Duocarmycin analogs

Abstract

The present invention relates to 2-methylbenzoxazole compounds of formula I, which are analogs of the DNA alkylating subunit of duocarmycin. The compounds of formula I can be used to synthesize DNA alkylating agents and antibody-drug conjugates and related compounds. The 2-methylbenzoxazole unit of formula I has favorable properties in combining high cytotoxicity, low lipophilicity and exceptional water stability, all of which are desirable for use as a payload in effective antibody-drug conjugates.

Description

1. Technical Field

[0001] The present invention generally relates to 2-methylbenzoxazole compounds and related compounds that are useful in the synthesis of antibody-drug conjugates (ADCs). 2. Background technology

[0002] Duocarmycin is a natural product with significant cytotoxic activity that binds to the minor groove of DNA and alkylates at the N3 position of adenine. The following two examples, Duocarmycin SA and CC-1065, illustrate the typical structure of Duocarmycin, which consists of a DNA-alkylating subunit and a DNA-binding subunit that non-covalently binds in the minor groove of the DNA helix.

[0003]

[0004] The mechanism of conjugation involves the addition of adenine to the cyclopropyl ring of the alkylating subunit, illustrated below in Scheme 1 using the general structure of this subunit. The reaction is rapid and selective for DNA, but is orders of magnitude slower for other nucleophiles such as water, such that in the absence of DNA, the alkylating subunit persists for very long times under physiological conditions in aqueous buffers.

[0005]

[0006] Solution 1

[0007] The cyclopropyl ring can be formed from a seco (i.e., ring-opened) precursor bearing a chloromethyl or other leaving group substituent, as shown in Scheme 1. The ring closure reaction occurs so readily under physiological conditions that both forms (ring-opened and cyclopropyl) exhibit essentially the same cytotoxicity. However, if the phenol of the ring-opening alkylated subunit is in a chemical form that prevents cyclization, the cytotoxicity is greatly reduced.

[0008] Natural products such as duocarmycin SA are isolated as single enantiomers, as shown. However, both enantiomers can alkylate DNA, although the non-natural enantiomer is generally less cytotoxic.

[0009] Many synthetic forms of the alkylating subunit of the duocarmycins have been reported. These generally vary in the nature of the ring fusion indicated by the dotted lines in the structure in Scheme 1 above. The nature of these ring fusions can have a strong effect on cytotoxicity.

[0010] For example, CBI (cyclopropylbenzindole) alkylation of the subunit (illustrated below in open-ring form in combination with the TMI (trimethoxyindole) side chain found in duocarmycin SA) produces duocarmycin analogs with cytotoxic potency similar to that of the natural product (J. Org. Chem. (1990) 55, 5823).

[0011]

[0012] In contrast, compounds comprising an alternative COI (cyclopropyloxazoloindole) alkylating subunit in combination with TMI were several hundred times less cytotoxic (Bioorg. Med. Chem. Lett. (2010) 20, 1854).

[0013] Other synthetic variations have also been reported. For example, amino-opened-CBIs are known, in which the amine group replaces the hydroxyl group of the phenol. These variants undergo the same spirocyclization and DNA alkylation reactions as their phenol analogs and have equal cytotoxic potency (ChemBioChem (2014) 15, 1998).

[0014] Further variations include linking the two alkylated subunits together in a manner that allows interstrand crosslinking of two adenines in DNA (J. Am. Chem. Soc. (1989) 111, 6428; Angew. Chem. (2010) 49, 7336). These dimers can even be more cytotoxic than the corresponding monoalkylating agents, although the activity of the dimer is not necessarily predicted by the activity of its component monoalkylated units.

[0015] The dimer of amino-ring-secreting-duocarmycin and the alkylated subunit is shown below.

[0016]

[0017] Several duocarmycin analogs have been studied as potential small molecule anticancer agents. However, clinical trials have been unsuccessful because toxicity limits the amount that can be administered at very low doses.

[0018] Recently, duocarmycin analogs have found application as payloads in antibody-drug conjugates (ADCs). ADCs are most commonly used for cancer therapy, in which a cytotoxic small molecule (payload) is linked to an antibody that recognizes a tumor-associated antigen via a linker (Nat. Rev. Drug Discov. (2017) 16, 315; Pharmacol. Rev. (2016) 68, 3).

[0019] ADCs are constructed by chemically linking a payload to a suitable linker, which itself is conjugated to the desired antibody. ADCs are designed to be stable in circulation, but typically release the payload at a predetermined target site after receptor binding and internalization into target cells. In this way, the cytotoxic effect of the payload is specifically directed to the site where damage is required, i.e., the tumor. Several such ADCs have been approved as anticancer treatments.

[0020] The ADC concept is an ingenious means to direct the payload specifically to its target cells, thereby minimizing the adverse effects associated with conventional nonspecific systemic delivery of toxic compounds in vivo. Unfortunately, major technical challenges have limited the successful application of the ADC concept.

[0021] The ADC concept limits the amount of payload that can be delivered to the target cell, such that the payload must be very cytotoxic for the ADC to have a therapeutic effect.

[0022] Due to their high cytotoxicity, duocarcin analogs have been studied as ADC payloads, as monoalkylating agents, or as components of dimers that can cross-link DNA (see, e.g., Mol. Pharm. (2015) 12, 1813; WO 2011 / 133039; Biopharm. Drug Dispos. (2016) 37, 93; Cancer Chemother. Pharmacol. (2016) 77: 155–162; J. Med. Chem. (2012) 55, 766; J. Med. Chem. (2005) 48, 1344; Cancer Res. (1995) 55, 4079; Bioorg. Med. Chem. (2000) 8, 2175; WO 2018 / 035391; WO 2015 / 038426; WO 2015 / 153401; WO 2015 / 023355; WO 2017 / 194960; WO 2018 / 071455).

[0023] In the vast majority of these examples, the alkylated subunit is the highly toxic open-ring-CBI, as shown below.

[0024]

[0025] Although the use of such alkylated subunits will result in ADCs with the desired cytotoxicity, open-CBI has a significant disadvantage in being highly lipophilic. Lipophilic payloads and their derivatives are not very soluble in the aqueous buffer used to conjugate the payload-linker component to the antibody, making the conjugation step difficult. The resulting ADCs also tend to aggregate. ADC aggregates must be removed before the ADC is used, adding to the complexity and cost of producing clinically useful ADCs.

[0026] Lipophilic payloads are also associated with faster clearance of ADCs from the bloodstream, which reduces their overall exposure and, therefore, their antitumor efficacy (Nat. Biotechnol. (2015) 33, 733–735).

[0027] Lipophilic payloads, particularly those that induce aggregation, can also produce ADCs that elicit an immune response in vivo, leading to unexpected toxicity or reduced therapeutic efficacy.

[0028] However, despite the issues related to lipophilicity, the high cytotoxicity of open-CBI compounds continues to make them attractive payloads for anticancer ADCs, with various strategies being employed to mitigate the drawbacks.

[0029] For example, to counteract the high lipophilicity of the open-ring-CBI, some researchers have resorted to modifying other component parts so that the ADC as a whole is less lipophilic. Some researchers have incorporated modified linkers (e.g., polyethylene glycol spacers, as in Mol. Pharm. (2015) 12, 1813; J. Med. Chem. (2005) 48, 1344). Others have constructed more water-soluble prodrug forms (e.g., J. Med. Chem. (2012) 55, 766, WO 2015 / 023355).

[0030] However, addressing the issue of payload lipophilicity by modifying other components of the ADC may itself have disadvantages. In addition to making the synthesis of the payload-linker component more complex and time-consuming, this technique may also limit the choice of linker.

[0031] The linker moiety attached to the payload has a significant impact on the efficacy and safety of the ADC in vivo (Bioanalysis (2015) 7 (13), 1561). It must have appropriate stability in the circulation, but cleave in vivo when needed. ADC design includes consideration of the physiological conditions under which the payload is released so that a suitable linker can be used. For example, a linker containing a hydrazone moiety is pH sensitive and will cleave in lower pH environments such as endosomes and lysosomes. Disulfide linkers release the payload in a reducing environment (e.g., the intracellular environment).

[0032] Some linker modifications made to mitigate the undesirable lipophilicity of the alkylated subunit can reduce the efficacy of the ADC in which they are used, such that the resulting ADC may not be the most potent product that can be made from a given payload and antibody.

[0033] Other solutions to the lipophilicity problem include ADC Bio's patented lock-and-release technology ( www.adcbio.com ), which immobilizes the antibody on a solid support before conjugation to the payload-linker component. Aggregation is prevented by physically separating them from each other during the manufacture of the ADC molecules. This allows the use of a wider range of linkers, but adds additional costs to the conjugation process without actually reducing the lipophilicity of the final ADC product.

[0034] Therefore, the lipophilicity of the highly toxic alkylated subunit is a problem that hinders the development of new ADCs.

[0035] Another potential disadvantage of known duocarcin analogs as ADC payloads is the high stability of the cyclopropyl form of the alkylated subunit. The stable payload released from the ADC into the circulation can last long enough to cause undesirable systemic toxicity. Unfortunately, high stability is a characteristic of almost all synthetic analogs of the CBI alkylated subunit that retain the required effective cytotoxicity. Previous studies have shown a correlation between aqueous stability and cytotoxicity, making most cytotoxic analogs stable in aqueous buffers at neutral pH (J Med Chem (2009) 52, 5271).

