Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
  • C07D491/08—Bridged systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/439—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

• This invention relates to enediyne compounds and conjugates thereof, methods for making and using such compounds and conjugates, and compositions comprising such compounds and conjugates.
• the enediynes are a family of antibiotics that possess a distinctive strained nine- or ten-member ring system comprising a Z-carbon-carbon double bond and two carbon- carbon triple bonds, usually arranged with the latter two flanking the former.
• the enediynes are potent damagers of DNA, causing single and double strand cuts. Their potency is attributed to their ability to bind to DNA and undergo a Bergmann rearrangement in which the strained ring system is converted into a highly reactive 1 ,4-benzenoid diradical, which damages the DNA by abstracting hydrogens from it.
• Uncialamycin is an enediyne isolated from a Streptomyces strain found on the lichen Cladonia uncialis (Davies et al. 2005; 2007). (Full citations for references cited in this specification by first named author or inventor and year are provided in the section entitled “REFERENCES” later herein.)
• Conjugates are an important method for the delivery of anti-cancer drugs, which are often highly cytotoxic and might otherwise be problematic to administer due to the risk of systemic toxicity.
• the drug is conjugated (covalently linked) to a targeting moiety that specifically or preferentially binds to a chemical entity characteristic of the cancer cell, thus delivering the drug there with high specificity. Further, the drug is held in an inactive form until released from the conjugate, usually by cleavage of the covalent linker.
• the targeting moiety is an antibody or an antigen-binding portion thereof, whose antigen is overexpressed or uniquely expressed by a cancer cell ("tumor associated antigen").
• tumor associated antigen an antibody or an antigen-binding portion thereof, whose antigen is overexpressed or uniquely expressed by a cancer cell
• the resulting conjugate is sometimes refered to as an “immunoconjugate” or an “antibody-drug conjugate” (ADC).
• ADC antibody-drug conjugate
• the tumor associated antigen is located on the surface of the cancer cell, but also can be one that is secreted into the vicinal extracellular space.
• the antigen-conjugate complex Upon binding, the antigen-conjugate complex is internalized and eventually finds its way inside a vesicular body such as a lysosome, where the covalent linker is cleaved, liberating active drug to exert its chemotherapeutic effect.
• the covalent linker is designed such that cleavage is caused by a factor prevalent inside a cancer cell but not in plasma.
• a factor prevalent inside a cancer cell but not in plasma is the low lysosomal pH, so that the covalent linker can be an acid-sensitive group such as a hydrazone.
• Another such factor is the generally higher intracellular concentration of glutathione, allowing for the cleavage of a disulfide covalent linker by a disulfide exchange mechanism.
• Yet another such factor is the presence of lysosomal enzymes such as cathepsin B, which can cleave peptide linkers designed to be preferred substrates (Dubowchik et al. 2002).
• Conjugates have been used to deliver enediyne drugs in oncology.
• Gemtuzumab ozogamicin (Mylotarg®) is a conjugate of an anti-CD33 monoclonal antibody and a derivative of the enediyne calicheamicin. It was approved for treatment of acute
• the present invention provides compounds based on an uncialamycin scaffold, which are potent cytotoxins having utility as chemotherapeutic drugs, whether used as such or in conjugates.
• R lb is -C 6 alkyl, (CH 2 ) n NH 2 ,
• R 3 is H or unsubstituted or substituted C1-C6 alkyl
• R 8 is the side chain residue of an a-amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, ⁇ -carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine;
• a-amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, ⁇ -carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phen
• R 9 is unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene, unsubstituted or substituted alkylarylene, unsubstituted or substituted cycloalkylene or unsubstituted or substituted heterocycloalkylene;
• each R 10 is independently unsubstituted or substituted Ci-Ce alkyl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocycloalkyl, unsubstituted or substituted arylalkyl, unsubstituted or substituted aryl; or unsubstituted or substituted heteroaryl; and
• n 2, 3, 4, 5, or 6;
• R° is NHR la .
• the group NHR la can be attached to any of the carbon atoms at positions 6, 7, 8, or 9 (see structural formula for uncialamycin, above, for numbering of carbon atoms).
• the structure of formula (I) can be equivalently depicted by formula ( ⁇ )
• R 11 groups where one of the R 11 groups is R° and the remaining R 11 groups are each H and R°, R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 are as defined above for formula (I).
• Uncialamycin is a potential candidate for the drug component in a conjugate, but it lacks functional groups that are readily usable as sites for conjugation to a targeting moiety without compromising biological activity.
• a R° group to the leftmost aromatic ring in the anthraquinone moiety, as shown in formula (I), without unacceptable loss of biological activity and, further, that the R° group is a versatile site for conjugation.
• this invention provides a conjugate comprising a compound according to formula (I) covalently linked to a targeting moiety that specifically or preferentially binds to a chemical entity on a target cell, which preferably is a cancer cell.
• the targeting moiety is an antibody - more preferably a monoclonal antibody and even more preferably a human monoclonal antibody - and the chemical entity is a tumor associated antigen.
• composition of matter comprising a compound of this invention and a linker moiety having a reactive functional group, suitable for conjugation to a targeting moiety.
• this invention provides a method for inhibiting the proliferation of cancer cells in a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of a compound of this invention or a conjugate thereof with a targeting moiety (particularly an antibody).
• the cancer cells can be leukemia, renal cancer, ovarian cancer, lung cancer, colon cancer, breast cancer, or prostate cancer cells.
• a method of treating a cancer in a subject suffering from such cancer comprising administering to the subject a therapeutically effective amount of a compound of this invention or a conjugate thereof with a targeting moiety (particularly an antibody).
• a targeting moiety particularly an antibody
• the cancer can be leukemia, renal cancer, ovarian cancer, lung cancer, colon cancer, breast cancer, or prostate cancer.
• FIGs. 1 through 6 show schemes for the synthesis of compounds of this invention.
• Figs. 7 through 10 show reaction schemes for the attachment of linker and reactive functional groups to compounds of this invention, in preparation for conjugation.
• Figs. 11a and lib show plots of the antiproliferative activity of a compound of this invention, compared to selected reference compounds.
• Figs. 12a and 12b show plots of the antiproliferative activity of additional compounds of this invention, compared to selected reference compounds.
• Figs. 13a, 13b, and 13c show plots of the antiproliferative activity of antibody- drug conjugates made from compounds of this invention. DETAILED DESCRIPTION OF THE INVENTION
• Antibody means whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chain variants thereof.
• a whole antibody is a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
• Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region comprising three domains, CHI, CH2 and CH3.
• Each light chain comprises a light chain variable region (VL or V k ) and a light chain constant region comprising one single domain, CL.
• the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved framework regions (FRs).
• CDRs complementarity determining regions
• Each VH and VL comprises three CDRs and four FRs, arranged from amino- to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
• the variable regions contain a binding domain that interacts with an antigen.
