Bifunctional prodrugs
11622958 · 2023-04-11
Assignee
Inventors
Cpc classification
A61K47/6889
HUMAN NECESSITIES
A61K47/6803
HUMAN NECESSITIES
A61K31/403
HUMAN NECESSITIES
C07H17/02
CHEMISTRY; METALLURGY
C07D209/60
CHEMISTRY; METALLURGY
International classification
A61K31/403
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
A61K47/68
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
C07D209/60
CHEMISTRY; METALLURGY
Abstract
A first aspect of the invention relates to novel compounds and more precisely to novel bifunctional prodrugs and drugs. An additional aspect of the invention relates to antibody compound conjugates, wherein the compound is a claimed compound, and to pharmaceutical compositions containing the compound or antibody compound conjugate. The invention lastly relates to the use of this compound or antibody compound conjugates according to the invention in order to treat tumour diseases, particularly in mammals.
Claims
1. A compound of the formula A-L-B, where A and B independently of one another are formed from ##STR00023## in which Hal is a halide selected from F, Cl, Br, and I; R is H; R.sub.1 is H or a C.sub.1-C.sub.4 alkyl group; X.sub.1 is O, S, NR.sub.5, where R.sub.5 is selected from H and C.sub.1-C.sub.4 alkyl; R.sub.2 is hydrogen; L is a linker for the covalent linkage of A and B, where L has the formula Z-Y-Z′; where Z and Z′ independently of one another are selected from C═O, OC═O, and where Y represents: ##STR00024## where n is 0 or 1; o and p independently of one another are selected from an integer from 0 to 5; where o and p may adopt the same value or a different value; G is hydrogen or selected from the group consisting of an alkyne group, an amino group, a hydroxyl group, or a thiol group, with the proviso that when n is 0, G is not hydrogen, and pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.
2. A compound as claimed in claim 1, where o and p independently of one another are an integral uneven number selected from the group consisting of 1, 3, and 5; and Z and Z′ are C═O.
3. A compound as claimed in claim 1, where Hal is Cl and R.sub.1 is H.
4. A compound as claimed in claim 1, where L is a compound of formula II, ##STR00025##
5. A pharmaceutical composition comprising a compound as claimed in claim 1.
6. The compound of claim 1 wherein n is zero and G is selected from the group consisting of an alkyne group, an amino group, a hydroxyl group, a thiol group.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DESCRIPTION OF THE INVENTION
(17) Provided in accordance with the invention are new compounds which represent bifunctional alkylating agents especially for use in a selective tumor therapy. A feature of the new compounds described herein is that these new dimers are more cytotoxic than the monomeric prodrugs or drugs. The IC.sub.50 of the compounds of the invention is typically in the pmol range. Furthermore, a much larger QIC.sub.50 is achieved. This means that the quotient between the cytotoxicity of the drug and the cytotoxicity of the prodrug is substantially greater. As a result, it is possible to achieve better therapeutic efficacy in conjunction with lower prodrug cytotoxicity and hence to achieve fewer side effects on administration to the patients.
(18) In a first aspect, compounds are provided of the general structure A-L-B, where A and B independently of one another are formed from the structure I
(19) ##STR00001##
in which Hal is a halide selected from F, Cl, Br, I;
R is H or an optionally substituted C.sub.1-C.sub.4 alkyl group, an optionally substituted C.sub.1-C.sub.4 alkoxy group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted C.sub.1-C.sub.4 alkylcarboxyl-C.sub.1-C.sub.4 alkyl group, Hal, CN, an optionally substituted C.sub.1-C.sub.4 alkylsulfanyl group, an optionally substituted arylsulfanyl group, a group NR.sub.z as defined below;
R.sub.1 is H or a C.sub.1-C.sub.4 alkyl group or a C.sub.1-C.sub.4 alkoxy group;
X.sub.1 is O, S, NR.sub.5, where R.sub.5 is selected from H and optionally substituted C.sub.1-C.sub.4 alkyl;
R.sub.2 is selected from hydrogen or a cleavable substrate, more particularly a substrate which is cleavable by chemical reaction;
G independently at each occurrence is absent or is hydrogen or a functional group selected more particularly from an alkyne group, an amino group, a hydroxyl group, a thiol group, a carboxyl group, an azide group or a polyglycine group, where the functional group G is present at least once in the compound A-L-B;
L is a linker for the covalent linkage of A and B, where L has the structure Z—Y—Z′;
where Z and Z′ independently of one another are selected from C═O, OC═O, SO.sub.2, NR.sub.z, NR.sub.ZC═O, C═ONR.sub.z, where each R.sub.Z independently of any other is selected from hydrogen and optionally substituted C.sub.1-C.sub.4 alkyl or optionally substituted C.sub.1-C.sub.4 acyl;
where Y is selected from a structure according to structure III or structure IV;
(20) ##STR00002##
where each R.sub.A independently of any other is selected from hydrogen or optionally substituted C.sub.1-C.sub.4 alkyl or optionally substituted C.sub.1-C.sub.4 acyl;
o and p independently of one another are selected from an integer from 1 to 20; where o and p may adopt the same value or a different value;
G is as defined above;
X.sub.2 is i) N or S or ii) an aryl or heteroaryl group, where (CR.sub.A).sub.o and (CR.sub.A).sub.p are located in meta position to this aryl group or to this heteroaryl group,
R.sub.3 is selected from C.sub.1-C.sub.10 alkyl, such as C.sub.1-C.sub.4 alkyl; C.sub.0-C.sub.4 alkylaryl C.sub.0-C.sub.10 alkyl group, such as C.sub.0-C.sub.4 alkylaryl C.sub.0-C.sub.4 alkyl group; C.sub.0-C.sub.4 alkylheteroaryl C.sub.0-C.sub.10 alkyl group, such as C.sub.0-C.sub.4 alkylheteroaryl C.sub.0-C.sub.4 alkyl group; or a cleavable substrate, more particularly a substrate cleavable by chemical reaction; or is not present;
R.sub.4 is i) absent or is a C.sub.1-C.sub.10 alkyl group, such as a C.sub.1-C.sub.4 alkyl group; a C.sub.0-C.sub.4 alkylaryl C.sub.0-C.sub.10 alkyl group, such as a C.sub.0-C.sub.4 alkylaryl C.sub.0-C.sub.4 alkyl group; a C.sub.0-C.sub.4 alkylheteroaryl C.sub.0-C.sub.10 alkyl group, such as a C.sub.0-C.sub.4 alkylheteroaryl C.sub.0-C.sub.4 alkyl group; or a cleavable substrate, more particularly a substrate cleavable by chemical reaction, if X.sub.2 is N or S, or ii) R.sub.4 is a C.sub.1-C.sub.10 alkyl group, such as C.sub.1-C.sub.4 alkyl group; or a cleavable substrate, more particularly a substrate cleavable by chemical reaction; or is absent if X.sub.2 is an aryl or heteroaryl group;
and pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof.
(21) Employed presently are glycosidic prodrugs of dimeric duocarmycin that themselves have only a low toxicity, and whose activity following uptake into the cell is developed by elimination of the sugar component. The same applies to prodrugs wherein, through reaction with the phenolic hydroxyl group, the cytotoxicity is lowered, by means of a methyl group or a benzyl group, for example.
(22) For the attachment of the toxins and their precursors (drugs and prodrugs) with monoclonal antibodies, in accordance with the invention, there are a number of possibilities.
(23) A distinction is made generally between two fundamental approaches: 1. Use of noncleavable linkers. The assumption here is that, following uptake of the ADC into the cell and transport into the lysosome, there is complete breakdown of the antibody by means of proteinases and related enzymes. This leaves the toxin or its prodrug with a functional group to which the antibody was bonded. 2. Use of cleavable linkers, where cleavage can take place, for example, via acid-catalyzed hydrolysis, enzymatic transformation, or glutathione-induced cleavage of disulfides. Hence the pH in the lysosome is 4.8, whereas in the cytosol a pH=7.4 is found. Acid-labile linkers may contain hydrazone, acetal, enol ether, and azomethine functions. For an enzymatically cleavable linker, suitable compounds are those which contain sugar components or the dipeptide valine-citrulline, which can be cleaved by cathepsin B, which is strongly expressed intracellularly. The glutathione-mediated reductive cleavage of a disulfide linker is based on the finding that intracellularly, glutathione occurs at much higher concentrations than extracellularly.