[0036] Therefore, there is a need for alternative DNA-alkylating subunits that facilitate the efficient synthesis of a range of ADCs while demonstrating the cytotoxicity and stability properties required for efficacy.

[0037] It is therefore an object of the present invention to go at least some way towards satisfying this need; and / or at least to provide the public with a useful choice. Other objects of the present invention may become apparent from the following description which is given by way of example only.

[0038] In this specification, reference has been made to external sources of information, including patent specifications and other documents, this is generally for the purpose of providing a context for discussing the features of the invention. Unless otherwise stated, reference to such sources of information should not be construed as an admission that such sources of information are prior art or form part of the common general knowledge in the art in any jurisdiction. 3. Summary of the invention

[0039] The present invention provides a novel alkylated subunit '2-methylbenzoxazole' and protected and prodrug derivatives of this subunit, which are suitable for attachment to a wide range of heteroaryl and aryl DNA binding moieties to generate highly cytotoxic duocarmycin analogs.

[0040] The present invention also provides duocarmycin analogs comprising a novel 2-methylbenzoxazolidinyl subunit conjugated to a DNA minor groove binding unit. When conjugated to an antibody via a linker group, these compounds can be used to generate ADCs.

[0041] Therefore, in a first aspect, the present invention provides a compound of formula I or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0042]

[0043] in:

[0044] LG is a leaving group;

[0045] X is a group selected from hydroxy, protected hydroxy, prodrug hydroxy, amine and protected amine; wherein amine is -NH2, or -NH(C1-C6)alkyl; and

[0046] Y is an N-protecting group.

[0047] In a second aspect, the present invention provides a compound of formula II or a pharmaceutically acceptable salt, hydrate or solvate thereof

[0048]

[0049] in:

[0050] LG is a leaving group;

[0051] X is a group selected from hydroxy, protected hydroxy, prodrug hydroxy, amine and protected amine; wherein amine is -NH2, or -NH(C1-C6)alkyl; and

[0052] DB is the DNA minor groove binding unit.

[0053] In one embodiment, DB is optionally substituted aryl or optionally substituted heteroaryl attached directly or via an alkenyl group.

[0054] In one embodiment, DB is an optionally substituted indole, azaindole, benzofuran, pyridine, pyrimidine, pyrrole, imidazole, thiophene, thiazole, oxazole, pyrazole, triazole, pyrazine, or pyridazinyl.

[0055] In a third aspect, the present invention provides a compound of formula III or a pharmaceutically acceptable salt, hydrate or solvate thereof

[0056]

[0057] in:

[0058] LG is a leaving group;

[0059] X is a group selected from hydroxy, protected hydroxy, prodrug hydroxy, amine, and protected amine; wherein the amine is -NH2 or -NH(C1-C6)alkyl; and

[0060] Y is selected from:

[0061] (a) N-protecting group;

[0062] (b)-C(O)-Ar 1

[0063] (c)-C(O)-Ar 1 -NH-C(O)-Ar 2

[0064] (d)-C(O)-Ar1 -NH-C(O)-CH=CH-Ar 3 ;or

[0065] (e)-C(O)-CH=CH-Ar 3

[0066] Among them, Ar 1 ,Ar 2 and Ar 3 each independently selected from heteroaryl or aryl, wherein each of the heteroaryl or aryl is optionally substituted with one or more of: -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -OH, -O-(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl;

[0067] wherein in each case -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -O-(C1-C6)alkyl, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl are independently optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0068] In one embodiment, Ar 1 ,Ar 2 and Ar 3 Independently selected from the group consisting of

[0069]

[0070] in represents the point of attachment to a -C(O) or C(O)-CH=CH- group, and each aryl or heteroaryl group may be substituted at the numbered positions with up to three substituents selected from -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -OH, -O-(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl;

[0071] wherein the substituents -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -O-(C1-C6)alkyl, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl may in each case independently be optionally substituted by one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0072] In a fourth aspect, the present invention provides a compound of formula IV or a pharmaceutically acceptable salt, hydrate or solvate thereof

[0073]

[0074] wherein V is Y or DB and X, Y and DB have the same meanings as defined for the compounds of formula I, II and III and X' is X minus H.

[0075] Compounds of formula IV can be formed by in vitro or in vivo rearrangement and concomitant elimination of H-LG from the corresponding ring-opening compounds of formula I, II and III. Unless the context dictates otherwise, all embodiments of the invention described herein for compounds of formula I, II or III, or any portion thereof, are also expressly contemplated as being part of the aspects of the invention relating to compounds of formula IV.

[0076] The following embodiments and preferences may relate to any aspect of the invention described herein either alone or in any combination of two or more.

[0077] In one embodiment, LG is selected from the group consisting of chloride, bromide, iodide, and -OSO2R 1 ; where R 1 Selected from (C1-C 10 )alkyl, (C1-C 10 ) heteroalkyl, (C1-C 10 )aryl or (C1-C 10 ) heteroaryl.

[0078] In one embodiment, LG is a halogen, preferably chlorine, and the configuration on the chiral carbon to which LG is attached is (S).

[0079] In one embodiment, X is selected from the group consisting of: -OH, -OBn, -OTf, -OMOM, -OMEM, -OBOM, -OTBDMS, -OPMB, -OSEM, piperazine-1-carboxylate, wherein the N at position 4 is replaced by (C1-C 10 )alkyl, -OP(O)(OH)2, -OP(O)(OR2 )2, -NH2, -N=C(Ph)2, -NHZ, NH(C1-C 10 )alkyl and -NZ(C1-C 10 ) alkyl substitution;

[0080] Where R 2 is t-Bu, Bn or allyl; and Z is selected from Boc, COCF3, Fmoc, Alloc, Cbz, Teoc and Troc.

[0081] In one embodiment, Y is an N-protecting group selected from Boc, COCF3, Fmoc, Alloc, Cbz, Teoc and Troc.

[0082] Numerical ranges disclosed herein (e.g., 1 to 10) also include reference to all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) as well as any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus, all subranges of all ranges explicitly disclosed herein are hereby explicitly disclosed. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited are considered to be expressly stated in this application in a similar manner.

[0083] Although the present invention is broadly defined above, it will be appreciated by those skilled in the art that the present invention is not limited thereto and that the present invention also includes embodiments for which the following description gives examples. 4. Description of the drawings

[0084] The present invention will now be described with reference to the accompanying drawings, in which:

[0085] Figure 1 LCMS analysis of the stability of open-CBI-TMI in neutral aqueous buffer is shown. The upper panel shows an example of a chromatogram (after 20 minutes of incubation time). The lower panel summarizes the changes in the composition of the mixture over a total incubation time of >300 min.

[0086] Figure 2 LCMS analysis of the stability of compound 23 in neutral aqueous buffer is shown. The upper panel shows an example of a chromatogram (after an incubation time of 200 minutes). The lower panel summarizes the changes in the composition of the mixture during a total incubation time of 500 minutes.

[0087] Figure 3 HPLC analysis showing the stability of compound 52 in neutral aqueous buffer. The experiment was monitored every hour for a total of 8 hours.

[0088] Figure 4HPLC analysis showing the stability of compound 59 in neutral aqueous buffer. The experiment was monitored every hour for a total of 8 hours.

[0089] Figure 5 HPLC analysis showing the stability of compound 66 in neutral aqueous buffer. The experiment was monitored every hour for 8 hours. 5. Specific implementation methods

[0090] 5.1 Definitions

[0091] The term "comprising" as used in the present specification and claims means "consisting at least in part of..." When interpreting each expression including the term "comprising" in the present specification and claims, features other than the term or those beginning with the term may also exist. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

[0092] As used herein, the term "and / or" means "and" or "or", or both.

[0093] As used herein, "(s)" following a noun is intended to refer to the plural and / or singular forms of that noun.

[0094] Asymmetric centers may be present in the compounds described herein. Asymmetric centers may be designated as (R) or (S), depending on the configuration of substituents in three-dimensional space at the chiral carbon atom. Unless a specific stereochemistry or isomeric form is indicated, all chiral, diastereoisomers, and racemic forms of the structure are intended to be included. All stereochemical isomeric forms of the compound, including diastereomeric, enantiomeric, and epimeric forms, as well as cis and trans isomers, and mixtures thereof, including mixtures of stereochemical isomers enriched in enantiomers and diastereoisomers enriched, are within the scope of the present invention.

[0095] Individual enantiomers can be prepared by commercially available enantiomerically pure raw material synthesis, or by preparing an enantiomeric mixture and splitting the mixture into individual enantiomers. The splitting method includes (a) separating the enantiomeric mixture by chromatography on a chiral stationary phase and (b) converting the enantiomeric mixture into a mixture of diastereomers and separating the diastereomers by, for example, recrystallization or chromatography, and any other suitable method known in the art. The raw material with defined stereochemistry can be commercially available or prepared, and if necessary, split by techniques well known in the art. It is preferred to have an enantiomer with a "native" configuration at a chiral carbon (a carbon with a CH2-LG moiety in an open ring form).

[0096] The compounds described herein may also exist as conformational or geometric isomers, including cis, trans, syn, anti, entgegen (E) and zusammen (Z) isomers. All of these isomers and any mixtures thereof are within the scope of the present invention.

[0097] Any tautomer or mixture thereof of the compound is also within the scope of the present invention. As will be appreciated by those skilled in the art, various functional groups and other structures may exhibit tautomerism. Examples include, but are not limited to, ketone / enol, imine / enamine and thioketone / enethiol tautomerism.