• the constant regions may mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
• An antibody is said to "specifically bind" to an antigen X if the antibody binds to antigen X with a KD of 5 x 10 ⁇ 8 M or less, more preferably 1 x 10 ⁇ 8 M or less, more preferably 6 x 10 ⁇ 9 M or less, more preferably 3 x 10 ⁇ 9 M or less, even more preferably 2 x 10 ⁇ 9 M or less.
• the antibody can be chimeric, humanized, or, preferably, human.
• the heavy chain constant region can be engineered to affect glycosylation type or extent, to extend antibody half-life, to enhance or reduce interactions with effector cells or the complement system, or to modulate some other property. The engineering can be accomplished by replacement, addition, or deletion of one or more amino acids or by replacement of a domain with a domain from another immunoglobulin type, or a combination of the foregoing.
• Antigen binding fragment and "antigen binding portion” of an antibody mean one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody, such as (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially an Fab with part of the hinge region (see, for example, Abbas et al, Cellular and Molecular Immunology, 6th Ed., Saunders Elsevier 2007); (iv) a Fd fragment consisting of the VH and CHI domains; (v) a Fv fragment consisting of the VL and
• the two domains of the Fv fragment, VL and VH are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv, or scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883 ⁇ ).
• Such single chain antibodies are also encompassed within the term "antigen- binding portion" of an antibody.
• an "isolated antibody” means an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds antigen X is substantially free of antibodies that specifically bind antigens other than antigen X).
• An isolated antibody that specifically binds antigen X may, however, have cross- reactivity to other antigens, such as antigen X molecules from other species.
• an isolated antibody specifically binds to human antigen X and does not cross- react with other (non-human) antigen X antigens.
• an isolated antibody may be substantially free of other cellular material and/or chemicals.
• “Monoclonal antibody” or “monoclonal antibody composition” means a preparation of antibody molecules of single molecular composition, which displays a single binding specificity and affinity for a particular epitope.
• Human antibody means an antibody having variable regions in which both the framework and CDR regions (and the constant region, if present) are derived from human germline immunoglobulin sequences. Human antibodies may include later modifications, including natural or synthetic modifications. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
• Human monoclonal antibody means an antibody displaying a single binding specificity, which has variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.
• human monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.
• Aliphatic means a straight- or branched-chain, saturated or unsaturated, non- aromatic hydrocarbon moiety having the specified number of carbon atoms (e.g., as in “C3 aliphatic,” “C 1 -C5 aliphatic,” or “Ci to C5 aliphatic,” the latter two phrases being
• Alkyl means a saturated aliphatic moiety, with the same convention for designating the number of carbon atoms being applicable.
• C 1 -C4 alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, ?-butyl, 1- butyl, 2-butyl, and the like.
• Alkylene means a divalent counterpart of an alkyl group, such as CH 2 CH 2 , CH 2 CH 2 CH 2 , and CH 2 CH 2 CH 2 CH 2 .
• alkenyl means an aliphatic moiety having at least one carbon-carbon double bond, with the same convention for designating the number of carbon atoms being applicable.
• C 2 -C 4 alkenyl moieties include, but are not limited to, ethenyl (vinyl), 2-propenyl (allyl or prop-2-enyl), cis-l-propenyl, trans- 1 -propenyl, E- (or Z-) 2-butenyl, 3-butenyl, 1,3-butadienyl (but-l,3-dienyl) and the like.
• Alkynyl means an aliphatic moiety having at least one carbon-carbon triple bond, with the same convention for designating the number of carbon atoms being applicable.
• C 2 -C 4 alkynyl groups include ethynyl (acetylenyl), propargyl (prop-2-ynyl), 1-propynyl, but-2-ynyl, and the like.
• Cycloaliphatic means a saturated or unsaturated, non-aromatic hydrocarbon moiety having from 1 to 3 rings, each ring having from 3 to 8 (preferably from 3 to 6) carbon atoms.
• Cycloalkyl means a cycloaliphatic moiety in which each ring is saturated.
• Cyclo- alkenyl means a cycloaliphatic moiety in which at least one ring has at least one carbon-carbon double bond.
• Cycloalkynyl means a cycloaliphatic moiety in which at least one ring has at least one carbon-carbon triple bond.
• cycloaliphatic moieties include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohe- xyl, cyclohexenyl, cycloheptyl, cyclooctyl, and adamantyl.
• Preferred cycloaliphatic moieties are cycloalkyl ones, especially cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
• Cycloalkylene means a divalent counterpart of a cycloalkyl group.
• Heterocycloaliphatic means a cycloaliphatic moiety wherein, in at least one ring thereof, up to three (preferably 1 to 2) carbons have been replaced with a heteroatom independently selected from N, O, or S, where the N and S optionally may be oxidized and the N optionally may be quaternized.
• heterocycloalkyl means a cycloalkyl, cycloalkenyl, or cycloalkynyl moiety, respectively, in which at least one ring thereof has been so modified.
• heterocycloaliphatic moieties include aziridinyl, azetidinyl, 1,3-dioxanyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl, piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrothiopyranyl sulfone, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolanyl, tetrahydro- 1 , 1 -dioxothienyl, 1,4-dioxanyl, thietanyl, and the like.
• Heterocycloalkylene means a divalent counterpart of a heterocycloalkyl group.
• Alkoxy means -O(alkyl), -O(aryl), -S(alkyl), and -S(aryl), respectively. Examples are methoxy, phenoxy, methylthio, and phenylthio, respectively.
• Halogen or "halo" means fluorine, chlorine, bromine or iodine.
• Aryl means a hydrocarbon moiety having a mono-, bi-, or tricyclic ring system wherein each ring has from 3 to 7 carbon atoms and at least one ring is aromatic.
• the rings in the ring system may be fused to each other (as in naphthyl) or bonded to each other (as in biphenyl) and may be fused or bonded to non-aromatic rings (as in indanyl or cyclohexyl- phenyl).
• aryl moieties include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthracenyl, and acenaphthyl.
• “Arylene” means a divalent counterpart of an aryl group, for example 1 ,2-phenylene, 1,3- phenylene, or 1,4-phenylene.
• Heteroaryl means a moiety having a mono-, bi-, or tricyclic ring system wherein each ring has from 3 to 7 carbon atoms and at least one ring is an aromatic ring containing from 1 to 4 heteroatoms independently selected from from N, O, or S, where the N and S optionally may be oxidized and the N optionally may be quaternized.
• Such at least one heteroatom containing aromatic ring may be fused to other types of rings (as in benzo- furanyl or tetrahydroisoquinolyl) or directly bonded to other types of rings (as in phenylpy- ridyl or 2-cyclopentylpyridyl).
• heteroaryl moieties include pyrrolyl, furanyl, thiophenyl (thienyl), imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl, N-oxopyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolynyl, quinazolinyl, cinnolinyl, quinozalinyl, naphthyridinyl, benzo- furanyl, indolyl, benzothiophenyl, oxadiazolyl, thiadiazolyl, phenothiazolyl, benzimidazolyl, benzotriazolyl, dibenzofuranyl, carbazolyl, dibenzothiophenyl,
• Heteroarylene means a divalent counterpart of an aryl group.