(24) The attachment of antibodies to the linker, in accordance with the invention, may be accomplished by the addition reaction of a lysine NH.sub.2 group or a cysteine SH group with a maleimidocaproyl function, with a maleimido-methylcyclohexanecarboxylate function, with a maleimidodioxacaproyl function, or with comparable functionalities. Further attachments are possible via the linking of two amino functionalities with quadratic acid diethyl ester and comparable functionalities to form a diamide. The addition reaction of nucleophiles with α,β-unsaturated carbonyl compounds has also been employed. Another linking method is the 1,3-dipolar cycloaddition reaction of linkers having an alkyne or an azide group to antibodies which carry an azide or an alkyne group, respectively. Lastly, enzymatically induced linking of polyglycine substituted toxins and/or precursors thereof (prodrugs) to an antibody may take place by means of a sortase.
(25) In one embodiment here, R.sub.2, R.sub.3 and/or R.sub.4 is selected from a substrate which by enzymatic cleavage, such as by proteolytic, oxidative or reductive enzymes, plasmin, cathepsin, cathepsin B, beta-glucuronidase, galactosidase, mannosidase, glucosidase, neuramidase, saccharosidase, maltase, fructosidase, glycosylases, prostate-specific antigen (PSA), urokinase-type plasminogen activator (u-PA), metalloproteinase, cytochrome P450, or an enzyme which can be cleaved specifically by means of directed enzyme prodrug therapy, such as ADEPT, VDEPT, MDEPT, GDEPT, or PDEPT; or a substituent which can be transformed or cleaved off under hypoxic conditions or by reduction by nitroreductase, R.sub.2, R.sub.3 and/or R.sub.4 being selected more particularly from a monosaccharide, disaccharide or oligosaccharide, more particularly hexoses, pentoses or heptoses, optionally as deoxy derivative or amino derivative and optionally substituted by halogen, C.sub.1-8 alkyl, C.sub.1-8 acyl, C.sub.1-8 heteroalkyl, C.sub.3-7 cycloalkyl, C.sub.3-7 heterocycloalkyl, C.sub.4-12 aryl or C.sub.4-12 heteroaryl, amino groups or amide groups, or by amino, amido or carboxyl units which may optionally be substituted by halogen, C.sub.1-8 alkyl, C.sub.1-8 acyl, C.sub.1-8 heteroalkyl, C.sub.3-7 cycloalkyl, C.sub.3-7 heterocycloalkyl, C.sub.4-12 aryl or C.sub.4-12 heteroaryl, amino radicals or amide radicals; dextran, dipeptide, tripeptide, tetrapeptide, oligopeptide, peptidomimetics, or combinations thereof; or a substrate which can be cleaved off by chemical reaction, where this substrate may comprise an acetal group, a benzyl group, or a substituted benzyl group.
(26) The expression “functional group” refers presently to a substituent which is able, with a further structure, the target structure, more particularly proteins, especially antibodies or antibody fragments, to enter into bonding, preferably covalent bonding, in order to bind the compounds of the invention to these target structures, in particular in order to produce antibody-compound conjugates of the invention.
(27) At the same time, after binding, these functional groups may also form a constituent of a cleavable substrate.
(28) The expression “cleavable substrate” or “substrate which can be cleaved off” as presently used refers to a structure, such as the target structure, which under appropriate conditions and/or using appropriate molecules is split off from the prodrug and ultimately from the active drug. As observed, such cleavage may take place physically or chemically, more particularly enzymatically.
(29) Target structures, in addition to the stated antibodies and antibody fragments, also include aptamers, lectins, biological response modifiers, enzymes, vitamins, growth factors, steroids, nutrients, sugar residues, oligosaccharide residues, hormones, and derivatives and combinations thereof.
(30) The expression “antibody” refers presently to a naturally occurring or recombinant antibody or else antibody fragments; in one embodiment the antibodies are humanized antibodies or antibody fragments. The skilled person is aware of suitable antibodies and antibody fragments and their production. These antibodies or antibody fragments may have been modified in such a way that binding can take place to the compounds of the invention by way of the presently described functional group.
(31) With the aid of the antibody-compound conjugates of the invention it is possible to guide the conjugates in a goal-directed manner to the target cells or target structures. In one embodiment the antibodies are directed at tumor-associated antigens or other cell surface constituents of the target structures, to allow target-driven introduction of the prodrugs into the cells.
(32) The expression “antibody-compound conjugate” refers presently to a conjugate made up of antibody and compound of the invention. The compound in this case is, for example, a prodrug or drug molecule. Accordingly, it is also possible presently to refer to an antibody-prodrug conjugate or antibody-drug conjugate. This conjugate is one form of a target structure-compound conjugate, with, for example, the above-stated target structures and the compounds of the invention.
(33) The linking of the compound of the invention to the antibody is accomplished via the functional group G, in order for the antibody of the invention, including antibody fragments, to be bonded covalently to the compound of the invention. This covalent bonding may optionally be parted again by means of suitable agents.
(34) In a further embodiment of the present invention, the compound is one where o and p are identical or independently of one another are an integral uneven number from 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19, Z and Z′ are C═O, and each R.sub.A is, independently of any other, hydrogen or CH.sub.3.
(35) Furthermore, one embodiment relates to a compound where R.sub.2 independently at each occurrence is a hydrogen or CH.sub.3.
(36) In accordance with the invention a compound is one where Hal is a Cl and R.sub.1 is H.
(37) One embodiment relates to a compound where L is a compound according to the structure II,
(38) ##STR00003##
where Y is defined as above.
(39) Furthermore, one embodiment of the invention is one with a compound where X.sub.2 is an aryl group, more particularly a benzyl group, Y is a structure IV where R.sub.4 is a C.sub.1-C.sub.4 alkyl group, and G is a functional group presently defined.
(40) Finally, one embodiment is a compound where the functional group G is presently only on the radical R.sub.2.
(41) In experiments in vitro, the compounds of the invention displayed excellent cytotoxicity values, with IC.sub.50 values in the pmol range, in some cases below the pmol range. Furthermore, the compounds displayed an excellent quotient of the IC.sub.50. The QIC.sub.50 of the compounds tested was above 1000, with particularly suitable compounds showing values of more than 100 000.
(42) This means that the compounds presently represented, which constitute new dimeric prodrugs and drugs of CC-1065 analogs, exhibit a very high cytotoxicity as a drug, whereas the prodrugs have only a relatively low cytotoxicity. As a result, these compounds ought to be significantly safer in therapeutic use. The compounds of the invention composed of dimers, furthermore, are substantially more cytotoxic than the monomeric prodrugs or drugs. The compounds are therefore distinguished by high activity in conjunction with fewer side effects in their application.
(43) Earlier bifunctional CC-1065 analogs and duocarmycin analogs displayed only low selectivity, and the cytotoxicity values as well were similar to those of the monomeric analogs, and in some cases even lower.
(44) A feature of the compounds of the invention is that they exhibit cytotoxicity values in the pmol range and have a very large quotient of the IC.sub.50 values of the drug to the prodrug.
(45) The expression “substituted” as used herein in relation in particular to alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, and acyl refers to the fact that these groups one or more substituents selected from the group encompassing OH, ═O, ═S, ═NR.sup.h, ═N—OR.sup.h, S.sup.h, NH.sub.2, NO.sub.2, NO, N.sub.3, CF.sub.3, ON, OCN, SCN, NCO, NCS, C(O)NH.sub.2, C(O)H, C(O)OH, halogen, R.sup.h, SR.sup.h, S(O)R.sup.h, S(O)OR.sup.h, S(O).sub.2R.sup.h, S(O).sub.2OR.sup.h, OS(O)R.sup.h, OS(O)OR.sup.h, OS(O).sub.2R.sup.h, OS(O).sub.2OR.sup.h, OP(O)(OR.sup.h)(OR.sup.i), P(O)(OR.sup.h)(OR.sup.i), OR.sup.h, NHR.sup.i, N(R.sup.h)R.sup.i, +N(R.sup.h)(R.sup.i)R.sup.i, Si(R.sup.h)(R.sup.i)R.sup.j, Si(R.sup.h)(R.sup.i)(R.sup.j), C(O)R.sup.h, C(O)OR.sup.h, C(O)N(R.sup.i)R.sup.h, OC(O)R.sup.h, OC(O)OR.sup.h, OC(O)N(R.sup.h)R.sup.i, N(R.sup.i)C(O)R.sup.h, N(R.sup.i)C(O)OR.sup.h, N(R.sup.i)C(O)N(R.sup.j)R.sup.h, and the thiol derivatives of these substituents, or a protonated or deprotonated form of these substituents, where R.sup.h, R.sup.i, and R.sup.j independently of one another are selected from H and optionally substituted C.sub.1-15 alkyl, C.sub.1-15 heteroalkyl, C.sub.3-15 cycloalkyl, C.sub.3-15 heterocycloalkyl, C.sub.4-15 aryl, or C.sub.4-15 heteroaryl or a combination thereof; two or more of R.sup.h, R.sup.i, and R.sup.j are optionally joined to one another to form one or more carbon ring systems or heterocyclic ring systems.