[0098] The compounds described herein may also exist in isotopologues and isotopologues, wherein one or more atoms in the compound are replaced by a different isotope. Suitable isotopes include, for example 1 H. 2 H(D), 3 H(T), 12 C. 13 C. 14 C. 16 O and 18 O. The procedures for incorporating such isotopes into the compounds described herein will be apparent to those skilled in the art. Isotopologues and isotopologues of the compounds described herein are also within the scope of the present invention.

[0099] Salts of the compounds described herein, including pharmaceutically acceptable salts, are also within the scope of the present invention. Such salts include acid addition salts, base addition salts, and quaternary salts of basic nitrogen-containing groups. Acid addition salts can be prepared by reacting the compound in free base form with an inorganic acid or an organic acid. Examples of inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid. Examples of organic acids include, but are not limited to, acetic acid, trifluoroacetic acid, propionic acid, succinic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, isethionic acid, sulfanilic acid, adipic acid, butyric acid, and pivalic acid. Base addition salts can be prepared by reacting the compound in free acid form with an inorganic or organic base. Examples of inorganic base addition salts include alkali metal salts, alkaline earth metal salts, and other physiologically acceptable metal salts, such as aluminum, calcium, lithium, magnesium, potassium, sodium, or zinc salts. Examples of organic base addition salts include amine salts, such as salts of trimethylamine, diethylamine, ethanolamine, diethanolamine and ethylenediamine. Quaternary salts of basic nitrogen-containing groups in the compounds can be prepared, for example, by reacting the compounds with alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides, dialkyl sulfates (such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate and diamyl sulfate), and the like.

[0100] The compounds described herein may form solvates with various solvents or exist as solvates. If the solvent is water, the solvate may be referred to as a hydrate, such as a monohydrate, a dihydrate, or a trihydrate. All solvated and non-solvated forms of the compounds described herein are within the scope of the present invention.

[0101] General chemical terms used herein have their usual meanings.

[0102] Standard abbreviations for chemical groups are well known in the art and their usual meanings are used, for example, Me = methyl, Et = ethyl, Bu = butyl, t-Bu = tert-butyl, Ph = phenyl, Bn = benzyl, Ac = acetyl, Boc = tert-butyloxycarbonyl, Fmoc = 9-fluorenylmethoxycarbonyl, Tf = trifluoromethanesulfonate, OMOM = methoxymethyl ether, OMEM = methoxyethoxymethyl ether, OBOM = benzyloxymethyl ether, OTBDMS = tert-butyldimethylsilyl ether, DPPA = diphenylphosphoryl azide, NBS = N- Bromosuccinimide, NIS = N-iodosuccinimide, OPMB = 4-methoxybenzyl ether, EDCI = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, HOBt = hydroxybenzotriazole, OSEM = [2-(trimethylsilyl)ethoxy]methyl ether, Alloc = allyloxycarbonyl, Cbz = benzyloxycarbonyl, Teoc = (2-(trimethylsilyl)ethoxycarbonyl, TEMPO = 2,2,6,6-tetramethyl-1-piperidinyloxy, Troc = 2,2,2-trichloroethylcarbonyl, etc.

[0103] Unless otherwise stated, these abbreviations apply to all examples below.

[0104] Unless otherwise indicated, the term "alkyl" as used herein alone or in combination with other terms refers to a straight or branched saturated or unsaturated acyclic hydrocarbon radical. In some embodiments, the alkyl group has 1 to 15, 1 to 13, 1 to 11, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 12, 2 to 9, 2 to 8, 2 to 6, 2 to 4, 3 to 9, 3 to 8, 4 to 9, 4 to 15, 6 to 15, 8 to 15, 10 to 15, or 1, or 2, or 3 carbon atoms. In some embodiments, the alkyl group is saturated. Examples of such alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl, -n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, -neopentyl, 2-methylbutyl, -isohexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl In some embodiments, the alkyl group is unsaturated. Examples of such alkyl groups include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, -ethynyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1-butynyl, and the like. The prefix "C x -C y ”, wherein x and y are each integers, when used in combination with the term “alkyl”, refers to the number of carbon atoms in the alkyl group. In some embodiments, the “alkyl” group may be substituted with one or more optional substituents as described herein.

[0105] Unless otherwise indicated, the term "aryl" as used herein alone or in combination with other terms refers to a cyclic aromatic hydrocarbon group that does not contain any ring heteroatoms. Aryl includes monocyclic, bicyclic and tricyclic ring systems. Examples of aryl include, but are not limited to, phenyl, azulenyl, heptenyl, biphenyl, fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl, pentalenyl and naphthyl. In some embodiments, aryl has 6 to 20, 6 to 14, 6 to 12 or 6 to 10 carbon atoms in the ring. In some embodiments, aryl is phenyl or naphthyl. Aryl includes aromatic carbocyclic fused ring systems. Examples include, but are not limited to, indanyl and tetrahydronaphthyl. The prefix "C x -C y ”, wherein x and y are each integers, when used in combination with the term “aryl”, refers to the number of ring carbon atoms in the aryl group. In some embodiments, the “aryl” group may be substituted with one or more optional substituents as described herein.

[0106] Unless otherwise indicated, the term "heteroaryl" as used herein alone or in combination with other terms refers to an aromatic ring system containing 5 or more ring atoms, one or more of which are heteroatoms. In some embodiments, the heteroatom is nitrogen, oxygen or sulfur. Heteroaryl is a variety of heterocyclic radicals with aromatic electronic structures. In some embodiments, heteroaryl includes mono-, bi- and tricyclic ring systems having 5 to 20, 5 to 16, 5 to 14, 5 to 12, 5 to 10, 5 to 8 or 5 to 6 ring atoms. Heteroaryl includes, but is not limited to, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridinyl), indazolyl, benzimidazolyl, pyrazolopyridinyl, triazolopyridinyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, imidazopyridinyl, imidazolyl, isoxazolopyridinylxanthenyl, amidinyl (guaninyl), quinolyl, isoquinolyl, tetrahydroquinolyl, quinoxalinyl and quinazolinyl. Heteroaryl includes fused ring systems in which all rings are aromatic, such as indolyl, and fused ring systems in which only one ring is aromatic, such as 2,3-dihydroindolinyl. When used in combination with the term "heteroaryl," the prefix "xy-membered" refers to the number of ring atoms in the heteroaryl, wherein x and y are each an integer. In some embodiments, a "heteroaryl" group may be substituted with one or more optional substituents as described herein.

[0107] The term "halo" or "halogen" is intended to include F, Cl, Br and I.

[0108] The term "heteroatom" is intended to include oxygen, nitrogen, sulfur, selenium, or phosphorus. In some embodiments, the heteroatom is selected from oxygen, nitrogen, and sulfur.

[0109] As used herein, the term "substituted" is intended to represent that one or more hydrogen atoms in the group shown are replaced by one or more independently selected suitable substituents, provided that the normal valence of each atom connected to the substituent is not exceeded, and the substitution produces a stable compound. In various embodiments, suitable optional substituents in the compounds described herein include, but are not limited to, halogen, -(C1-C6) alkyl, -CO-(C1-C6) alkyl, -CONH(C1-C6) alkyl, -CON(C1-C6) alkyl(C1-C6) alkyl, -OH, -O-(C1-C6) alkyl, -NH2, -NH(C1-C6) alkyl, -N(C1-C6) alkyl(C1-C6) alkyl and -NHC(O)-(C1-C6) alkyl, -NMe2, -NHMe, -NH2, -OH, morpholine and -SH. Unless otherwise indicated, the term "stable" herein refers to a compound having stability sufficient to allow manufacture and maintaining its integrity for a period of time sufficient for purposes described herein.

[0110] The term "antibody" as used herein refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule containing an antigen binding site that immunospecifically binds to an antigen or a portion thereof of a target of interest, the target including but not limited to cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The term "antibody" includes complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed by at least two complete antibodies, and antibody fragments, as long as they exhibit the desired biological activity. Structurally, antibodies are generally Y-shaped proteins consisting of four amino acid chains, two heavy chains, and two light chains. Each antibody has two main regions: a variable region and a constant region. The variable region at the end of the Y arm binds to and interacts with the target antigen. The variable region includes a complementary determining region (CDR), which recognizes and binds to a specific binding site on a specific antigen. The constant region at the tail of the Y is recognized and interacts with the immune system (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). The target antigen usually has many binding sites, also called epitopes, which are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, one antigen can have more than one corresponding antibody.

[0111] As used herein, the term "reactive moiety" means a functional group that can react with a second functional group under relatively mild conditions without the need for pre-functionalization. The reaction between the reactive moiety and the second functional group only requires the application of heat, pressure, catalyst, acid and / or base. Examples of reactive moieties include carbamoyl halides, acyl halides, active esters, anhydrides, α-haloacetyl groups, α-haloacetamides, maleimides, isocyanates, isothiocyanates, disulfides, thiols, hydrazines, hydrazides, sulfonyl chlorides, aldehydes, methyl ketones, vinyl sulfones, halomethyl groups, and methyl sulfonates. The second functional group is typically a linker or a ligand.

[0112] As used herein, the term "ligand" means a ligand that binds or reactively associates or complexes with a receptor, antigen, or other receptive moiety associated with a given target cell population. Ligands are typically bound by intermolecular forces such as hydrogen bonds, ionic bonds, and van der Waals forces. Ligands typically deliver payloads to target cell populations to which the ligand binds by binding to cells expressing specific antigens or cell surface receptors. For example, a ligand can bind to a cell surface receptor or surface protein that is overexpressed in diseased cells such as cancer cells.