• Arylalkyl (heterocycloaliphatic)alkyl
• arylalkenyl “arylalkynyl”
• biasalkyl and the like mean an alkyl, alkenyl, or alkynyl moiety, as the case may be, substituted with an aryl, heterocycloaliphatic, biaryl, etc., moiety, as the case may be, with the open (unsatisfied) valence at the alkyl, alkenyl, or alkynyl moiety, for example as in benzyl, phenethyl, N-imidazoylethyl, N-morpholinoethyl, and the like.
• alkylaryl means an aryl, cycloalkyl, etc., moiety, as the case may be, substituted with an alkyl, alkenyl, etc., moiety, as the case may be, for example as in methylphenyl (tolyl) or allylcyclohexyl.
• Hydrophilalkyl means an alkyl, aryl, etc., moiety, as the case may be, substituted with one or more of the identified substituent (hydroxyl, halo, etc., as the case may be).
• C 1 -C4 alkyl cyano, nitro, halo, and Ci-C 4 alkoxy.
• “Pharmaceutically acceptable ester” means an ester that hydrolyzes in vivo (for example in the human body) to produce the parent compound or a salt thereof or has per se activity similar to that of the parent compound.
• Suitable esters include C 1 -C5 alkyl, C 2 -Cs alkenyl or C 2 -Cs alkynyl esters, especially methyl, ethyl or n-propyl.
• “Pharmaceutically acceptable salt” means a salt of a compound suitable for pharmaceutical formulation. Where a compound has one or more basic groups, the salt can be an acid addition salt, such as a sulfate, hydrobromide, tartrate, mesylate, maleate, citrate, phosphate, acetate, pamoate (embonate), hydroiodide, nitrate, hydrochloride, lactate, methyl- sulfate, fumarate, benzoate, succinate, mesylate, lactobionate, suberate, tosylate, and the like.
• an acid addition salt such as a sulfate, hydrobromide, tartrate, mesylate, maleate, citrate, phosphate, acetate, pamoate (embonate), hydroiodide, nitrate, hydrochloride, lactate, methyl- sulfate, fumarate, benzoate, succinate, mesylate, lactobionate
• the salt can be a salt such as a calcium salt, potassium salt, magnesium salt, meglumine salt, ammonium salt, zinc salt, piperazine salt, tromethamine salt, lithium salt, choline salt, diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodium salt, tetramethylammonium salt, and the like.
• a salt such as a calcium salt, potassium salt, magnesium salt, meglumine salt, ammonium salt, zinc salt, piperazine salt, tromethamine salt, lithium salt, choline salt, diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodium salt, tetramethylammonium salt, and the like.
• Polymorphic crystalline forms and solvates are also encompassed within the scope of this invention.
• a preferred embodiment according to formula (I) is a compound having a structure represented by formula (la), or a pharmaceutically acceptable salt thereof.
• a group NHR la is attached to C6, wherein R la is as defined hereinabove in the context of formula (I):
• a more preferred embodiment is a compound having a structure represented by formula (lb), or a pharmaceutically acceptable salt thereof, where R la is as defined hereinabove in the context of formula (I).
• R la is as defined hereinabove in the context of formula (I).
• the stereochemistry of the C27- methyl corresponds to that of the naturally occurring uncialamycin (see structural formula for uncialamycin, supra).
• R° R la , R lb . R 2 , R 3 , R 4 , R 5 , R 6 . and R 7 where they occur in formula (I) or other formulae elsewhere in this specification, where an alkyl, alkylene, aryl, arylene, heteroaryl, heteroarylene, cycloalkyl, cycloalkylene, heterocycloalkyl, or
• heterocycloalkylene group is indicated as being either unsubstituted or substituted, the unsubstituted embodiment is preferred.
• R 2 preferably is H or C1-C3 alkyl, more preferably H.
• R 3 preferably is Ci- C3 alkyl, more preferably Me.
• R 6 preferably is H.
• R 9 preferably is
• R 9 is
• R 10 preferably is Ci-Ce alkyl, cyclohexyl, cyclopentyl, phenyl, furanyl, or pyridyl. More preferably, R 10 is methyl, ethyl, propyl, or isopropyl.
• R 8 preferably is the side chain residue of an a-amino acid selected from the group consisting of arginine, aspartic acid, citrulline, glutamic acid, glutamine, glycine, histidine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine. More preferably, R 8 is the side chain residue of glycine, lysine, citrulline, or serine. Preferably, the stereochemistry at the chiral carbon corresponds to that of a naturally occurring proteogenic a-amino acid, i.e. the L-isomer.
• Another embodiment of this invention comprises a compound having a structure represented by formula (I), (la), (lb), (Ila), (lib), (lie), (lid), (He), (Ilg), or (Ilh) is conjugated to a targeting moiety that specifically or preferentially binds to a chemical entity on a cancer cell.
• the targeting moiety is an antibody or antigen binding portion thereof and the chemical entity is a tumor associated antigen.
• the conjugation is effected through a chemical bond to the group R°.
• D is a cytotoxic compound according to this invention (e.g. , a compound according to formula (I), (la), or (lb)); and -(X D ) a C(X z ) b - are collectively referred to as a "linker moiety" or "linker” because they link Z and D.
• C is a cleavable group designed to be cleaved at or near the site of intended biological action of compound D;
• X D and X z are referred to as spacer moieties (or “spacers") because they space apart D and C and C and Z, respectively; subscripts a and b are independently 0 or 1 (that is, the presence of X D and/or X z are optional); and subscript m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (preferably 1, 2, j , or 4).
• D, X D , C, X z and Z are more fully described hereinbelow.
• Ligand Z - for example an antibody - serves a targeting function.
• ligand Z directs the conjugate there.
• the target tissue or cell is a cancer tissue or cell and the antigen or receptor is a tumor-associated antigen, that is, an antigen that is uniquely expressed by cancerous cells or is overexpressed by cancer cells, compared to non-cancerous cells. Cleavage of group C at the target tissue or cell releases compound D to exert its cytotoxic effect locally.
• the conjugate is internalized into a target cell by endocytosis and cleavage takes place within the target cell.
• compound D is normally biologically inactive (or significantly less active) in its conjugated state, thereby reducing undesired toxicity against non-target tissue or cells. As anticancer drugs are often highly toxic to cells in general, this is an important consideration.
• each molecule of ligand Z can conjugate with more than one compound D, depending on the number of sites ligand Z has available for conjugation and the experimental conditions employed.
• a preparation of the conjugate may analyze for a non-integer ratio of compounds D to ligand Z, reflecting a statistical average.
• ligand Z is an antibody.
• the detailed subsequent discussion herein about the conjugation of ligand Z is written in the context of its being an antibody, but those skilled in the art will understand that other types of ligand Z can be conjugated, mutatis mutandis.
