic to cancer cells. Exemplary cytotoxins include, by way of example only and not limitation, combretastatins, duocarmycins, CC-1065 antitumor antibiotics, anthracyclines and related compounds. Other cytotoxins include mycotoxins, ricin and its analogs, calicheamicins, doxirubicin, and maytansinoids.

The term "marker" is intended to mean a compound that can be used for the identification of tumors or other medical condition features, such as diagnosis, progression of tumors, and the determination of factors secreted by tumor cells. Markers are considered a subset of "diagnostic agents".

The term "targeting group" is intended to mean a moiety that (1) is capable of directing an entity (e.g., a therapeutic agent or label) to which it is attached to a target cell, such as a particular type of tumor cell, or (2) is preferentially activated in a target tissue, such as a tumor. The directing group may be a small molecule, which is intended to include non-peptides and peptides. The targeting group can also be a macromolecule, including sugars, lectins, receptors, ligands for receptors, proteins (e.g., BSA), antibodies, and the like.

The term "cleavable group" is intended to mean a moiety that is labile in vivo. Preferably, the "cleavable group" allows the label or therapeutic agent to be activated by cleavage from the remainder of the conjugate. A useful definition is that the linking group is preferably cleaved in vivo by the biological environment. Cleavage can be from any process, e.g., enzymatic, reductive, pH, etc., without limitation. Preferably, the cleavable group is selected such that activation occurs at a desired site of action, which may be a site within or near a target cell (e.g., cancer cell) or tissue, such as a site of therapeutic action or marker activity. Such cleavage is enzymatic cleavage, and exemplary enzymatically cleavable groups include natural amino acids or peptide sequences terminating in natural amino acids, linked at their carboxy terminus to a linker group. Although the degree of enhancement of the cleavage rate is not critical to the present invention, a preferred example of a cleavable linking group is one in which at least about 10% of the cleavable group, and most preferably at least about 35%, is cleaved in the blood stream within 24 hours of administration. Preferred cleavable groups are peptide bonds, ester bonds and disulfide bonds.

The terms "polypeptide", "peptide" and protein "are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. These terms also encompass the term "antibody. "

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ -carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a hydrogen-bonded alpha carbon, carboxyl, amino, and R groups, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds having a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The term "unnatural amino acid" is intended to represent the "D" stereochemical configuration of the above twenty naturally occurring amino acids. It will be further understood that the term unnatural amino acid includes homologs of natural amino acids and synthetically modified forms of natural amino acids. Synthetic modifications include, but are not limited to, amino acids in which the alkylene chain is shortened or lengthened by up to two carbon atoms, amino acids comprising optionally substituted aryl groups, and amino acids comprising halogenated groups, preferably halogenated hydrocarbyl groups and aryl groups. When attached to a linking group or conjugate of the invention, an amino acid is an "amino acidA form of side chain "in which the carboxylic acid group of the amino acid has been replaced by a keto group (C (O)). Thus, for example, the alanine side chain is-C (O) -CH (NH)₂)-CH₃And so on.

"nucleic acid" means deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless indicated to the contrary, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19: 5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-. The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

(symbol)The vertical position, whether used as a bond or showing a bond, indicates the point at which the shown moiety is attached to the rest of the molecule, solid support, etc.

The term "hydrocarbyl" by itself or as part of another substituent means-unless specified to the contrary-a straight or branched chain or cyclic hydrocarbon radical or combination thereof, which may be fully saturated, mono-or poly-unsaturated, may include di-and poly-valent radicals having the specified number of carbon atoms (that is to say C)₁-C₁₀Representing one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated hydrocarbon radicals are those having one or more double or triple bonds. Examples of unsaturated hydrocarbon groups include, but are not limited to, ethenyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and higher homologs and isomers. The term "hydrocarbyl" is meant to include, unless otherwise noted, those hydrocarbyl derivatives defined in detail below, such as "heterohydrocarbyl". "hydrocarbyl groups limited to hydrocarbon groups are also referred to as" homohydrocarbyl ".

The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkane, such as, but not limited to, -CH₂CH₂CH₂CH₂Further included are those groups of the "heteroalkylene" groups described below. Generally, hydrocarbyl (or alkylene) groups will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. "lower alkyl" or "lower alkylene" is a short chain hydrocarbon or alkylene group, typically having eight or fewer carbon atoms.

The term "heterocarbyl" by itself or in combination with another term means-unless specified to the contrary-a stable straight or branched chain or cyclic hydrocarbon radical, or combinations thereof, consisting of the specified number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S, wherein the nitrogen, carbon and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom O, N, S and Si can be located anywhere within the heterohydrocarbyl group or where the hydrocarbyl group is attached to the rest of the molecule. Examples include, but are not limited to-CH₂-CH₂-O-CH₃、-CH₂-CH₂-NH-CH₃、-CH₂-CH₂-N(CH₃)-CH₃、-CH₂-S-CH₂-CH₃、-CH₂-CH₂-S(O)-CH₃、-CH₂-CH₂-S(O)₂-CH₃、-CH＝CH-O-CH₃、-Si(CH₃)₃、-CH₂-CH＝N-OCH₃and-CH ═ CH-N (CH)₃)-CH₃. Up to two heteroatoms may be consecutive, e.g. -CH₂-NH-OCH₃and-CH₂-O-Si(CH₃)₃. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from a heterohydrocarbyl group, such as, but not limited to, -CH₂-CH₂-S-CH₂-CH₂-and-CH₂-S-CH₂-CH₂-NH-CH₂-. With regard to heteroalkylene groups, heteroatoms can also occupy one or both of the chain termini (e.g., hydrocarbylene oxy, hydrocarbylene dioxy, hydrocarbylene amino, hydrocarbylene diamino, and the like). The terms "heterohydrocarbyl" and "heteroalkylene" encompass poly (ethylene glycol) and its derivatives (see, e.g., Shearwater Polymers Catalog, 2001). Further, with respect to alkylene and heteroalkylene linking groups, the direction in which the structural formula of the linking group is written does not imply orientation of the linking group. For example, of the formula-C (O)₂R' -represents-C (O)₂R '-and-R' C (O)₂-。

The term "lower" in conjunction with the term "hydrocarbyl" or "heterohydrocarbyl" denotes a moiety having from 1 to 6 carbon atoms.

The terms "hydrocarbyloxy", "hydrocarbylamino" and "hydrocarbylthio" (or thioalkyloxy) are used in their conventional sense to denote those hydrocarbyl groups attached to the remainder of the molecule via an oxygen atom, an amino group or a sulfur atom, respectively.

In general, "acyl substituent" is also selected from the groups described above. The term "acyl substituent" as used herein means a group attached to and meeting the valence of the carbonyl carbon attached, directly or indirectly, to the polycyclic core of a compound of the invention.

The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic variations of substituted or unsubstituted "alkyl" and substituted or unsubstituted "heteroalkyl", respectively. In addition, with respect to the heterocyclic hydrocarbon group, the heteroatom may occupy the position at which the heterocyclic ring is attached to the rest of the molecule. Examples of cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1, 2, 5, 6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. The heteroatoms and carbon atoms of the cyclic structure are optionally oxidized.

The term "halo" or "halogen" by itself or as part of another substituent means, unless stated to the contrary, a fluorine, chlorine, bromine or iodine atom. In addition, the term "halogenated hydrocarbon group" and the like is meant to include monohalogenated hydrocarbon groups and polyhalogenated hydrocarbon groups. For example, the term "halo (C)₁-C₄) Hydrocarbyl "is meant to include, but is not limited to, trifluoromethyl, 2,2, 2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term "aryl" means, unless specified to the contrary, a substituted or unsubstituted, polyunsaturated, aromatic hydrocarbon substituent which can be a single ring or a plurality of rings (preferably 1 to 3 rings) which are fused together or linked covalently. The term "heteroaryl" denotes an aryl (or ring) containing one to four heteroatoms selected from N, O and S, wherein the nitrogen, carbon and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. The heteroaryl group may be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, etc, Purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalyl, 5-quinoxalyl, 3-quinolyl and 6-quinolyl. The substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. "aryl" and "heteroaryl" also encompass ring systems in which one or more non-aromatic ring systems are fused or bonded to an aryl or heteroaryl system.

For convenience, the term "aryl" when used in conjunction with other terms (e.g., aryloxy, arylthio, arylalkyl) includes aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to include radicals wherein an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like), including alkyl groups wherein a carbon atom (e.g., methylene) has been replaced, for example, by an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl, and the like).

Each of the above terms (e.g., "hydrocarbyl," "heterohydrocarbyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

Substituents for hydrocarbyl and heterohydrocarbyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are generally referred to as "hydrocarbyl substituents" and "heterohydrocarbyl substituents," respectively: they may be one or more of various groups selected from, but not limited to: -OR ', -O, ═ NR ', -N-OR ', -NR ' R ", -SR ', -halogen, -SiR ' R '" -oc (O) R ', -c (O) R ', -CO₂R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R*、-NR”C(O)₂R’、-NR-C(NR’R”R*)＝NR””、-NR-C(NR’R”)＝NR*、-S(O)R’、-S(O)₂R’、-S(O)₂NR’R”、-NRSO₂R', -CN and-NO₂The amount ranges from 0 to (2 m' +1) Wherein m' is the total number of carbon atoms in such radicals. R ', R ", R'" and R "" each preferably independently represent hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy, or arylalkyl. When the compounds of the invention include more than one R group, for example, each R group is independently selected, as are more than one R ', R ", R'", and R "" groups when present. When R' and R "are attached to the same nitrogen atom, they may combine with the nitrogen atom to form a 5-, 6-or 7-membered ring. For example, -NR' R "is meant to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. As will be understood by those skilled in the art from the foregoing discussion of substituents, the term "hydrocarbyl" is meant to include groups in which a carbon atom is bonded to a group other than hydrogen, such as halogenated hydrocarbyl (e.g., -CF)₃and-CH₂CF₃) And acyl (e.g., -C (O) CH)₃、-C(O)CF₃、-C(O)CH₂OCH₃Etc.).

Similar to the substituents described with respect to the hydrocarbyl radical, the substituents for aryl and heteroaryl are generally referred to as "aryl substituents" and "heteroaryl substituents," respectively: and are each different, for example selected from: halogen, -OR ', -O, ═ NR ', -N-OR ', -NR ' R ", -SR ', -halogen, -SiR ' R '" -oc (O) R ', -c (O) R ', -CO₂R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R*、-NR”C(O)₂R’、-NR-C(NR’R”)＝NR*、-S(O)R’、-S(O)₂R’、-S(O)₂NR’R”、-NRSO₂R’、-CN、-NO₂、-R’、-N₃、-CH(Ph)₂Fluoro (C)₁-C₄) Hydrocarbyloxy and fluoro (C)₁-C₄) A hydrocarbyl group in an amount ranging from zero to the total number of open valences on the aromatic ring system; wherein R ', R ", R'" and R "" preferably independently represent hydrogen, (C)₁-C₈) Alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl) - (C)₁-C₄) Hydrocarbyl and (not)Substituted aryl) oxy- (C)₁-C₄) A hydrocarbyl group. When the compounds of the invention include more than one R group, for example, each R group is independently selected, as are more than one R ', R ", R'", and R "" groups when present.

Two aryl substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be substituted by a group of formula-T-C (O) - (CRR')_q-U-substituent, wherein T and U are independently-NR-, -O-, -CRR' -or a single bond, and q is an integer from 0 to 3. Alternatively, two substituents on adjacent atoms of an aryl or heteroaryl ring may be optionally substituted by a group of formula-A- (CH)₂)_r-B-substituent substitution, wherein A and B are independently-CRR' -, -O-, -NR-, -S (O)₂-、-S(O)₂NR' -or a single bond, r is an integer of 1 to 4. One of the single bonds of the new ring thus constituted may optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally substituted by a group of formula- (CRR')_s-X-(CR”R*)_d-substituent substitution, wherein S and d are independently integers from 0 to 3, X is-O-, -NR' -, -S (O)₂-or-S (O)₂NR'. The substituents R, R ', R "and R'" are preferably independently selected from hydrogen or substituted or unsubstituted (C)₁-C₆) A hydrocarbyl group.

The term "heteroatom" as used herein includes oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

The symbol "R" is a general abbreviation representing a substituent selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclyl.

The term "pharmaceutically acceptable salts" includes salts of the active compounds which are prepared using relatively non-toxic acids or bases, depending on the particular substituents on the compounds described herein. When the compounds of the present invention contain relatively acidic functionalities, base addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functionalities, acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydroiodic, or phosphoric acids and the like, as well as salts derived from relatively nontoxic organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like. Also included are Salts of amino acids such as arginine Salts and the like, and Salts of organic acids such as glucuronic acid or galacturonic acid (see, for example, Berge et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the invention contain both basic and acidic functionalities, allowing the compounds to be converted into base or acid addition salts.

The neutral form of the compound is preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of this invention a salt is equivalent to the parent form of the compound.

In addition to salt forms, the present invention provides compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Alternatively, prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir containing a suitable enzyme or chemical agent.

Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in polymorphic or amorphous forms. In general, all physical forms are equivalent in the uses to which the invention pertains and are intended to be within the scope of the invention.

Some of the compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers and individual isomers are all encompassed within the scope of the present invention.

The compounds of the present invention may also contain an isotope of an atom of a non-natural moiety in one or more of the atoms that make up such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as tritium (A), (B), (C), (D), (C), (D³H) Iodine-125 (¹²⁵I) Or carbon-14 (¹⁴C) In that respect All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

The term "linking moiety" or "moiety attached to a directing group" means a moiety that allows the directing group to be attached to a linking group. Typical linking groups include, for illustration only and not by way of limitation, hydrocarbyl, aminohydrocarbyl, aminocarbonylhydrocarbyl, carboxyhydrocarbyl, hydroxyhydrocarbyl, hydrocarbyl-maleimide, hydrocarbyl-N-hydroxysuccinimide, poly (ethylene glycol) -maleimide, and poly (ethylene glycol) -N-hydroxysuccinimide, all of which may be further substituted. The linking group may also be such that the linking moiety is in fact attached to the directing group.

The term "leaving group" as used herein means the portion of the reactant that is cleaved from the reactant in the reaction.

An "antibody" generally refers to a polypeptide comprising a framework region from an immunoglobulin or fragment thereof that specifically binds to and recognizes an antigen. The immunoglobulins identified include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. Kappa or lambda belongs to the light chain. γ, μ, α, δ or ε belongs to the heavy chain, which in turn defines the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) denote these light and heavy chains, respectively.

Antibodies exist, for example, as intact immunoglobulins or as a large number of well-identified fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the hinge region disulfide bond, producing f (ab)' 2, a dimer of Fab, which is itself a light chain linked to VH-CH1 by a disulfide bond. F (ab) ' 2 can decompose under mild conditions, breaking the disulfide bonds of the hinge region, thereby converting the f (ab) ' 2 dimer to Fab ' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (Fundamental Immunology, Paul ed., 3rd ed. 1993). Although various antibody fragments are defined in terms of the digestion of intact antibodies, the skilled artisan will appreciate that such fragments may be synthesized de novo using chemical or recombinant DNA methods. Thus, the term antibody as used herein also includes antibody fragments produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methods (e.g., single chain Fv).

For the preparation of Monoclonal or polyclonal antibodies, any technique known in the art may be used (see, for example, Kohler & Milstein, Nature 256: 495-.

Methods for the production of polyclonal antibodies are known to those skilled in the art. Syngeneically bred mice (e.g., BALB/C mice) or rabbits are immunized with the protein using standard adjuvants, e.g., Freund's adjuvant, and standard immunization protocols. The immune response of the animal to the immunogen preparation is monitored by performing an experimental bleed and determining the reactive titer to the beta subunit. When a suitably high titer of antibody to immunogen is obtained, blood from the animal is collected and antisera are prepared. Further isolation of antisera can be performed, if desired, to enrich for antibodies reactive with the protein.

Monoclonal antibodies can be obtained by various processes familiar to those skilled in the art. Briefly, splenocytes from animals immunized with the desired antigen are immortalized, generally by fusion with myeloma cells (Kohler & Milstein, Eur. J. Immunol.6: 511-519 (1976)). Alternative methods of immortalization include transformation with epstein-barr virus, oncogenes, or retroviruses, or other methods well known in the art.

In a further preferred embodiment, the antibody is a human or humanized antibody. "humanized" refers to non-human polypeptide sequences that have been modified to minimize immunoreactivity to humans, typically by altering the amino acid sequence to mimic existing human sequences, without substantially altering the function of the polypeptide sequence (see, e.g., Jones et al, Nature 321: 522-525(1986), and published UK patent application No. 8707252). "human" antibodies consist entirely of polypeptide sequences derived from human antibody genes, such as may be obtained by phage display or derived from mice genetically modified to contain human immunoglobulin genes.

As used herein, "solid support" refers to a material that is substantially insoluble in the selected solvent system or can be readily separated from the selected soluble solvent system (e.g., by precipitation). Solid supports useful in the practice of the present invention may include activated or activatable groups to allow selected species to bind to the solid support. The solid support may also be a substrate, such as a chip, wafer or well, to which the individual or more than one compound of the invention is bound.

As used herein, "reactive functional group" means a group including, but not limited to, alkenes, alkynes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazos, nitro groups, nitriles, thiols, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acids, isocyanides, amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids, thiohydroxamic acids, propadienes, orthoesters, sulfites, enamines, alkynylamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates, imines, azides, azo compounds, azoxy compounds, and nitroso compounds. Reactive functional groups also include those used to prepare bioconjugates, such as N-hydroxysuccinimide esters, maleimides, and the like (see, e.g., Hermanson, bioconjugate techniques, Academic press, San Diego, 1996). Methods for preparing each of these Functional groups are well known in the art, and their use for a particular purpose or modification according to a particular purpose is within the ability of those skilled in the art (see, e.g., Sandler and karo, eds. organic Functional Group precursors, Academic Press, San Diego, 1989).

The compounds of the present invention are prepared as single isomers (e.g., enantiomers, cis-trans, positional, diastereomers) or mixtures of isomers. In a preferred embodiment, the compounds are prepared as substantially single isomers. Methods for preparing substantially isomerically pure compounds are known in the art. For example, enantiomerically enriched mixtures and pure enantiomeric compounds can be prepared by using enantiomerically pure synthetic intermediates in combination with reactions that maintain the stereochemistry of the chiral center unchanged or that result in its complete inversion. Alternatively, the final product or an intermediate along the synthesis may be resolved into individual stereoisomers. Processes for inverting a particular stereocenter or maintaining it unchanged and processes for resolving stereoisomeric mixtures are in the artIt is well known to the person skilled in the art to select suitable methods according to the particular situation, within his ability. See generally, Furniss et al (eds.), Vogel's encyclopedia of Practical Organic Chemistry 5^th ed.，Longman Scientific andTechnical Ltd.，Essex，1991，pp.809-816；Heller，Acc.Chem.Res.23：128(1990)。

Cytotoxins

Many therapeutic agents, particularly those that are particularly effective for cancer chemotherapy, often exhibit acute in vivo toxicity, especially bone marrow and muscle toxicity, as well as chronic cardiac and neurological toxicity. Such high toxicity limits their use. The development of more and safer specific therapeutic agents, in particular anti-tumor agents, requires greater effectiveness on tumor cells and a reduction in the number and severity of the side effects (toxicity, destruction of non-tumor cells, etc.) of these products.