[0113] Examples of ligands for use in the present invention include antibodies (which may be monoclonal, bispecific, chimeric or humanized antibodies, or antibody fragments of any of these), growth factors, hormones, cell / tissue targeting peptides, aptamers and small molecules such as imaging agents, cofactors or cytokines.

[0114] Unless otherwise indicated, the term "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of a compound described herein. For example, the compound described herein may contain an amino group and thus may form an acid addition salt with the amino group. Examples of salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, sugarates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pamoates (i.e., 1,1′-methylenebis(2-hydroxy-3-naphthoic acid)). Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as acetate ions, succinate ions, or other counterions. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt can have more than one charged atom in its structure. Cases where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.

[0115] Unless otherwise indicated, the term "pharmaceutically acceptable solvate" or "solvate" as used herein refers to the association of one or more solvent molecules with a compound described herein. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0116] 5.2 Compounds of the present invention

[0117] The compounds of the present invention provide novel DNA-alkylation units with highly desirable properties that can be used to synthesize a wide range of highly potent ADCs using conventional techniques.

[0118] Specifically, the compounds of the present invention comprise:

[0119] (a) an N-protected 2-methylbenzoxaxole DNA alkylation unit (wherein Y is an N-protecting group) and

[0120] (b) 2-Methylbenzoxazole DNA alkylating unit linked to the DNA minor groove binding unit.

[0121] The N-protected 2-methylbenzoxazole DNA alkylating units of the present invention can be easily linked to DNA minor groove binding units to provide highly cytotoxic duocarmycin analogs. They can also be conjugated with other chemical moieties to form new biologically active compounds, including ADCs.

[0122] The compound of the present invention wherein the DNA alkylation subunit is linked to the DNA minor groove binding unit can be converted into an ADC by connecting an antibody or other ligand via a linker. The linker can be directly linked to the DNA alkylation subunit via a hydroxyl or amine group X, or indirectly linked to the DNA alkylation subunit via the DNA minor groove binding unit at the Y position.

[0123] In a first aspect, the present invention provides a compound of formula I or a pharmaceutically acceptable salt, hydrate or solvate thereof

[0124]

[0125] in:

[0126] LG is a leaving group;

[0127] X is a group selected from hydroxy, protected hydroxy, prodrug hydroxy, amino and protected amino; wherein amino is -NH2, or -NH(C1-C6)alkyl; and

[0128] Y is an N-protecting group.

[0129] The compounds of formula I can be used to synthesize compounds of formula II and III, as well as for ADCs and their precursors.

[0130] The present invention also relates to compounds of formula Ia,

[0131]

[0132] wherein LG, X and Y are as defined in Formula I.

[0133] In a second aspect, the present invention provides a compound of formula II or a pharmaceutically acceptable salt, hydrate or solvate thereof

[0134]

[0135] in:

[0136] LG is a leaving group;

[0137] X is a group selected from hydroxy, protected hydroxy, prodrug hydroxy, amino and protected amino; wherein amino is -NH2, or -NH(C1-C6)alkyl; and

[0138] DB is the DNA minor groove binding unit.

[0139] The present invention also relates to compounds of formula IIa,

[0140]

[0141] wherein LG, X and DB are as defined for Formula II.

[0142] In one embodiment, DB is optionally substituted aryl or optionally substituted heteroaryl attached directly or via an alkenyl group.

[0143] In one embodiment, DB is an optionally substituted indole, azaindole, benzofuran, benzene, pyridine, pyrimidine, pyrrole, imidazole, thiophene, thiazole, oxazole, pyrazole, triazole, pyrazine, or pyridazinyl.

[0144] As described below, there is extensive information available in the art regarding the design and synthesis of DNA minor groove binding units and methods for establishing their DNA binding mode and strength. Thus, one skilled in the art can readily determine whether a particular chemical entity constitutes a DNA minor groove binding unit.

[0145] In one embodiment DB comprises a reactive moiety RM that is compatible with a complementary reactive site on a linker group or a component of a linker group, wherein the linker group is attached to, or is suitable for attachment to, a ligand, such as an antibody.

[0146] In one embodiment, RM is a reactive moiety selected from the group consisting of: azide, alkyne, carbamoyl halide, acyl halide, active ester, anhydride, α-haloacetyl, α-haloacetamide, maleimide, isocyanate, isothiocyanate, disulfide, thiol, hydrazine, hydrazide, sulfonyl chloride, aldehyde, methyl ketone, vinyl sulfone, halomethyl and methyl sulfonate.

[0147] In some embodiments of the invention, such as compounds of Formula II, the 2-methylbenzoxazole DNA alkylating subunit binds to the DNA minor groove binding unit (DB). Suitable DNA binding moieties for use in the invention have an affinity for binding in the minor groove of double-stranded DNA. A wealth of information is available on methods for designing and synthesizing these molecules and determining their DNA binding mode and strength, for example, as described in Drug-Nucleic Acid Interactions, edited by JB Haires and MJ Waring (Methods in Enzymology Vol 340, Academic Press, 2001); Molecular Recognition of DNA by Small Molecules (Bioorg. Med. Chem. (2001) 9, 2215); Structure-Based DNA-Targeting Strategies with Small Molecule Ligands for Drug Discovery (Med. Res. Rev. (2013) 33, 1119); and A Fluorescent Intercalator Displacement Assay for Establishing DNA Binding Selectivity and Affinity. Affinity) (Chem. Rev. (2004) 37,61).

[0148] The DNA binding moieties used to link to the DNA-alkylating unit are heteroaryl or aryl groups that can be substituted with other functional groups. In general, planar aryl and heterocyclic ring systems have appropriate physicochemical properties for binding within the minor groove, which is usually driven by a combination of H-bonding and van der Waals interactions with the DNA components at the walls and bottom of the groove. Aryl and heteroaryl ring substituents that enhance these interactions increase the strength of binding.

[0149] Binding is further facilitated by linking two or more ring systems together to create longer and more extensive interactions with the minor groove, as long as the overall structure maintains the correct curvature and distortion to match the curvature and distortion of the minor groove into which it binds. This is most advantageously achieved when there is minimal distortion of the small molecule ligand or DNA; that is, when there is a high degree of shape complementarity. The use of amide bonds to link the ring systems is an advantageous motif because the amides themselves can participate in hydrogen bonding interactions with DNA while also providing sufficient flexibility to accommodate the required distortion and curvature.

[0150] Another factor to consider, particularly for DNA minor groove binders of extended length, is maintaining the correct alignment or positioning between the H-bond donor and acceptor on the ligand and on the DNA. In some cases, this can be achieved by varying the nature of the substituents on the aryl or heteroaryl rings or by changing the position of the substituents. A large library of DNA minor groove binding ligands has been constructed and their binding affinities determined (e.g., Total Synthesis of Distamycin A and 2640 Analogs: A Solution-Phase Combinatorial Approach to the Discoveryof New, Bioactive DNA Binding Agents and Development of a Rapid, High-Throughput Screen for Determining Relative DNA Binding Affinity or DNA Binding Sequence Selectivity). Sequence Selectivity), J. Am. Chem. Soc. (2000) 122, 6382), and these libraries have been used to prepare duocarmycin analogs (e.g., Parallel Synthesis and Evaluation of 132(+)-1,2,9,9a-Tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) Analogues of CC-1065 and the Duocarmycins Defining the Contribution of the DNA-Binding Domain), J. Org. Chem. (2001) 66, 6654).

[0151] In specific embodiments of the compounds of Formula II and Ha, the DNA minor groove binding unit comprises a heteroaryl group which may be linked to a second heteroaryl group or aryl group via an amide bond.

[0152] In other embodiments of the compounds of Formula II and Ha, the DNA minor groove binding unit comprises a single aryl or heteroaryl group linked to the DNA alkylating unit through an alkenyl group (-CH=CH-).

[0153] In a third aspect, the present invention provides a compound of formula III or a pharmaceutically acceptable salt, hydrate or solvate thereof

[0154]

[0155] in:

[0156] LG is a leaving group;

[0157] X is a group selected from hydroxy, protected hydroxy, prodrug hydroxy, amino and protected amino; wherein amino is -NH2, or -NH(C1-C6)alkyl; and

[0158] Y is selected from:

[0159] (a) N-protecting group;

[0160] (b)-C(O)-Ar 1

[0161] (c)-C(O)-Ar 1 -NH-C(O)-Ar 2

[0162] (d)-C(O)-Ar 1 -NH-C(O)-CH=CH-Ar 3 ;or

[0163] (e)-C(O)-CH=CH-Ar 3

[0164] Among them, Ar 1 ,Ar 2 and Ar 3 each independently selected from heteroaryl or aryl, wherein each of the heteroaryl or aryl is optionally substituted with one or more of: -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -OH, -O-(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl;

[0165] wherein in each case -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -O-(C1-C6)alkyl, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl are independently optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0166] The present invention also relates to compounds of formula IIIa,

[0167]

[0168] wherein LG, X and DB are as defined for Formula III.

[0169] The following embodiments apply to compounds of Formula I, Ia, II, IIa, III and IIIa.