• conjugates with folic acid as the ligand can target cells having the folate receptor on their surfaces (Vlahov et al, Bioorg. Med. Chem. Lett. 2008, 18(16), 4558-4561; Leamon et al, Cancer Res. 2008, 68 (23), 9839-9844).
• the detailed discussion below is primarily written in terms of a 1 : 1 ratio of antibody Z to compound D.
• ligand Z is an antibody against a tumor associated antigen, allowing a conjugate comprising such a ligand Z to selectively target cancer cells.
• antigens include: mesothelin, prostate specific membrane antigen (PSMA), CD 19, CD22, CD30, CD70, CD200 (also known as OX-2), B7H4 (also known as 08E), protein tyrosine kinase 7 (PTK7), RG1, CTLA-4, and CD44.
• PSMA prostate specific membrane antigen
• CD19 CD22, CD30, CD70, CD200 (also known as OX-2), B7H4 (also known as 08E), protein tyrosine kinase 7 (PTK7), RG1, CTLA-4, and CD44.
• the antibody can be animal (e.g., murine), chimeric, humanized, or, preferably, human.
• the antibody preferably is monoclonal, especially a monoclonal human antibody.
• Ligand Z can also be an antibody fragment or antibody mimetic, such as an affibody, a domain antibody (dAb), a nanobody, a unibody, a DARPin, an anticalin, a versabody, a duocalin, a lipocalin, or an avimer.
• dAb domain antibody
• a nanobody a unibody
• a DARPin a domain antibody
• an anticalin a versabody
• a duocalin a lipocalin
• avimer an avimer
• Any one of several different reactive groups on ligand Z can be a conjugation site, including ⁇ -amino groups in lysine residues, pendant carbohydrate moieties, carboxylic acid groups, disulfide groups, and thiol groups.
• Each type of reactive group represents a trade-off, having some advantages and some disadvantages.
• For reviews on antibody reactive groups suitable for conjugation see, e.g., Garnett, Adv. Drug Delivery Rev. 53 (2001), 171-216 and Dubowchik and Walker, Pharmacology & Therapeutics 83 (1999), 67-123, the disclosures of which are incorporated herein by reference.
• ligand Z is conjugated via a lysine ⁇ -amino group.
• Most antibodies have multiple exposed lysine ⁇ -amino groups, which can be conjugated via amide, urea, thiourea, or carbamate bonds using techniques known in the art, including modification with a heterobifunctional agent (as further described hereinbelow).
• a heterobifunctional agent as further described hereinbelow.
• conjugation may cause neutralization of a protonated ⁇ -amino group important for maintaining the antibody's native conformation or may take place at a lysine near or at the antigen binding site, neither being a desirable occurrence.
• ligand Z can be conjugated via a carbohydrate side chain, as many antibodies are glycosylated.
• the carbohydrate side chain can be oxidized with periodate to generate aldehyde groups, which in turn can be reacted with amines to form an imine group, such as in a semicarbazone, oxime, or hydrazone.
• the imine group can be converted to a more stable amine group by reduction with sodium cyanoborohydride.
• conjugation via carbohydrate side chains see, e.g., Rodwell et al, Proc. Nat'l Acad. Sci. USA 83, 2632-2636 (1986); the disclosure of which is incorporated herein by reference.
• lysine ⁇ -amino groups there are concerns regarding
• ligand Z can be conjugated via a carboxylic acid group.
• a terminal carboxylic acid group is functionalized to generate a carbohydrazide, which is then reacted with an aldehyde-bearing conjugation moiety. See Fisch et al, Bioconjugate Chemistry 1992, 3, 147-153.
• antibody Z can be conjugated via a disulfide group bridging a cysteine residue on antibody Z and a sulfur on the other portion of the conjugate.
• Some antibodies lack free thiol (sulfhydryl) groups but have disulfide groups, for example in the hinge region. In such case, free thiol groups can be generated by reduction of native disulfide groups. The thiol groups so generated can then be used for conjugation. See, e.g., Packard et al, Biochemistry 1986, 25, 3548-3552; King et al, Cancer Res. 54, 6176-6185 (1994); and Doronina et al, Nature Biotechnol. 21(7), 778-784 (2003); the disclosures of which are incorporated herein by reference. Again, there are concerns regarding conjugation site location and stoichiometry and the possible disruption of antibody native conformation.
• a number of methods are known for introducing free thiol groups into antibodies without breaking native disulfide bonds, which methods can be practiced with a ligand Z of this invention. Depending on the method employed, it may be possible to introduce a predictable number of free sulfhydryls at predetermined locations.
• mutated antibodies are prepared in which a cysteine is substituted for another amino acid. See, for example, Eigenbrot et al, US 7,521,541 B2 (2009); Chilkoti et al, Bioconjugate Chem. 1994, 5, 504-507; Urnovitz et al, US 4,698,420 (1987);skyl et al, J. Biol.
• lysine ⁇ -amino groups can be modified with hete- robifunctional reagents such as 2-iminothiolane or N-succinimidyl-3-(2-pyridyldithio)- propionate (SPDP), converting an ⁇ -amino group into a thiol or disulfide group - creating a cysteine surrogate, as it were.
• SPDP N-succinimidyl-3-(2-pyridyldithio)- propionate
• ligand Z is conjugated via the nucleophi-lic addition product of a thiol group to an acceptor moiety.
• a preferred acceptor moiety is a maleimide group, whose reaction with an antibody thiol group is generically illus-trated below.
• the thiol group can be a native one, or one introduced as described above.
• Ligand Z can also be conjugated via a functional group adapted for use with "click" chemistry, as discussed hereinbelow.
• the linker portion of a conjugate of this invention comprises up to three elements: a cleavable group C and optional spacers X z and X D .
• Cleavable group C is a group cleavable under physiological conditions, preferably selected such that it is relatively stable while the conjugate is in general circulation in the blood plasma, but is readily cleaved once the conjugate reaches its site of intended action, that is, near, at, or within the target cell.
• the conjugate is internalized by endocytosis by a target cell upon binding of antibody Z to an antigen displayed on the surface of the target cell. Subsequently, cleavage of group C occurs in a vesicular body of the target cell (an early endosome, a late endosome, or, especially, a lysosome).
• group C is a pH sensitive group.
• the pH in blood plasma is slightly above neutral, while the pH inside a lysosome is acidic, circa 5.
• a group C whose cleavage is acid catalyzed will cleave at a rate several orders of magnitude faster inside a lysosome than in the blood plasma rate.
• suitable acid-sensitive groups include cis-aconityl amides and hydrazones, as described in Shen et al, US 4,631,190 (1986); Shen et al, US 5, 144,01 1 (1992); Shen et al, Biochem. Biophys. Res. Commun.
• group C is a disulfide.