Research on more selective cytotoxic agents has been extremely active for decades, with dose-limiting toxicity (i.e., undesirable activity of cytotoxins on normal tissues) being one of the major causes of failure of cancer therapy. For example, CC-1065 and duocarmycin are known to be extremely potent cytotoxins. Numerous attempts have been made to evaluate analogs of these compounds; however, many have been shown to exhibit undesirable toxicity at therapeutic doses. Thus, the goal is to improve the specificity of antineoplastic agents, increase the effectiveness against tumor cells, while reducing undesirable side effects such as toxicity and destruction of non-tumor cells.

There has been research focused on developing new therapeutic agents in the form of prodrugs, i.e. compounds that can be converted in vivo into drugs (active therapeutic compounds) by some chemical or enzymatic modification of their structure. For the purpose of reducing toxicity, such transformation is preferably limited to the site of action or target tissue rather than the circulatory system or non-target tissue. However, even prodrugs are problematic because many are characterized by low stability in blood and serum due to the presence of enzymes that degrade or activate the prodrug before it reaches the desired site in the patient.

Thus, despite advances in the art, there remains a need to develop improved therapeutic agents for the treatment of mammals, particularly humans, more specifically cytotoxins and related prodrugs, which exhibit higher specificity of action, reduced toxicity and increased blood stability relative to known compounds of similar structure.

In a first aspect, the present invention provides a cytotoxic compound having the structure of formula I:

wherein ring system a is a member selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl. Exemplary ring systems include phenyl and pyrrole.

The symbols E and G represent H, substituted or unsubstituted hydrocarbyl, substituted or unsubstituted heterohydrocarbyl, a heteroatom or a single bond. E and G are optionally joined to form a ring system selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl.

The symbol X represents a radical selected from O, S and NR²³Is a member of (1). R²³Is a member selected from the group consisting of H, substituted or unsubstituted hydrocarbyl, substituted or unsubstituted heterohydrocarbyl, and acyl.

Symbol R³Represents selected from (═ O), SR¹¹、NHR¹¹And OR¹¹Wherein R is¹¹Is H, substituted or unsubstituted hydrocarbyl, substituted or unsubstituted heterohydrocarbyl, acyl, C (O) R¹²、C(O)OR¹²、C(O)NR¹²R¹³、C(O)OR¹²、P(O)(OR¹²)₂、C(O)CHR¹²R¹³、C(O)OR¹²、SR¹²Or SiR¹²R¹³R¹⁴. Symbol R¹²、R¹³And R¹⁴Independently represent H, substituted or notSubstituted hydrocarbyl, substituted or unsubstituted heterohydrocarbyl and substituted or unsubstituted aryl, wherein R¹²And R¹³Optionally joined together with the nitrogen atom to which they are attached to form a 4-to 6-membered substituted or unsubstituted heterocycloalkyl ring system, optionally containing two or more heteroatoms. One or more R¹²、R¹³Or R¹⁴A cleavable group may be included within its structure.

R⁴And R⁵Is independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted arylalkyl, halogen, NO₂、NR¹⁵R¹⁶、NC(O)R¹⁵、OC(O)NR¹⁵R¹⁶、OC(O)OR¹⁵、C(O)R¹⁵、SR¹⁵And OR¹⁵Is a member of (1). R¹⁵And R¹⁶Independently represent H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, arylalkyl, and substituted or unsubstituted peptidyl, wherein R is¹⁵And R¹⁶Optionally joined together with the nitrogen atom to which they are attached to form a 4-to 6-membered substituted or unsubstituted heterocycloalkyl ring system, optionally containing two or more heteroatoms.

R⁴、R⁵、R¹¹、R¹²、R¹³、R¹⁵And R¹⁶Optionally containing one or more cleavable groups within their structure. Exemplary cleavable groups include, but are not limited to, peptides, amino acids, and disulfides.

In another exemplary embodiment, the invention provides a compound according to formula I, wherein at least one R is⁴、R⁵、R¹¹、R¹²、R¹³、R¹⁵And R¹⁶Comprises the following steps:

wherein R is³⁰Is a member selected from the group consisting of H, substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl. Symbol R³¹And R³²Independently represent H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, or R³¹And R³²Together, are:

wherein R is³³And R³⁴Independently represents H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. Symbol R³⁵Represents a substituted or unsubstituted hydrocarbon group, a substituted or unsubstituted heterohydrocarbon group or NR³⁶。R³⁶Is a member selected from the group consisting of H, substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl. X⁵Is O or NR³⁷Wherein R is³⁷Is a member selected from the group consisting of H, substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl. In a further embodiment, at least one R is³³And R³⁴Is selected from L⁵X⁶Wherein "L" and "X" are generally as described herein.

In an exemplary embodiment, at least one R in the above structure³¹、R³²、R³³And R³⁴Is an aryl or heteroaryl moiety substituted with a moiety that includes a reactive functional group, either protected or unprotected, a directing agent, or a detectable label.

In a further exemplary embodiment, at least one R⁴、R⁵、R¹¹、R¹²、R¹³、R¹⁵And R¹⁶Carrying a reactive group suitable for conjugating a compound according to formula I with another molecule. In a further exemplary embodiment, R⁴、R⁵、R¹¹、R¹²、R¹³、R¹⁵And R¹⁶Independently selected from substituted hydrocarbyl and substituted heterohydrocarbyl, and having a free terminal end of a hydrocarbyl or heterohydrocarbyl moietyA reactive functional group. One or more R⁴、R⁵、R¹¹、R¹²、R¹³、R¹⁵And R¹⁶May be conjugated to another molecule, such as a targeting agent, a detectable label, a solid support, and the like.

As is apparent from the discussion herein, when at least one R is¹⁵And R¹⁶When a reactive functional group, the group may be a component of a bond between a compound according to formula I and another molecule. In exemplary embodiments, at least one R therein¹⁵And R¹⁶Is a bond between a compound of formula I and another molecule, at least one R¹⁵And R¹⁶Is the part that is cleaved by the enzyme.

In a further exemplary embodiment, at least one R⁴And R⁵The method comprises the following steps:

in the above formula, symbol X²And Z¹Represents independently selected from O, S and NR²³Is a member of (1). Radical R¹⁷And R¹⁸Independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, NO₂、NR¹⁹R²⁰、NC(O)R¹⁹、OC(O)NR¹⁹、OC(O)OR¹⁹、C(O)R¹⁹、SR¹⁹OR OR¹⁹。

Symbol R¹⁹And R²⁰Independently represent a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, a substituted or substituted heteroaryl group, a substituted or unsubstituted heterocycloalkyl group, a substituted or unsubstituted peptidyl group, wherein R¹⁹And R²⁰Together with the nitrogen atom to which they are attached optionally form a 4-to 6-membered substituted or unsubstituted heterocycloalkyl ring system, optionally containing two or more heteroatoms, with the proviso that when Z is¹When is NH, R¹⁷And R¹⁸Are not both H, R¹⁷Is not NH₂. Symbol R throughout the specification¹⁹And R²⁰Also encompassed are R⁴And R⁵The group is described. Thus, for example, it is within the scope of the present invention to provide compounds having two or more linked fused phenyl-heterocyclic ring systems or a combination of fused rings and linking groups as just described above. Also, in those embodiments where a linking group is present, the linking group may be represented by R⁴Or R⁵In the form of substituents or R¹⁷Or R¹⁸Substituted forms exist.

R⁶Is a single bond, which may or may not be present. When R is⁶When present, R⁶And R⁷The linkage forms a cyclopropyl ring. R⁷Is CH₂-X¹or-CH₂-. When R is⁷is-CH₂When it is a component of a cyclopropane ring. Symbol X¹Represents a leaving group. The skilled person will understand R⁶And R⁷In a manner that does not violate the principle of valence.

The curve within a six-membered ring indicates that the ring may have one or more unsaturations and may be aromatic. Thus, for example, the following isocyclic structures and related structures are within the scope of formula I:and

in an exemplary embodiment, the ring system a is a substituted or unsubstituted phenyl ring. Ring system a is preferably substituted with one or more aryl substituents as described in the definition section herein. In a preferred embodiment, the phenyl ring is substituted with a CN moiety.

In another exemplary embodiment, the present invention provides a compound having the structure of formula IV:

in this embodiment, the radical R³、R⁴、R⁵、R⁶、R⁷And X is a substituent as described above. The symbols Z are independently selected from O, S and NR²³Is a member of (1). Symbol R²³Represents a member selected from H, substituted or unsubstituted hydrocarbyl, substituted or unsubstituted heterohydrocarbyl and acyl. When X and Z are both NR²³When each R is²³Are independently selected. Symbol R¹Represents H, substituted or unsubstituted lower alkyl or C (O) R⁸。R⁸Is selected from NR⁹R¹⁰、NR⁹NHR¹⁰And OR⁹Is a member of (1). R⁹And R¹⁰Independently selected from H, substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl. Radical R²Is H or substituted or unsubstituted lower alkyl. It is generally preferred when R is²When substituted hydrocarbyl, it is not perfluoroalkyl, e.g. CF₃。

As discussed above, X¹May be a leaving group. Useful leaving groups include, but are not limited to, halides, azides, sulfonates (e.g., hydrocarbyl sulfonyl, arylsulfonyl), oxonium ions, hydrocarbyl perchlorates, ammonium alkanesulfonates, hydrocarbyl fluorosulfonates, and fluoro compounds (e.g., triflate, nonaflatate, tosylate), and the like. The selection of these and other leaving groups suitable for a particular set of reaction conditions is within the ability of those skilled in the art (see, e.g., March J, Advanced Organic Chemistry, 2nd Edition, John Wiley and Sons, 1992; Sandler SR, Karo W, Organic functional group Preparations, 2nd Edition, Academic Press, Inc., 1983; WaderLG, company of Organic Synthetic Methods, John Wiley and Sons, 1980).

In exemplary embodiments, R¹Is an ester moiety, e.g. CO₂CH₃. In a further exemplary embodiment, R²Is lower alkyl, which may be substituted or unsubstituted. The presently preferred lower hydrocarbyl group is CH₃. In a further embodiment, R¹Is CO₂CH₃，R²Is CH₃。

In another exemplary embodiment, R⁴And R⁵Is independently selected from H, halogen, NH₂、O(CH₂)₂N(Me)₂And NO₂Is a member of (1). R⁴And R⁵Preferably not H or OCH₃。

In another exemplary embodiment, the present invention provides compounds having the structures of formulas V and VI:and

in the above formula, X is preferably O; z is preferably O.

The compounds according to formula I may also comprise a peptidyl linker as substituent. The linking group may be located at any desired position on the compound. In exemplary embodiments, at least one R⁴、R⁵、R¹¹、R¹²、R¹³、R^1SAnd R¹⁶Having a structure according to formula VII:

in the discussion that follows, the linking group according to formula VII is, for example, R¹¹As exemplified. The focus of the discussion is merely for clarity, it will be apparent to those skilled in the art that the linking group may be in any position on the compounds of the invention.

In the formula VII, the symbol X³Representing a protected or unprotected reactive functional group, a detectable label or a directing agent. Group L¹And L²Is a linking group selected from substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl. Exemplary linking group L¹And L²Comprising a poly (ethylene glycol) moiety. The linking group may or may not be present, and thus, q and v are integers independently selected from 0 and 1. Symbol AA¹、AA^bAnd AA^b+1Represents natural and unnatural alpha-amino acids. AA¹And AA^bThe dashed line in between indicates that any number of amino acids can be located between two recited amino acids. In exemplary embodiments, the total number of amino acids ("b") in parentheses is from about 0 to about 20. In a further exemplary embodiment, "b" is an integer from about 1 to about 5.

An exemplary linking group according to formula VII is described in formula VIII:

wherein the symbol R²¹And R²²Independently represent substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, a detectable label, and a targeting agent. Radical R¹²And R²⁵Independently selected from the group consisting of H, substituted or unsubstituted lower alkyl, an amino acid side chain, a detectable label, and a targeting agent. In this structure by AA¹、AA^bAnd AA^b+1The amino acid moieties represented are substantially similar to formula VII.

In another embodiment, a compound according to formula I includes a linking group having a structure of formula IX:

wherein the symbol X⁴Representing a protected or unprotected reactive functional group, a detectable label or a directing agent. Symbol L³And L⁴Represents a linking group which is a substituted or unsubstituted hydrocarbyl group or a substituted or unsubstituted heterohydrocarbyl group. The amino acid portion of the linking group is substantially similar to that described for formula VII. Exemplary linking groups include poly (ethylene glycol) analogs within their framework. Each linking group may or may not be present, and thus, p and t are integers independently selected from 0 and 1.

The linking group according to formula IX may be substituted at any position of the molecule according to formula I. In an exemplary embodiment, the rootAccording to formula IX the linking group is selected from R⁴、R⁵、R¹¹、R¹²、R¹³、R¹⁵And R¹⁶Is a member of (1). One skilled in the art will appreciate that the linking group may also be one or more R¹⁷Or R¹⁸Or in a similar position in a higher homologue of a compound of formula I.

Exemplary linking groups according to formula IX are described in formula X:

in the formula X, R²⁷And R²⁸Is a member independently selected from the group consisting of H, substituted or unsubstituted lower alkyl, an amino acid side chain, a detectable label, and a targeting agent. The symbol "s" represents an integer selected to provide a linking group of any desired length. Presently preferred are linking groups wherein "s" is an integer from 0 to 6, more preferably between 1 and 5.

In another exemplary embodiment, the invention provides molecules according to formula I, which are substituted with one or more linking groups comprising a cleavable disulfide moiety in their structure, for example as described in formula XI:

wherein X⁴Is a member selected from the group consisting of protected reactive functional groups, unprotected reactive functional groups, detectable labels and targeting agents. L is³Is a linking group selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl. L is⁴Is a linking group selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl. The symbols p and t represent integers independently selected from 0 and 1.

In an exemplary embodiment according to formula XI, the linking groupL⁴Is a substituted or unsubstituted ethylene moiety.

Group X⁴Is selected from R²⁹、COOR²⁹、C(O)NR²⁹And C (O) NNR²⁹Wherein R is²⁹Is a member selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heteroaryl.

In another exemplary embodiment, R²⁹Is selected from H; OH; NHNH₂；And

wherein R is³⁰Represents a substituted or unsubstituted hydrocarbon group terminating in a reactive functional group, a substituted or unsubstituted heteroaryl group terminating in a functional group and- (L)³)_pX⁴Wherein each L³、X⁴And p are independently selected.

Multivalent compounds of the present invention are also within the scope of the present invention, including, for example, dimers, trimers, tetramers and higher homologs of the compounds of the present invention or reactive analogs thereof. The multivalent species may be assembled from a single or more than one compound of the invention. For example, a dimeric construct may be "homodimeric" or "heterodimeric". Also, multivalent constructs in which a compound of the invention or a reactive analog thereof is attached to an oligomeric or polymeric framework (e.g., polylysine, dextran, hydroxyethyl starch, etc.) are within the scope of the invention. The framework is preferably multifunctional (e.g., having a set of reactive sites for attachment of the compounds of the invention). Furthermore, the framework may be derivatized with a single or more compounds of the invention.

Furthermore, the present invention includes compounds that, when functionalized, provide compounds with enhanced water solubility relative to similar compounds that have not been similarly functionalized. Thus, any of the substituents described herein may be replaced by a similar group that is more water soluble. For example, it is within the scope of the invention to replace the hydroxyl groups with diols, or to replace the amines with quaternary amines, hydroxylamines, or similar moieties that are more water soluble. In preferred embodiments, sites not essential for the activity of the ion channels of the compounds described herein are substituted with moieties that enhance the water solubility of the parent compound, imparting additional water solubility. Methods for enhancing the water solubility of organic compounds are known in the art. Such methods include, but are not limited to, functionalization of the organic core with permanently charged moieties, such as quaternary amines, or with groups that are charged at physiologically relevant pH, such as carboxylic acids, amines. Other methods include the addition of hydroxyl or amine containing groups, such as alcohols, polyols, polyethers, and the like, to the organic core. Representative examples include, but are not limited to, polylysine, polyethyleneimine, poly (ethylene glycol), and poly (propylene glycol). Suitable functionalization chemistries and strategies for these compounds are known in the art. See, for example, Dunn, R.L., et al, eds. polymeric Drugs and Drug delivery systems, ACS Symposium Series Vol.469, American Chemical Society, Washington, D.C. 1991.

Exemplary cytotoxins of the present invention are described in fig. 2.

Prodrugs and cleavable linkers

In addition to the linkers exemplified in detail in the above section that are attached to the cytotoxins of the present invention, the present invention also provides a cleavable linker arm that is suitable for attaching essentially any molecule. The linker arm aspects of the invention are exemplified herein with reference to their attachment to a therapeutic moiety. However, it will be readily apparent to those skilled in the art that the linking group may be attached to different species, including but not limited to diagnostic agents, analytical agents, biomolecules, targeting agents, detectable labels, and the like.

The present invention relates in one aspect to linking groups useful for linking targeting groups to therapeutic agents and labels. The present invention provides, in another aspect, linking groups that confer stability to compounds, reduce their toxicity in vivo, or beneficially affect their pharmacokinetics, bioavailability and/or pharmacodynamics. It is generally preferred that in such embodiments, once the drug is released to its site of action, the linking group is cleaved, releasing the active drug. Thus, in one embodiment of the invention, the linking group of the invention is traceless such that once removed from the therapeutic agent or label (e.g., during activation), there is no longer a trace of the linking group.

In another embodiment of the invention, the linking groups are characterized by their ability to be cleaved at a site within or near the target cell, such as a site of therapeutic action or marker activity. Such cleavage is preferably an enzymatic action in nature. This property helps to reduce systemic activation of the therapeutic agent or marker, reducing toxicity and systemic side effects.

The linking group also serves to stabilize the therapeutic agent or marker against degradation during circulation. This property provides a significant benefit because such stabilization extends the circulatory half-life of the attached drug or marker. The linking group also serves to attenuate the activity of the attached drug or label so that the conjugate is relatively benign in circulation and has the desired effect, e.g., is toxic, upon activation at the desired site of action. With respect to therapeutic agent conjugates, this property of the linking group serves to increase the therapeutic index of the drug.