(46) The expression “alkyl” as used herein relates to straight-chain or branched, saturated or unsaturated hydrocarbonyl substituents; examples of the alkyl groups include a methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, decyl, isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, pentenyl, and the like.
(47) The expression “cycloalkyl” or “carbon ring systems” as used herein relates to saturated or unsaturated, nonaromatic hydrocarbon ring systems which may consist of one, two or more rings. Examples hereof include the following: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexonyl, etc.
(48) The expression “heteroalkyl” as used herein relates to straight-chain or branched, saturated or unsaturated hydrocarbonyl substituents in which at least one carbon has been replaced by a heteroatom. The heteroatoms are preferably selected from S, N, O, and P.
(49) The expression “aryl” as used herein relates to aromatic substituents which may consist of one or more rings fused with one another. Examples of aryl include the following: phenyl, naphthyl, and anthracenyl.
(50) The expression “heteroaryl” as used herein relates to aromatic substituents which may consist of one or more rings fused with one another. In this case at least one carbon atom in the aromatic ring group has been replaced by a heteroatom. Examples of heteroaryl groups include the following: pyridyl, furanyl, pyrrolyl, triazolyl, pyrazolyl, imidazolyl, thiophenyl, indolyl, benzofuranyl, benzimidazolyl, indazolyl, benzotriazolyl, benzisoxazolyl, and quinolyl.
(51) The expression “heterocycloalkyl” or “heterocyclic ring systems” as used herein relates to saturated or unsaturated, nonaromatic, cyclic hydrocarbonyl substituents which may consist of one or more rings fused with one another, where at least one carbon atom in one of the rings has been replaced by a heteroatom. Examples of heterocycloalkyls include the following: tetrahydrofuranyl, pyrrolidinyl, piperidyl, 1,4-dioxanyl, morpholinyl, piperazinyl, oxyzolidinyl, decahydroquinolyl.
(52) The expression “acyl” as used herein relates to the functional group having the general structure R.sub.AC—CH═O—where R.sub.AC represents an optionally substituted carbon radical, more particularly a hydrocarbon chain having C.sub.1 to C.sub.8 carbon atoms.
(53) If expressions such as “optionally substituted” are used, these expressions refer to all of the following radicals, unless otherwise stated.
(54) Therefore, the expression “optionally substituted alkyl, heteroalkyl, aryl, acyl” should be read as “optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, optionally substituted acyl”.
(55) Depending on the substituents, especially depending on the substituent R.sub.2, R.sub.3 and/or R.sub.4 and the antibodies conjugated via G, the compounds can be introduced simply and in a directed way into cells. In one embodiment, R.sub.2, R.sub.3 and/or R.sub.4 is preferably hydrogen.
(56) In a further preferred embodiment, R.sub.2, R.sub.3 and/or R.sub.4 represents a substrate which is cleavable, for example, enzymatically, in order to convert prodrugs featuring a cleavable product on the R.sub.2, R.sub.3 and/or R.sub.4 substituent into drugs.
(57) In one preferred embodiment, therefore, the R.sub.2, R.sub.3 and/or R.sub.4 in the substrate comprises a cleavable product. Cleavage may be accomplished by chemical reaction, as for example on the basis of a change in the ambient conditions, such as pH, concentration of particular ions, etc.
(58) In one preferred embodiment, R.sub.2, R.sub.3 and/or R.sub.4 in the substrate comprises a cleavable substrate. This cleavable substrate is preferably one which by proteolytic enzymes, plasmin, cathepsin, cathepsin B, beta-glucuronidase, galactosidase, mannosidase, glucosidase, neuramidase, saccharosidase, maltase, fructosidase, glycosylases, prostate-specific antigen (PSA), urokinase-type plasminogen activator (u-PA), metalloproteinase, or an enzyme which can be cleaved specifically by means of directed enzyme prodrug therapy, such as ADEPT, VDEPT, MDEPT, GDEPT, or PDEPT; or a substituent which can be transformed or cleaved off under hypoxic conditions or by reduction by nitroreductase.
(59) The radical R.sub.2, R.sub.3 and/or R.sub.4 preferably comprises one selected from the group encompassing monosaccharide, disaccharide or oligosaccharide, more particularly hexoses, pentoses or heptoses, optionally as deoxy derivative or amino derivative, and optionally substituted by halogen, C.sub.1-8 alkyl, C.sub.1-8 acyl, C.sub.1-8 heteroalkyl, C.sub.3-7 cycloalkyl, C.sub.3-7 heterocycloalkyl, C.sub.4-12 aryl or C.sub.4-12 heteroaryl, amino groups or amide groups, or by amino, amido or carboxyl units which may optionally be substituted by halogen, C.sub.1-8 alkyl, C.sub.1-8 acyl, C.sub.1-8 heteroalkyl, C.sub.3-7 cycloalkyl, C.sub.3-7 heterocycloalkyl, C.sub.4-12 aryl or C.sub.4-12 heteroaryl, amino radicals or amide radicals; dextran, dipeptide, tripeptide, tetrapeptide, oligopeptide, peptidomimetics, or combinations thereof.
(60) It is possible, furthermore, to utilize substituents which are labile under certain ambient conditions, such as hemiacetals and acetals, benzyl groups and substituted benzyl groups.
(61) With the aid of the substrate R.sub.2, R.sub.3 and/or R.sub.4, targeting of the compounds of the invention to target structures may be possible. In other words, goal-directed coupling of the compounds of the invention to target structures, for the targeting of these conjugates, for example, into the target cells or target tissue, is possible, and the corresponding introduction of these compounds of the invention into, for example, selected cells and cell types is possible. The compounds of the invention wherein R.sub.2, R.sub.3 and/or R.sub.4 is a substrate other than H contain a cleavable or transformable group. The cleavage or the transformation of the compound of the invention with R.sub.2, R.sub.3 and/or R.sub.4 other than H may be accomplished by chemical, photochemical, physical, biological or enzymatic processes under the corresponding conditions. These conditions include, for example, the provision of appropriate enzymes, the modification of the ambient medium, the action of, for example, radiation, such as UV light, etc. The skilled person is aware of corresponding methods. The substrate is correspondingly amenable to known methods such as ADEPT (Antibody Directed Enzyme Prodrug Therapy), PDEPT (Polymer-Directed Enzyme Prodrug Therapy) or MDEPT (Macromolecular-Directed Enzyme Prodrug Therapy), VDEPT (Virus-Directed Enzyme Prodrug Therapy) or GDEPT (Gene-Directed Enzyme Prodrug Therapy).
(62) Correspondingly, a further aspect of the present invention is directed to an antibody-compound conjugate where an antibody is linked via the functional group G to the above-defined compound of the invention.
(63) In one embodiment of this antibody-compound conjugate, the conjugate is one where the antibody is directed against a tumor antigen or an antigen which is expressed onto a target structure, such as a cell.
(64) In a further embodiment, the antibody-compound conjugate is one where the antibody is one of an antibody, an antibody fragment, such as F(ab′).sub.2, F(ab′), Fab, Fv, sFv, scFv.
(65) The present patent application is directed, furthermore, to pharmaceutical compositions which comprise the compounds of the invention, optionally with pharmaceutically acceptable excipients or diluents. The compounds of the invention may in this case be present in the form of pharmaceutically acceptable salts or solvates. This means that the pharmaceutical composition comprises a compound of the invention or an antibody-compound conjugate of the invention.
(66) Pharmaceutically acceptable salts are, in particular, acid addition salts which are formed correspondingly on amine groups. Equally possible are base addition salts or corresponding zwitter addition salts.
(67) The expression “pharmaceutically acceptable solvates” refers to the association of one or more solvent molecules and a compound of the invention.
(68) Examples of those solvent molecules which form pharmaceutically acceptable solvates are the following: water, isopropyl alcohol, ethanol, methanol, DSMO, ethyl acetate, and acetic acid.
(69) The compounds of the invention are suitable particularly for producing pharmaceutical compositions suitable in tumor therapy. The monomers of the bifunctional compounds of the invention are known to be cytotoxic compounds suitable for tumor therapy. The present invention includes pharmaceutical compositions which in addition to the customary excipients or diluents comprise the compounds of the invention. The pharmaceutical preparations referred to above are conventionally produced by known methods, as for example by mixing the active ingredient or ingredients or excipient or excipients.
(70) In general the compounds of the invention can be administered in total amounts of 0.5 to about 500, preferably 1 to 150 mg/kg body weight per 24 hours, optionally in the form of a plurality of individual doses, in order to obtain the desired outcomes. The skilled person is well aware of the possibilities for determining the dosage. It may be done as a function of the age, the body weight, the nature and severity of the disease in the patient, the type of preparation and administration of the medicinal product, and also the period or the interval of administration.