• Disulfides can be cleaved by a thiol-disulfide exchange mechanism, at a rate dependent on the ambient thiol concentration. As the intracellular concentration of glutathione and other thiols is higher than their serum concentrations, the cleavage rate of a disulfide will be higher intracellularly.
• the rate of thiol-disulfide exchange can be modulated by adjustment of the steric and electronic characteristics of the disulfide (e.g., an alkyl-aryl disulfide versus an alkyl-alkyl disulfide; substitution on the aryl ring, etc.), enabling the design of disulfide linkages that have enhanced serum stability or a particular cleavage rate.
• disulfide cleavable groups in conjugates see, e.g., Thorpe et al, Cancer Res.
• a preferred group C comprises a peptide bond that is cleaved, preferentially by a protease at the intended site of action, as opposed to by a protease in the serum.
• group C comprises from 1 to 20 amino acids, preferably from 1 to 6 amino acids, more preferably from 1 to 3 amino acids.
• the amino acid(s) can be natural and/or unnatural a- amino acids. Natural amino acids are those encoded by the genetic code, as well as amino acids derived therefrom, e.g., hydroxyproline, ⁇ -carboxyglutamate, citrulline, and O- phosphoserine. The term amino acid also includes amino acid analogs and mimetics.
• Analogs are compounds having the same general H 2 (R)CHC0 2 H structure of a natural amino acid, except that the R group is not one found among the natural amino acids.
• analogs include homoserine, norleucine, methionine-sulfoxide, and methionine methyl sulfonium.
• An amino acid mimetic is a compound that has a structure different from the general chemical structure of an a-amino acid but functions in a manner similar to one.
• the term "unnatural amino acid” is intended to represent the "D" stereochemical form, the natural amino acids being of the "L” form.
• group C contains an amino acid sequence that is a cleavage recognition sequence for a protease.
• cleavage recognition sequences are known in the art. See, e.g., 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); and Bouvier et al.
• a group C can be chosen such that it is cleaved by a protease present in the extracellular matrix in the vicinity of the target tissue, e.g., a protease released by nearby dying cells or a tumor-associated protease.
• exemplary extracellular tumor-associated proteases are matrix metalloproteases (MMP), thimet oligopeptidase (TOP) and CD 10.
• group C preferably comprises an amino acid sequence selected for cleavage by an endosomal or lysosomal protease, especially the latter.
• proteases include cathepsins B, C, D, H, L and S, especially cathepsin B.
• Cathepsin B preferentially cleaves peptides at a sequence -AA 2 -AA X - where AA 1 is a basic or strongly hydrogen bonding amino acid (such as lysine, arginine, or citrulline) and AA 2 is a hydrophobic amino acid (such as phenylalanine, valine, alanine, leucine, or isoleucine), for example Val-Cit (where Cit denotes citrulline) or Val-Lys. (Herein, amino acid sequences are written in the N-to-C direction, as in
• Group C is a peptide comprising the two-amino acid sequence -AA 2 -AA X - wherein AA 1 is lysine, arginine, or citrulline and AA 2 is phenylalanine, valine, alanine, leucine or isoleucine.
• C consists of a sequence of one to five amino acids, selected from the group consisting of Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Lys, Cit, Ser, and Glu.
• Group C can also be a photocleavable one, for example a nitrobenzyl ether that is cleaved upon exposure to light.
• Group C can be bonded directly to antibody Z or compound D; that is, spacers X z and X D , as the case may be, can be absent.
• group C is a disulfide, one of the two sulfurs can be a cysteine residue or its surrogate on antibody Z.
• group C can be a hydrazone bonded to an aldehyde on a carbohydrate side chain of the antibody.
• group C can be a peptide bond formed with a lysine ⁇ -amino group of antibody Z.
• compound D is directly bonded to group C via a peptidyl bond to a carboxyl or amine group in compound D.
• spacer X z provides spatial separation between group C and antibody Z, lest the former sterically interfere with antigen binding by latter or the latter sterically interfere with cleavage of the former. Further, spacer X z can be used to confer increased solubility or decreased aggregation properties to conjugates.
• a spacer X z can comprise one or more modular segments, which can be assembled in any number of combinations. Examples of suitable segments for a spacer X z are:
• Spacer X if present, provides spatial separation between group C and compound D, lest the latter interfere sterically or electronically with cleavage of the former. Spacer X D also can serve to introduce additional molecular mass and chemical functionality into a conjugate. Generally, the additional mass and functionality will affect the serum half-life and other properties of the conjugate. Thus, through judicious selection of spacer groups, the serum half-live of a conjugate can be modulated. Spacer X D also can be assembled from modular segments, as described above in the context of spacer X z .
• Spacers X z and/or X D where present, preferably provide a linear separation of from 5 to 15 atoms, more preferably from 5 to 20 atoms, between Z and C or D and C, respectively.
• Either spacer X z or X D can comprise a self-immolating moiety.
• a self- immolating moiety is a moiety that (1) is bonded to group C and either antibody Z or cytotoxin D and (2) has a structure such that cleavage from group C initiates a reaction sequence resulting in the self-immolating moiety disbonding itself from antibody Z or cytotoxin D, as the case may be.
• reaction at a site distal from antibody Z or cytotoxin D causes the X z -Z or the X D -D bond to rupture as well.
• a self-immolating moiety is desirable in the case of spacer X D because, if, after cleavage of the conjugate, spacer X D or a portion thereof were to remain attached to cytotoxin D, the biological activity of the latter may be impaired.
• the use of a self- immolating moiety is especially desirable where cleavable group C is a polypeptide.
• the self-immolating moiety is the structure between dotted lines a and b, with adjacent structural features shown to provide context.
• Self-immolating moieties (i) and (v) are bonded to a compound D-NH 2 (i.e., compound D is conjugated via an amino group), while self-immolating moieties (ii), (iii), and (iv) are bonded to a compound D-OH (i.e., compound D is conjugated via a hydroxyl or carboxyl group).
• Cleavage of the amide bond at dotted line b releases the amide nitrogen as an amine nitrogen, initiating a reaction sequence that results in the cleavage of the bond at dotted line a and the consequent release of D-OH or D-NH 2 , as the case may be.
• self-immolating moieties see Carl et al, J. Med. Chem., 24 (3), 479-480 (1981); Carl et al., WO 81/01145 (1981); Dubowchik et al, Pharmacology & Therapeutics, 83, 67-123 (1999); Firestone et al, US 6,214,345 Bl (2001); Toki et al, J. Org. Chem.
• an antibody targeting moiety and the cytotoxic compound D are linked by a non-cleavable linker. Degradation of the antibody eventually reduces the linker to a small appended moiety that does not interfere with the biological activity of cytotoxic compound D.
• conjugation is preferably effected through a bond to a group R° as defined in formula (I).
• R° is NHR la .
• R la is H or alkyl
• the bond may be to the nitrogen of R la NH.
• R la is (CH 2 ) n NH 2 ,
• R 12 is d-C 6 alkyl and R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and n are as defined in respect of formula (I).