[0170] The term "leaving group" as used herein refers to a group that leaves a carbon center in a substitution reaction. Such groups are usually stable in anionic form. Examples of leaving groups are well known in the art and include, but are not limited to, halogen groups and sulfonate groups, such as optionally substituted (C1-C6) alkanesulfonates (e.g., mesylate, triflate, and triflate) and optionally substituted benzenesulfonates.

[0171] In one embodiment, LG is selected from the group consisting of chloride, bromide, iodide, and -OSO2R 1 ; where R 1 Selected from (C1-C 10 )alkyl, (C1-C 10 ) heteroalkyl, (C1-C 10 )aryl or (C1-C 10 ) heteroaryl. In one embodiment, LG is a halogen group, preferably chlorine. Scheme 2 below shows the synthesis of DNA alkylating subunits containing different leaving groups.

[0172] In the compounds of the present invention, group X may be a free hydroxyl or free amino group, or may be a hydroxyl or amino group protected by a suitable protecting group (protected hydroxyl or protected amino group, respectively). X may also be a prodrug form of a hydroxyl group (prodrug hydroxyl group).

[0173] The term "prodrug hydroxyl" means a group that is converted in vivo by the action of a biochemical such as an enzyme to provide a free OH group. Conventional procedures for selecting and preparing suitable prodrug hydroxyl groups that can be used are described in "Design of Prodrugs" edited by H. Bundgaard, Elsevier in 1985. Examples are well known in the art and include, but are not limited to, phosphates, carbamates, and glycosides.

[0174] Compounds of the invention wherein X is a prodrug hydroxyl group may be used in ADCs in which the linker will be attached to the DNA minor groove binding unit rather than through X. This will result in a prodrug form of the ADC. One skilled in the art, designing an ADC incorporating a compound of the invention, will be able to select the appropriate position for the tethered antibody component and determine the desirability of a particular prodrug form for the intended application of the ADC.

[0175] As used herein, the term "protected hydroxyl" refers to a hydroxyl that has been protected from undesirable reactions during a synthetic procedure. When the hydroxyl reaction is no longer needed and / or allowed, the protected hydroxyl is easily converted into a free hydroxyl. Hydroxyl protecting groups are described in the protective groups in organic synthesis edited by TW Greene et al. (John Wiley & Sons, 1999). As used herein, the term "protected hydroxyl" also includes groups such as OTf, which contain good leaving groups and can be used to synthesize derivatives in which the OH group is replaced by a substituted group. Examples of hydroxyl protecting groups that can be used for compounds of the present invention include -OBn, -OTf, -OMOM, -OMEM, -OBOM, -OTBDMS, -OPMB, -OSEM.

[0176] The term "protected amino group" as used herein refers to an amino group that has been protected from undesirable reactions during a synthetic procedure. When the amino group is no longer needed and / or allowed to react, the protected amino group is easily converted into a free amino group. In the compounds of the present invention, the amino group at position X is selected from -NH2 and -NH(C1-C6)alkyl. Amino protecting groups are described in Protecting Groups in Organic Synthesis edited by TWGreene et al. (John Wiley & Sons, 1999) and 'Amino Acids-Protecting Groups' edited by Fernando Albericio (with Albert Isidro-Llobet and Mercedes Alvarez) Chemical Reviews 2009 (109) 2455-2504.

[0177] Examples of amino protecting groups that can be used in the compounds of the present invention include, but are not limited to, acyl and acyloxy groups, such as acetyl, chloroacetyl, trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxyacetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, picolinyl, aminocaproyl, benzoyl, methoxy-carbonyl, 9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-carbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2,4-dichloro-benzyloxycarbonyl, etc. Further examples include nitrobenzenesulfonyl (o- or p-nitrobenzenesulfonyl), Bpoc (2-(4-biphenyl)isopropoxycarbonyl) and Dde (1-(4,4-dimethyl-2,6-dioxohexylidene)ethyl).

[0178] In one embodiment, X is selected from the group consisting of: -OH, -OBn, -OTf, -OMOM, -OMEM, -OBOM, -OTBDMS, -OPMB, -OSEM, piperazine-1-carboxylate, wherein the N at position 4 is replaced by (C1-C 10 )alkyl, -OP(O)(OH)2, -OP(O)(OR 2 )2, -NH2, -N=C(Ph)2, -NHZ, NH(C1-C 10 )alkyl and -NZ(C1-C 10 ) alkyl substitution;

[0179] Where R 2 is t-Bu, Bn or allyl; and Z is selected from Boc, COCF3, Fmoc, Alloc, Cbz, Teoc and Troc.

[0180] In one embodiment, X is -OH or -NH2.

[0181] In one embodiment, X is protected or prodrug -OH.

[0182] In one embodiment, X is protected -NH2.

[0183] In one embodiment, X is selected from -OBn, -OTf, -OMOM and -OMEM.

[0184] A method for preparing compounds of the present invention wherein X is hydroxy, protected hydroxy or prodrug hydroxy is shown below in Scheme 2. A method for preparing compounds of the present invention wherein X is amino or protected amino is shown below in Scheme 3.

[0185] The compounds of the present invention wherein Y is an N-protecting group provide a 2-methylbenzoxazole DNA-alkylating unit which can be easily linked to a DNA minor groove binding unit to provide a highly cytotoxic duocarmycin analog.

[0186] The term "N-protecting group" as used herein refers to a group that can be easily removed to provide a free N and protects the N atom from undesired reactions during synthetic procedures. Such protecting groups are described in Protecting Groups in Organic Synthesis edited by TW Greene et al. (John Wiley & Sons, 1999) and in 'Amino Acids-Protecting Groups' edited by Fernando Albericio (with Albert Isidro-Llobet and Mercedes Alvarez) Chemical Reviews 2009 (109) 2455-2504. Examples include, but are not limited to, acyl and acyloxy groups such as acetyl, chloroacetyl, trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxyacetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, picolinyl, aminocaproyl, benzoyl, methoxy-carbonyl, 9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-carbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2,4-dichloro-benzyloxycarbonyl, etc. Further examples include nitrobenzenesulfonyl (o- or p-nitrobenzenesulfonyl), Bpoc (2-(4-biphenyl)isopropoxycarbonyl) and Dde (1-(4,4-dimethyl-2,6-dioxohexylidene)ethyl).

[0187] In one embodiment, Y is an N-protecting group selected from Boc, COCF3, Fmoc, Alloc, Cbz, Teoc and Troc.

[0188] One skilled in the art will be able to select protecting groups and prodrug hydroxyl moieties appropriate to the particular synthetic scheme employed and the desired end product.

[0189] In the compound of formula III, the DNA minor groove binding unit comprises a heteroaryl or aryl group, which may be linked to a second heteroaryl or aryl group via an amide bond or via -NH-C(O)-CH=CH-.

[0190] In other embodiments, the DNA minor groove binding unit comprises a single aryl or heteroaryl group.

[0191] In one embodiment, Ar 1 ,Ar 2 and Ar 3 Independently selected from the group consisting of

[0192]

[0193] in represents the point of attachment to a -C(O) or C(O)-CH=CH- group, and each aryl or heteroaryl group may be substituted at the numbered positions by up to three substituents selected from -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -OH, -O-(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl.

[0194] As will be appreciated by those skilled in the art, 5-azaindole, 6-azaindole, 7-azaindole, imidazole, thiazole, oxazole and pyrazole groups may be substituted with up to two groups and triazole with only one.

[0195] When Ar 1 Connected to Ar via NH-C(O) 2 When, or when Ar 1 Connected to Ar via -NH-C(O)-CH=CH 3 When Ar 1 The connection point on can be any numbered position. As will be appreciated by those skilled in the art, such connection will reduce Ar 1 One of the possible number of substituents.

[0196] The substituents -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -O-(C1-C6)alkyl, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl may independently be optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH in each case.

[0197] In one embodiment, Ar 1 In one embodiment, the heteroaryl group is indole, azaindole, benzofuran or benzothienyl, which is linked to the DNA alkylation unit via -C(O) or C(O)-CH=CH- at the 2-position of the heteroaryl group.

[0198] In one embodiment, Ar 1 Connected to Ar via NH-C(O) 2 or Ar 1 Connected to Ar via -NH-C(O)-CH=CH 3In one embodiment, Ar 1 The point of attachment is at the 5-position of the indole, azaindole, benzofuran or benzothiophenyl group.

[0199] In one embodiment, Ar 2 Selected from the group consisting of indole, azaindole, benzene, benzofuran, pyridine, pyrimidine, pyrrole, imidazole, thiophene, thiazole, oxazole, pyrazole, triazole, pyrazine or pyridazine.

[0200] In one embodiment, Ar 2 Selected from indole, azaindole, benzene, benzofuran, pyrrole or imidazole.

[0201] In one embodiment, Ar 3 is selected from benzene, pyridine, pyrimidine and pyridazine. In one embodiment, Ar 3 is benzene or pyridine, preferably benzene.

[0202] In one embodiment Y is -C(O)-Ar 1 , where Ar 1 is heteroaryl or aryl, which is optionally substituted by one or more of the following: -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -OH, -O-(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl or -NHC(O)-(C1-C6)alkyl,

[0203] wherein in each case -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -O-(C1-C6)alkyl, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl are independently optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0204] In one embodiment, Ar 1 is substituted at one position on the heteroaryl ring or the aryl ring.

[0205] In one embodiment, Ar 1 It is a heteroaryl group.