The stabilizing group is preferably selected to limit clearance and metabolism of the therapeutic agent or marker by enzymes that may be present in the blood or non-target tissue, and is further selected to limit transport of the drug or marker into the cell. The stabilizing group acts to retard the degradation of the drug or label and may also provide other physical characteristics of the drug or label. The stabilizing group may also improve the stability of the drug or label during storage, whether in formulated or unformulated form.

Ideally, if the stabilizing group acts to protect the drug or label from degradation, then when the drug or label is stored in human blood at 37 ℃ for 2 hours, less than 20%, preferably less than 2%, of the drug or label is cleaved by the enzymes present in the human blood under the given assay conditions, indicating that it can be used to stabilize the therapeutic agent or label.

The invention also relates to conjugates containing these linking groups. More specifically, the invention relates to prodrugs that can be used in the treatment of diseases, especially cancer chemotherapy. In particular, the use of linking groups as described herein provides prodrugs with higher specificity of action, reduced toxicity and improved blood stability relative to prodrugs of similar structure.

Thus, the linking group provided may contain as part of its chain any of a variety of groups that will cleave in vivo, for example in the bloodstream, at a higher rate than a construct lacking such groups. Conjugates of the linker arm with therapeutic and diagnostic agents are also provided. The linking group may be used to create prodrug analogs of the therapeutic agent, reversibly linking the therapeutic or diagnostic agent to a targeting agent, detectable label or solid support. The linking group may be incorporated into a complex comprising a cytotoxin of the invention.

In one embodiment, the invention provides a linking group having the general formula set forth in formula II:

in the above formula, the symbol E represents an enzymatically cleavable moiety (e.g., peptide, ester, etc.). Symbol R, R^I、R^IIAnd R^IIIRepresentative, for example, include H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, poly (ethylene glycol), acyl, targeting agent, detectable label member. In exemplary embodiments, the oxygen of the carboxyl moiety is attached to a detectable label, a therapeutic moiety, or a solid support. The carbonyl moiety may be further attached to oxygen, sulfur, nitrogen or carbon at a position where the fragment is truncated. In a further exemplary embodiment, the carbonyl moiety is a component of a urethane. Oxygen attachment targeting agents, cytotoxins, immobilizations attached to a carbonyl moietyBulk carriers, and the like.

Peptide linker

In exemplary embodiments, the enzymatically cleavable group is an amino acid or a peptide sequence terminating in an amino acid linked at its carboxy terminus to the remainder of the linker group. Presently preferred amino acids or peptides are those that are activated by tumors. The tumor-activated peptide is an enzymatically cleavable group that is specifically cleaved at the tumor site. Specific peptides associated with the selected tumor, which are activated by specific enzymes, may be used; a large number of such peptides are known in the art. The amino acids used in the linking group may be natural or unnatural amino acids. In a preferred embodiment, at least one amino acid in the sequence is a natural amino acid. Exemplary preparation of linkers incorporating amino acid moieties the conjugation of Combrestatin to the linkers of the invention is described in scheme 1.

Scheme 1

In scheme 1, EDCI-mediated dehydration coupling of primary amines to t-Boc protected leucine gives protected leucine-amine conjugates. The conjugate was coupled to combrestin activated with p-nitrophenyl carbonate and the product was deprotected by cleavage of the t-Boc group with trifluoroacetic acid.

In another exemplary embodiment, the linking group is a cyclic amino carbamate as described in formula XII.

The radicals in the above formula are essentially the same as those described for the linear linking group. R, R ', R ", and R'" together with the atoms to which they are attached form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl moiety.

Exemplary synthetic routes for the cyclic amino carbamates of the invention are shown in the scheme2, the preparation method is as follows.

Scheme 2

In scheme 2, p-methoxybenzyl protected phenylenediamine is coupled with t-Boc protected leucine on the unprotected aniline nitrogen using EDCI. The p-methoxybenzyl group is removed by the action of DDQ and the linker arm is coupled to Combrestatin using Combrestatin activated with p-nitrophenyl carbonate. The t-Boc group was removed with TFA to give a Combrestin-linker complex.

In another embodiment, there is provided an amino carbamate benzyl alcohol linking group as described in formula XIII below.

The radicals in the above structures are substantially similar to those described above. R "" represents any of the substituents of the aryl moiety discussed above. When more than one R "" group is present, each R "" group is independently selected and z is an integer from 0 to 5.

An exemplary synthesis of the amino carbamate benzyl alcohol linking group of the present invention is depicted in scheme 3.

Scheme 3

In scheme 3, a tert-butyldimethylsilyl O-protected phenol having an activated benzylic position is coupled to a therapeutic or diagnostic agent. The conjugate was treated with tetrabutylammonium fluoride to remove the tert-butyldimethylsilyl protecting group. The free OH groups were converted to the active carbonate with p-nitrophenyl chloroformate. Activated carbonate intermediates for coupling protected BocLeuNH (CH)₂)₂NHEt reacts with OH groups to form a linker-drug conjugate.

Peptide linker-duocarmycin conjugates

CC-1065 and duocarmycins are known to be extremely potent anti-tumor cytotoxins and exhibit undesirable toxicity at therapeutic doses. By linking the peptide activated by the tumor to the cytotoxin, systemic toxicity is reduced and the therapeutic index is increased. Thus, the invention also provides prodrug conjugates of duocarmycins and conjugates between duocarmycins and targeting or other agents of formula I.

An exemplary synthetic scheme for the conjugates of the invention is depicted in scheme 4. Additional synthetic routes are provided in the appended examples.

a: p-nitrophenyl chloroformate; b: beta-Ala (FMOC) -Leu-Ala-Leu-NH (CH)₂)₂NHEt; c: piperidine derivatives

Scheme 4

In scheme 4, the cytotoxin is converted to the active carbonate using p-nitrophenylchloroformate, and the activated derivative is contacted with FMOC-protected, tumor-activated peptide to form a conjugate. The conjugate was treated with piperidine to remove the FMOC group to give the desired compound.

Many peptide sequences that are cleaved by enzymes in serum, liver, intestine, etc. are known in the art. Exemplary peptide sequences of the invention include peptide sequences that are cleaved by proteases. The following focused discussion on the use of protease-sensitive sequences is provided for clarity only and does not serve to limit the scope of the invention.

When the enzyme that cleaves a peptide is a protease, the linker generally comprises a peptide comprising the cleavage recognition sequence of the protease. The cleavage recognition sequence of a protease is a specific amino acid sequence recognized by the protease during protein cleavage. Many protease cleavage sites are known in the art, and these and other cleavage sites may be included in the linker moiety. See, for example, Matayoshi et al, Science 247: 954 (1990); dunn et al, meth.enzymol.241: 254 (1994); seidah et al, meth. enzymol.244: 175 (1994); thornberry, meth. enzymol.244: 615 (1994); weber et al, meth.enzymol.244: 595 (1994); smith et al, meth.enzymol.244: 412 (1994); bouvier et al, meth.enzymol.248: 614(1995), Hardy et al, Amyloid Protein recursor in Development, Aging, and Alzheimer's Disease, ed. masters et al, pp.190-198 (1994).

Proteases have been implicated in cancer metastasis. In many cancers, increased synthesis of the protease urokinase correlates with increased metastatic capacity. Urokinase activates plasminogen to plasmin, which is generally located in the extracellular space, and its activation results in the degradation of proteins in the extracellular matrix, by which metastasizing tumor cells invade. Plasmin can also activate collagenases, promoting the degradation of collagen in the basement membrane surrounding the capillary and lymphatic systems, thereby allowing tumor cells to invade the target tissue (Dano et al, adv. cancer res., 44: 139 (1985)). Thus, it is within the scope of the present invention to employ peptide sequences that are cleaved by urokinase as the linking group.

The invention also provides the use of a peptide sequence that is sensitive to tryptase cleavage. Human mast cells express at least four different tryptases, designated α β I, β II and β III. These enzymes are not controlled by plasma protease inhibitors and cleave only a few physiological substrates in vitro. The tryptase family of serine proteases has been implicated in a variety of allergic and inflammatory diseases involving mast cells, as elevated levels of tryptase are found in biological fluids of patients suffering from these disorders. However, the exact role of tryptase in the pathophysiology of the disease remains to be delineated. The biological function and the corresponding physiological consequences of tryptase are essentially defined by their substrate specificity.

Tryptase is a potent activator of the plasminogen activator of pro-urokinase (uPA), a zymogen form of a protease involved in tumor metastasis and invasion. Activation of the plasminogen cascade, which leads to destruction of the extracellular matrix for extracellular extravasation and migration, may be a function of tryptic activation of the prourokinase plasminogen activator at the P4-P1 sequence Pro-Arg-Phe-Lys (Stack, et al, Journal of Biological Chemistry 269 (13): 9416-9419 (1994)). Vasoactive intestinal peptides, neuropeptides, are involved in the regulation of vascular permeability and are also cleaved by tryptase, mainly in the Thr-Arg-Leu-Arg sequence (Tam, et al, am.J.Respir.cell mol.biol.3: 27-32 (1990)). The receptor PAR-2 coupled to the G protein is capable of being cleaved and activated by tryptase at the Ser-Lys-Gly-Arg sequence, driving fibroblast proliferation, while the receptor PAR-1 activated by thrombin is inactivated by tryptase at the Pro-Asn-Asp-Lys sequence (Molino et al, Journal of Biological Chemistry 272 (7): 4043-. Taken together, this evidence suggests a central role for tryptase in tissue remodelling as a consequence of disease. This is consistent with the profound changes observed in several mast cell mediated disorders. One indication of chronic asthma and other long-term respiratory diseases is fibrosis and thickening of diseased tissue, which may be the result of activation of its physiological targets by tryptase. Similarly, a series of reports have shown that angiogenesis is associated with mast cell density, tryptase activity and poor prognosis in various cancers (Coissens et al, Genesand Development 13 (11): 1382-97 (1999)); takanami et al, Cancer 88 (12): 2686-92 (2000); Toth-Jakatics et al, Human Pathology 31 (8): 955- > 960 (2000); ribatti et al, International Journal of Cancer 85 (2): 171-5(2000)).

Methods for assessing whether a particular protease cleaves a selected peptide sequence are known in the art. For example, the use of 7-amino-4-methylcoumarin (AMC) fluorescent peptide substrates is a well established method for protease-specific assays (Zimmerman, M., et al, (1977) analytical biochemistry 78: 47-51). Specific cleavage of the N-acylamide linkage releases the fluorescent AMC leaving group, allowing simple determination of the cleavage rate of individual substrates. Recently, N-terminal specificity of proteases has been rapidly mapped by taking extensive samples in a single experiment using a panel of AMC peptide substrate libraries (Lee, D., et al, (1999) Bioorganic and Medicinal Chemistry Letters 9: 1667-72) and position-scanning libraries (Rano, T.A., et al, (1997) Chemistry and Biology 4: 149-55). Thus, one skilled in the art can readily evaluate a set of peptide sequences to determine their utility in the present invention without resorting to undue experimentation.

Disulfide linking group

In a further aspect, the invention provides cleavable linker arms based on disulfide moieties. Thus, there is provided a compound having the structure of formula III:by the symbol R, R^I、R^II、R^III、R^IV、R^VAnd R^VIThe atomic group represented is R, R above^I、R^IIAnd R^IIIThe method is as follows.

In another embodiment, the invention provides disulfide carbamate linking groups, for example as described in formula XIV:

by the symbol R, R^I、R^II、R^III、R^IV、R^VAnd R^VIThe radicals represented are as described above.

As discussed above, the linking groups of the present invention may be used to generate conjugates that include a cytotoxin as a therapeutic agent, such as combretastatin or duocarmycin. duocarmycins are unstable in plasma. The linker of the invention finds particular utility in stabilising duocarmycins in circulation, releasing the drug once the desired site of action is reached (activation). In addition, both the linking group and the directing group are preferably removed in order to obtain again the maximum activity of combretastatin or duocarmycin after activation. Thus, in one embodiment of the invention, the linking group is a traceless linking group. Conjugates comprising a cytotoxin, such as duocarmycin, as a therapeutic agent are also of particular interest. The linker of the invention serves to stabilize duocarmycin in circulation and to release the optimally potent cytotoxin in or near the target cell after activation. Since the cytotoxin is cleaved within or near the target cell, systemic toxicity due to random activation is reduced. Furthermore, the increased circulatory stability also increases the half-life and overall effectiveness of the cytotoxin.

An exemplary route for preparing the disulfide linker arm-cytotoxin conjugates of the invention is depicted in scheme 5.

Scheme 5

In scheme 5, the amine-protected thiol a is reacted with 2, 2' -dipyridyl disulfide b to form an amine-protected, activated disulfide c. Contacting the activated disulfide with a carboxylic acid ester bearing a free sulfhydryl group, d, to eliminate the pyridyl thiol, to produce an amine-protected carboxylic acid ester including the disulfide moiety, e. The methyl ester is cleaved by the action of LiOH to give the corresponding carboxylic acid f. The carboxylic acid is coupled to a heterobifunctional PEG molecule, comprising a maleimide group and an amine, by the action of EDCI, to yield compound g. The PEG derivative is contacted with the activated carbonate h of combretastatin to form the conjugate i. If desired, conjugate i may be attached to a targeting agent, detectable label, or the like, via a maleimide moiety.

In another embodiment, the present invention provides a disulfide carbamate linking group, wherein the non-carbonyl oxygen of the urethane linkage is derived from an aryl group. Representative linking groups of the present invention are described by formula XV:

the radicals are essentially as described above. R^VIIIs an aryl substituent as described in the definition section. The symbol w represents an integer of 0 to 4. When more than one R is present^VIIWhen each group is independently selected.

An exemplary route for the compound of formula XV is depicted in scheme 6.

Scheme 6

In scheme 6, TBS-alcohol protected benzyl bromide derivatives are reacted with Q-OH under alkylation conditions to give b. Compound b is deprotected by tetrabutylammonium fluoride to give c, acylated with d to give carbonate e. Reacting carbonate e with heterobifunctional PEG derivative i from scheme 5 above. The resulting PEG adduct f can be conjugated to another molecule through a maleimide moiety.

Disulfide linker-duocarmycin conjugates

As discussed above, the disulfide linker of the invention is also a useful component for stabilizing therapeutic or diagnostic moieties, generating prodrugs and conjugating drugs with targeting agents and detectable labels in vivo. Thus, the present invention provides in a further aspect a conjugate between duocarmycin and a disulfide linker of formula I of the present invention. Scheme 7 provides a facile route to the conjugates of the invention.

Scheme 7

In scheme 7, duocarmycin cytotoxin a of the present invention is converted to activated carbonate b using p-nitrophenyl chloroformate. Compound b is coupled to the heterobifunctional PEG linker from scheme 5 to produce compound d, which can then be coupled to an antibody via a maleimide-thiol coupling reaction to produce conjugate e.

As discussed above, the therapeutic efficacy of certain toxic agents is dramatically enhanced by a strategy that selectively releases the drug to the desired site and/or maintains the essentially inactive form of the drug until it is released to the desired site of action. The invention also provides linker arms which function by directing the drug to a selected site and/or inactivating the biologically active drug until it reaches the desired site.

Thus, in certain embodiments, the invention provides conjugates of the above cytotoxins and other drugs with a linker arm having useful properties. In one embodiment, the linker arm is conjugated to the therapeutic or diagnostic moiety with an agent that selectively releases the former to a desired site in vivo. The linking group between the moiety and the targeting agent may be stable in vivo or may be cleaved. If the agent is cleaved, it is preferably cleaved predominantly after reaching the desired site of action.

In another embodiment, the invention provides a linker that does not link a diagnostic or therapeutic moiety to another agent, but essentially inactivates the moiety until it reaches the desired active site; the reactive species cleaves the linker at the desired reactive site, preferably restoring the reactive form of the moiety. This strategy provides a means to mitigate the systemic toxicity of many toxic and very useful drugs.

The urethane and disulfide linkers of the present invention are exemplified in conjugation with representative duocarmycin analogs of the present invention. See fig. 1 and 3, respectively.

Orientation agent

The linker arms and cytotoxins of the invention can be linked to targeting agents that selectively release the cargo to a cell, organ or body region. Exemplary targeting agents, such as antibodies (e.g., chimeric, humanized and human antibodies), ligands for receptors, lectins, sugars, antibodies, and the like, are recognized in the art and can be used in the practice of the invention without limitation. Other targeting agents include a class of compounds that do not include specific molecular recognition motifs, including macromolecules such as poly (ethylene glycol), polysaccharides, polyamino acids, and the like, which increase the molecular mass of the cytotoxin. The additional molecular mass affects the pharmacokinetics of the cytotoxin, such as serum half-life.

In an exemplary embodiment, the invention provides a cytotoxin, a linker, or a conjugate of a cytotoxin-linker and a targeting agent, which is a biomolecule, such as an antibody, a receptor, a peptide, a lectin, a sugar, a nucleic acid, or a combination thereof. Exemplary conjugate routes of the invention are described in the schemes above.

Biomolecules useful in the practice of the present invention may be derived from any source. Biomolecules can be isolated from natural sources or can be prepared synthetically. The protein may be a native protein or a mutated protein. Mutagenesis may be performed by chemical mutagenesis, site directed mutagenesis, or other means of inducing mutations known to those skilled in the art. Proteins useful in the practice of the present invention include, for example, enzymes, antigens, antibodies, and receptors. The antibody may be polyclonal or monoclonal. Peptides and nucleic acids may be isolated from natural sources, or may be otherwise wholly or partially synthetic.

In those embodiments in which the recognition moiety is a protein or antibody, the protein may be linked to the SAM component or spacer arm through any available reactive peptide residues on the protein surface. In a preferred embodiment, the reactive group is an amine or a carboxylic acid ester. In a particularly preferred embodiment, the reactive group is the epsilon-amine group of a lysine residue. Furthermore, these molecules can be adsorbed on the surface of the substrate or SAM by non-specific interactions (e.g., chemisorption, physisorption).

Antibody recognition moieties can be used to recognize analytes, which are proteins, peptides, nucleic acids, sugars, or small molecules, such as drugs, herbicides, pesticides, industrial chemicals, and ammunition. Methods for eliciting the activity of antibodies against specific molecules are well known to those skilled in the art. See U.S. patent No.5/147,786 issued to Feng et al, dated 15, 9, 1992; U.S. patent No.5/334,528 issued to Stanker et al, 8/2, 1994; U.S. patent No.5/686,237 issued 11.1997 to Al-bayti, m.a.s. and U.S. patent No.5/573,922 issued 11.12.1996 to Hoess et Al. Methods for attaching antibodies to surfaces are also known in the art. See Delamarche et al, Langmuir 12: 1944-1946(1996).

The directing agent may be attached to the linking group of the present invention through any available reactive group. For example, the peptide may be attached via an amine, carboxyl, thiol or hydroxyl group. Such a group may be located at the end of the peptide chain or at an internal position. Nucleic acids can be attached via reactive groups on bases (e.g., exocyclic amines) or available hydroxyl groups (e.g., 3 '-or 5' -hydroxyl groups) on sugar moieties. Peptide and nucleic acid strands may further be derivatized at one or more positions to attach appropriate reactive groups on the strands. See Chrisey et al, Nucleic Acids Res.24: 3031-3039(1996).