• Corresponding structures can be derived from formulae (la), (lb), or (Ila) through (Ilh), mutatis mutandis.
• D is N-(0,1]
• Conjugates of this invention preferably are prepared by first joining a compound
• R 31 is a functional group suitable for reacting with a functional group on antibody Z to form the conjugate.
• suitable groups R 31 include azide, cyclooctyne,
• R is CI, Br, F, mesylate, or tosylate and R is CI, Br, I, F, OH, -O-N-succinimidyl, -0-(4-nitrophenyl), -O-pentafluorophenyl, or -O-tetrafluorophenyl.
• reactive functional group -R 31 is -NH 2 , -OH, -C0 2 H, -SH, maleimido, cyclooctyne, azido (-N 3 ), hydroxylamino (-ONH 2 ) or N-hydroxysuccinimido.
• An -OH group can be esterified with a carboxy group on the antibody, for example, on an aspartic or glutamic acid side chain.
• a -C0 2 H group can be esterified with a -OH group or amidated with an amino group (for example on a lysine side chain) on the antibody.
• N-hydroxysuccinimide group is functionally an activated carboxyl group and can conveniently be amidated by reaction with an amino group (e.g., from lysine).
• a maleimide group can be conjugated with an -SH group on the antibody (e.g., from cysteine or from the chemical modification of the antibody to introduce a sulfhydryl functionality), in a Michael addition reaction.
• an -SH group on the antibody e.g., from cysteine or from the chemical modification of the antibody to introduce a sulfhydryl functionality
• An -SH group is particularly useful for conjugation where the antibody has been modified to introduce a maleimide group thereto, in a Michael addition reaction that is the "mirror image" of that described above.
• Antibodies can be modified to have maleimide groups with N-succinimidyl 4-(maleimidomethyl)-cyclohexanecarboxylate (SMCC) or its sulfonated variant sulfo-SMCC, both reagents being available from Sigma-Aldrich.
• Azide and cyclooctyne are complementary functional groups that can effect conjugation via so-called copper-free "click chemistry," in which the azide adds across the strained alkyne bond of the cyclooctyne to form an 1,2,3-triazole ring.
• the azide can be the reactive functional group R 31 in formula (IV) and the cyclooctyne can be situated on the antibody or antigen binding portion thereof, or vice-versa.
• a cyclooctyne group can be provided by a DIBO reagent (available from Invitrogen/Molecular Probes, Eugene, Oregon).
• non-natural amino acids into antibodies can be utilized, with the non-natural amino acid providing a functionality for conjugation with the reactive functional group.
• the non-natural amino acid p-acetylphenylalanine can be incorporated into an antibody, as taught in Tian et al, WO 2008/030612 A2 (2008).
• the ketone group in p-acetylphenyalanine can be a conjugation site by the formation of an oxime with a hydroxylamino reactive functional group.
• An amine (NH 2 ) group can be used for conjugation using the enzyme transglutaminase, as taught in Jeger et al, Angew. Chem. Int. Ed. 2010, 49, 9995 -9997 ' .
• Conjugation can also be effected using the enzyme Sortase A, as taught in Levary et al, PLoS One 2011, 6(4), el 8342; Proft, Biotechnol. Lett. 2010, 32, 1-10; Ploegh et al, WO 2010/087994 A2 (2010); and Mao et al, WO 2005/051976 A2 (2005).
• the Sortase A recognition motif (typically LPXTG, where X is any natural amino acid) may be located on the ligand Z and the nucleophilic acceptor motif (typically GGG) may be the group R 31 in formula (IV), or vice-versa.
• compositions according to formula (IV) include those having structures represented by formulae (IVa)-(IVg):
• a thiolation level of about three thiol groups per antibody is desirable. For example, with some antibodies this can be achieved by adding a 15-fold molar excess of 2-iminothiolane followed by incubation at RT for 1 h. The antibody is then incubated with 2-iminothiolane at the desired molar ratio and then desalted into conjugation buffer (50 mM pH 6.0 HEPES buffer containing 5 mM glycine and 2 mM DTPA). The thiolated material is maintained on ice while the number of thiols introduced is quantitated as described above.
• conjugation buffer 50 mM pH 6.0 HEPES buffer containing 5 mM glycine and 2 mM DTPA
• the drug-linker moiety is added at a 3 -fold molar excess per thiol.
• the conjugation reaction is allowed to proceed in conjugation buffer also containing a final concentration of 5% dimethylsulfoxide (DMSO), or similar alternative solvent.
• DMSO dimethylsulfoxide
• the drug-linker stock solution is dissolved in 100% DMSO.
• the stock solution is added directly to the thiolated antibody, which has enough DMSO added to bring the final concentration to 10%, or pre-diluted in conjugation buffer containing a final concentration of 10% DMSO, followed by addition to an equal volume of thiolated antibody.
• the conjugation reaction mixture is incubated at RT for 2 h with stirring.
• the conjugation reaction mixture is centrifuged and filtered through a 0.2 ⁇ filter.
• Purification of the conjugate can be achieved through chromatography using a number of methods.
• the conjugate is purified using size-exclusion chromatography on a SEPHACRYLTM S200 column pre-equilibrated with 50 mM pH 7.2 HEPES buffer containing 5 mM glycine and 50 mM NaCl. Chromatography is carried out at a linear flow rate of 28 cm/h. Fractions containing conjugate are collected, pooled and concentrated.
• purification can be achieved through ion-exchange chromatography. Conditions vary from antibody to antibody and should to be optimized in each case.
• antibody-drug conjugate reaction mix is applied to an SP- SEPHAROSETM column pre-equilibrated in 50 mM pH 5.5 HEPES containing 5mM glycine,.
• the antibody conjugate is eluted using a gradient of 0-1 M NaCl in equilibration buffer at pH 5.5.
• Relevant fractions containing the conjugate are pooled and dialyzed against formulation buffer (50 mM pH 7.2 HEPES buffer containing 5 mM glycine and 100 mM NaCl).
• the present disclosure provides a pharmaceutical composition
• a pharmaceutical composition comprising a compound of the present invention, or of a conjugate thereof, formulated together with a pharmaceutically acceptable carrier or excipient. It may optionally contain one or more additional pharmaceutically active ingredients, such as an antibody or another drug.
• the pharmaceutical compositions can be administered in a combination therapy with another therapeutic agent, especially another anti-cancer agent.
• the pharmaceutical composition may comprise one or more excipients.
• Excipients that may be used include carriers, surface active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof.
• the selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.
• a pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
• the active compound may be coated in a material to protect it from the action of acids and other natural conditions that may inactivate it.
• parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
• the pharmaceutical composition can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
• compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in a microemulsion, liposome, or other ordered structure suitable to achieve high drug concentration. The compositions can also be provided in the form of lyophilates, for reconstitution in water prior to administration.
• the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration and will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety -nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent of active ingredient in combination with a pharmaceutically acceptable carrier.