[0206] In one embodiment, the heteroaryl group is indole, azaindole, benzofuran or benzothienyl, which is linked to the DNA alkylating unit via -C(O) or C(O)-CH=CH- at the 2-position of the heteroaryl group.

[0207] In one embodiment, Ar 1 for

[0208]

[0209] wherein A is NH, O or S, and

[0210] R 10 , R 11 and R 12 independently selected from H, -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -OH, -O-(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl,

[0211] wherein in each case -(C1-C6)alkyl, -O-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NH-C(O)-(C1-C6)alkyl are independently optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0212] In one embodiment, A is NH.

[0213] In one embodiment, R 10 , R 11 and R 12 It is OMe.

[0214] In one embodiment, R 10 is -O-(C1-C6)alkyl, which is optionally substituted by one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH; R 11 and R 12 All are H.

[0215] In one embodiment, R 10 is -NHC(O)-(C1-C6)alkyl, which is optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH; R 11 and R 12 All are H.

[0216] In one embodiment, R 10 -NH-C(O)-Ar 2 , where Ar 2is optionally substituted indole, azaindole, benzene, benzofuran, pyrrole or imidazolyl; R 11 and R 12 All are H.

[0217] In one embodiment, R 10 -NH-C(O)-Ar 2 , where Ar 2 is an optionally substituted indolyl group; R 11 and R 12 All are H. In one embodiment, the indole group is substituted with -O-(C1-C6)alkyl, optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0218] In one embodiment, R 10 -NH-C(O)-Ar 2 , where Ar 2 is an optionally substituted phenyl group; R 11 and R 12 All are H. In one embodiment, the phenyl group is substituted with -OH, -NH2 or -O-(C1-C6)alkyl, wherein -O-(C1-C6)alkyl is optionally substituted with -NMe2.

[0219] In one embodiment, R 10 is -NH-C(O)-CH=CH-Ar 3 , where Ar 3 is optionally substituted benzene, pyrimidinyl or pyrrolyl; R 11 and R 12 All are H. In one embodiment, benzene, pyrimidinyl or pyrrolyl is substituted by -O-(C1-C6)alkyl, -NH2 or -NHC(O)-(C1-C6)alkyl, wherein -O-(C1-C6)alkyl is optionally substituted by morpholine.

[0220] In some embodiments, Ar 1 Select from the group consisting of:

[0221]

[0222] Where R 10 , R 11 and R 12(when present) are independently selected from H, -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -OH, -O-(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl,

[0223] wherein in each case -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -O-(C1-C6)alkyl, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl are independently optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0224] In one embodiment, R 10 , R 11 and R 12 It is OMe.

[0225] In one embodiment, R 10 , R 11 and R 12 One of them is -O-(C1-C6)alkyl, which is optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0226] In one embodiment, R 10 , R 11 and R 12 One of them is -NHC(O)-(C1-C6)alkyl, which is optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0227] In one embodiment, R 10 , R 11 and R 12 One of them is -NH-C(O)-Ar 2 , where Ar 2 is optionally substituted indole, azaindole, benzene, benzofuran, pyrrole or imidazolyl.

[0228] In one embodiment, R 10 , R 11 and R 12 One of them is -NH-C(O)-Ar 2 , where Ar 2In one embodiment, the indolyl group is substituted with -O-(C1-C6)alkyl, which is optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0229] In one embodiment, R 10 , R 11 and R 12 One of them is -NH-C(O)-Ar 2 , where Ar 2 In one embodiment, phenyl is substituted with -OH, -NH2 or -O-(C1-C6)alkyl, wherein -O-(C1-C6)alkyl is optionally substituted with -NMe2.

[0230] In one embodiment, R 10 , R 11 and R 12 One of them is -NH-C(O)-CH=CH-Ar 3 , where Ar 3 is optionally substituted benzene, pyrimidinyl or pyrrolyl. In one embodiment, benzene, pyrimidinyl or pyrrolyl is substituted by -O-(C1-C6)alkyl, -NH2 or -NHC(O)-(C1-C6)alkyl, wherein -O-(C1-C6)alkyl is optionally substituted by morpholine.

[0231] In one embodiment Y is -C(O)-Ar 1 -NH-C(O)-Ar 2 , where Ar 1 and Ar 2 is heteroaryl or aryl, which is optionally substituted by one or more of the following: -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -OH, -O-(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl or -NHC(O)-(C1-C6)alkyl,

[0232] wherein in each case -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -O-(C1-C6)alkyl, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl are independently optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0233] In one embodiment, Ar 1 is a heteroaryl group and Ar 2 is a heteroaryl group or an aryl group.

[0234] In one embodiment, Ar 1 is indole, azaindole, benzofuran or benzothienyl, which is linked to the DNA alkylating unit at the 2-position of the heteroaryl group.

[0235] In one embodiment, Ar 2 Selected from the group consisting of indole, azaindole, benzene, benzofuran, pyridine, pyrimidine, pyrrole, imidazole, thiophene, thiazole, oxazole, pyrazole, triazole, pyrazine or pyridazine.

[0236] In one embodiment, Ar 2 is selected from indole, azaindole, benzene, benzofuran, pyrrole or imidazolyl.

[0237] In one embodiment, Ar 1 is indole, azaindole, benzofuran or benzothienyl, which is linked to the DNA alkylation unit at the 2-position of the heteroaryl group, and Ar 2 is selected from indole, azaindole, benzene, benzofuran, pyridine, pyrimidine, pyrrole, imidazole, thiophene, thiazole, oxazole, pyrazole, triazole, pyrazine or pyridazine.

[0238] In one embodiment, Ar 1 The point of attachment is at the 5-position of the indole, azaindole, benzofuran or benzothiophenyl group.

[0239] In one embodiment Y is -C(O)-Ar 1 -NH-C(O)-CH=CH-Ar 3 , where Ar 2 and Ar 3 is heteroaryl or aryl, which is optionally substituted by one or more of the following: -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -OH, -O-(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl or -NHC(O)-(C1-C6)alkyl,

[0240] wherein in each case -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -O-(C1-C6)alkyl, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl are independently optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0241] In one embodiment, Ar 1 is a heteroaryl group and Ar 3 is a heteroaryl group or an aryl group.

[0242] In one embodiment, Ar 1 is indole, azaindole, benzofuran or benzothienyl, which is linked to the DNA alkylating unit at the 2-position of the heteroaryl group.

[0243] In one embodiment, Ar 3 is selected from benzene, pyridine, pyrimidine and pyridazine. In one embodiment, Ar 3 is benzene or pyridine, preferably benzene.

[0244] In one embodiment, Ar 1 is indole, azaindole, benzofuran or benzothienyl, which is linked to the DNA alkylation unit at the 2-position of the heteroaryl group, and Ar 3 Selected from benzene, pyridine, pyrimidine and pyridazine.

[0245] In one embodiment, the point of attachment on Ar1 is at the 5 position of the indole, azaindole, benzofuran or benzothiophenyl group.

[0246] In one embodiment Y is -C(O)-CH=CH-Ar 3 , where Ar 3 is heteroaryl or aryl, which is optionally substituted by one or more of the following: -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -OH, -O-(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl or -NHC(O)-(C1-C6)alkyl,

[0247] wherein in each case -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -O-(C1-C6)alkyl, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl are independently optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0248] In one embodiment, Ar 3 is selected from benzene, pyridine, pyrimidine and pyridazine. In one embodiment, Ar 3 It is benzene or pyridine, preferably benzene.

[0249] In one embodiment, Ar 3 is phenyl optionally substituted by -O-(C1-C6)alkyl, which is optionally substituted by one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

[0250] Compounds of formula I, II and III are ring-opened precursors of the compound of formula IV, which are believed to be active agents in vivo.

[0251] In a fourth aspect, the present invention provides a compound of formula IV or a pharmaceutically acceptable salt, hydrate or solvate thereof

[0252]

[0253] wherein V is Y or DB and X, Y and DB have the same meanings as defined for the compounds of formula I, II and III and X' is X minus H.

[0254] Compounds of formula IV can be formed by in vitro or in vivo rearrangement and concomitant elimination of H-LG from the corresponding ring-opening compounds of formula I, II and III. Unless the context dictates otherwise, all embodiments of the invention described herein for compounds of formula I, II or III, or any portion thereof, are also expressly contemplated as being part of the aspects of the invention relating to compounds of formula IV.

[0255] The compounds of Formula I, II and III contain a leaving group (LG) which promotes cyclopropyl ring formation under physiological conditions to form the compound of Formula IV.

[0256] In one aspect, the invention provides a compound selected from any one of compounds 49, 18, 50, 51, 23, 52, 53, 57, 58, 59, 63, 64, 65, 66, 70, 74, 79, 80, 81, 82, 83, 24, 26, 85, 87, 88, 89, 90, 91 and 93.

[0257] In one embodiment, the invention provides a compound selected from any one of compounds 49, 18, 50, 51, 23, 52, 57, 53, 58, 59, 63, 64, 65, 66, 70, 74, 79, 80, 81, 82, 83, 24, 85, 88 and 90.

[0258] In one embodiment, the present invention provides a compound selected from the group consisting of:

[0259]

[0260] In one embodiment, the present invention provides a compound selected from the group consisting of:

[0261]

[0262]

[0263] In one embodiment, the present invention provides a compound selected from the group consisting of:

[0264]

[0265]

[0266] 5.3 Synthesis of the compounds of the present invention

[0267] The compounds of the present invention can be prepared using the methods and procedures described herein, or methods and procedures analogous thereto. Other suitable methods for preparing the compounds of the present invention will be apparent to those skilled in the art.