(71) The seco-duocarmycin derivatives presently described carry, for example, alkyne, carboxyl, amino, azide, thiol, and hydroxyl groups, i.e., functional groups G which can be linked to antibodies very easily by means of the techniques discussed above.
(72) One out of many possible typical procedures for attaching an antibody to the bifunctional seco-duocarmycin analogs is shown in
(73) The use of duocarmycin analogs for the production of ADCs has the great advantage of their high cytotoxicity; in the case of the monofunctional derivatives, the latter is approximately IC.sub.50=10 pM, whereas in the case of the bifunctional compounds it is even possible to achieve IC.sub.50 values=150 fM. Furthermore, the compounds can be detoxified very efficiently by glycosidation, with figures of around 6000 in the case of the monofunctional and around 1 000 000 in the case of the bifunctional compounds. With other toxins, an approach of this kind is possible only with great difficulty. There are a number of pathways which can be taken for the formation of the ADCs: 1. In the case of the bifunctional duocarmycin analogs, there are three pathways that can be traveled a) insertion on the chain of a functionality which links the two CPI units to one another. In this case, however, there is a certain reduction in the cytotoxicity. Thus the compound 1 has a slightly increased IC.sub.50 of 60 pM. For attachment via a cleavable linker of known type, it is possible to use functionalized benzene-1,3-diacetates.
(74) ##STR00004##
where n is an integer from 0 to 10 and o and p is an integer from 0 to 5; the chains may additionally contain one or two oxygen atoms, one or two nitrogen atoms with an alkyl or aryl group, or one or two sulfur atoms.
(75) Other bifunctional CBI derivatives as well, with, for example, an N atom in the chain which links the two CBI units, can be used in order to insert monoclonal antibodies,
(76) ##STR00005##
where n is an integer from 0 to 10 and o and p is an integer from 0 to 5; the chains ( ).sub.o and ( ).sub.p may additionally contain one or two oxygen atoms, one or two nitrogen atoms with an alkyl or aryl group, or one or two sulfur atoms.
(77) All compounds can be detoxified by means, for example, of benzylation or glycosidation of the phenolic hydroxyl groups. The toxins are then released in the lysosome by oxidative cleavage with P 450 or by reaction with a glycohydrolase: b) attachment of a monoclonal antibody via the benzene ring of a benzyl protective bifunctional duocarmycin derivative. The advantage of this approach is that on elimination of the benzyl group with the antibody by P450 in the lysosome, the primary compound is released with an IC.sub.50=150 fM (R═H, r=1). Moreover, the compounds are stable in the serum and the conjugates display a very low toxicity.
(78) ##STR00006## c) attachment of a monoclonal antibody via an acid-labile sugar acetal of a glycolized bifunctional duocarmycin derivative. The advantage of this approach is that on acid-catalyzed cleavage of the acetal with the antibody, and the elimination of the sugar component by glycohydrolases in the lysosome, the primary compound is released with an IC.sub.50=150 fM. Moreover, the compounds are stable in the serum and the conjugates display a very low toxicity.
(79) ##STR00007##
(80) In the compounds, r may be an integer of 0-5, the radical ( ).sub.r may, moreover, contain one or two oxygen atoms, one or two sulfur atoms, or one or two NR.sub.Z groups, where R.sub.Z is as defined above. Alternatively, the radical ( ).sub.r may be a radical Y, as defined above.
(81) In order to have the possibility of attachment of the dimeric duocarmycin derivatives, dicarboxylic acid components were developed which carry, centrally, a functionality. The functionality may be an alkyne group, an amino group, an OH group, an SH group, or a polyglycine group. There are numerous techniques known for attaching functionalities of these kinds to monoclonal antibodies.
(82) The following substances are suitable for attachment to tumor-specific monoclonal antibodies: therein, R may be an alkyl of 0 to 5 atoms, halogen, OH, C0 to C5 alkoxy, N—C0 to C5 alkyl and/or S—C0 to C5 alkyl and/or S-aryl.
(83) ##STR00008## ##STR00009## ##STR00010## ##STR00011##
(84) Described, finally, is the use of a compound of the invention or of an antibody-compound conjugate of the invention for treating tumoral diseases, especially in mammals.
(85) In accordance with the invention it is possible to utilize the compounds and antibody-compound conjugates described in order to introduce the prodrugs into the target cells or the target tissue in order there to operate a tumor treatment or to treat precursors thereof.
(86) The tumoral diseases include, in particular, solid tumors.
EXPERIMENTAL SECTION
2,2′-(1,3-Phenylene)bis(1-((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)ethan-1-one) (2)
(87) ##STR00012##
(88) 4M HCl/EtOAc (12 ml) was added to the CBI derivative 1 (100 mg, 300 μmol, 1.0 eq.) and the mixture was stirred at room temperature for 4 h, after which the excess of HCl/EtOAc was removed by application of a high vacuum for 1 h. The residue was dissolved in DMF (12 ml) and pyridine (48 μl, 600 μmol, 2.0 eq.), then admixed at 0° C. with 2,2′-(1,3-phenylene)diacetyl chloride (34 mg, 150 μmol, 0.5 eq.), and the resulting mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure on a rotary evaporator and the residue was purified by chromatography on silica gel (MeOH/CH.sub.2Cl.sub.2 1:19). Further purification took place by preparative TLC (TLC (MeOH/CH.sub.2Cl.sub.2 1:40) and preparative HPLC. The desired product 2 was obtained as a white foam (16 mg, 25.6 μmol, 17%).
(89) R.sub.f: 0.5 (MeOH/CH.sub.2Cl.sub.2 1:19).
(90) Optical rotation: [α].sub.D.sup.20=−22.0 (c 0.5, DMSO).
(91) IR(KBr): ν [cm.sup.−1]=3182, 2360, 1635, 1584, 1421, 1391, 1250, 1133, 857, 772, 754, 715.
(92) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ [ppm]=2.58 (t, J=9.0 Hz, 2H, 2′-H.sub.a, 6′-H.sub.a), 2.99 (t, J=9.0 Hz, 2H, 2′-H.sub.b, 6′-H.sub.b), 3.54 (dd, J=9.0, 3.0 Hz, 2H, 2×10-H.sub.a), 3.68 (t, J=9.0 Hz, 2H, 2×10-H.sub.b), 3.75-4.04 (m, 6H, 2×1-H, 2×2-H.sub.a, 2×2-H.sub.b), 7.08 (d, J=9.0 Hz, 2H, 2×9-H), 7.28-7.37 (m, 4H, 2×7-H, 2×15-H), 7.42-7.47 (m, 2H, 2×8-H), 7.59 (t, J=9.0 Hz, 1H, 16-H), 7.93 (s, 3H, 2×4-H, 14-H), 8.0 (d, J=6.0 Hz, 2H, 6-H), 9.91 (bs, 2H, 2×OH). .sup.13C-NMR (125 MHz, CDCl.sub.3): δ [ppm]=41.2 (2×C-1), 43.4 (2×C-12), 46.4 (2×C-10), 53.3 (2×C-2), 100.2 (2×C-4), 113.9 (2×C-2), 122.1 (2×C-9), 122.2 (2×C-9.sub.b), 122.9 (2×C-7), 123.2 (2×C-6), 126.3 (2×C-8), 128.7 (2×C-15), 129.0 (C-16), 129.3 (2×C-9.sub.a), 130.6 (C-14), 133.8 (2×C-13), 140.6 (2×C-3), 154.4 (2×C-5), 170.3 (2×C═O).
(93) MS (ESI): m/z (%) 647.2 [M+Na].sup.+(100).
(94) C.sub.36H.sub.30Cl.sub.2N.sub.2O.sub.4 (624.16) calc.: 623.1510
(95) found.: 623.1502, [M−H].sup.−
(96) (ESI-HRMS).
(97) HPLC (analytical):
(98) Column: Kromasil® 100 C18, 250×4 mm, 5 μm
(99) Mobile phase: 30/70 H.sub.2O/MeOH
(100) Flow rate: 0.8 ml min.sup.−1
(101) λ=254 nm
(102) t.sub.R: 3.4 min
1,5-bis((S)-1-(Chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)-3-phenylpentane-1,5-dione (3)
(103) ##STR00013##
(104) 4M HCl/EtOAc (6 ml) was added to the CBI derivative 1 (50 mg, 150 μmol, 1.0 eq.) and the mixture was stirred at room temperature for 4 h, after which the excess of HCl/EtOAc was removed by application of a high vacuum for 1 h. The residue was dissolved in DMF (6 ml) and pyridine (24 μl, 300 μmol, 2.0 eq.), then admixed at 0° C. with 3-phenylpentanedioyl dichloride (15 mg, 60 μmol, 0.4 eq.), and the resulting mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure on a rotary evaporator and the residue was purified by chromatography on silica gel (EtOAc/Hex 1:1). Further purification took place by preparative HPLC. The desired product 3 was obtained as a white foam (30 mg, 47 μmol, 78%).