• Dosage regimens are adjusted to provide a therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic response, in association with the required pharmaceutical carrier.
• the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
• dosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
• Exemplary treatment regimens are administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every three to 6 months.
• Preferred dosage regimens include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks.
• dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 ⁇ g/mL and in some methods about 25-300 ⁇ g /mL.
• a "therapeutically effective amount" of a compound of the invention preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom- free periods, or a prevention of impairment or disability due to the disease affliction.
• a pharmaceutically effective amount for the treatment of tumor-bearing subjects, a
• therapeutically effective amount preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.
• a therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject, which is typically a human but can be another mammal.
• the pharmaceutical composition can be a controlled or sustained release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
• Therapeutic compositions can be administered via medical devices such as (1) needleless hypodermic injection devices (e.g., US 5,399, 163; 5,383,851; 5,312,335;
• the pharmaceutical composition can be formulated to ensure proper distribution in vivo.
• the therapeutic compounds of the invention can be formulated in liposomes, which may additionally comprise targeting moieties to enhance selective transport to specific cells or organs. See, e.g. US 4,522,81 1; 5,374,548; 5,416,016; and 5,399,331; V.V. Ranade (1989) J. Clin. Pharmacol. 29:685; Umezawa et al, (1988) Biochem. Biophys. Res. Commun.
• cancers of the head and neck which include tumors of the head, neck, nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oropharynx, larynx, hypopharynx, salivary glands, and paragangliomas; cancers of the liver and biliary tree, particularly hepatocellular carcinoma; intestinal cancers, particularly colorectal cancer; ovarian cancer; small cell and non-small cell lung cancer (SCLC and NSCLC); breast cancer sarcomas, such as fibrosarcoma, malignant fibrous histiocytoma, embryonal rhabdomyosarcoma, leiomysosarcoma, neurofibrosarcoma, osteosarcoma, synovial sarcoma, liposarcoma, and alveolar soft part sarcoma;
• diseases such as, but not limited to, hyperproliferative diseases, including: cancers of the head and neck which include tumors of the head, neck
• compositions described herein will result in a reduction in the size or number of the cancerous growth and/ or a reduction in associated symptoms (where applicable).
• Pathologically practice of the method and use of compositions described herein will produce a pathologically relevant response, such as: inhibition of cancer cell proliferation, reduction in the size of the cancer or tumor, prevention of further metastasis, and inhibition of tumor angiogenesis.
• the method of treating such diseases comprises administering a therapeutically effective amount of an inventive combination to a subject. The method may be repeated as necessary.
• the cancer can be colorectal cancer, liver cancer, prostate cancer, breast cancer, melanoma, glioblastoma, lung cancer, pancreatic cancer, ovarian cancer, multiple myeloma, renal cancer, leukemia (especially ALL, APL, or AML), or lymphoma.
• Compounds of this invention or their conjugates can be administered in combination with other therapeutic agents, including antibodies, alkylating agents, angiogenesis inhibitors, antimetabolites, DNA cleavers, DNA crosslinkers, DNA intercalators, DNA minor groove binders, enediynes, heat shock protein 90 inhibitors, histone deacetylase inhibitors, immunomodulators, microtubule stabilizers, nucleoside (purine or pyrimidine) analogs, nuclear export inhibitors, proteasome inhibitors, topoisomerase (I or II) inhibitors, tyrosine kinase inhibitors, and serine/threonine kinase inhibitors.
• other therapeutic agents including antibodies, alkylating agents, angiogenesis inhibitors, antimetabolites, DNA cleavers, DNA crosslinkers, DNA intercalators, DNA minor groove binders, enediynes, heat shock protein 90 inhibitors, histone deacetylase inhibitors, immunomodulators, microtubule stabilizers, nucleoside (pur
• Specific therapeutic agents include adalimumab, ansamitocin P3, auristatin, bendamustine, bevacizumab, bicalutamide, bleomycin, bortezomib, busulfan, callistatin A, camptothecin, capecitabine, carboplatin, carmustine, cetuximab, cisplatin, cladribin, cytarabin, cryptophycins, dacarbazine, dasatinib, daunorubicin, docetaxel, doxorubicin, duocarmycin, dynemycin A, epothilones, etoposide, floxuridine, fludarabine, 5-fluorouracil, gefitinib, gemcitabine, ipilimumab, hydroxyurea, imatinib, infliximab, interferons, interleukins, ⁇ -lapachone, lenali
• variants of compound (Ila) with the amino group located at other ring positions can be made by using as a starting material variants of compound 1 with its amino group elsewhere in the ring or replaced by a different group, as in:
• FIG. 2 shows illustrative procedures for preparing compounds (lib), (Hh), and (Ilg), via this procedure.
• compounds (lib), (Ilh), and (Ilg) are labeled 13a, 13b, and 13c', respectively.
• Fmoc-Gly-NH-uncialamycin-OTES 11a Aminouncialamycin-OTES 8 (5 mg) and Fmoc -protected glycinyl chloride 10a (Chem-Impex, 8.4 mg, 3 equiv) were dissolved in acetonitrile (2 mL) and stirred in the presence of AgCN (7 mg, 6 equiv) overnight. Reaction was complete as monitored by TLC and HPLC. Concentration and purification on a
• Boc-protected citrulline 6a (Chem- Impex, 0.726 g, 2.64 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 0.578g, 2.9 mmol) in DCM:DMF (17:3,20 mL) was stired at RT for 30 min. Then aminocyanophthalide 6 (0.23 g, 1.32 mmol) was added and stirred at RT for 18 h. The reaction mixture was worked up with ethyl acetate, washed with saturated aHC03 solution, and further washed with water and brine.
• Fig. 4 shows a scheme for the synthesis of compound (lie), labeled 22 in the figure.
• Fig. 5 shows a scheme for the synthesis of compound (He), labeled 25 in the figure.
• Fig. 6 shows a scheme for the synthesis of compound (lid), labeled 30 in the figure.
• Fig. 7 shows reaction schemes for adapting compound (lib) for conjugation.
• Fig. 8 shows reaction schemes for adapting compound (Ilf) for conjugation.
• reaction mixture was absorbed onto silica gel and purified with a COMBIFLASHTM unit using 30% methanol in DCM eluent to afford product 37 (60% yield).
• Fig. 9 shows reaction schemes for adapting compound (lie) for conjugation.
• Fig. 10 shows a reaction scheme for adapting compound (lid) (labeled 30) for conjugation.
• the monoclonal anti-mesothelin antibody 6A4 (Terrett et al. , WO 2009/045957 Al), at a concentration of 5.3 mg/mL in 100 mM sodium phosphate, 150 mM NaCl, pH 8.0, was thiolated with a 10-fold molar excess of 2-iminothiolane. The thiolation reaction was allowed to proceed for 1 hour at RT with continuous stirring.