[0268] It should be understood that where typical or preferred process conditions (e.g., reaction temperatures, times, molar ratios of reactants, solvents, pressures, etc.) are indicated, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the specific reactants used.

[0269] The starting materials for the processes and reactions are either commercially available or can be prepared by known procedures or modifications thereof, such as those described in standard reference texts, such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley and Sons, 1991), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, Fourth Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0270] The various starting materials, intermediates and compounds can be isolated and purified using conventional techniques such as precipitation, filtration, crystallization, evaporation, distillation and chromatography as appropriate. The characterization of the compounds can be carried out using conventional methods, for example by melting point, mass spectrometry, nuclear magnetic resonance and various other spectroscopic analyses.

[0271] Conventional protecting groups may be necessary to prevent some functional groups from experiencing undesirable reactions. The need for protection and deprotection and the selection of appropriate protecting groups are easily determined by those skilled in the art. Suitable protecting groups for various functional groups and suitable conditions for protecting and deprotecting specific functional groups are well known in the art (see, for example, TW Greene and GM Wuts, Protecting Groups in Organic Synthesis, 3rd edition, Wiley, New York, 1999).

[0272] The individual enantiomers of the effective load containing 2-methylbenzoxazole alkylated subunits can be prepared using the chiral HPLC separation of suitable intermediates (e.g., compounds 49 or 18). Suitable columns include those such as CBI (J.Am.Chem.Soc. (1994) 116, 7996), CTI (Bioorg.Med.Chem.Lett. (2009) 19, 6962), iso-DSA (J.Am.Chem.Soc. (2009) 131, 1187), CImI (Bioorg.Med.Chem. (2016) 24, 4779) for resolving related alkylated subunits. The intermediates of the separation can be converted into effective loads and drug-linker conjugates by methods similar to those described for racemates.

[0273] A general method for preparing 2-methylbenzoxazolidinylating subunits where X = protected OH is shown in Scheme 2. Compounds 5 and 7 in this scheme are readily converted to 2-methylbenzoxazolidinylating subunits where X = free OH and X = prodrug OH.

[0274]

[0275] Solution 2

[0276] In this scheme, 3,4-dihydroxy-5-nitrobenzoic acid esters 1 are used as starting materials. These can be prepared, for example, by oxidation of 3,4-dihydroxy-5-nitrobenzaldehyde to the corresponding acid followed by esterification with alcohol ROH. Alternatively, 1 can be prepared by nitration of 4-hydroxy-3-methoxybenzoic acid followed by dealkylation of the methoxy group using reagents such as HBr or BBr3 followed by esterification with alcohol ROH.

[0277] The nitro group of 1 is reduced to the corresponding amine by exposure to appropriate conditions (e.g., hydrogenation over a Pd or Pt catalyst, or exposure to an Fe(III) salt under acidic conditions), and the product is treated with a trialkyl orthoacetate (e.g., trimethyl orthoacetate) under acidic conditions to induce cyclization to the substituted 2-methylbenzoxazole 2.

[0278] At this point in the synthesis, various X=protected -OH can be introduced. For example, a benzyl protecting group can be introduced by reacting 2 with benzyl bromide or benzyl chloride under alkaline conditions to give 3, where X=OBn. Similarly, a MOM protecting group can be introduced by reacting with MOMCl; a MEM protecting group can be introduced by reacting with MEMCl; a BOM protecting group can be introduced by reacting with BOMCl; a TBDMS protecting group can be introduced by reacting with TBDMSCl; a PMB protecting group can be introduced by reacting with PMBCl; and a SEM protecting group can be introduced by reacting with SEMCl. For different protecting groups, different reaction conditions (e.g., base, solvent, temperature, chloride or bromide reagent, additives such as iodide salts) can be appropriately selected to optimize the yield of 3.

[0279] The ester of 3 is then hydrolyzed under standard conditions to give the corresponding acid, which is converted to the NHY group of 4. Where Y represents a carbamate protecting group, the second of these steps can be conveniently performed in a single pot using a diphenylphosphoryl azide (DPPA) reagent in combination with an organic base such as trimethylamine and a suitable alcohol. For example, DPPA and t-BuOH will give 4, where Y = Boc. Similarly, the use of benzyl alcohol will give Y = Cbz, the use of 2-(trimethylsilyl)ethanol will give Y = Teoc, and the use of 2,2,2-(trichloro)ethanol will give Y = Troc. Alternatively, the intermediate isocyanate can be reacted with water rather than an alcohol to produce the corresponding unprotected amine. This can then be converted to a protected form under standard conditions, for example reaction with trifluoroacetic anhydride will give Y = COCF3, while reaction with benzyl chloroformate will give Y = Cbz.

[0280] Compound 4 can be selectively halogenated at the 4-position by reaction with a suitable reagent, such as NBS to introduce bromide at the 4-position and NIS to introduce iodide at the 4-position. These reactions are best performed with a single equivalent of the halogenating agent to minimize dihalogenation at the 4- and 6-positions. To prepare the alkylated subunit whose leaving group is chloro, i.e., 5, the halogenated intermediate is then reacted with 1,3-dichloropropene in the presence of a suitable base such as NaH or K2CO3, which serves to deprotonate the NHY group and direct the chloroallylation to this position. The intermediate is then treated with a suitable reagent such as tributyltin hydride or tris(trimethylsilyl)silane to abstract the halogen atom and initiate a free radical-mediated cyclization at the pendant chloroallyl group, which generates the product 5.

[0281] Alkylated subunits where the leaving group is a halide or sulfonate can be prepared via intermediate 6. Compound 6 can be prepared from 4 by modifying the above procedure. Allyl bromide is used instead of 1,3-dichloropropylene, and a free radical-mediated cyclization is performed in the presence of a spin trap TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy). This produces a product containing an N-O bond, which is cleaved by exposure to Zn and an acid such as acetic acid to produce 6. The primary alcohol of 6 is converted to a LG=halide or sulfonate by using standard reagents, for example, bromine and triphenylphosphine can be used to prepare LG=Br, while methanesulfonyl chloride and triethylamine can be used to prepare LG=OSO2Me.

[0282] The protecting groups of X and Y can be selectively removed and replaced at the stage of compounds 5 and 7. This may be a preferred method of introduction for several Y protecting groups that may be less stable to the conditions used in the synthetic steps after compound 4. For example, Y=COCF3 may be unstable to the basic conditions used in the allylation reaction, but can be introduced by deprotecting 5 or 7 (where, for example, Y=Boc) and then reacting with trifluoroacetic anhydride. Similarly, Y=Fmoc may also be unstable under the same basic conditions, while Y=Alloc may interfere with free radical-mediated cyclization. However, these alternative protecting groups can be very useful in subsequent reactions of 5 and 7 where stability to different reaction conditions is required. This is illustrated below as an example in the synthesis of an alkylated subunit where X=protected NH2.

[0283] For 5 and 7, the group X = protected OH can also be converted to free OH (i.e., compound 8 below), and thereby to the prodrug OH. The latter conversion is carried out via well-established synthetic methods. For example, 8 is reacted with 4-alkylpiperazinecarbonyl chloride to give 5 and 7, where X = piperazine-1-carboxylate, where the N at the 4-position is substituted with an alkyl group. In another example, the free OH of 8 is reacted with the general structure R2NP (OR 2 )2, wherein R is a lower alkyl group, such as ethyl or isopropyl, R 4 is t-Bu, Bn or allyl. The intermediate is then oxidized with a suitable reagent such as hydrogen peroxide or an organic peracid to give 5 and 7, wherein X = OP(O)(OR 2 )2.

[0284] The general method for preparing 2-methylbenzoxazolidine alkylated subunits is shown in Scheme 3, where X = NH2, protected NH2, NHR 1 , or protected NHR 1 , where R 1 It is a (C1–C6) alkyl group.

[0285]

[0286] Solution 3

[0287] Compound 8 containing a free OH group is reacted with trifluoromethanesulfonic anhydride and a suitable base such as triethylamine to give compound 9. This compound is reacted with benzophenone imine using a suitable catalyst such as Pd(OAc)2 and a ligand such as BINAP to achieve amination and produce compound 10. The benzophenone imine is cleaved under acidic conditions to produce compound 11. The required acidic conditions mean that some protecting groups Y are more suitable than others. For example, Y=COCF3 is more suitable than Y=Boc, so a convenient approach requires exchanging Y=Boc for COCF3 at the stage of compound 7. Other combinations of protecting groups are also possible and may provide different synthetic advantages, depending on the specific target compound.

[0288] Compound 11 can be further converted into the alkylated subunit 12, which has an NHR at the X position. 1 (where R 1 is a (C1-C6) alkyl) substituent. For example, 11 can be reacted with acetic anhydride to produce a formylated intermediate, which is reduced using borane to produce 12, where R 1 =Me. Alternatively, 11 can be combined with aldehyde R 1 CHO condensation, where R 1 is (C1-C6) alkyl, to give an imine, which is reduced with a reagent such as sodium borohydride or sodium cyanoborohydride to give 12, wherein R 1 =(C1-C6)alkyl. Compounds 11 and 12 can be converted to protected analogs 13 and 14 using standard conditions as described above, for example reaction with trifluoroacetic anhydride will yield X=NHZ and NZR 1 , where Z = COCF3 and R 1 is (C1–C6)alkyl, and the reaction with fluorenylmethoxycarbonyl chloride will produce X=NHZ and NZR 1 , where Z = Fmoc and R 1 It is a (C1–C6) alkyl group.