When the peptide or nucleic acid is a fully or partially synthesized molecule, reactive groups or masked reactive groups may be incorporated during the synthesis process. Many derivatized monomers suitable for binding reactive groups in peptides and nucleic acids are known to those skilled in the art. See, for example, The Peptides: analysis, Synthesis, Biology, vol.2: "Special Methods in peptide Synthesis", Gross, E.and Meleneffer, J., eds., academic Press, New York (1980). Many useful monomers are commercially available (Bachem, Sigma, etc.). This masked group can then be removed after synthesis, at which time it can be reacted with a compound component of the invention.

In another exemplary embodiment, the directing group is attached to the compound of the present invention via an inclusion complex. For example, a compound or linking group of the invention may include a moiety such as a cyclodextrin or a modified cyclodextrin. Cyclodextrins are a class of cyclic oligosaccharides produced by a large number of microorganisms. Cyclodextrins have a ring structure with a basket-like shape. This shape allows cyclodextrins to include a wide variety of molecules in their internal cavity. See, for example, Szejtli, j., cyclodexinsand Their Inclusion Complexes; akadeniai Klado, Budapest, 1982; and Bender et al, Cyclodextrin Chemistry, Springer-Verlag, Berlin, 1978.

Cyclodextrins are capable of forming inclusion complexes with a group of organic molecules, including, for example, pharmaceuticals, pesticides, herbicides, and ammunition. See renjarla et al, j.pharm.sci.87: 425-429 (1998); zughul et al, pharm. 43-53(1998) and Albers et al, Crit. Rev. Ther. drug Carrier Syst.12: 311-337(1995). Importantly, cyclodextrins are able to distinguish the enantiomers of a compound in their inclusion complexes. Thus, in a preferred embodiment, the present invention provides for the detection of a specific enantiomer in a mixture of enantiomers. See Koppenhoefer et al j.chromanogr.a 793: 153-164(1998). Numerous routes for linking cyclodextrins with other molecules are known in the art. See, e.g., Yamamoto et al, j.phys.chem.b 101: 6855-6860 (1997); and Sreenivasan, k.j.appl.polym.sci.60: 2245-2249(1996).

The cytotoxin-targeting agent conjugates of the invention are further e.g. antisense oligonucleotide-cytotoxin conjugates. Focusing on the cytotoxin-oligonucleotide conjugates is for clarity of illustration only and does not limit the scope of targeting agents that can be conjugated to the cytotoxins of the present invention.

Exemplary nucleic acid targeting agents include aptamers, antisense compounds, and nucleic acids that make up a triple helix. Typically, the hydroxyl group of the sugar residue, the amino group from the base residue, or the phosphate oxygen of the nucleotide serves as the necessary chemical functionality to couple the nucleotide targeting agent to the cytotoxin. However, one skilled in the art will readily appreciate that other "non-native" reactive functionalities can be attached to nucleic acids using conventional processes. For example, the hydroxyl group of a sugar residue can be converted to a sulfhydryl group or an amino group using procedures well known in the art.

Aptamers (or nucleic acid antibodies) are single-or double-stranded DNA or single-stranded RNA molecules that bind to a specific molecular target. In general, aptamers function by inhibiting the action of molecular targets, such as proteins, binding to a pool of targets circulating in the blood. Aptamers have chemical functionality and are therefore capable of covalent bonding to cytotoxins, as described herein.

Although various molecular targets are capable of forming non-covalent and specific associations with aptamers, including small molecule drugs, metabolites, cofactors, toxins, carbohydrate drugs, nucleotide drugs, glycoproteins, and the like, in general, molecular targets will comprise proteins or peptides, including serum proteins, kinins, eicosanoids, cell surface molecules, and the like. Examples of aptamers include Gilead's antithrombin inhibitor GS 522 and its derivatives (Gilead Science, Foster City, Calif.). See also Macaya et al proc.natl.acad.sci.usa 90: 3745-9 (1993); bock et al, Nature (London) 355: 564-566(1992) and Wang et al, biochem.32: 1899-904(1993).

Aptamers specific for a given biomolecule can be identified using techniques known in the art. See, e.g., Toole et al, (1992) PCT publication No. WO 92/14843; tuerk and gold (1991) PCT publication No. WO 91/19813; PCT publication No. WO 92/05285 and Ellington and Szostak, Nature 346: 818(1990). Briefly, these processes generally involve the complexation of molecular targets with random mixtures of oligonucleotides. The aptamer-molecular target complex is separated from uncomplexed oligonucleotides. Recovering the aptamer from the separated complex, and amplifying. This cycle is repeated to identify the aptamer sequence with the highest affinity for the molecular target.

With respect to diseases caused by inappropriate expression of genes, specifically preventing or reducing expression of such genes represents an ideal therapy. In principle, the production of a particular gene product can be inhibited, reduced or cleaved by hybridization of single-stranded deoxynucleotides or ribonucleotides that are complementary to sequences within the mRNA that are available or within the transcript necessary for pre-mRNA processing, or complementary to sequences within the gene itself. This paradigm of genetic control is often referred to as antisense or antigene suppression. Conjugation of alkylating agents to nucleic acids confers additional efficacy, such as those alkylating agents of the present invention.

Antisense compounds are nucleic acids designed to bind to, disable, or prevent the production of mRNA, which is responsible for the production of a particular protein. Antisense compounds include antisense RNA or DNA, single-or double-stranded, oligonucleotides or analogs thereof, which are capable of specifically hybridizing to individual mRNAs, preventing transcription and/or RNA processing of the mRNAs and/or translation of the encoded polypeptides, thereby reducing the amount of the respective encoded polypeptide (Ching et al, Proc. Natl. Acad. Sci. U.S.A.86: 10006-10010 (1989); Broder et al, Ann. int. Med.113: 604-618 (1990); Loreau et al, FEBS Letters 274: 53-56 1990); Holcberg et al, WO 91/11535; WO 91/09865; WO 91/04753; WO 90/13641; WO 91/13080; WO 91/06629 and EP 386563). Due to their exquisite target sensitivity and selectivity, antisense oligonucleotides can be used to release therapeutic agents to a desired molecular target, such as the cytotoxins of the present invention.

Others have reported that nucleic acids are capable of binding to double-stranded DNA via triple-helix formation, inhibiting transcription and/or DNA synthesis. Triple-helical compounds (also known as triple-stranded drugs) are oligonucleotides which bind to sequences of double-stranded DNA and are intended to selectively inhibit the transcription of pathogenic genes, such as viral genes, for example HIV and herpes simplex virus, and oncogenes, that is to say they terminate protein production on the nucleus. These drugs bind directly to double-stranded DNA in the genome of the cell, forming triple helices, preventing the cell from making the target protein. See, for example, PCT publication Nos. WO 92/10590, WO 92/09705, WO 91/06626, and U.S. Pat. No.5,176,996. Thus, the cytotoxins of the present invention are also conjugated to nucleic acid sequences that make up the triple helix.

The site specificity of nucleic acids (e.g., antisense compounds and triple helix drugs) is not significantly affected by modification of the phosphodiester bond or chemical modification of the oligonucleotide termini. Thus, these nucleic acids can be chemically modified; enhance overall binding stability, increase stability with respect to chemical degradation, increase the rate of transport of the oligonucleotide into the cell, confer chemical reactivity to the molecule. The general methods for constructing various nucleic acids useful for antisense therapy have been summarized in van der krol et al, Biotechniques 6: 958-976(1988) and Stein et al, Cancer Res.48: 2659-2668(1988). Thus, in exemplary embodiments, the cytotoxins of the present invention are conjugated to nucleic acids through modification of the phosphodiester linkage.

Furthermore, aptamers, antisense compounds and triple helix drugs carrying the cytotoxins of the present invention may also include substitution, addition, deletion or displacement of nucleotides, as long as the functional properties of the oligonucleotide remain as a result of specific hybridization or association with the target sequence of interest. For example, some embodiments will employ phosphorothioate analogs that are more resistant to degradation by ribozymes than their naturally occurring phosphodiester counterparts and thus are expected to have greater in vivo persistence and greater potency (see, e.g., Campbell et al, J.biochem. Biophys. methods 20: 259-267 (1990)). Phosphoramidate derivatives of oligonucleotides are also known to bind complementary polynucleotides, having the ability to otherwise accommodate covalently linked ligands, and would be suitable for use in the methods of the invention. See, for example, Froehler et al, Nucleic Acids Res.16 (11): 4831(1988).

In some embodiments, aptamers, antisense compounds, and triple helix drugs will comprise O-methyl ribonucleotides (EP publication No. 360609). Chimeric oligonucleotides (Dagle et al, Nucleic Acids Res.18: 4751(1990)) may also be used. For some applications, antisense oligonucleotides and triple helices may comprise polyamide nucleic acids (Nielsen et al, Science 254: 1497(1991) and PCT publication No. WO 90/15065) or other cationic derivatives (Letsinger et al, J.Am.chem.Soc.110: 4470-4471 (1988)). Other applications may utilize oligonucleotides in which one or more phosphodiester bonds have been replaced by an isosteric group, for example an internucleotide linkage of 2-4 atoms in length, as described in PCT publication Nos. WO 92/05186 and 91/06556, or by a formacetal group (Matteucci et al, J.Am.chem.Soc.113: 7767-7768(1991)) or an amide group (Nielsen et al, Science 254: 1497-1500 (1991)).

In addition, nucleotide analogs may be employed in the present invention, for example, where the sugar or base is chemically modified. "analog" forms of purines and pyrimidines are those well known in the art, and many of them are useful as chemotherapeutic agents. An exemplary but non-exhaustive list includes 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl-uracilPyrimidine, dihydrouracil, inosine, N⁶Isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5' -methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N⁶-isopentenyladenine, uracil-5-oxoacetic acid methyl ester, uracil-5-oxoacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxoacetic acid methyl ester, uracil-5-oxoacetic acid (v), pseudouracil, queosine, 2-thiocytosine, and 2, 6-diaminopurine. In addition, conventional bases such as halogenated bases. In addition, the 2' -furanose position on the base may be substituted with an uncharged bulky group. Examples of uncharged bulky groups include branched hydrocarbyl groups, sugars, and branched sugars.

Terminal modifications also provide useful processes for conjugating cytotoxins to nucleic acids, modifying the cell type specificity, pharmacokinetics, nuclear permeability, and absolute cellular uptake rate of oligonucleotide drugs. For example, substitution at the 5 'and 3' ends to include reactive groups is known, which allows for covalent attachment of cytotoxins. See, for example, Oligodeoxynucleotides: AntisenseInhibitors of Gene Expression, (1989) Cohen, Ed., CRC Press; prospectra for Antisense Nucleic acids Therapeutics for Cancer and IDS, (1991), Wickstrom, Ed., Wiley-Liss; gene Regulation: biologyof Antisense RNA and DNA (1992), Erickson and IZANT, eds., ravenPress and Antisense RNA and DNA (1992), Murray, Ed., Wiley-Liss. For a general approach involving Antisense compounds, see Antisense RNA and DNA, (1988), d.a. melton, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, n.y.

Targeting agents are typically coupled to the cytotoxin via a covalent bond. The covalent bond may be irreversible, partially reversible, or fully reversible. The degree of reversibility is comparable to the susceptibility of the targeting agent-cytotoxin complex to degradation in vivo.

In preferred embodiments, the linkage is reversible (e.g., readily cleavable) or partially reversible (e.g., partially or slowly cleavable). Cleavage of the bond may occur by biological or physiological processes. The physiological/biological process cleaves the bond at any selected position within the complex (e.g., removes an ester group or other protecting group coupled to a different sensitive chemical functionality), followed or preceded by cleavage of the bond between the cytotoxin and the linking group, resulting in a partially degraded complex. Other cleavage may also occur, for example between the linking group and the directing agent.

For rapid degradation of the complex after administration, it is generally relied upon that enzymes (e.g., amidases, reductases) circulating in the plasma cleave the dendrimer on the drug. These enzymes may include non-specific aminopeptidases and esterases, dipeptidyl carboxypeptidases, proteases of the coagulation cascade, etc.

Alternatively, cleavage is performed by a non-enzymatic process. For example, the pH difference experienced by the complex after release may initiate chemical hydrolysis. In such a case, the complex may be characterized by a high degree of chemical instability at physiological pH 7.4, while exhibiting greater stability at acidic or basic pH in the delivery vehicle. An exemplary complex that is cleaved in such a process is one having a N-Mannich base bond incorporated in its framework.

In most cases, cleavage of the complex will occur during or shortly after administration. However, in other embodiments, cleavage does not occur until the complex reaches the site of action of the drug.

The susceptibility of the cytotoxin-targeting agent complex to degradation can be determined by studies of hydrolytic or enzymatic conversion of the complex to unbound drug. In general, a good correlation between in vitro and in vivo activity was found using this method. See, for example, philips et al, j.pharm.sciences 78: 365(1989). The rate of conversion is readily determined, for example, by spectrophotometry or gas-liquid or high performance liquid chromatography. Half-life and other kinetic parameters can then be calculated using standard procedures. See, for example, Lowry et al, mechanics and Theory in Organic Chemistry, 2nd Ed., Harper & Row, Publishers, New York (1981).

Spacer group ("Lx")

In addition to the cleavable group, one or more linking groups are optionally introduced between the cytotoxin and the targeting agent. The spacer group contains at least two reactive functional groups. Typically, one chemical functionality of the spacer group is bonded to a chemical functionality of the cytotoxin, while another chemical functionality of the spacer group is used to bond to a chemical functionality of the targeting agent or cleavable linking group. Examples of chemical functionalities of the spacer group include hydroxyl, thiol, carbonyl, carboxyl, amino, ketone, and thiol. The spacer group may also be a component of the cleavable linking group, in which case it is generally denoted Lx, where "x" is an integer.

The linking group represented by Lx is typically a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, or a substituted or unsubstituted heteroalkyl group.

Exemplary spacer groups include, for example, 6-aminohexanol, 6-mercaptohexanol, 10-hydroxydecanoic acid, glycine and other amino acids, 1, 6-hexanediol, beta-alanine, 2-aminoethanol, cysteamine (2-aminoethylene glycol), 5-aminopentanoic acid, 6-aminocaproic acid, 3-maleimidobenzoic acid, o-hydroxymethylbenzoate, alpha-substituted o-hydroxymethylbenzoate, carbonyl, aminal esters, nucleic acids, peptides, and the like.

The spacer group can serve to introduce additional molecular mass and chemical functionality to the cytotoxin-targeting agent complex. In general, the additional mass and functionality will affect the serum half-life and other properties of the complex. Thus, by careful selection of the spacer group, cytotoxic complexes having a range of serum half-lives can be prepared.

Reactive functional group

For clarity, the subsequent discussion focuses on the conjugation of the cytotoxins of the present invention with targeting agents. Taking one embodiment of the present invention as an example, other embodiments can be easily derived by those skilled in the art. The discussion is focused on a single embodiment and does not limit the invention.

Exemplary compounds of the invention carry a reactive functional group, which is typically located in a substituted or unsubstituted hydrocarbyl or heterohydrocarbyl chain, allowing them to be readily attached to another group. The usual position of the reactive group is the terminal position of the chain.

Reactive groups and reactive species useful in the practice of the present invention are generally those well known in the art of bioconjugate chemistry. The types of reactions currently favored that can be performed with reactive cytotoxic analogs are those performed under relatively mild conditions. They include, but are not limited to, nucleophilic substitutions (e.g., reaction of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions), and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reactions, Diels-Alder additions). These and other useful reactions are discussed, for example, in March, Advanced organic chemistry, 3rd ed., John Wiley & Sons, New York, 1985; hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996 and Feeney et al, Modification of Proteins, Advances in Chemistry Series, Vol.198, American Chemical Society, Washington, D.C., 1982.

Exemplary reaction types include the reaction of carboxyl groups and their various derivatives including, but not limited to, N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acyl halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters. The hydroxyl group can be converted into an ester, an ether, an aldehyde, etc. The halogenated hydrocarbon groups are converted into new groups, for example by reaction with amines, carboxylic acid anions, thiol anions, carbanions or alcoholate ions. Dienophile (e.g., maleimide) groups participate in the Diels-Alder reaction. The aldehyde or ketone group can be converted to an imine, hydrazone, semicarbazone or oxime, or via a Grignard addition or alkyllithium addition mechanism, among other mechanisms. The sulfonyl halides readily react with amines, for example to form sulfonamides. The amine or thiol group is, for example, acylated, alkylated or oxidized. Olefins can be converted to a new group of compounds using cycloaddition, acylation, Michael addition, and the like. Epoxides react readily with amines and hydroxyl compounds.

Those skilled in the art will readily appreciate that many of these keys can be generated in a variety of ways and with a variety of conditions. For the preparation of esters, see, e.g., March, supra, 1157; for the preparation of thioesters, see March, supra, 362-363, 491, 720-722, 829, 941, and 1172; for the preparation of carbonates, see March, supra, 346-; for the preparation of carbamates, see March, supra, 1156-57; for the preparation of amides, see March, supra, 1152; for the preparation of ureas and thioureas, see March, supra, 1174; for the preparation of acetals and ketals, see Greene et al, supra, 178-210 and March, supra, 1146; for the preparation of acyloxyalkyl derivatives see produgs: topical and Ocular Drug Delivery, K.B.Sloan, ed., Marcel Dekker, Inc., New York, 1992; for the preparation of enol esters, see March, supra, 1160; for the preparation of N-sulfonimide acid salts, see Bundgaard et al, j.med.chem., 31: 2066 (1988); for anhydride preparation, see March, supra, 355-56, 636-37, 990-91, and 1154; for the preparation of N-acyl amides, see March, supra, 379; for the preparation of N-Mannich bases, see March, supra, 800-02 and 828; for the preparation of hydroxymethyl ketoesters, see Petracek et al, Annals NY acad.sci., 507: 353-54 (1987); for disulfide preparation see March, supra, 1160; the preparation of phosphonates and aminophosphonates.

The reactive functional groups may be selected such that they do not participate in or interfere with the reactions necessary to assemble the reactive autoinducer analogs. Alternatively, the reactive functional group may be protected from the reaction by the presence of a protecting group. One skilled in the art will understand how to protect a particular functional group from interfering with a selected set of reaction conditions. For examples of useful protecting Groups, see Greene et al, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

Typically, the targeting agent is covalently linked to the cytotoxin through their respective chemical functionalities using standard chemical techniques. Optionally, the dendrimer or drug is coupled to the agent via one or more spacer groups. The spacer groups, when used in combination, may be the same or different.