• antibody 6A4 was buffer-exchanged into conjugation buffer (50 mM HEPES, 5 mM glycine, pH 7.0) via a PD10 column (Sephadex G-25). The concentration of the thiolated antibody was determined by UV spectroscopy at 280 nm. The thiol concentration was measured using the dithiodipyridine assay.
• the conjugated antibody 6A4 was filtered through a 0.2 ⁇ filter and then subjected to cation-exchange (CEX) chromatographic purification.
• CEX cation-exchange
• a SP Sepharose High Performance CEX column was regenerated with 5 column volumes (CVs) of 50 mM HEPES, 5 mM glycine, 1M NaCl, pH 7.0 buffer. Following regeneration, the column was equilibrated with 3 CVs of equilibration buffer (50 mM HEPES, 5 mM glycine, pH 7.0).
• the antibody 6A4 conjugate with compound (IVe) or (IVf), as the case might be, was loaded onto the column and the column was washed once with the equilibration buffer.
• the conjugate was eluted with 50 mM HEPES, 5 mM glycine, 1 10 mM NaCl, pH 7.0. Eluate was collected in fractions. The column was then regenerated with 50 mM HEPES, 5 mM glycine, 1M NaCl, pH 7.0, to remove protein aggregates and any unreacted compound (IVe) or (IVf). [00173] Eluate fractions containing monomeric antibody conjugate were pooled. Antibody conjugate concentration and substitution ratios were determined by measuring absorbance at 280 and 560 nm.
• the purified CEX eluate pool of conjugate was buffer exchanged into 50 mM HEPES, 5 mM glycine, 100 mM NaCl, 0.01% TWEEN 80TM, pH 7.0, by dialysis using a 10 MWCO membrane. Post-dialysis, antibody conjugate concentration and substitution ratios are determined by measuring absorbance at 280 and 560 nm. The characteristics of the conjugates obtained are summarized in Table 1 , below:
• Example 12 Conjugation with an anti-CD70 antibody
• TWEEN-80TM pH 7.5
• 2-iminothiolane a 15-fold molar excess of 2-iminothiolane.
• the thiolation reaction was allowed to proceed for 1 h at RT with continuous stirring.
• antibody 2H5 was buffer-exchanged into conjugation buffer (50 mM HEPES, 5 mM glycine, pH 7.0) via a PD10 column (Sephadex G-25). The concentration of the thiolated antibody was determined by UV spectroscopy at 280 nm. The thiol concentration was measured using the dithiodipyridine assay.
• a 2 mM stock solution of compound (IVe) or (IVf), as the case might be, in DMSO was added at a 1.5-fold molar excess per thiol group in antibody 2H5.
• DMSO was added to make a final concentration of 20% and TWEEN-80TM to a final concentration of 0.1%.
• the reaction medium was stirred for 2 h at RT.
• 100 mM NEM in DMSO was added at a 10-fold molar excess over thiol groups in antibody 6A4 to quench any unreacted thiol groups. The quenching reaction was allowed to proceed for one h at RT with continuous stirring.
• the conjugated antibody 2H5 was filtered through a 0.2 ⁇ filter and then subjected to CEX chromatographic purification.
• a SP Sepharose High Performance CEX column was regenerated with 5 CVs of 50 mM HEPES, 5 mM glycine, 1M NaCl, pH 7.0 buffer. Following regeneration, the column was equilibrated with 3 CVs of equilibration buffer (50 mM HEPES, 5 mM glycine, pH 7.0).
• the antibody 2H5 conjugate with compound (IVe) or (IVf), as the case might be, was loaded onto the column and the column was washed once with the equilibration buffer.
• the conjugate was eluted with 50 mM HEPES, 5 mM glycine, 110 mM NaCl, pH 7.0. Eluate was collected in fractions. The column was then regenerated with 50 mM HEPES, 5 mM glycine, 1M NaCl, pH 7.0, to remove protein aggregates and any unreacted compound (IVe) or (IVf).
• the 3 H thymidine plates were pulsed with 1.0 ⁇ of 3 H-thymidine per well for the last 24 hours of the total incubation period, harvested, and read on a Top Count Scintillation Counter (Packard Instruments, Meriden, CT). ATP levels in the ATP plates were measured using the CELLTITER-GLO
• Luminescent Cell Viability kit following the manufacturer's manual and read on a
• GLOMAX ® 20/20 luminometer both from Promega, Madison, WI, USA.
• Fig. 11a is a plot of the antiproliferative activity of compound (Ila) against HL-60 leukemia cells, as measured by the ATP assay using a 72 h incubation period, compared against three reference compounds: doxorubicin (adriamycin), uncialamycin, and Compound A, which is a DNA alkylating agent having the following structure:
• Fig. lib is an analogous plot of antiproliferative activity against a doxorubicin- resistant ovarian cancer cell line (Adr), also using the ATP assay and a 72 h incubation period.
• the corresponding EC5 0 values are shown in Table 4.
• the potency of compound (Ila) - again even greater than that of uncialamycin itself - is noteworthy in view of the loss of potency of doxorubicin and Compound A when confronted with a resistant cell line.
• Table 5 shows additional antiproliferative data for Compound (Ila), compared to two other toxins that have been used in conjugates: Compound A and doxorubicin.
• the assay method was the ATP method.
• the cancer cell lines tested against were A2780 (ovarian), A549 (lung), CCRF-CEM (acute lymphoblastic leukemia), COLO205 (colon), DU4475 (breast), H2087 (lung, non-small cell), H661 (lung, large cell), HCTl 16 (colon), LNCaP (prostate), LS 174T (colon), MDA MB468 (breast), MDA MB231 (breast), and SET2 (leukemia).
• the antiproliferative effects are reported as ICso's, i.e., the concentration of toxin that produces a 50% inhibitory effect.
• Fig. 12a shows additional antiproliferative data, for compounds (Ila), (lie), (lid), and (He), with doxorubicin as a comparative compound, against 786-0 renal cancer cells.
• the EC5 0 values derived from Fig. 12a are provided in Table 6.
• the ATP assay was used, with a 72 h incubation period.
• Fig. 12b is a similar antiproliferative plot, but against H226 lung cancer cells.
• the derived EC5 0 values are provided in Table 7.
• the ATP assay was used, with a 72 h incubation period.
• Table 7 Antiproliferative Activity of Compounds against H226 Cells
• Fig. 13a shows the antiproliferative activity against 786-0 cells, using the 3 H thymidine incorporation assay (72 h incubation), of four conjugates made from compounds of this invention: (a) a conjugate of antibody 2H5 (anti-CD70, Terrett et al, US
• Fig. 13b shows the antiproliferative activity of the same four conjugates against H226 cells, again using the 3 H thymidine incorporation assay and a 72 h incubation period.
• the derived EC50 values are shown in Table 9.
• Fig. 13c is another plot of the antiproliferative activity of conjugates 2H5-(IVf) and 6A4-(rVf) against H226 cells, but measured using the ATP assay, with a 72h incubation period.
• the derived EC5 0 values were 6.630 and 0.1548 nM, respectively.

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