(105) R.sub.f: 0.6 (EtOAc/Hex 1:1).
(106) Optical rotation: [α].sub.D.sup.20=−26.6 (c 0.82, DMSO).
(107) IR (KBr): ν [cm.sup.−1]=3122, 2360, 1630, 1583, 1422, 1392, 1255, 1132, 856, 756, 718, 700.
(108) .sup.1H-NMR (300 MHz, CDCl.sub.3): δ [ppm]=2.27-2.35 (bm, 0.5H.sub.minor, 12-H.sub.a), 2.53-2.74 (bm, 1.5H, 12-H.sub.a) 2.81-3.17 (m, 4H, 2×12-H.sub.b, 2×1-H), 3.22-3.47 (m, 2H, 2×10-H.sub.a), 3.48-3.66 (m, 2H, 2×10-H.sub.b), 3.74-4.04 (m, 2H, 2×2-H.sub.a), 4.06-4.39 (m, 2H, 2×2-H.sub.b), 4.58 (t, J=9.0 Hz, 0.3H.sub.minor, 13-H), 4.76 (t, J=9.0 Hz, 0.7H, 13-H), 6.74 (bd, J=6.0 Hz, 1H, 17-H), 6.87-7.17 (m, 2H, 2×16-H), 7.31-7.46 (m, 5H, 2×7-H, 2×9-H, 8-H), 7.47-7.59 (bm, 3H, 8-H, 2×15-H), 8.04-8.39 (m, 4H, 2×4-H, 2×6-H), 10.23 (bs, 0.6H, OH), 10.50 (bs, 0.4H.sub.minor, OH), 10.80 (bs, 0.2H.sub.minor, OH), 11.32 (bs, 0.8H, OH).
(109) .sup.13C-NMR (125 MHz, CDCl.sub.3): δ [ppm]=35.7 (C-13), 40.6 (2×C-1.sub.minor), 41.0 (2×C-1), 41.3 (2×C-1′), 45.8 (2×C-12), 45.9 (2×C-12.sub.minor), 46.3 (2×C-12′), 46.7 (2×C-10), 46.8 (2×C-10.sub.minor), 47.0 (2×C-10′), 53.4 (2×C-2), 53.6 (2×C-2.sub.minor), 53.9 (2×C-2′), 54.1 (2×C-2′.sub.minor), 101.0 (2×C-4.sub.minor), 101.3 (2×C-4), 101.7 (2×C-4′), 102.1 (2×C-4′.sub.minor), 114. 1 (2×C-2.sub.minor), 114.5 (2×C-2), 114.6 (2×C-2′), 115.2 (2×C-2′.sub.minor), 121.9 (2×C-9.sub.b minor), 122.1 (2×C-9.sub.b), 122.3 (2×C-7), 122.4 (2×C-7′), 122.7 (2×C-6), 122.9 (2×C-6′), 123.0 (2×C-9), 123.3 (2×C-9′), 126.3 (C-17), 126.4 (2×C-15), 126.5 (2×C-8), 127.2 (2×C-16), 129.3 (2×C-9.sub.a), 129.4 (2×C-16′), 140.3 (2×C-3), 140.6 (2×C-3.sub.minor), 140.8 (2×C-3′.sub.minor), 141.2 (2×C-3′), 146.0 (C-14), 146.4 (C-14.sub.minor), 153.3 (2×C-5), 153.6 (2×C-5.sub.minor), 154.2 (2×C-5′.sub.minor), 154.6 (2×C-5′), 169.5 (2×C═O.sub.minor), 169.7 (2×C=O), 171.0 (2×C═O.sub.minor), 171.1 (2×C═O),
(110) MS (ESI): m/z (%) 661.2 [M+Na]+(100).
(111) C.sub.37H.sub.32Cl.sub.2N.sub.2O.sub.4 (661.2) calc.: 661.1637.
(112) found.: 661.1618, [M+Na].sup.+.
(113) (ESI-HRMS).
(114) HPLC (analytical):
(115) Column: Kromasil® 100 C18, 250×4 mm, 5 μm
(116) Mobile phase: 30/70 H.sub.2O/MeOH
(117) Flow rate: 0.8 ml min.sup.−1
(118) λ=254 nm
(119) t.sub.R: 21.1 min
(120) The attached
(121)
(122)
(123) General Methods
(124) Experimental Methods:
(125) Unless otherwise stated, the reactions were carried out under an inert gas atmosphere in flame-dried vessels and the reaction materials were introduced by syringe or transfer cannula under argon pressure. All solvents were of analytical purity and were stored over molecular sieve. All reagents obtained from commercial sources were used without further purification. Long-term cooling was carried out using a Haake EK 90 cryostat. Thin-layer chromatography was carried out using precoated silica gel plates SI 60 F.sub.254 from Merck. Silica gel 60 (0.040 0.063 mm) from Merck was used for the flash chromatography. Staining was carried out by means of phosphomolybdic acid hydrate from Sigma-Aldrich (in MeOH). Yields are based on isolated and purified compounds, unless otherwise stated.
(126) NMR Spectroscopy:
(127) NMR spectra were recorded using Varian Mercury-300, Unity-300 and Inova-600 spectrometers in CDCl.sub.3 or CD.sub.3OD or d.sup.6-DMSO; chemical shifts are reported in ppm relative to tetramethylsilane (TMS), coupling constants J in hertz. The mobile phase signals were used as references and the chemical shifts were converted into the TMS scaling (CHCl.sub.3: δ.sub.H=7.26 ppm, δ.sub.C=77.0 ppm; CD.sub.3OD: δ.sub.H=3.34 ppm, δ.sub.C=49.86 ppm and d.sup.6-DMSO: δ.sub.H=2.54 ppm, δ.sub.C=40.45 ppm). The first-order multiplicities were designated as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), and so on. Higher-order signals were designated as m (multiplet).
(128) IR Spectroscopy:
(129) IR spectra were recorded using Bruker Vektor 22 spectrometers;
(130) UV Spectroscopy:
(131) UV spectra were recorded using JASCO V-630 spectrometers,
(132) Mass Spectrometry:
(133) ESI-MS and ESI-HRMS spectra were recorded using Bruker Daltonik Apex IV.
Further Syntheses
1,3-Phenylenebis(((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)methanone) (51)
(134) ##STR00014##
(135) 4M HCl/EtOAc (12 ml) was added to tbutoxycarbonylseco-CBI (50 mg, 150 μmol, 1.0 eq.) at room temperature and the mixture was stirred at the same temperature for three hours. Excess HCl/EtOAc was removed by evaporation under reduced pressure and drying was carried out under reduced pressure for one hour. The residue obtained was dissolved in DMF (12 ml) and admixed at 0° C. with pyridine (48 μl, 600 μmol, 4.0 eq.) followed by isophthaloyl dichloride (15 mg, 75 μmol, 0.5 eq.), and the reaction mixture was stirred at room temperature for 16 hours. Evaporation of the solvent under a high pressure gave a crude product which was purified by flash chromatography (EtOAc:PE 2:3 to EtOAc) in order to give 51 (38 mg, 85%).
(136) R.sub.f: 0.6 (EtOAc).
(137) HPLC (analytical):
(138) Column: Kromosil® 100 C18, 250×4 mm, 5 m
(139) Mobile phase: MeOH/THF (1:1): H.sub.2O=70:30
(140) Flow rate: 0.8 ml min.sup.−1
(141) λ: 254 nm
(142) t.sub.R: 9.6 min
(143) Optical rotation: [α].sub.D.sup.20=−35.0 (c 1.0, DMSO).
(144) .sup.1H-NMR (500 MHz, DMSO-d.sub.6, 75° C.): δ [ppm]=3.88 (dd, J=11.0, 7.4 Hz, 2H, 2×10-H.sub.a), 3.98 (dd, J=10.8, 3.1 Hz, 2H, 2×10-H.sub.b), 4.05-4.23 (m, 4H, 2×1-H, 2×2-H.sub.a), 4.38 (dd, J=9.0, 2.0 Hz, 2H, 2×2-H.sub.b), 6.93 (d, J=0.8 Hz, 1H, 3′-H), 7.40 (ddd, J=8.1, 6.9, 1.1 Hz, 2H, 2×7-H), 7.55 (ddd, J=8.3, 6.8, 1.3 Hz, 2H, 2×8-H), 7.66 (brs, 1H, 4-H), 7.72-7.77 (m, 1H, 3′-H), 7.79-7.88 (m, 4H, 6′-H, 8′-H, 2×9-H), 7.91 (t, J=1.6 Hz, 1H, 4-H), 8.18 (d, J=8.1 Hz, 2H, 2×6-H), 10.24 (s, 2H, 2×5-OH).