Generally, at least one chemical functional group will be activated prior to the formation of a bond between the cytotoxin and the targeting agent (or other agent) and optional spacer group. One skilled in the art will appreciate that various chemical functional groups, including hydroxyl, amino, and carboxyl groups, can be activated using various standard methods and conditions. For example, the hydroxyl group of the cytotoxin or directing agent may be activated by treatment with phosgene to form the corresponding chloroformate, or by treatment with p-nitrophenyl chloroformate to form the corresponding carbonate.

In an exemplary embodiment, the present invention employs a directing agent that includes carboxyl functionality. The carboxyl group can be activated, for example, by conversion to the corresponding acid halide or active ester. The reaction can be carried out under various conditions, as described by March, supra, pp.388-89. In an exemplary embodiment, the acid halide is prepared by reaction of a carboxyl-containing group with oxalyl chloride. The activated agent is reacted with a cytotoxin or a combination of cytotoxin-linker arms to produce a conjugate of the invention. Those skilled in the art will appreciate that the use of carboxyl-containing targeting agents is merely illustrative and that agents having a wide variety of other functional groups can be conjugated to the dendrimers of the present invention.

When the compound of the invention is conjugated to a detectable label, the label is preferably a member selected from the group consisting of: radioisotopes, fluorescers, fluorescer precursors, chromophores, enzymes, and combinations thereof. Methods for conjugating various groups to antibodies are well known in the art. For example, a detectable label often conjugated to an antibody is an enzyme, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose oxidase.

Detectable label

The particular label or detectable group used in the compounds and methods of the invention is generally not critical to the invention, so long as it does not significantly interfere with the activity or utility of the compounds of the invention. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels are well established in the field of immunoassays, and in general most any label useful in such methods can be adapted for use in the present invention. Thus, a label is any composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the present invention include magnetic beads (e.g., DYNABAEDS), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, etc.), radioactive labels (e.g., fluorescent dyes, fluorescein isothiocyanate, etc.), radioactive labels, etc³H、¹²⁵I、³⁵S、¹⁴C or³²P), enzymes (e.g. horseradish peroxidase, alkaline phosphatase and other enzymes commonly used in ELISA), and colorimetric labels, such as colloidal gold or coloured glass or plastic beads (e.g. polystyrene, polypropylene, latex etc.).

Labels may be coupled directly or indirectly to the compounds of the invention according to methods well known in the art. As indicated above, a variety of labels can be used, the choice of label depending on the sensitivity desired, ease of conjugation with the compound, stability requirements, available instrumentation and processing rules.

The non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bonded to the conjugate component. The ligand is then bound to another molecule (e.g., streptavidin) that is otherwise detectable or covalently bonded to a signaling system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.

The components of the conjugates of the invention may also be conjugated directly to a signal-generating compound, for example to an enzyme or fluorophore. The enzyme concerned as a label will be primarily a hydrolase, specifically a phosphatase, esterase or glycosidase, or an oxidase, specifically a peroxidase. The fluorescent compound includes fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2, 3-dihydrophthalazinedione, such as luminol. For a review of the various marking systems or signal generating systems that may be used, see U.S. patent No.4,391,904.

Means for detecting the label are well known to those skilled in the art. Thus, for example, where the label is a radioactive label, the detection means may comprise a scintillation counter or a photographic film, as in autoradiography. If the label is a fluorescent label, it can be detected by exciting a fluorescent dye with light of the appropriate wavelength and detecting the resulting fluorescence. Fluorescence can be detected visually, via photographic films, using electron detectors such as Charge Coupled Devices (CCDs) or photomultiplier tubes, and the like. Similarly, an enzyme label may be detected by providing the enzyme with an appropriate substrate and detecting the resulting reaction product. Finally, simple colorimetric labels can be detected simply by observing the color associated with the label. Thus, in various dipstick assays, the conjugated gold often appears pink, while various conjugated beads appear in the color of the beads.

Fluorescent labels are presently preferred because they have the advantage of requiring little precautions in operation and are suitable for high throughput imaging processes (optical analysis, including the digitization of the analysis image in an integrated system containing a computer). Preferred markers typically have one or more of the following characteristics: high sensitivity, high stability, low background, low environmental sensitivity and high specificity in labeling. Many fluorescent labels are commercially available: SIGMA Chemical Company (Saint Louis, MO), Molecular Probes (Eugene, OR), R & D systems (Minneapolis, MN), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analyta (Fluka Chemie AG, Buchs, Switzeand) and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to those skilled in the art. Furthermore, the skilled person will confirm how to select the appropriate fluorophore according to the particular application and will be able to resynthesize the necessary fluorophore or modify a commercially available fluorescent compound by synthetic means to obtain the desired fluorescent label, if not readily commercially available.

In addition to small molecule fluorophores, naturally occurring fluorescent proteins and artificial analogs of such proteins can also be used in the present invention. Such proteins include, for example, the green fluorescent protein of cNidarians (Ward et al, Photochem. Photobiol.35: 803- & 808 (1982); Levine et al, Comp. biochem. Physiol., 72B: 77-85(1982)), yellow fluorescent protein from the species Fischeri of the genus Vibrio (Baldwin et al, Biochemistry 29: 5509-15(1990)), Peridinin-chlorophyll from Symbiodinium sp. of the order Anacardiales (Morris et al, plantamolecular Biology 24: 673-77(1994)), phycobiliproteins (e.g., phycoerythrin and phycocyanin) from marine cyanobacteria (e.g., Synechococcus) (Wilbanks et al, J. biol. chem.268: 1226-35(1993)), and the like.

Pharmaceutical preparation

In another preferred embodiment, the present invention provides a pharmaceutical formulation comprising a compound of the present invention and a pharmaceutically acceptable carrier.

In a further preferred embodiment, the invention provides a pharmaceutical formulation comprising a pharmaceutically acceptable carrier and a conjugate of a targeting agent of the invention and a cytotoxin.

The compounds described herein, or pharmaceutically acceptable addition salts or hydrates thereof, can be delivered to a patient using a variety of routes or modes of administration. Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, enteral and parenteral administration, including intramuscular, subcutaneous and intravenous injection.

The term "administration" as used herein is intended to encompass all means of directly and indirectly releasing a compound to its intended site of action.

The compounds described herein, or pharmaceutically acceptable salts and/or hydrates thereof, can be administered alone, in combination with other compounds of the invention, and/or in the form of cocktails with other therapeutic agents. Of course, the choice of therapeutic agent that can be co-administered with the compounds of the present invention will depend, in part, on the condition being treated.

For example, when administered to a patient suffering from a disease state caused by an organism that is dependent on an autoinducer, the compounds of the invention may be administered in the form of a cocktail containing drugs for the treatment of pain, infections and other symptoms and side effects associated with the disease. Such drugs include, for example, analgesics, antibiotics, and the like.

When administered to a patient receiving cancer treatment, the compounds may be administered in the form of a cocktail containing anti-cancer agents and/or supplemental enhancers. The compounds may also be administered in the form of cocktails with drugs that treat the side effects of radiation therapy, such as antiemetics, radioprotectors, and the like.

Supplemental enhancers which can be co-administered with the compounds of the invention include, for example, tricyclic antidepressants (e.g., imipramine, desipramine, amitriptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine, and maprotiline); non-tricyclic antidepressants (e.g., sertraline, trazodone and citalopram); ca²⁺Antagonists (e.g., verapamil, nifedipine, nitrendipine, and caroverine); amphotericin; tripareol analogs (e.g., tamoxifen); antiarrhythmic agents(e.g., quinidine); antihypertensive agents (e.g., reserpine); thiol scavengers (e.g., buthionine and sulfoximine); and calcium folinate.

The active compounds of the present invention are administered as such, or in the form of pharmaceutical compositions, wherein the active compound is in admixture with one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmaceutical compositions used in accordance with the invention are generally formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The appropriate formulation will depend on the route of administration chosen.

For injection, the medicaments of the invention may be formulated as aqueous solutions, preferably in physiologically compatible buffers, such as Hank's solution, Ringer's solution or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds may be readily formulated by combining the active compound with a pharmaceutically acceptable carrier, as is well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral administration to a patient to be treated. The oral pharmaceutical preparations can be obtained by mixing with solid excipients, optionally grinding the resulting mixture, processing the mixture of granules, if desired with addition of suitable auxiliaries, to give tablets or dragee cores. Suitable excipients are in particular fillers, for example sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, for example maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate.

Suitable coatings are provided for the lozenge cores. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, Carbopol gel, polyethylene glycol, and/or titanium dioxide; a lacquer solution; and a suitable organic solvent or solvent mixture. Dyestuffs or pigments may be added to the tablets or dragee cores for identification or to distinguish different active compound dose combinations.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules can contain a mixture of the active ingredient with: fillers, such as lactose; binders, such as starch; and/or lubricants, such as talc or magnesium stearate; and optionally a stabilizer. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or dragees, formulated in conventional manner.

For administration by inhalation, the compounds for use according to the invention are suitably delivered in the form of an aerosol from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve for metered release. Capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, for example bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents, for example cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, for example sodium alginate.

Pharmaceutical preparations for parenteral administration comprise aqueous solutions of water-soluble active compounds. Oily injection suspensions of the active compounds may be prepared as appropriate. Suitable lipophilic solvents or vehicles include fatty oils, for example sesame oil, or synthetic fatty acid esters, for example ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds in order to prepare highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the foregoing formulations, the compounds may also be formulated as a library preparation. Such long acting formulations may be administered by implantation or transdermal delivery (e.g. subcutaneous or intramuscular), intramuscular injection or transdermal patch. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.

The pharmaceutical compositions may also contain suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

Libraries

Also included within the scope of the invention are cytotoxins of the invention, libraries of cytotoxin-linking group and drug-linking group conjugates of cytotoxins and linking groups of the invention. An exemplary library comprises at least 10 compounds, more preferably at least 100 compounds, even more preferably at least 1,000 compounds, even more preferably at least 100,000 compounds. The format of the library is easily interrogated for specific properties, such as cytotoxicity, cleavage of the linker by an enzyme or other cleaving agent. Exemplary formats include chip format, microarray, etc.

The main objective of parallel or combinatorial synthesis is to generate libraries of different molecules that share a common property, referred to as scaffolding in this specification. By substituting different portions on each variable portion of the scaffold molecule, the amount of space that can be developed in the library is increased. The concept of space occupied by theory and modern medicinal chemistry advocates is a key factor in determining the efficacy of a given compound on a given biological target. By creating different molecular libraries, developing a large proportion of the space targeted, the chances of developing efficient lead compounds are dramatically increased.

Parallel synthesis is generally carried out on a solid support, such as a polymeric resin. Scaffolding or other suitable intermediates are cleavably attached to the resin by a chemical linker. A reaction is performed to modify the scaffold attached to the resin. Variations in reagents and/or reaction conditions produce structural diversity, which is the quality of each library.

Parallel synthesis of "small" molecules (non-oligomers with molecular weight 200-. See, for example, Camps et al, Annaks de Quimica, 70: 848(1990). More recently, Ellmann disclosed the solid-phase supported parallel (also called "combinatorial") synthesis of eleven benzodiazepine  analogs as well as some prostaglandins and β -mimetics. Such as U.S. Pat. No.5,288,514. Another relevant publication for the parallel synthesis of small molecules can be found in U.S. Pat. No.5,324,483. This patent discloses that 4 to 40 compounds were synthesized in each of sixteen different scaffolds. Chen et al also used an organic synthesis strategy to synthesize non-peptide libraries using a multi-step process on polymeric supports (Chen et al, J.Am.chem.Soc., 116: 2661-2662 (1994)).

Once a library of unique compounds is prepared, a library of immunoconjugates or antibodies can be prepared using the methods described herein using the library of autoinducers as a starting point.

Reagent kit

The invention provides in another aspect a kit comprising one or more compounds or compositions of the invention and instructions for using the compounds or compositions. In an exemplary embodiment, the present invention provides a kit for conjugating a linker arm of the invention to another molecule. The kit includes a linking group and instructions for linking the linking group to a particular functional group. Other kit formats will be apparent to those skilled in the art and are within the scope of the invention.

Method

In addition to the compositions and constructs described above, the present invention also provides a number of methods by which the compounds and conjugates of the invention can be utilized.

Purification of

In another exemplary embodiment, the invention provides a method for isolating a molecular target of a cytotoxin of the invention that binds a molecule having a group according to formula I as part of its structure. The method preferably comprises contacting a cell preparation comprising the target with an immobilized compound of formula I, thereby forming a complex between the receptor and the immobilized compound.

The cytotoxins of the present invention may be immobilized on an affinity carrier by any art-recognized means. Alternatively, the cytotoxin may be immobilized using one or more of the linking groups of the present invention.

In another exemplary embodiment, the present invention provides an affinity purification matrix comprising a linking group of the present invention.

The methods of the invention for separating a target will generally employ one or more affinity chromatography processes. Affinity chromatography makes use of highly selective recognition sites of biomolecules or biopolymers for certain supported chemical structures, enabling their efficient separation. There are many articles, books and periodicals in the literature relating to affinity chromatography, including the subject matter of affinity chromatography supports, cross-linking members, ligands and their preparation and use. Examples of these references include: ostrove, Methods Enzymol.182: 357-71 (1990); ferent, bioeng.70: 199-; huang et al, J.Chromatogr.492: 431-69 (1989); "Purification of enzymes by heparin-Sepharose affinity chromatography", J.chromatography, 184: 335-45 (1980); farooqi, Enzyme eng, 4: 441-2 (1978); nishikawa, chem.technol., 5 (9): 564-71 (1975); guilford et al, practice.high Performance.liq.Chromatogr., Simpson (ed.), 193-206 (1976); nishikawa, Proc. int. Workshop technol. protein Sep. Improv. bloodPlasma Fraction, Sandberg (ed.), 422-35 (1977); "Affinity chromatography of enzymes", Affinity chromatography, Proc. int. Symp.25-38(1977) (Pub.1978); affinity Chromatography: a practical approach, Dean et al, (ed.), IRL Press Limited, Oxford, England (1985). Those skilled in the art will have a number of teachings in developing specific affinity chromatography methods that employ the materials of the present invention.

In the present method, affinity chromatography media of various chemical structures can be used as supports. For example, sepharose and cross-linked sepharose can be used as support materials because their hydrophilicity makes them relatively free of non-specific bonding. Other useful supports include, for example, Controlled Pore Glass (CPG) beads, cellulose particles, polyacrylamide gel beads, and Sephadex made from dextran and epichlorohydrin^TMGel beads.

Treatment of disease

The cytotoxins of the present invention are active, potent duocarmycin derivatives. The parent drugs are exceptionally potent antitumor antibiotics whose biological effects result from reversible, stereoelectronically controlled, sequence-selective DNA alkylation (Boger et al, J.org.chem.55: 4499 (1990); Boger et al, J.Am.chem.Soc.112: 8961 (1990); Boger et al, J.Am.chem.Soc.113: 6645 (1991); Boger et al, J.Am.chem.Soc.115: 9872 (1993); Boger et al, bioorg.Med.chem.Lett.2: 759 (1992)). After the initial disclosure of duocarmycins, extensive efforts have been made to elucidate the selectivity of DNA alkylation and its structural origin of duocarmycins.

In a further embodiment, the invention provides a method of killing a cell. The method comprises administering to the cell an amount of a compound of the invention sufficient to kill the cell. In an exemplary embodiment, the compound is administered to a subject carrying the cell. In a further exemplary embodiment, the administration serves to delay or stop the growth of a tumor comprising the cell.

Effective dose

Pharmaceutical compositions suitable for use in the present invention include compositions comprising a therapeutically effective amount of the active ingredient, i.e., an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. For example, when administered in a method of reducing sickle cell dehydration and/or delaying the onset of red blood cell sickling or deformation in situ, such compositions will contain an amount of the active ingredient effective to achieve such a result. Determination of an effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure herein.

With respect to any of the compounds described herein, a therapeutically effective amount can be initially determined from a cell culture assay. The target plasma concentration will be that concentration of the active compound which is capable of inhibiting the growth or differentiation of cells. In a preferred embodiment, cellular activity is inhibited by at least 25%. Target plasma concentrations of active compounds capable of inducing inhibition of cellular activity of at least about 50%, 75% or even 90% or more are presently preferred. The percent inhibition of cellular activity in a patient can be monitored to assess the appropriateness of the achieved plasma drug concentration, and the dosage can be adjusted up or down to achieve the desired percent inhibition.

As is well known in the art, a therapeutically effective amount for use in humans can also be determined from animal models. For example, human dosages can be formulated to achieve circulating concentrations that have been found to be effective in animals. As described above, the dose for human use can be adjusted by monitoring cytostatic effects and adjusting the dose up or down.

A therapeutically effective amount may also be determined from human data on compounds known to exhibit similar pharmacological activity. The dosage used may be adjusted based on the relative bioavailability and potency of the administered compound compared to known compounds.

It is well within the ability of the ordinarily skilled artisan to adjust dosages to achieve maximal efficacy in humans based on the methods described above and other methods well known in the art.

In the case of topical administration, the systemic circulating concentration of the administered compound will not be particularly important. In such cases, the compound is administered in order to achieve a concentration in the local area effective to achieve the desired effect.

For use in the prevention and/or treatment of diseases involving abnormal cell proliferation, the circulating concentration of the administered compound is preferably about 0.001 μ M to 20 μ M, and about 0.01 μ M to 5 μ M is preferable.

The compounds described herein are typically administered to a patient orally at a dose of from about 1 mg/day to about 10,000 mg/day, more typically from about 10 mg/day to about 1,000 mg/day, and most typically from about 50 mg/day to about 500 mg/day. Typical dosages will range from about 0.01 to about 150 mg/kg/day, more usually from about 0.1 to about 15 mg/kg/day, and most usually from about 1 to about 10 mg/kg/day, depending on the weight of the patient.

With respect to other modes of administration, adjustments in dosage and interval may vary from person to provide plasma levels of the administered compound effective for the particular clinical indication being treated. For example, in one embodiment, the compounds according to the present invention may be administered in relatively high concentrations, multiple times per day. Alternatively, it may be preferable to administer the compounds of the invention at a minimum effective concentration and with less frequent dosing regimens. This will provide a treatment regimen commensurate with the severity of the individual's disease.

Using the teachings provided herein, an effective therapeutic treatment regimen can be planned that results in neither substantial toxicity, but is completely effective in treating the clinical symptoms exhibited by a particular patient. Such planning should involve careful selection of the active compound taking into account the following factors: compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred mode of administration and toxicity profile of the drug selected.

The following examples further illustrate the compounds, compositions and methods of the present invention. These examples are for illustration only and do not limit the claimed invention.

Examples

Example 1

1.1 materials and methods

In the following examples, temperatures are given in degrees Celsius (. degree. C.) unless otherwise specified; the operation is carried out at room or ambient temperature (generally about 18-25 ℃); the evaporation of the solvent is carried out using a rotary evaporator under reduced pressure (generally 4.5-30mmHg) at bath temperatures up to 60 ℃; the course of the reaction is usually followed by TLC, the reaction time being given by way of example only; melting points are uncorrected; the product performed satisfactorily¹H-NMR and/or microanalysis data; the yields are given by way of example only; the following conventional abbreviations are also used: mp (melting point), L (L), mL (mL), mmol (mmol), g (g), mg (mg), min (min), LC-MS (liquid chromatography-gas chromatography) and h (h).

¹H-NMR spectra were measured on a Varian Mercury 300MHz spectrometerConsistent with the specified structure. Chemical drift is reported in parts per million (ppm) deviation from tetramethylsilane. Electrospray mass spectra were recorded on a Perkin Elmer Sciex API 365 mass spectrometer. Elemental analysis was performed by Robertson Microlit Laboratories, Madison, NJ. Silica gel for flash chromatography is grade E.Merck (230- & 400 mesh). Reverse phase analytical HPLC was performed on an HP 1100 or Varian ProStar 210 instrument with a Phenomenex Luna 5. mu. m C-18(2)150 mm. times.4.6 mm column or a Varian Microsorb-MV 0.1. mu. m C-18150 mm. times.4.6 mm column. The flow rate was 1mL/min, which was a gradient of 0% to 50% buffer B over 15 minutes or a gradient of 10% to 100% buffer B over 10 minutes, UV detection at 254 nm. Buffer a was 20mM ammonium formate + 20% acetonitrile or acetonitrile containing 0.1% trifluoroacetic acid and buffer B was 20mM ammonium formate + 80% acetonitrile or 0.1% aqueous trifluoroacetic acid. Reverse phase preparative HPLC was performed on a Varianprostar 215 instrument with a Waters Delta Pak 15. mu. m C-18300 mm. times.7.8 mm column.

1.2 Synthesis method

1.2a Synthesis of Compound 134

The compounds of formula I are readily prepared by reacting the appropriate spirocyclopropyl cyclohexadienyl analog (compound B) with the activated heterocyclic compound A in N, N-Dimethylformamide (DMF) or Tetrahydrofuran (THF) using sodium hydride. The resulting compound 28 is then converted to compound 29 by treatment with an appropriate halogenated acid, such as hydrochloric acid. Compound 29 is reduced by catalytic hydrogenation to give compound 65, which is coupled with an activated ester to give compound 134, a compound of formula I.

Other compounds of formula I are prepared according to the disclosed procedures, modified using procedures well known to those skilled in the art to prepare additional analogs, such as reduction, oxidation, addition, aqueous extraction, evaporation, and purification.

1.2b Synthesis of Compound A

To a solution of 5-nitro-2-carboxylic acid (0.83g, 4.0mmol) in N, N-dimethylformamide (60mL) at 0 deg.C was added EDC (1.15g, 6.0 mmol. the resulting suspension was stirred at 0 deg.C for 45min, at which point the EDC had completely dissolved, 4-nitrophenol (0.83g, 6.0mmol) and DMAP (0.73g, 6.0mmol) were added, the resulting mixture was stirred at ambient temperature for 13 h, after which the mixture was diluted with ethyl acetate, washed twice with 10% aqueous citric acid, then with water and brine, then over Na₂SO₄Dried, filtered and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (7% ethyl acetate in dichloromethane) to give 1.02g (78%) of a as a yellow solid:¹H MR(CDCl₃)δ9.0(brs，1H)，8.2(d，2H)，7.8(m，2H)，7.4(d，2H)，7.3(s，1H)，6.8(s，1H).

1.2c Synthesis of Compound 28

To a solution of B (20mg, 0.08mmol) in N, N-dimethylformamide (1.0mL) at-40 deg.C was added a suspension of sodium hydride (4.0mg, 0.1mmol, 60% oil in N, N-dimethylformamide (1.0 mL). The resulting mixture was allowed to warm slowly (1.5h) to 0 ℃ and then cooled back to-40 ℃. A (37mg, 0.1mmol) was added and the mixture was slowly warmed (1.5h) to 0 ℃ for 20 min. The mixture was cooled to-30 ℃, quenched with acetic acid (10 μ Ι _), stirred for 10min, diluted with ethyl acetate, then washed with water and brine. The organic layer was separated over MgSO₄Dried, filtered and concentrated in vacuo. Purification by flash column chromatography on silica gel (50% to 100% ethyl acetate in dichloromethane) afforded 16.3mg (43%) 28 as a pale yellow solid:

¹H NMR(CDCl₃)δ11.3(brs，1H)，9.4(brs，1H)，7.4(m，2H)，7.1(s，1H)，6.95(s，1H)，6.8(s，1H)，4.4(s，2H)，3.8(s，3H)，3.8(m，1H)，2.6(s，3H)，2.4(dd，1H)，1.4(m，1H).ESMS m/z 490(M-H)^-.

1.2d Synthesis of Compound 29

A solution of 28(50mg, 0.103mmol) in N, N-dimethylformamide (1.0mL) was treated with 1mL of anhydrous hydrochloric acid (1.0M in dioxane). The resulting solution was stirred at ambient temperature for 30min, then the solvent was concentrated. The resulting residue was purified by flash column chromatography on silica gel (50% to 100% ethyl acetate in dichloromethane) to give 50mg (100%) of 29 as a light yellow solid:¹H NMR(CDCl₃)δ11.3(brs，1H)，9.4(brs，1H)，7.4(m，2H)，7.1(s，1H)，6.95(s，1H)，6.8(s，1H)，4.4(s，2H)，3.8(s，3H)，3.8(m，1H)，2.6(s，3H)，2.4(dd，1H)，1.4(m，1H).

1.2d Synthesis of Compound 65

To a solution of 29(110mg, 0.184mmol) in 1: 1 methanol: dichloromethane (20ml) was added 10% palladium on carbon (100 mg). The mixture was hydrogenated on a Parr apparatus at 50psi for one hour. The mixture was filtered through celite, washed with dichloromethane, then concentrated in vacuo to give 94mg (90%) of 65 as a yellow solid:

¹H MR(CDCl₃)δ7.8-7.3(m，5H)，4.4(m，3H)，3.8(m，5H)，3.4(s，3H).ESMS m/z454(M-H)^-.

1.2 Synthesis of e Compound 134

Compound C (19mg, 0.044mmol) and HATU (50mg, 0.132mmol) were dissolved in dimethylformamide (2mL) and N-methylmorpholine (19.3. mu.L, 0.176mmol) was added. After 15 min, a solution of 65(20mg, 0.044mmol) in dimethylformamide (1mL) was added. The reaction mixture was stirred at room temperature for 16 hours, then concentrated in vacuo. The resulting solid was washed with water and saturated aqueous sodium bicarbonate, dried under vacuum, and then washed with ethyl acetate. The crude product was purified by flash chromatography on silica, eluting with 5% methanol in dichloromethane, to give 15mg (39%) of 134:

¹H NMR(DMSO)：δ11.4(brs，1H)，11.0(brs，1H)，10.9(s，1H)，8.5(s，1H)，8.3(brs，2H)，8.0(m，2H)，7.9(s，1H)，7.7(m，3H)，7.5(d，1H)，7.3(t，2H)，4.6(m，2H)，4.5(d，2H)，4.3(m，1H)，3.8(s，3H)，3.7(d，1H)，3.5(t，1H)，2.7(s，3H)，1.7(m，2H)，0.9(d，6H).

the following compounds were prepared in a similar manner: 46:¹H NMR(CDCl₃)：δ8.9(s，1H)，8.7(s，1H)，8.4(dd，1H)，8.1(brs，1H)，7.75(d，1H)，7.65(s，1H)，4.8(d，1H)，4.55(m，2H)，3.9(s，3H)，3.85(s，1H)，3.7(m，4H)，3.4(t，1H)，2.7(s，3H)，2.5(brs，4H)，2.4(s，3H).95：¹H NMR(DMSO)：δ12(s，1H)，10.6(d，1H)，8.25(s，1H)，8.2(brs，1H)，8.1(s，1H)，7.7(m，5H)，7.5(d，1H)，4.6(t，1H)，4.5(d，1H)，4.4(m，2H)，4.1(m，1H)，3.9(d，2H)，3.8(s，3H)，3.3(m，10H)，2.8(s，3H)，2.6(s，3H)，1.6(brs，3H)，0.9(s，6H).47：¹H NMR(CDCl₃)：δ9.1(brs，1H)，8.1(brs，1H)，7.4(t，2H)，6.9(s，1H)，6.8(dd，1H)，4.8(d，1H)，4.5(m，2H)，3.9(s，3H)，3.85(m，3H)，3.7(m，2H)，3.4(t，1H)，2.7(s，3H)，2.6(brs，4H)，2.4(s，3H).52：¹H NMR(DMSO)：δ12.5(s，1H)，11.8 (s，1H)，10.4 (s，1H)，8.4(s，1H)，7.8 (m，5H)，7.5(m，2H)，7.3(t，1H)，7.1(t，1H)，6.7(s，1H)，4.6(m，4H)，3.8(s，3H)，2.5(s，3H).108：¹H NMR(DMSO)：δ10.9(s，1H)，10.7(s，1H)，10.0(s，1H)，8.5(s，1H)，8.3(s，1H)，8.1(m，5H)，7.8(m，5H)，7.5(m，2H)，7.3(m，5H)，7.1(m，5H)，5.0(m，2H)，4.8(m，1H)，4.6(m，2H)，4.3(m，2H)，4.1(t，2H)，3.9(m，1H)，3.7(m，4H)，3.0(m，6H)，2.6(s，2H)，2.3(t，1H)，1.8(s，3H)，1.5(m，9H)，1.3(m，4H)，0.8(m，6H).43：¹H NMR(DMSO)：δ12.1(s，2H)，11.8(s，1H)，10.5 (d，1H)， 8.3(s，1H)，8.0(s，1H)，7.8(m，5H)，7.6(s，1H)，7.2(d，2H)，4.7(m，2H)，4.5(m，3H)，3.8(s，3H)，3.5(m，2H)，3.2(m，2H)，2.9(s，3H)，2.7(s，3H)，2.3(s，4H).153：¹H NMR (DMSO)：δ12.3(brs，1H)，11.7(brs，1H)，10.5(brs，1H)，10.0(brs，1H)，8.3(m，2H)，7.9(m，4H)，7.5(s，1H)，7.4(d，1H)，7.0(m，1H)，4.5(m，5H)，4.1(m，1H)，3.9(d，1H)，3.8(s，3H)，3.4(m，8H)，2.9(brs，3H)，2.8(s，3H)，2.7(s，3H)，1.8(s，2H)，1.6(brs，4H)，1.4(m，2H)，1.2(d，4H)，0.9(m，16H).45：¹H NMR(DMSO)：δ12.0(s，1H)，11.6(s，1H)，10.8(s，1H)，8.4(s，1H)，8.3(d，2H)，8.0(s，1H)，7.8(m，3H)，7.6(s，1H)，7.4(t，1H)，4.6(m，5H)，3.8(s，3H)，3.4(m，8H)，2.9(s，3H)，2.7(s，3H).115：¹H NMR(CDCl₃)：δ9.1(brs，1H)，8.4(s，1H)，8.3(s，1H)，7.7(d，1H)，7.5(m，3H)，7.2(m，3H)，6.9(s，2H)，4.7(d，1H)，4.5(m，4H)，3.9(s，3H)，3.5(m，14H)，2.6(s，3H)，1.3(t，3H).109：¹H NMR(DMSO)：δ11.9 (s，11.9)，10.5(s，1H)，10.2(d，1H)，8.2(s，1H)，7.7(m，6H)，7.2(d，1H)，7.1(t，1H)，6.8(d，1H)，4.6(m，1H)，4.4(d，2H)，4.3(m，2H)，3.7(s，3H)，2.6(s，3H).135：¹H NMR(DMSO)：δ11.4(brs，1H)，11.0(brs，1H)，10.9(s，1H)，8.5(s，1H)，8.3(brs，2H)，8.0(m，2H)，7.9(s，1H)，7.7(m，3H)，7.5(d，1H)，7.3(t，2H)，4.6(m，2H)，4.5(d，2H)，4.3(m，1H)，3.8(s，3H)，3.7(d，1H)，3.5(t，1H)，2.7(s，3H)，1.7(m，2H)，0.9(d，6H).24：¹H NMR(DMSO)：δ10.8(s，1H)， 8.6(s，1H)，8.3(m，5H)，7.9(s，1H)，7.8(d，2H)，7.7(d，1H)，7.65(s，1H)，7.6 (d，2H)，7.4(m，5H)，5.3(s，2H)，4.9(t，1H)，4.7(d，1H)，4.4(m，1H)，4.0(m，2H).ESMS m/z 696(M-H)^-.25：¹H NMR(DMSO)：δ8.6(s，1H)，8.3(m，5H)，7.8(m，3H)，7.6(m，3H)，7.4(m，1H)，4.8(m，1H). 4.6(m，1H)，4.3(m，1H)，4.1(m，2H).ESMS m/z 605 (M-H)^-.27：¹H NMR(DMSO)：δ10.9(s，1H)，10.3(s，1H)，8.6(s，1H)，8.3(s，1H)，8.2(d，1H)，8.1(m，2H)，7.8(m，3H)，7.6(d，1H)，7.3(s，1H)，6.9(d，1H)，6.8(t，1H)，6.4(d，1H)，4.8(t，1H)，4.6(d，1H)，4.3(m，1H)，4.0(m，2H).154：¹H NMR(DMSO)：δ10.7(s，1H)，10.0(s，1H)，8.6(s，1H)，8.4(s，1H)，8.2(s，1H)，8.1(m，3H)，7.9(m，5H)，7.7(m，1H)，7.6(m，3H)，7.3(m，5H)，5.0(s，2H)，4.8(t，1H)，4.6(d，1H)，4.3(m，3H)，4.1(d，1H)，3.9(m，1H)，3.1(s，1H)，3.0(m，1H)，2.7(s，1H)，2.3(m，5H)，1.6(m，5H)，1.4(t，2H)，1.2(d，3H)，0.9(m，12H).ESMS m/z 1134(M-H)^-.162：¹H NMR(DMSO)：δ11.6(s，1H)，10.9(s，1H)，10.5(s，1H)，9.9(s，1H)，8.6(s，1H)，8.3(s，1H)，8.2(d，1H)，8.0(m，5H)，7.8(m，4H)，7.6(d，1H)，7.5(m，3H)，7.1(t，1H)，4.8(t，1H)，4.6(d，2H)，4.3(m，3H)，4.1(d，1H)，3.9(m，1H)，2.4(m，2H)，2.3(m，3H)1.5(m，9H)，1.2(m，3H)，0.9(m，12H).ESMS m/z 1044 (M-H)^-.79：¹H NMR(CDCl₃)：δ9.4(s，1H)，8.5(s，1H)，8.1(s，2H)，8.0(brs，1H)，7.9(d，2H)，7.6(s，2H)，7.5(s，1H)，7.0(d，2H)，6.9(d，3H)，4.7(d，1H)，4.5(m，2H)，4.2(m，1H)，3.95(s，3H)，3.85(s，3H)，3.7(m，4H)，3.4(m，1H)，2.7(s，3H)，2.5(s，3H)，2.4(t，2H)，1.1(s，9H)，0.4(brs，6H).80：¹H NMR(CDCl₃)：δ9.3 (brs，1H)，8.3(s，1H)，8.2(brs，1H)，8.0(m，3H)，7.5(m，4H)，7.4(m，2H)，7.0(m，4H)，4.7(d，1H)，4.6(m，1H)，4.4(m，1H)，4.2(m，5H)，3.9(s，3H)，3.4(m，1H)，2.7(s，3H)，2.9(s，3H)，2.3(m，2H)，1.1(s，9H)，0.4(brs，6H).81：¹H NMR (CDCl₃)：δ10.5(s，1H)，8.8(s，1H)，8.6(d，2H)，8.0(d，2H)，7.8(d，2H)，7.4(m，3H)，7.3(d，2H)，7.0(m，2H)，6.9(d，2H)，4.6(m，3H)，4.4(m，2H)，3.9(m，4H)，3.4(m，1H)，2.7(s，3H)，2.5(s，3H)，1.0(s，9H)，0.3(s，6H).82：¹H NMR(CDCl₃)：δ8.5(s，2H)，8.4(s，1H)，8.2(s，1H)，8.0(m，4H)，7.6(m，4H)，7.5(s，1H)，7.3(s，1H)，7.1(s，2H)，4.7(m，3H)，4.55(m，1H)，4.45(m，1H)，3.9(m，4H)，3.4(m，1H)，2.7(s，3H)，2.5(s，3H)，1.0(s，9H)，0.4(brs，6H).83：¹H NMR(CDCl₃)：δ9.6(s，1H)，8.4(s，1H)，8.1(s，2H)，8.0(m，1H)，7.8(s，1H)，7.6(m，2H)，7.5(s，1H)，7.35(q，2H)，7.0(s，1H)，4.7(d，1H)，4.55(m，1H)，4.45(m，1H)，3.9(m，4H)，3.4(m，1H)，2.7(s，3H)，1.0(s，9H)，0.4(brs，6H).89：¹H NMR(CDCl₃)：δ9.3(brs，1H)，8.4(brs，1H)，8.2(s，1H)，8.0(brs，2H)，7.5(m，9H)，7.2(s，1H)，7.1(d，1H)，6.9(s，1H)，5.1(s，2H)，4.7(d，1H)，4.55(m，1H)，4.45(m，1H)，3.9(m，4H)，3.4(m，1H)，2.7(s，3H)，1.05(s，9H)，0.4(brs，6H).90：¹H NMR(CDCl₃)：δ9.6(brs，1H)，8.5(brs，1H)，8.2(s，1H)，8.0(m，2H)，7.5(m，8H)，7.3(m，2H)，7.1(d，1H)，6.6(d，1H)，5.2(s，2H)，4.7(d，1H)，4.55(m，1H)，4.45(m，1H)，3.9(m，4H)，3.4(m，1H)，2.7(s，3H)，1.05(s，9H)，0.3(brs，6H).163：¹H NMR(DMSO)：δ12.0(s，1H)，11.6(s，1H)，10.4(s，1H)，10.2(s，1H)，8.4(s，1H)，8.1(m，4H)，7.9(m，3H)，7.5(m，3H)，7.2(d，2H)，4.9(s，2H)，4.7(m，1H)，4.6(m，1H)，4.5(m，1H)，3.9(m，4H)，3.6(m，1H)，2.7(s，3H)，2.5(s，3H)，1.1(s，9H)，0.7(s，6H).92：¹H NMR(DMSO)：δ12.0(s，1H)，10.4(s，1H)，10.2(s，1H)，8.3(s，1H)，7.9(s，1H)，7.8(m，4H)，7.5(d，2H)，7.25(s，1H)，7.15(s，1H)，6.9(m，3H)，4.7(m，3H)，4.6(m，1H)，4.5(m，1H)，3.9(m，2H)，3.8(m，4H)，3.5(m，1H)，3.2(m，2H)，2.9(s，3H)，2.7(s，3H)，2.6(s，3H).98：¹H NMR(CDCl₃)：δ8.3(brs，1H)，8.1(d，1H)，8.0(brs，1H)，7.95(s，1H)，7.85(d，2H)，7.65(s，1H)，7.6(d，2H)，7.5(s，1H)，7.4(m，2H)，7.0(s，1H)，6.85(d，2H)，4.8(m，3H)，4.55(m，1H)，4.45(m，1H)，3.9(m，6H)，3.4(m，1H)，2.7(s，3H)，2.5(s，3H)，2.4(m，2H)，1.05(s，9H)，0.4(brs，6H).110：¹H NMR(C₃D₆O)：δ11.3(brs，1H)，9.7(s，1H)，9.6(d，1H)，8.2(s，1H)，7.9(m，2H)，7.7(d，2H)，7.6(m，3H)，7.5(d，1H)，7.2(m，3H)，6.9(m，3H)，6.8(s，1H)，4.9(m，2H)，4.7(m，1H)，4.6(m，1H)，4.5(m，1H)，3.8(m，25H)，3.5(m，1H)，3.2(m，2H)，2.7(s，3H)，2.3(m，4H)，2.0(s，3H).113：¹H NMR(C₃D₆O)：δ11.3(brs，1H)，10.1(s，1H)，9.9(s，1H)，8.4(s，1H)，7.9(m，3H)，7.7(m，2H)，7.5(m，3H)，7.4(m，1H)，7.2(s，1H)，7.1(d，2H)，6.9(t，1H)，5.0(s，2H)，4.6(d，1H)，4.5(m，1H)，4.4(m，1H)，3.9(d，1H)，3.7(s，3H)，3.4(m，1H)，2.6(s，3H)，2.4(s，3H).114：¹H NMR(DMSO)：δ11.9(brs，1H)，11.7(s，1H)，10.4(d，1H)，10.2(t，1H)，8.3(s，1H)，7.9(s，1H)，7.8(m，7H)，7.5(m，2H)，7.0(m，6H)，4.9(s，2H)，4.6(m，1H)，4.5(m，1H)，4.4(m，1H)，3.9(d，1H)，3.8(s，3H)，3.7(m，2H)，3.5(m，25H)，2.9(s，3H)，2.7(s，3H)，2.2(m，2H).159：¹H NMR(DMSO)：δ12.0(m，1H)，11.9(brs，1H)，8.3(m，2H)，8.0(m，4H)，7.6(m，2H)，7.3(s，1H)，7.1(d，1H)，6.9(s，1H)，4.5(m，3H)，4.3(m，2H)，3.9(d，1H)，3.8(s，6H)，3.5(m，8H)，3.2(m，4H)，2.7(s，3H)，2.3(m，4H)，1.4(m，9H)，1.1(m，3H)，0.8(m，12H).131：¹H NMR(DMSO)：δ11.9(s，1H)，10.2(s，1H)，7.7(m，1H)，7.5(s，1H)，7.0(m，3H)，6.7(d，1H)，4.7(m，1H)，4.4(d，1H)，4.3(m，2H)，3.9(d，1H)，3.8(s，3H)，2.6(s，3H).129：¹H NMR(DMSO)：δ11.9(s，1H)，10.2(s，1H)，7.8(m，4H)，4.5(m，2H)，4.3(m，1H)，3.9(s，3H)，3.8(d，1H)，3.7(s，3H)，3.4(t，1H)，2.5(s，3H).136：¹H NMR(DMSO)：δ11.9(s，1H)，10.2(s，1H)，7.65(d，2H)，7.55(s，1H)，7.3(d，1H)，7.1(dd，1H)，4.6(m，1H)，4.5(m，1H)，4.3(m，1H)，3.85(m，1H)，3.8(s，3H)，3.75(s，3H)，3.5(m，1H)，2.6(s，3H).137：¹H NMR(DMSO)：δ11.9(s，1H)，10.2(s，1H)，8.0(m，1H)，7.5(m，3H)，7.2(s，1H)，7.0(m，1H)，4.6(m，1H)，4.5(m，1H)，4.4(m，1H)，3.9(m，2H)，3.8(d，1H)，3.7(s，3H)，3.4(m，1H)，2.6(s，3H)，2.4(m，2H)，1.3(s，9H).143：¹H NMR(DMSO)：δ11.9(s，1H)，10.2(s，1H)，8.0(m，2H)，7.7(m，3H)，7.3(s，1H)，7.1 (d，1H)，4.6(m，1H)，4.5(m，1H)，4.3(m，1H)，4.2(m，2H)，3.9(d，1H)，3.8(s，3H)，3.4(m，1H)，3.3(m，2H)，2.6(s，3H).148：¹H NMR(DMSO)：δ11.9(s，1H)，10.1(s，1H)，7.7(d，2H)，7.5(s，1H)，7.3(s，1H)，7.1(d，1H)，4.5(m，1H)，4.4(d，1H)，4.3(m，3H)，3.8(m，1H)，3.7(s，3H)，3.5(m，3H)，2.8(s，6H)，2.5(s，3H).150：¹H NMR(DMSO)：δ10.3(s，1H)，9.4(s，1H)，7.8(m，1H)，7.5(m，2H)，7.1(s，1H)，6.9(dd，1H)，4.5(m，4H)，4.3(m，1H)，3.8(s，3H)，3.75(d，1H)，3.5(m，1H)，3.1(m，2H)，2.75(s，6H)，2.65(s，3H)，2.1(m，2H).151：¹H NMR(DMSO)：δ11.9(s，1H)，10.2(s，1H)，7.7(d，2H)，7.65(s，1H)，7.55(s，1H)，7.25(d，1H)，4.6(m，1H)，4.5(m，1H)，4.4(m，1H)，3.9(d，1H)，3.8(s，3H)，3.5(m，1H)，3.3(brm，8H)，2.9(s，3H)，2.6(s，3H).251：¹H NMR(CDCl₃)：δ12.0(s，1H)，7.9(m，1H)，7.7(d，1H)，7.6(s，1H)，7.4(s，1H)，7.2(dd，1H)，4.6(m，3H)，4.4(m，2H)，3.9(m，1H)，3.8(s，3H)，3.7(m，1H)，3.6(m，2H)，3.4(m，4H)，3.2(m，2H)，3.0(m，4H)，2.9(s，6H)，2.7(m，4H)，2.6(s，2H)，1.6(m，2H)，1.3(m，8H)，0.9(q，2H).227：¹H NMR(CDCl₃)：δ10.3(s，1H)，8.7(s，1H)，7.7(d，2H)，7.5(m，3H)，7.1(m，2H)，6.9(d，2H)，4.6(m，5H)，4.25(m，2H)，4.15(m，2H)，3.9(s，3H)，3.4(m，1H)，3.1(s，2H)，2.9(m，12H)，2.7(s，3H)，2.5(s，6H)，2.3(s，3H).230：¹H NMR(CDCl₃)：δ10.3(s，1H)，8.6(s，1H)，7.7(d，2H)，7.5(m，3H)，7.1(m，2H)，6.9(d，2H)，6.7(s，2H)，4.7(m，4H)，4.1(m，4H)，3.9(s，3H)，3.5(m，2H)，3.4(m，1H)，3.2(m，4H)，3.1(s，2H)，2.8(m，5H)，2.4(m，10H)，2.2(m，7H).166：ESMS m/z 532(M-H)^-.165：ESMS m/z 920(M-H)^-.9：ESMS m/z 966(M-H)^-.17：ESMS m/z 753(M-H)^-.19：ESMS m/z 696(M-H)^-.50：ESMS m/z 800(M-H)^-.174：¹H NMR(CDCl₃)：δ8.4(s，1H)，7.9(m，1H)，7.5(m，2H)，7.2(m，2H)，4.75(m，3H)，4.6(m，1H)，4.45(m，1H)，3.9(m，4H)，3.4(m，1H)，2.7(s，3H)，1.05(s，9H)，0.4(brs，6H)；ESMS m/z 627(M-H)^-.176：¹H NMR(CDCl₃)：δ8.4(s，1H)，7.9(m，1H)，7.5(m，2H)，7.1(m，2H)，4.7(m，3H)，4.6(m，1H)，4.45(m，1H)，3.9(m，4H)，3.4(m，1H)，2.7(s，3H)，1.05(s，9H)，0.4(brs，6H).94：¹H NMR(CDCl₃)：δ8.5(d，2H)，8.2(d，2H)，7.9(s，2H)，7.8(d，2H)，7.5(m，2H)，7.4(m，4H)，6.8(d，2H)，4.7(d，1H)，4.5(m，1H)，4.4(m，1H)，3.9(m，6H)，3.3(m，1H)，2.9(t，2H)，2.7(s，3H)，2.5(s，3H)，2.1(m，2H)，1.6(s，6H)，1.0(s，9H)，0.3(brs，6H).ESMS m/z 1038(M-H)^-EXAMPLE 22.1 Synthesis method