(145) .sup.13C-NMR (126 MHz, DMSO-d.sub.6, 75° C.): 3 [ppm]=41.4 (2×C-1), 47.8 (2×C-10), 55.9 (2×C-2), 100.7 (C-4), 116.0 (2×C-5a), 122.9 (2×C-9b), 123.2 (2×C-9), 123.6 (2×C-7), 123.8 (2×C-6), 125.4 (C-3′), 125.9 (C-4), 127.9 (2×C-8), 129.3 (C-6′, C-8′), 129.7 (C-7′), 130.6 (2×C-9a), 137.9 (C-2′, C-4′), 142.1 (2×C-3a), 154.8 (2×C-5), 167.7 (2×CON).
(146) HRMS (ESI): m/z calculated for C.sub.34H.sub.26Cl.sub.2N.sub.2O.sub.4 619.1167 [M+Na].sup.+. found 619.1132.
5-Nitro-1,3-phenylene)bis(((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)methanone) (52)
(147) ##STR00015##
(148) 4M HCl/EtOAc (6 ml) was added to tbutoxycarbonylseco-CBI (50 mg, 150 μmol, 1.0 eq.) at room temperature and the resulting solution was stirred at the same temperature for 3 hours. Excess HCl/EtOAc was removed by evaporation under reduced pressure and the residue obtained was dried under a strong vacuum for 1 hour. The seco-CBI formed was dissolved in DMF (6 ml), which was admixed with stirring at 00° C. with pyridine (48 μl, 600 mol, 4.0 eq.) and 5-nitroisophthaloyl dichloride (20 mg, 75 mol, 0.5 eq.), and the stirring was continued at room temperature for 16 hours. Evaporation of the solvent under a high pressure gave the crude product, which was purified by flash chromatography (EtOAc:PE 1:1) to give 52 (44 mg, 88%).
(149) R.sub.f: 0.5 (EtOAc:PE=1:1).
(150) HPLC (analytical):
(151) Column: Kromosil® 100 C18, 250×4 mm, 5 m
(152) Mobile phase: MeOH/THF (1:1): H.sub.2O=70:30
(153) Flow rate: 0.8 ml min.sup.−1
(154) λ: 254 nm
(155) t.sub.R: 10.7 min
(156) Optical rotation: [α].sub.D.sup.20=−6.6 (c 0.6, DMSO).
(157) .sup.1H-NMR (500 MHz, DMSO-d.sub.6, 75° C.): δ [ppm]=3.87-4.05 (m, 4H, 2×10-H.sub.a, 2×10-H.sub.b), 4.07-4.27 (m, 4H, 2×1-H, 2×2-H.sub.b), 4.40-4.57 (m, 2H, 2×2-H.sub.a), 7.41 (ddd, J=8.1, 6.7, 1.1 Hz, 2H, 2×7-H), 7.56 (ddd, J=8.3, 6.8, 1.4 Hz, 3H, 2×8-H, 4-H.sub.b), 7.76-7.97 (m, 3H, 2×9-H, 3′-H), 8.18 (d, J=8.3 Hz, 2H, 2×6-H), 8.37 (s, 1H, 4-H.sub.a), 8.63 (d, J=1.5 Hz, 2H, 6′-H, 8′-H), 10.28 (s, 2H, 2×5-OH).
(158) .sup.13C-NMR (126 MHz, DMSO-d.sub.6, 75° C.): δ [ppm]=41.5 (2×C-1), 47.8 (2×C-10), 55.7 (2×C-2), 100.4 (C-4a), 116.3 (2×C-5a), 123.1 (2×C-9a), 123.3 (2×C-9), 123.8 (2×C-7), 123.9 (2×C-6), 124.1 (C-7′), 125.4 (C-4.sub.b), 128.0 (C-3′), 130.6 (2×C-8), 131.8 (2×C-9a), 139.4 (C-2′, C-4′), 141.6 (2×C-3a), 148.9 (C-6′, C-8′), 154.9 (2×C-5), 165.4 (2×CON).
(159) HRMS (ESI): m/z calculated for C.sub.34H.sub.25Cl.sub.2N.sub.3O.sub.6 640.1042 [M−H].sup.+. found 640.1022.
5-Amino-1,3-phenylene)bis(((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzol[e]indol-3-yl)methanone) (53)
(160) ##STR00016##
(161) Compound 52 ((18 mg, 28 μmol) was dissolved in THF (20 ml) and passed twice over H-Cube 10% Pd/C system under full H.sub.2 mode at room temperature. After the end of the reaction, the solvent was evaporated under reduced pressure to give the crude product, which was then purified by flash column chromatography (MeOH:CH.sub.2Cl.sub.2 1:4) to give 53 (13 mg, 76%).
(162) R.sub.f: 0.4 (EtOAc).
(163) HPLC (analytical):
(164) Column: Kromosil® 100 C18, 250×4 mm, 5 m
(165) Mobile phase: MeOH/THF (1:1): H.sub.2O=62:38
(166) Flow rate: 0.8 ml min.sup.−1
(167) λ: 254 nm
(168) t.sub.R: 19.8 min
(169) Optical rotation: [α]D.sup.20=−27.5 (c 0.4, DMSO).
(170) .sup.1H-NMR (500 MHz, DMSO-d.sub.6, 50° C.): δ [ppm]=3.46-4.26 (m, 8H, 2×10-H.sub.a, 2×10-H.sub.b, 2×1-H, 2×2-H.sub.a), 4.40 (s, 2H, 2×2-H.sub.b), 6.95 (s, 1H, 3′-H), 6.92 (d, J=1.0 Hz, 2H, 6′-H, 8′-H), 6.57 (d, J=1.0 Hz, 2H.sub.minor, 6′-H, 8′-H) 7.30-7.45 (m, 2H, 2×7-H), 7.47-7.61 (m, 2H, 2×8-H), 7.62-8.07 (m, 4H, 2×9-H, 2×4-H), 8.08-8.28 (m, 2H, 2×6-H), 10.35 (s, 2H, 2×5-OH).
(171) .sup.13C-NMR (126 MHz, DMSO-d.sub.6, 50° C.): δ [ppm]=40.9 (2×C-1), 48.1 (2×C-10), 56.0 (2×C-2), 100.8 (C-4), 114.5 (C-3′), 116.0 (2×C-5b), 122.9 (2×C-9b), 123.4 (2×C-7), 123.7 (C-6′, C-8′), 123.9 (2×C-9), 125.6 (2×C-6), 128.0 (2×C-8), 128.7 (C-2′, C-4′), 130.8 (2×C-9a), 139.9 (2×C-3a), 152.2 (C-7′), 154.9 (2×C-5), 168.6 (2×CON).
(172) HRMS (ESI): m/z calculated for C.sub.34H.sub.27Cl.sub.2N.sub.3O.sub.4 634.1276 [M+Na].sup.+. found 634.1277.
5-Iodo-1,3-phenylene)bis(((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)methanone) (54)
(173) ##STR00017##
(174) 4M HCl/EtOAc (12 ml) was added to tbutoxycarbonylseco-CBI (50 mg, 150 μmol, 1.0 eq.) at room temperature and the mixture was stirred at the same temperature for 3 hours. Excess HCl/EtOAc was removed by evaporation under reduced pressure and the residue was dried under a strong vacuum for one hour. The resulting residue was dissolved in DMF (12 ml) and admixed with stirring at 0° C. with pyridine (48 μl, 600 μmol, 4.0 eq.) and 5-iodoiso-phthaloyl dichloride (24 mg, 75 μmol, 0.5 eq.). Stirring was continued at room temperature for 16 hours. After evaporation of the solvent under a high pressure, a crude product was obtained which was purified by flash chromatography (EtOAc:PE 1:1 to EtOAc) to give 54 (49 mg, 90%).
(175) R.sub.f: 0.3 (EtOAc:PE 1:1).
(176) HPLC (analytical):
(177) Column: Kromosil® 100 C18, 250×4 mm, 5 m
(178) Mobile phase: MeOH/THF (1:1): H.sub.2O=70:30
(179) Flow rate: 0.8 ml min.sup.−1
(180) λ: 254 nm
(181) t.sub.R: 14.7 min
(182) Optical rotation: [α].sub.D.sup.20=−22.5 (c 0.8, DMSO).
(183) .sup.1H-NMR (500 MHz, DMSO-d.sub.6, 75° C.): δ [ppm]=3.75-4.04 (m, 4H, 2×10-H.sub.a, 2×2-H.sub.b), 4.05-4.34 (m, 4H, 2×2-H.sub.b, 2×1-H), 4.44 (t, J=10.0 Hz, 2H, 2×2-H.sub.a), 6.93 (s, 1H, 3′-H), 7.40 (ddd, J=8.0, 7.0, 1.0 Hz, 2H, 7-H), 7.55 (ddd, J=8.0, 6.5, 1.0 Hz, 2H, 8-H), 7.69 (brs, 1H, 4-H), 7.84 (d, J=8.3 Hz, 2H, 2×9-H), 7.93 (s, 1H, 4-H), 8.12-8.39 (m, 4H, 6′-H, 8′-H, 2×6-H), 10.27 (s, 2H, 2×5-OH).
(184) .sup.13C-NMR (126 MHz, DMSO-d.sub.6, 75° C.): δ [ppm]=40.8 (2×C-1), 47.8 (2×C-10), 55.8 (2×C-2), 95.4 (C-7′), 100.5 (2×C-4b), 116.1 (2×C-5a), 123.0 (2×C-9a), 123.3 (2×C-9), 123.7 (2×C-7), 123.8 (2×C-6), 125.1 (C-4a), 125.4 (C-3′), 127.9 (2×C-8), 130.6 (2×C-9a), 137.7 (C-6′, C-8′), 139.6 (C-2′, C-4′), 141.8, (2×C-3a), 154.8 (2×C-5), 166.0 (2×CON).
(185) HRMS (ESI): m/z calculated for C.sub.34H.sub.25Cl.sub.2IN.sub.2O.sub.4 745.0134 [M+Na]+. found 745.0100.
5-((Trimethylsilyl)ethynyl)-1,3-phenylene)bis(((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)methanone) (55)
(186) ##STR00018##
(187) A solution of 54 (79 mg, 105 mol, 1.0 eq.) in triethylamine (420 μl) was admixed with tetrakis(triphenylphosphine)palladium(0) ((6 mg, 5 μmol, 5 mol %) and copper iodide (2 mg, 10 μmol, 10 mol %) and then ethynyl trimethylsilane (22 μl, 158 μmol, 1.5 eq.) was added at room temperature and the stirring was continued at the same temperature for 16 hours. Evaporation of the solvent under a high pressure gave the crude product, which was then purified by flash chromatography (EtOAc:PE 2:3 to EtOAc:PE 3:2) to give 55 (61 mg, 88%).
(188) R.sub.f: 0.4 (EtOAc:PE 1:1).
(189) Optical rotation: [α].sub.D.sup.20=−43.8 (c 0.73, DMSO).
(190) .sup.1H-NMR (500 MHz, DMSO-d.sub.6, 75° C.): δ [ppm]=3.82-3.93 (m, 2H, 2×10-H.sub.a), 3.93-4.02 (m, 2H, 2×10-H.sub.b), 4.02-4.23 (m, 4H, 2×2-H.sub.a, 2×1-H), 4.44 (dd, J=11.2, 8.5 Hz, 2H, 2×2-H.sub.b), 6.44 (s, 1H.sub.minor, 3′-H) 6.93 (s, 1H, 3′-H), 7.40 (ddd, J=8.1, 6.8, 1.2 Hz, 2H, 2×7-H), 7.55 (ddd, J=8.2, 6.8, 1.4 Hz, 2H, 2×8-H), 7.69 (brs, 1H, 4-H), 7.80-8.02 (m, 5H, 4-H, 6′-H, 8′-H, 2×9-H), 8.17 (d, J=8.4 Hz, 2H, 2×6-H), 10.25 (s, 2H, 2×5-OH).
(191) .sup.13C-NMR (126 MHz, DMSO-d.sub.6, 75° C.): δ [ppm]=0.3 (CH.sub.3), 41.4 (2×C-1), 47.8 (2×C-10), 55.7 (2×C-2), 97.2 (Si—C≡), 100.5 (C-4), 104.2 (Ar-C≡), 116.1 (2×C-5a), 123.0 (2×C-9b), 123.3 (2×C-9), 123.7 (2×C-7), 123.8 (2×C-6), 124.0 (C-7′), 125.4 (C-3′), 126.0 (2×C-4), 127.9 (2×C-8), 130.6 (2×C-9a), 132.1 (C-6′, C-8′), 138.5 (C-2′, C-4′), 139.6 (2×C-3a), 141.8, 154.8 (2×C-5), 166.6 (2×CON).
(192) HRMS (ESI): m/z calculated for C.sub.39H.sub.34Cl.sub.2N.sub.2O.sub.4Si 691.1587 [M−H].sup.+. found 691.1555.
5-Ethynyl-1,3-phenylene)bis(((S)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benzo[e]indol-3-yl)methanone) (56)
(193) ##STR00019##
(194) A stirred, precooled (0° C.) solution of 55 (61 mg, 881 μmol, 1.0 eq.) in THF (2 ml) was admixed with 70% hydrogen fluoride/pyridine (63 μl, 2.20 mmol, 25 eq.) and the stirring was continued at 35° C. for 12 hours. Additional 70% hydrogen fluoride/pyridine (63 μl, 2.20 mmol, 25 eq.) was added at 0° C. and the stirring was continued at 35° C. for a further 24 hours. The solvent was stripped off under a high pressure to give the crude product, which was then purified by flash chromatography (EtOAc:PE 2:3 to EtOAc:PE 1:1) to give 56 (39 mg, 71%). Starting material recovered (17 mg, 28%).
(195) R.sub.f: 0.3 (EtOAc:PE 1:1).
(196) HPLC (analytical):
(197) Column: Kromosil® 100 C18, 250×4 mm, 5 m
(198) Mobile phase: MeOH/THF (1:1): H.sub.2O=70:30
(199) Flow rate: 0.8 mL min.sup.−1
(200) λ: 254 nm
(201) t.sub.R: 11.0 min
(202) Optical rotation: [α].sub.D.sup.20=−15.2 (c 0.46, DMSO).
(203) .sup.1H-NMR (500 MHz, DMSO-d.sub.6, 75° C.): δ [ppm]=3.90 (dd, J=11.0, 7.4 Hz, 2H, 2×10-H.sub.a), 3.98 (d, J=10.8 Hz, 2H, 2×10-H.sub.b), 4.04-4.19 (m, 4H, 2×2-H.sub.a, 2×1-H), 4.33 (s, 1H, CH), 4.44 (dd, J=11.1, 8.7 Hz, 2H, 2×2-H.sub.b), 6.93 (s, 1H, 3′-H), 7.40 (ddd, J=8.2, 6.8, 1.1 Hz, 2H, 2×7-H), 7.55 (ddd, J=8.2, 6.8, 1.4 Hz, 2H, 2×8-H), 7.67 (s, 1H, 4-H.sub.a), 7.84 (d, J=8.2 Hz, 2H, 2×9-H), 7.88-7.98 (m, 3H, 4-H.sub.b, 6′-H, 8′-H), 8.18 (d, J=8.3 Hz, 2H, 2×6-H), 10.26 (s, 2H, 2×5-OH).
(204) .sup.13C-NMR (126 MHz, DMSO-d.sub.6, 75° C.): δ [ppm]=41.4 (2×C-1), 47.8 (2×C-10), 55.8 (2×C-2), 82.7 (≡CH), 83.0 (Ar-C≡), 100.6 (C-4), 116.1 (2×C-5a), 123.0 (2×C-9b), 123.3 (2×C-9), 123.5 (C-7′), 123.7 (2×C-7), 123.8 (2×C-6), 125.4 (2×C-3′), 126.2 (C-4), 127.9 (2×C-8), 130.6 (2×C-9a), 132.2 (C-6′, C-8′), 138.5 (C-2′, C-4′), 139.6 (2×C-3a), 154.9 (2×C-5), 166.6 (2×CON).
(205) HRMS (ESI): m/z calculated for C.sub.36H.sub.26Cl.sub.2N.sub.2O.sub.4 621.1348 [M+H].sup.+. found 621.1324.
(2R,3S,4S,5R,6S)-2-(Acetoxymethyl)-6-(((S)-1-(chloromethyl)-3-(3-((S)-1-(chloromethyl)-5-(((2R,3S,4R,5R,6S)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-2,3-dihydro-1H-benzo[e]indole-3-carbonyl)-5-nitrobenzoyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (LT-SH016) (57)
(206) ##STR00020##
(207) A solution of tbutoxycarbonylseco-CBI (0.336 mmol, 223 mg) in dry CH.sub.2Cl.sub.2 (20 ml) under an argon atmosphere was admixed with molecular sieve (4 Å) and O-(2,3,4,6-tetra-O-acetyl-/3-d-galactopyranosyl) trichloroacetimidate (170 mg, 0.344 mmol, 1.05 eq.). The solution was cooled to 0° C. and a solution of boron trifluoride-diethyletherate (0.504 mmol, 62 μl) in dry CH.sub.2Cl.sub.2 (2 ml) was added with stirring. The reaction mixture warmed up to room temperature and was stirred for a further 4 hours. The mixture was subsequently filtered and the filtrate was concentrated under reduced pressure. 5-Nitroisophthaloyl dichloride acyl chloride (0.168 mmol, 42 mg) and dry DMF (4 ml) were added to the crude material under an argon atmosphere. The mixture obtained was cooled to 00° C. and pyridine (1.344 mmol, 109 μl) was cautiously added. The reaction mixture warmed slowly to room temperature and was stirred overnight. After the end, the mixture was concentrated under reduced pressure and purified by flash chromatography ((PET/EtOAc, 1:0 to 1:1) to give the dimer 57 as an orange solid (0.066 mmol, 86 mg), 39% yield.