2.1a Synthesis of Compound 1

The compounds of formula I are readily prepared by reacting the appropriate spirocyclopropyl cyclohexadienyl analog (compound B) with the activated heterocyclic compound A in N, N-Dimethylformamide (DMF) or Tetrahydrofuran (THF) using sodium hydride. The resulting compound 28 is then converted to compound 29 by treatment with an appropriate halogenated acid, such as hydrochloric acid. Coupling with compound 29 by in situ activation gives compound 1, i.e. a compound of formula I.

Other compounds of formula I are prepared according to the disclosed procedures, modified using procedures well known to those skilled in the art to prepare additional analogs, such as reduction, oxidation, addition, aqueous extraction, evaporation, and purification.

2.1b Synthesis of Compound A

To a solution of 5-nitro-2-carboxylic acid (0.83g, 4.0mmol) in N, N-dimethylformamide (60mL) at 0 deg.C was added EDC (1.15g, 6.0 mmol). The resulting suspension was stirred at 0 ℃ for 45min, at which point EDC had completely dissolved. 4-Nitrophenol (0.83g, 6.0mmol) and DMAP (0.73g, 6.0mmol) were added and the resulting mixture was stirred at ambient temperature. After 13 hours, the mixture was diluted with ethyl acetate, washed twice with 10% aqueous citric acid,then washed with water and brine, then over Na₂SO₄Dried, filtered and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (7% ethyl acetate in dichloromethane) to give 1.02g (78%) of a as a yellow solid:¹H NMR(CDCl₃)δ 9.0(brs，1H)，8.2(d，2H)，7.8(m，2H)，7.4(d，2H)，7.3(s，1H)，6.8(s，1H).

2.1c Synthesis of Compound 28

To a solution of B (20mg, 0.08mmol) in N, N-dimethylformamide (1.0mL) at-40 deg.C was added a suspension of sodium hydride (4.0mg, 0.1mmol, 60% oil in N, N-dimethylformamide (1.0 mL). The resulting mixture was allowed to warm slowly (1.5h) to 0 ℃ and then cooled back to-40 ℃. A (37mg, 0.1mmol) was added and the mixture was slowly warmed (1.5h) to 0 ℃ for 20 min. The mixture was cooled to-30 ℃, quenched with acetic acid (10 μ Ι _), stirred for 10min, diluted with ethyl acetate, then washed with water and brine. The organic layer was separated over MgSO₄Dried, filtered and concentrated in vacuo. Purification by flash column chromatography on silica gel (50% to 100% ethyl acetate in dichloromethane) afforded 16.3mg (43%) 28 as a pale yellow solid:

¹H NMR(CDCl₃)δ11.3(brs，1H)，9.4(brs，1H)，7.4(m，2H)，7.1(s，1H)，6.95(s，1H)，6.8(s，1H)，4.4(s，2H)，3.8(s，3H)，3.8(m，1H)，2.6(s，3H)，2.4(dd，1H)，1.4(m，1H).ESMS m/z 490(M-H)^-.

2.1d Synthesis of Compound 29

A solution of 28(50mg, 0.103mmol) in N, N-dimethylformamide (1.0mL) was treated with 1mL of anhydrous hydrochloric acid (1.0M in dioxane). The resulting solution was stirred at ambient temperature for 30min, then the solvent was concentrated. The resulting residue was purified by flash column chromatography on silica gel (50% to 100% ethyl acetate in dichloromethane) to give 50mg (100%) of 29 as a light yellow solid:¹H NMR(CDCl₃)δ11.3(brs，1H)，9.4(brs，1H)，7.4(m，2H)，7.1(s，1H)，6.95(s，1H)，6.8(s，1H)，4.4(s，2H)，3.8(s，3H)，3.8(m，1H)，2.6(s，3H)，2.4(dd，1H)，1.4(m，1H).

2.1e Synthesis of Compound 1

To a solution of 29(66mg, 0.136mmol) in dry dichloromethane (10mL) at-70 deg.C was added 4-nitrophenyl chloroformate (55mg, 0.273mmol) followed by triethylamine (27mg, 0.273 mmol). The resulting mixture was allowed to warm slowly. After 2 h, the mixture was placed in an ice bath and amine C (82mg, 0.273mmol) was added in one portion. The resulting mixture was stirred at ambient temperature overnight. After 22 hours, the mixture was poured into saturated NaHCO₃In aqueous solution. The aqueous layer was separated and extracted with dichloromethane. The combined organic extracts were then dried over MgSO₄Dried, filtered and concentrated in vacuo. Purification by flash column chromatography on silica gel (1% to 2% methanol in dichloromethane) afforded 42mg (38%) 1 as a pale yellow solid:

¹H MR(CDCl₃)δ11.0(s，1H)，8.6(s，1H)，8.4(d，1H)，8.2(brd，1H)，7.6(m，2H)，6.8(m，1H)，4.8(m，1H)，4.7(m，1H)，4.5(m，1H)，4.1(m，2H)，3.9(s，3H)，3.4(m，5H)，2.7(d，3H)，1.6(s，6H)，1.4(s，9H)，1.2(m，4H)，0.9(m，9H).

the following compounds were prepared in a similar manner: 220:¹H NMR(CDCl₃)：δ11.4(d，1H)，8.7(s，1H)，8.4(，dd，1H)，8.1(brs，1H)，7.75(d，1H)，7.65(s，1H)，5.8(brs，2H)，4.7(m，1H)，4.5(m，2H)，4.2(m，1H)，3.9(s，3H)，3.4(m，1H)，3.0(s，2H)，2.75(s，3H)，2.65(s，3H).ESMS m/z 613(M-H)^-.222：¹H NMR(CDCl₃)：δ10.3(s，1H)，8.7(d，2H)，8.4(d，1H)，8.1(m，1H)，7.7(d，2H)，7.6(m，1H)，6.9(d，2H)，4.9(d，1H)，4.7(m，1H)，4.5(m，3H)，3.95(s，3H)，3.85(s，3H)，3.6(m，1H)，3.4(m，1H)，3.1(s，3H)，2.7(s，3H)，2.3(s，3H).ESMS m/z 745(M-H)^-.224：ESMS m/z 899(M-H)^-. 235：ESMS m/z 913(M-H)^-.229：ESMS m/z 598(M-H)^-. 8：ESMS m/z 856(M-H)^-.

232： ESMS m/z 612(M-H)^-.233：ESMS m/z 816(M-H)^-.

234：ESMS m/z 952(M-H)^-EXAMPLE 33.1 Synthesis method

3.1a Synthesis of Compound 239

The compounds of formula I are readily prepared by reacting the appropriate spirocyclopropyl cyclohexadienyl analog (compound B) with the activated heterocyclic compound A in N, N-Dimethylformamide (DMF) or Tetrahydrofuran (THF) using sodium hydride. The resulting compound 28 is then converted to compound 29 by treatment with an appropriate halogenated acid, such as hydrochloric acid. Compound 29 is activated to 4-nitrophenyl ester, which is coupled with compound C to give compound 239, a compound of formula I.

Other compounds of formula I are prepared according to the disclosed procedures, modified using procedures well known to those skilled in the art to prepare additional analogs, such as reduction, oxidation, addition, aqueous extraction, evaporation, and purification.

3.1b Synthesis of Compound A

To a solution of 5-nitro-2-carboxylic acid (0.83g, 4.0mmol) in N, N-dimethylformamide (60mL) at 0 deg.C was added EDC (1.15g, 6.0 mmol). The resulting suspension was stirred at 0 ℃ for 45min, at which point EDC had completely dissolved. 4-Nitrophenol (0.83g, 6.0mmol) and DMAP (0.73g, 6.0mmol) were added and the resulting mixture was stirred at ambient temperature. After 13 hours, the mixture was diluted with ethyl acetate, washed twice with 10% aqueous citric acid, then with water and brine, thenPassing through Na2SO₄Dried, filtered and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (7% ethyl acetate in dichloromethane) to give 1.02g (78%) of a as a yellow solid:¹H NMR(CDCl₃)δ9.0(brs，1H)，8.2(d，2H)，7.8(m，2H)，7.4(d，2H)，7.3(s，1H)，6.8(s，1H).

3.1c Synthesis of Compound 28

To a solution of B (20mg, 0.08mmol) in N, N-dimethylformamide (1.0mL) at-40 deg.C was added a suspension of sodium hydride (4.0mg, 0.1mmol, 60% oil in N, N-dimethylformamide (1.0 mL). The resulting mixture was allowed to warm slowly (1.5h) to 0 ℃ and then cooled back to-40 ℃. A (37mg, 0.1mmol) was added and the mixture was slowly warmed (1.5h) to 0 ℃ for 20 min. The mixture was cooled to-30 ℃, quenched with acetic acid (10 μ Ι _), stirred for 10min, diluted with ethyl acetate, then washed with water and brine. The organic layer was separated over MgSO₄Dried, filtered and concentrated in vacuo. Purification by flash column chromatography on silica gel (50% to 100% ethyl acetate in dichloromethane) afforded 16.3mg (43%) 28 as a pale yellow solid:

¹H NMR(CDCl₃)δ11.3(brs，1H)，9.4(brs，1H)，7.4(m，2H)，7.1(s，1H)，6.95(s，1H)，6.8(s，1H)，4.4(s，2H)，3.8(s，3H)，3.8(m，1H)，2.6(s，3H)，2.4(dd，1H)，1.4(m，1H).ESMS m/z 490(M-H)^-.

3.1 d Synthesis of Compound 29

A solution of 28(50mg, 0.103mmol) in N, N-dimethylformamide (1.0mL) was treated with 1mL of anhydrous hydrochloric acid (1.0M in dioxane). The resulting solution was stirred at ambient temperature for 30min, then the solvent was concentrated. The resulting residue was purified by flash column chromatography on silica gel (50% to 100% ethyl acetate in dichloromethane) to give 50mg (100%) of 29 as a light yellow solid:

¹H NMR(CDCl₃)δ11.3(brs，1H)，9.4(brs，1H)，7.4(m，2H)，7.1(s，1H)，6.95(s，1H)，6.8(s，1H)，4.4(s，2H)，3.8(s，3H)，3.8(m，1H)，2.6(s，3H)，2.4(dd，1H)，1.4(m，1H).