(208) R.sub.f 0.25 (PET/EtOAc, 6:4)
(209) Mp 172° C.
(210) Optical rotation [α].sub.D.sup.20=
(211) IR (film): ν [cm.sup.−1]=
(212) UV (CH.sub.3CN): λ.sub.max (Ig ε)=
(213) .sup.1H-NMR (600 MHz, DMSO-d.sub.6, 75° C.): δ 8.60 (d, J=1.5 Hz, 2H), 8.38 (s, 1H), 8.18 (br s, 2H), 8.03 (d, J=8.5 Hz, 2H), 7.95 (d, J=8.4 Hz, 2H), 7.61 (m, 2H), 7.49 (m, 2H), 5.56 (d, J=7.5 Hz, 2H), 5.49-5.40 (m, 6H), 4.57-4.47 (m, 4H), 4.22 (m, 2H), 4.19-4.04 (m, 6H), 4.02-3.97 (m, 2H), 3.94 (dd, J=11.0, 7.4 Hz, 2H), 2.19 (s, 6H), 2.04 (s, 6H), 2.01 (s, 6H), 1.98 (s, 6H).
(214) .sup.13C-NMR (126 MHz, DMSO-d.sub.6, 75° C.): δ 169.30, 169.24, 168.83, 168.70, 164.53, 152.57, 147.86, 140.44, 138.18, 130.38, 129.15, 127.36, 124.21, 123.13, 122.67, 122.62, 121.72, 119.48, 101.88, 98.93, 70.47, 69.67, 68.48, 67.10, 61.01, 55.02, 46.71, 40.82, 20.06, 19.91, 19.90, 19.85.
(215) HRMS (ESI) m/z calculated for C.sub.62H.sub.61Cl.sub.2N.sub.3NaO.sub.24 [M+Na].sup.+: 1324.2914. found 1324.2909
(2R,3S,4S,5R,6S)-2-(Acetoxymethyl)-6-(((S)-3-(3-amino-5-((S)-1-(chloromethyl)-5-(((2R,3S,4R,5R,6S)-3,4,5-triacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-2,3-dihydro-1H-benzo[e]indole-3-carbonyl)benzoyl)-1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (LT-SH025) (58)
(216) ##STR00021##
(217) The dimer 57 (0.037 mmol, 48 mg) was dissolved in a mixture of MeOH/EtOAc (7:3, 5 ml) and conveyed twice over an H-Cube system (Pd/C 10%, full H.sub.2 mode, 1 ml/min, room temperature). The mixture obtained was concentrated under reduced pressure and flash chromatography was carried out (CH.sub.2Cl.sub.2/MeOH, 98:2) to give the amine 58 as a white solid (0.034 mmol, 43 mg) with a yield of 92%.
(218) R.sub.f 0.17 (CH.sub.2Cl.sub.2/MeOH, 96:4)
(219) Mp 96° C.
(220) .sup.1H-NMR (600 MHz, DMSO-d.sub.6, 75° C.): δ 8.00 (d, J=8.4 Hz, 2H), 8.00 (br s, 2H), 7.91 (d, J=8.4 Hz, 2H), 7.58 (m, 2H), 7.45 (m, 2H), 6.95 (m, 3H), 5.59-5.48 (m, 4H), 5.48-5.36 (m, 6H), 4.49 (m, 2H), 4.42 (dd, J=11.0, 9.2 Hz, 2H), 4.21-4.07 (m, 8H), 4.00 (dd, J=11.0, 2.8 Hz, 2H), 3.89 (dd, J=11.0, 7.1 Hz, 2H), 2.18 (s, 6H), 2.02 (s, 6H), 2.01 (s, 6H), 1.98 (s, 6H).
(221) .sup.13C-NMR (126 MHz, DMSO-d.sub.6, 75° C.): δ 169.55, 169.47, 169.06, 168.94, 167.65, 152.63, 149.06, 140.98, 137.42, 129.37, 127.38, 124.03, 122.65, 122.40, 121.78, 119.17, 113.42, 111.50, 101.95, 98.97, 70.43, 69.73, 68.45, 67.11, 60.98, 55.00, 46.89, 40.42, 20.05, 19.94, 19.92, 19.86.
(222) HRMS (ESI) m/z calculated for C.sub.62H.sub.64Cl.sub.2N.sub.3O.sub.22 [M+H].sup.+: 1272.3353. found 1272.3349 and m/z calculated for C.sub.62H.sub.63Cl.sub.2N.sub.3NaO.sub.22 [M+Na].sup.+: 1294.3172, found 1294.3169
(3-Amino-5-((S)-1-(chloromethyl)-5-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-2,3-dihydro-1H-benzo[e]indole-3-carbonyl)phenyl)((S)-1-(chloromethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1,2-dihydro-3H-benzo l[e]indol-3-yl)methanone (59) (LT-SH032)
(223) ##STR00022##
(224) Aniline 58 (0.024 mmol, 31 mg) was taken up in MeOH (10 mil), and NaOMe (5.4 M in MeOH, 18 μl) was added at 0° C. with stirring. The reaction mixture warmed up to room temperature and was stirred for a further two hours. The mixture was subsequently diluted with H.sub.2O (1 mil) and neutralized (pH 7) by dropwise addition of 0.1 M aqueous HCl at 0° C. The mixture obtained was concentrated under reduced pressure and the residue was taken up with DMF (1.4 ml) and purified via preparative HPLC to give the above-stated compound as a white solid (0.0160 mmol, 15 mg) with a 66% yield.
(225) HPLC analytical (Kromasil 100 C18, 250×4.0 mm, 5 μm)
(226) H.sub.2O/MeOH
(227) 0.8 ml/min
(228) 0-65 min 40:60 to 0:100
(229) 65-72 min 0:100
(230) 72-72 min 0:100 to 40:60
(231) 72-90 min 40:60
(232) λ: 254 nm
(233) t.sub.R: 30.5 min
(234) HPLC preparative (Kromasil 100 C18, 50×20 mm, 5 μm+Kromasil 100 C18, 250×20 mm, 7 μm)
(235) H.sub.2O/MeOH
(236) 16 ml/min
(237) 0-75 min 40:60 to 0:100
(238) 75-82 min 0:100
(239) 82-83 min 0:100 to 40:60
(240) 83-100 min 40:60
(241) λ: 254 nm
(242) t.sub.R: 31.5 min
(243) .sup.1H-NMR (600 MHz, DMSO-d.sub.6, 75° C.): δ 8.33 (d, J=8.1 Hz, 2H), 7.86 (d, J=8.4 Hz, 2H), 7.86 (br s, 2H), 7.55 (ddd. J=8.3, 6.8, 1.3 Hz, 2H), 7.41 (ddd, J=8.2, 6.8, 1.1 Hz, 2H), 6.98-6.93 (m, 3H), 5.50 (s, 2H), 5.05 (d, J=5.3 Hz, 2H), 4.89 (d, J=7.3 Hz, 2H), 4.58 (d, J=5.8 Hz, 2H), 4.40 (dd, J=11.1, 8.9 Hz, 2H), 4.37-4.30 (m, 4H), 4.18-4.09 (m, 4H), 3.99 (dd, J=11.1, 2.5 Hz, 2H), 3.88 (dd, J=11.1, 7.3 Hz, 2H), 3.84 (t, J=3.8 Hz, 2H), 3.79 (ddd, J=9.4, 7.7, 5.3 Hz, 2H), 3.69-3.62 (m, 2H), 3.61-3.55 (m, 4H), 3.55-3.49 (m, 2H),
(244) .sup.13C-NMR (126 MHz, DMSO-d.sub.6, 75° C.): δ 167.66, 153.60, 149.02, 141.03, 137.52, 129.41, 127.16, 123.42, 123.13, 122.87, 122.34, 118.00, 113.53, 111.79, 102.09, 101.52, 75.04, 73.10, 70.41, 67.64, 59.73, 54.88, 46.97, 40.42.
(245) HRMS (ESI) m/z calculated for C.sub.46H.sub.47Cl.sub.2KN.sub.3O.sub.14 [M+K].sup.+: 974.2067. found 974.2059
(246)