3.1 Synthesis of e Compound 239

To a suspension of 29(24mg, 0.05mmol) in dichloromethane (5mL) at-78 deg.C was added 4-nitrophenyl chloroformate (40mg, 0.2mmol) and triethylamine (28. mu.L, 0.2 mmol). The reaction mixture was allowed to warm to room temperature and then concentrated in vacuo. The resulting residue was washed with diethyl ether and then dried in vacuo to give 9 as a yellow solid. Yellow solid 9(19mg, 0.029mmol) was dissolved in dichloromethane (3mL), amine C (20mg, 0.029mmol) was added, followed by triethylamine (8.3. mu.L, 0.06 mmol). The reaction was stirred for 16 hours, then concentrated in vacuo. The resulting residue was purified by flash chromatography on silica, eluting with 40: 1 dichloromethane: methanol, to give 12mg (45%) 239 as a yellow solid:¹HNMR(CDCl₃)：δ9.9(s，1H)，9.7(s，1H)，8.7(s，1H)，8.4(dd，1H)，8.3(d，1H)，8.1(brs，1H)，7.75(d，1H)，7.65(s，1H)，6.7(m，2H)，4.75(m，1H)，4.55(m，2H)，3.9(m，4H)，3.8(m，4H)，3.5(m，18H)，3.0(m，2H)，2.7(s，3H)，2.5(m，4H)，1.7(m，2H)，1.4(m，10H)，1.0(m，2H).ESMS m/z 1100(M-H)^-.

the following compounds were prepared in a similar manner:

238：¹H NMR(CDCl₃)：δ10.3(s，1H)，8.7(s，1H)，8.5(m，1H)，8.4(d，1H)，8.2(brs，1H)，7.7(m，4H)，7.2(m，1H)，4.8(m，1H)，4.6(m，2H)，3.9(m，4H)，3.7(m，2H)，3.4(m，1H)，3.2(m，2H)，2.9(s，3H)，2.5(s，3H).

ESMSm/z 710(M-H)^-.

242：¹H NMR(CDCl₃)：δ9.7(brs，1H)，9.0(brs，1H)，8.6(s，1H)，8.4(d，1H)，8.1(brs，1H)，7.7(m，1H)，7.5(m，1H)，7.4(m，1H)，4.7(d，1H)，4.5(m，2H)，3.9(s，4H)，3.8(m，1H)，3.6(m，1H)，3.4(m，1H)，3.0(m，5H)，2.6(s，3H)，2.5(m，2H)，1.5(m，9H)，1.4(m，6H).

244：¹H NMR(CDCl₃)：δ8.6(brs，1H)，8.4(d，1H)，8.1(m，1H)，7.7(m，6H)，6.9(m，2H)，4.75(m，1H)，4.55(m，2H)，4.1(m，2H)，3.9(m，4H)，3.7(m，12H)，3.5(m，2H)，3.4(m，3H)，3.2(m，3H)，3.1(m，5H)，2.7(s，6H)，2.1(m，2H)，1.5(s，6H).

248：¹H NMR(CDCl₃)：δ9.9(s，1H)，9.3(brs，1H)，8.1(m，1H)，7.5(m，3H)，7.1(m，2H)，4.8(d，1H)，4.5(m，2H)，3.95(s，3H)，3.85(s，3H)，3.8(m，1H)，3.7(m，2H)，3.4(m，1H)，3.0(m，8H)，2.5(m，2H)，1.5(dd，6H)，1.3(s，9H).

250：¹H NMR(CDCl₃)：δ9.4(s，1H)，8.6(s，1H)，8.3(m，1H)，8.1(m，1H)，7.9(m，2H)，7.5(m，7H)，7.1(m，4H)，4.8(m，1H)，4.5(m，2H)，4.3(m，1H)，4.95(s，3H)，4.85(s，3H)，4.7(m，1H)，3.4(m，1H)，3.1(m，4H)，2.7(s，3H)，2.2(s，3H)，1.5(s，6H).

272：¹H NMR(DMSO)：δ12.0(m，1H)，8.8(brs，1H)，8.3(m，1H)，7.9(m，3H)，7.7(m，3H)，6.9(m，2H)，4.85(s，1H)，4.7(m，1H)，4.5(m，3H)，3.8(m，4H)，3.6(m，4H)，3.4(m，5H)，3.1(m，6H)，2.7(s，3H)，2.2(m，2H)，1.4(s，6H).

example 4

Proliferation assay

The assays selected for measuring the biological activity of cytotoxic compounds are well established³H-thymidine proliferation assay. This is a method suitable for quantifying cell proliferation because it is measured by exogenous radiolabelling³Binding of H-thymidine assessed DNA synthesis. This assay is nonIs reproducible and can accommodate a large number of compounds.

Leukemia cells, HL-60, of promyelocytes were cultured in RPMI medium containing 10% heat-inactivated Fetal Calf Serum (FCS). On the day of the study, cells were harvested, washed, and plated at 0.5X 10⁶The cells/mL were resuspended in RPMI containing 10% FCS. 100 μ L of cell suspension was added to a 96-well plate. Serial dilutions (3-fold increments) of doxorubicin or test compound were performed, adding 100 μ L of compound per well. Finally, 10. mu.L of 100. mu. Ci/mL was added to each well³H-thymidine, plates were incubated for 24 hours. Plates were harvested using a 96-well harvester (Packard Instruments) and counted on a Packard Top Count counter. Determination from four-parameter logic curves³Binding of H-thymidine as a function of molar concentration of drug, IC was determined using Prism software₅₀The value is obtained.

IC of Compounds of the invention in the above assays₅₀Values are generally from about 1pM to about 100nM, preferably from about 10pM to about 10 nM.

Each patent application, patent, publication, and other published document referred to or referred to in this specification is herein incorporated by reference in its entirety to the same extent as if each individual patent application, patent, publication, or other published document was specifically and individually indicated to be incorporated by reference.

While the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention and the appended claims. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to fall within the scope of the appended claims.

Claims (47)

1. A compound having the structure:

wherein

X and Z are independently selected from O, S and NR²³Is a member of (a) a group of (b),

wherein

R²³Is a member selected from the group consisting of H, substituted or unsubstituted hydrocarbyl, substituted or unsubstituted heterohydrocarbyl, and acyl;

R¹is H, substituted or unsubstituted lower alkyl, or C (O) R⁸Wherein R is⁸Is selected from NR⁹R¹⁰And OR⁹Is a member of (a) a group of (b),

wherein

R⁹And R¹⁰Is a member independently selected from the group consisting of H, substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl;

R²is H, or substituted or unsubstituted lower alkyl;

R³is selected from (═ O) and OR¹¹Is a member of (a) a group of (b),

wherein

R¹¹Is selected from H, substituted or unsubstituted hydrocarbyl, substituted or unsubstituted heterohydrocarbyl, acyl, C (O) R¹²R¹³、C(O)OR¹²、C(O)NR¹²R¹³、C(O)OR¹²、P(O)(OR¹²)₂、C(O)CHR¹²R¹³、C(O)OR¹²And SiR¹²R¹³R¹⁴Is a member of (a) a group of (b),

wherein

R¹²、R¹³And R¹⁴Is a member independently selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted aryl, wherein R is¹²And R¹³Optionally joined together with the nitrogen atom to which they are attached to form a 4-to 6-membered substituted or unsubstituted heterocycloalkyl ring system, optionally containing two or more heteroatoms, and one or more R¹²、R¹³And R¹⁴Optionally comprising an enzyme cleavable group;

R⁴and R⁵Is independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, NO₂、NR¹⁵R¹⁶、NC(O)R¹⁵、OC(O)NR¹⁵R¹⁶、OC(O)OR¹⁵、C(O)R¹⁵、OR¹⁵Is a member of (a) a group of (b),

wherein

R¹⁵And R¹⁶Independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substitutedOr unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted peptidyl, wherein R is¹⁵And R¹⁶Optionally joined together with the nitrogen atom to which they are attached to form a 4-to 6-membered substituted or unsubstituted heterocycloalkyl ring system, optionally containing two or more heteroatoms;

R⁶is a single bond, which may or may not be present, and when present, R⁶And R⁷Are linked to form a cyclopropyl ring;

R⁷is CH₂-X¹or-CH₂-, with R in the cyclopropyl ring⁷Is connected, wherein X¹Is a leaving group.

2. A compound according to claim 1, wherein R²Is not CF₃。

3. The compound according to claim 1, wherein said leaving group is a member selected from the group consisting of azide, halogen, alkylsulfonyl and arylsulfonyl.

4. A compound according to claim 3, wherein the leaving group is Cl or Br.

5. A compound according to claim 1, wherein R¹Is CO₂CH₃。

6. A compound according to claim 1, wherein R²Is CH₃。

7. A compound according to claim 1, wherein R¹Is CO₂CH₃，R²Is CH₃。

8. A compound according to claim 7, wherein R⁴And R⁵Is independently selected from H, halogen, NH₂、O(CH₂)₂N(Me)₂And NO₂Is a member of (1).

9. A compound according to claim 1, wherein at least one R⁴And R⁵Is not selected from H and OCH₃Is a member of (1).

10. A compound according to claim 1, wherein at least one R⁴、R⁵、R¹⁵And R¹⁶Comprising a cleavable disulfide group.

11. A compound according to claim 1, wherein at least one R⁴、R⁵、R¹⁵And R¹⁶Comprises the following steps:wherein

R³⁰Is a member selected from the group consisting of H, substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl;

R³¹and R³²Is a member independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, or R³¹And R³²Together, are:wherein

R³³And R³⁴Is a member independently selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;

R³⁵is selected from substituted or unsubstituted hydrocarbyl, substituted or unsubstituted heterohydrocarbyl and NR³⁶Is a member of (a) a group of (b),

wherein

R³⁶Is a member selected from the group consisting of H, substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl;

X⁵is O or NR³⁷，

Wherein R is³⁷Is selected from the group consisting of H, substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heteroA member of a hydrocarbyl group.

12. The compound according to claim 11, wherein at least one R³¹、R³²、R³³And R³⁴Substituted with a member selected from the group consisting of a targeting agent and a detectable label.

13. The compound of claim 12, wherein said targeting agent is an antibody.

14. The compound according to claim 1, wherein at least one is selected from R⁴、R⁵、R¹⁵And R¹⁶Comprises a targeting agent or a detectable label.

15. The compound of claim 14, wherein said targeting agent is an antibody.

16. The compound according to claim 7, having the structure:and

17. the compound according to claim 7, wherein X is O and Z is O.

18. A compound according to claim 1, wherein R is selected from⁴And R⁵The members of (a) are:

wherein

X²And Z¹Is independently selected from O, S and NR²³A member of (a);

R¹⁷and R¹⁸Is independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstitutedHeteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, NO₂、NR¹⁹R²⁰、NC(O)R¹⁹、OC(O)NR¹⁹、OC(O)OR¹⁹、C(O)R¹⁹、OR¹⁹Is a member of (a) a group of (b),

wherein

R¹⁹And R²⁰Independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or substituted heteroaryl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted peptidyl, wherein R is¹⁹And R²⁰Together with the nitrogen atom to which they are attached optionally form a 4-to 6-membered substituted or unsubstituted heterocycloalkyl ring system, optionally containing two or more heteroatoms, and one or more is selected from R¹⁹And R²⁰Comprises a cleavable group.

19. A compound according to claim 18 wherein when Z is¹When is NH, R¹⁷And R¹⁸Are not both H, R¹⁷Is not NH₂。

20. A compound according to claim 18, wherein X²Is O, Z¹Is selected from O and NR²³Is a member of (1).

21. A compound according to claim 1, wherein R¹¹Is a peptidyl moiety having the following structure:

wherein

X³Is a member selected from the group consisting of protected or unprotected reactive functional groups, detectable labels and targeting agents;

L¹is a linking group selected from substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl;

AA¹、AA^band AA^b+1Is a member independently selected from natural and non-natural alpha-amino acids;

L²is a linking group selected from substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl;

q and v are integers independently selected from 0 and 1;

b is an integer of 0 to 20.

22. The compound according to claim 21, wherein at least one is selected from L³And L⁴Comprises a poly (ethylene glycol) moiety.

23. A compound according to claim 21, wherein the peptidyl moiety has the following structure:

wherein

R²¹And R²²Is a member independently selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, a detectable label, and a targeting agent;

R²⁵is a member selected from the group consisting of H, substituted or unsubstituted lower alkyl, an amino acid side chain, a detectable label, and a targeting agent;

s is an integer from 0 to 20.

24. The compound according to claim 21, wherein c is an integer between 1 and 5.

25. The compound according to claim 21, wherein at least one is selected from R¹⁵And R¹⁶Has the following structure:

wherein

X⁴Is a member selected from the group consisting of protected or unprotected reactive functional groups, detectable labels and targeting agents;

L³is a linking group selected from substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl;

AA¹、AA^cand AA^c+1Is a member independently selected from natural and non-natural alpha-amino acids;

L⁴is a linking group selected from substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl;

R²⁴is a member selected from the group consisting of H, substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl;

p and t are integers independently selected from 0 and 1;

c is an integer of 0 to 20.

26. The compound according to claim 25, wherein at least one is selected from L¹And L²Comprises a poly (ethylene glycol) moiety.

27. The compound according to claim 25, wherein said member has the structure:wherein

R²⁷And R²⁸Is a member independently selected from the group consisting of H, substituted or unsubstituted lower alkyl, an amino acid side chain, a detectable label, and a targeting agent.

28. The compound according to claim 25, wherein c is an integer from 1 to 5.

29. A compound according to claim 1 or 25, wherein R¹¹Has the following structure:wherein

X⁴Is a member selected from the group consisting of protected reactive functional groups, unprotected reactive functional groups, detectable labels and targeting agents;

L³is a linking group selected from substituted or unsubstituted hydrocarbyl and substituted or unsubstituted heterohydrocarbyl;

L⁴is a linking group selected from substituted or unsubstituted hydrocarbyl andsubstituted or unsubstituted heterohydrocarbyl;

p and t are integers independently selected from 0 and 1.

30. The compound according to claim 29, wherein L⁴Is a substituted or unsubstituted ethylene moiety.

31. A compound according to claim 29, wherein X⁴Is selected from R²⁹、COOR²⁹、C(O)NR²⁹And C (O) NNR²⁹Is a member of (a) a group of (b),

wherein

R²⁹Is a member selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heteroaryl.

32. The compound according to claim 31, wherein R²⁹Is selected from H; OH; NHNH₂；And

is a member of (a) a group of (b),

wherein

R³⁰Represents a substituted or unsubstituted hydrocarbon group terminating in a reactive functional group, a substituted or unsubstituted heteroaryl group terminating in a functional group and- (L)³)_pX⁴Wherein each L³、X⁴And p are independently selected.

33. The compound according to claim 21, 25 or 29, wherein the detectable label is a fluorophore.

34. The compound according to claim 21, 25 or 29, wherein the targeting agent is a biomolecule.

35. The compound of claim 34, wherein said biomolecule is a member selected from the group consisting of antibodies, receptors, peptides, lectins, sugars, nucleic acids, and combinations thereof.

36. A compound according to claim 1, wherein at least one R¹⁵And R¹⁶Carrying a reactive group suitable for conjugating the compound to another molecule.

37. The compound according to claim 36, wherein at least one R¹⁵And R¹⁶Is a member selected from the group consisting of substituted hydrocarbyl and substituted heterohydrocarbyl, said member having said reactive functional group as its free terminus.

38. The compound according to claim 36, wherein said compound is conjugated to said another molecule via said reactive functional group.

39. A compound according to claim 1, wherein R¹⁵And R¹⁶One contains a cleavable moiety.

40. The compound according to claim 27, wherein at least one R²¹And R²²Carrying a reactive group suitable for conjugating the compound to another molecule.

41. The compound according to claim 36, wherein at least one R²¹And R²²Is a member selected from the group consisting of substituted hydrocarbyl and substituted heterohydrocarbyl, said member having said reactive functional group as its free terminus.

42. The compound according to claim 36, wherein said compound is conjugated to said another molecule via said reactive functional group.

43. A pharmaceutical formulation comprising a compound according to claim 1 and a pharmaceutically acceptable carrier.

44. A method of killing a cell, the method comprising administering to the cell an amount of a compound according to claim 1 sufficient to kill the cell.

45. A method of killing cancer cells in a subject carrying said cells, said method comprising administering to said subject an amount of a compound according to claim 1 sufficient to kill said cells.

46. A method of retarding or stopping tumor growth in a mammalian subject, said method comprising administering to said subject an amount of a compound according to claim 1 sufficient to retard or stop said growth.

47. A compound having the structure:

wherein

Ring system a is a member selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl;

e and G are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, a heteroatom, or a single bond, E and G optionally joined to form a ring system selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocycloalkyl;

x is selected from O, S and NR²³Member of (A), R²³Is a member selected from the group consisting of H, substituted or unsubstituted hydrocarbyl, substituted or unsubstituted heterohydrocarbyl, and acyl;

R³is selected from (═ O), SR¹¹And OR¹¹Is a member of (a) a group of (b),

wherein

R¹¹Is selected from H, substituted or unsubstituted hydrocarbyl, substituted or unsubstituted heterohydrocarbyl, acyl, C (O) R¹²、C(O)OR¹²、C(O)NR¹²R¹³、C(O)OR¹²、P(O)(OR¹²)₂、C(O)CHR¹²R¹³、C(O)OR¹²Or SiR¹²R¹³R¹⁴Member of (A), R¹¹Optionally comprising an enzymatically cleavable group,

wherein

R¹²、R¹³And R¹⁴Is a member independently selected from the group consisting of H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted aryl, wherein R is¹²And R¹³Optionally joined together with the nitrogen atom to which they are attached to form a 4-to 6-membered substituted or unsubstituted heterocycloalkyl ring system, optionally containing two or more heteroatoms, and one or more R¹²、R¹³And R¹⁴Optionally comprising an enzyme cleavable group;

R⁴and R⁵Is independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, halogen, NO₂、NR¹⁵R¹⁶、NC(O)R¹⁵、OC(O)NR¹⁵R¹⁶、OC(O)OR¹⁵、C(O)R¹⁵、SR¹⁵And OR¹⁵Is a member of (a) a group of (b),

wherein

R¹⁵And R¹⁶Independently represent H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted peptidyl, wherein R is¹⁵And R¹⁶Optionally joined together with the nitrogen atom to which they are attached to form a 4-to 6-membered substituted or unsubstituted heterocycloalkyl ring system, optionally containing two or more heteroatoms, and one or more R⁴、R⁵、R¹⁵And R¹⁶Optionally comprising an enzymatically cleavable group.