[0289] A general method for preparing DNA alkylating agents incorporating a 2-methylbenzoxazolyl alkylating subunit and a DNA minor groove binding moiety (DB) is shown in Scheme 4.

[0290]

[0291] Solution 4

[0292] In the case where 15 contains X═OH, the protecting group Y is removed by an appropriate method (e.g., for Y═Boc, by treatment with HCl), and the resulting intermediate is ligated to the DNA minor groove binding unit Ar 1 CO2H or Ar 3CH=CHCO2H is reacted in the presence of a suitable amide coupling agent such as EDCI or HOBt. Other activated forms of DNA minor groove binding units, such as acyl chlorides, can also be used. This method directly produces 17 where X=OH.

[0293] In 15, X = protected OH or protected NH2 or protected NHR 1 In the case of , the synthesis proceeds in 2 steps via intermediate 16. It will be clear to those skilled in the art that the protecting groups in X and Y should be appropriately selected to allow Y to be deprotected in the presence of the protecting group used in X. For example, if Y is Boc, then X should not be NHBoc or NRBoc, or any other protecting group that is too sensitive to acid, such as NHTeoc or NRTeoc. Suitable combinations of orthogonal protecting groups are readily available by following the general scheme shown above. Amide coupling is performed in the same general manner as above to form compound 16, wherein Y = -C(O)Ar 1 or -C(O)-CH=CH-Ar 3 , and 16 is converted to 17 by removal of the protecting group in X. In the case where compound 17 contains X=NH2, then benzophenone imine can be used as a suitable protected precursor, ie, 15 where X=N=C(Ph)2.

[0294] The same approach can be used to prepare payloads where the side chain contains two linked aromatic rings by substituting the appropriate side chain acid, i.e., by using Ar 2 C(O)NHAr 1 CO2H or Ar 3 CH=CHC(O)NHAr 1 CO2H.

[0295] Scheme 5 shows a prophetic example of the synthesis of compounds of the invention wherein X is NH2.

[0296]

[0297] Solution 5

[0298] In the synthesis proposed in Scheme 5, the 2-methylbenzoxazolidinyl subunit containing a free amino group (general structure 22) can be prepared by methods similar to those reported for the ring-opened-CBI compounds (Bioorg. Med. Chem. (2016) 24, 6075). In particular, compound 18 is converted to the triflate 19 using trifluoromethanesulfonic anhydride and triethylamine. Compound 19 is aminized using benzophenone imine and Pd(OAc)2 catalyst and BINAP ligand to give 20. Selective cleavage of the Boc protecting group using HCl in methanol or TFA in dichloromethane and reaction of the resulting intermediate with a suitable side chain acid using EDCI as a coupling agent gives amide 21. Removal of the benzophenone protecting group using aqueous acetic acid gives the desired product 22. This method can also be applied to the R and S enantiomers of 18 to give the corresponding R and S enantiomers of 22.

[0299] Many DNA minor groove binding monomers with suitable substituents 1 CO2H or Ar 3 CH=CHCO2H is a commercially available compound or can be easily prepared from a commercially available compound by simple functional group changes, such as esters and nitriles can be hydrolyzed to carboxylic acids, nitro substituents can be reduced to amino substituents, which can be further alkylated or acylated, and halide substituents can undergo a series of metal-mediated reactions and / or displacement reactions to obtain various desired substituents. In addition, many other aryl and heteroaryl compounds for preparing suitable DNA minor groove binding unit acids are known compounds or can be prepared by modification of known synthetic methods. Many of these methods are described in "Comprehensive Heterocyclic Chemistry III", edited by AR Katritzky, CA Ramsden, EF V Scriven and R J K Taylor, Elsevier, 2008. For example, the synthesis of indoles is reviewed in Section 3.03 (Vol 3, pp 269-351), the synthesis of azaindoles is reviewed in Section 10.06 (Vol 10, pp 263-338), the synthesis of benzofurans is reviewed in Section 3.07 (Vol 3, pp 497-569), and the synthesis of benzothiophenes is reviewed in Section 3.11 (Vol 3, pp 834-930), all of which are incorporated herein by reference. There are many other reviews of the synthesis of substituted heteroaryl compounds, for example, indole synthesis is reviewed in the following publications: Chem. Rev. (2006) 106, 2875; Chem. Rev. (2005) 105, 2873; Perkin Trans 1 (2000), 1045.

[0300] More specifically, for use as DNA minor groove binding monomers, methods are known to produce heteroaryl compounds with a carboxylate substituent at the desired 2-position. Many of these are included in the reviews cited above, but specific examples are also given here: for indole-2-carboxylates Org. Lett. (2016) 18, 3586; Org. Lett. (2004) 6, 2953; for azaindole-2-carboxylates WO 2009 / 030725; for benzofuran-2-carboxylates Org. Lett. (2013) 15, 3876; J. Chem. Soc., Perkin Trans. 2 (1998) 1063; and for benzothiophene-2-carboxylates Org. Lett. (2013) 15, 3876; Org. Lett. (2012) 14, 5334.

[0301] Many of these methods have been used to synthesize specific DNA minor groove binding monomer acids for the preparation of duocarmycin analogs. For example, the synthesis of 72 different substituted indole-2-carboxylates and their coupling with the ring-opening-CBI alkylation subunit is described in Bioorg. Med. Chem. (2003) 11, 3815. Other examples of a series of substituted indole-2-carboxylates for the preparation of duocarmycin analogs are described in J. Med. Chem. (2017) 60, 5834; Chem Med Chem (2014) 9, 2193; Eur. J. Org. Chem. (2006) 2314; and J. Am. Chem. Soc. (1997) 119, 4977; while similar uses of various substituted cinnamic acids are described in J. Med. Chem. (1997) 40, 972. It has also been reported that Ar 1 Connected to aryl or heteroaryl Ar 3 The synthesis of DNA minor groove binding monomer acids of the type described is described, for example, in WO 2011 / 133039; J. Med. Chem. (2003) 46, 634; and J. Org. Chem. (2001) 66, 6654 (which describes the synthesis of such side chains 132 that combine ring systems such as indole, benzofuran, benzothiophene, pyrrole, thiophene, imidazole and thiazole).

[0302] Those skilled in the art will recognize that Ar 1 with Ar 2 or Ar 3 Many other side chains can be attached by using standard methods and coupling reagents on the Ar containing amino substituents. 1 Unit and Ar containing carboxylic acid substituent 2 or Ar 3The amide bond is formed between the units and is prepared using appropriate protecting groups suitable for the other substituents involved in the specific embodiment. Therefore, there is a lot of information and precedents describing the preparation of acids Ar that react with DNA alkylating units in the synthesis of the compounds of the present invention. 1 CO2H or Ar 3 CH=CHCO2H or Ar 2 CONHAr 1 CO2H or Ar 3 CH=CHCONHAr 1 General and specific methods for CO2H.

[0303] DNA alkylating agents incorporating 2-methylbenzoxazolidine alkylated subunits and minor groove binding side chains can be used as the effective load of ADC. To this end, a linker must be used to connect the effective load to the antibody. The linker can be connected to the effective load in several different ways. For example, the linker can be connected at the X position, which can be via a carbamate (-OC (O) NHR or -OC (O) NR2 or -NHC (O) OR or -NRC (O) OR ') or via an ether (-OR) functional group. These types of linkers need to be fragmented after ADC metabolism to release X = OH or NH2 or NHR, and one type of connection is called a 'traceless' linker. Several examples of suitable linker types are known, which are usually incorporated into self-consumable spacers, such as p-aminobenzyl ether or p-aminobenzyl carbamate, which can be further substituted in a manner that affects its cleavage rate, or connected to additional cleavable spacers, such as N, N-dialkyl-1,2-diaminoethane.

[0304] An illustrative example of the synthesis of a representative drug-linker compound comprising a traceless linker to the X position is shown below.

[0305] Scheme 6 outlines the preparation of compounds of the invention wherein the linker is attached via a phenolic carbamate.

[0306]

[0307] Solution 6

[0308] In this scheme, compound 23 is reacted with 4-nitrophenyl chloroformate, and the product is then reacted with mono-Boc-protected N,N'-dimethyl-1,2-diaminoethane to give 24. The Boc group of 24 is deprotected using TFA to give the corresponding TFA salt. Activated maleimide-valine-citrulline-PABA compound 25 is prepared as described (Mol. Pharm. (2015) 12, 1813). The TFA salt and 25 react under slightly alkaline conditions to give drug-linker 26.

[0309] Scheme 7 outlines the preparation of compounds of the invention wherein the linker is attached via a phenol ether.

[0310]

[0311] Solution 7

[0312] In this scheme, phenol 18 is deprotonated using a suitable base such as MeLi and then reacted with the known valine-alanine-PAB-bromide compound 27 (WO 2018 / 035391) to give compound 28. Removal of both Boc protecting groups by treatment with TFA followed by replacement of the Boc protecting groups on the aliphatic amine by treatment with (Boc)2O in the presence of DIPEA gives compound 29. This compound is reacted with 5-(2-(dimethylamino)ethoxy)-1H-indole-2-carboxylic acid using EDCI as a coupling agent to produce 30. Removal of the Boc protecting groups with TFA and reaction of the amine with commercially available NHS ester 31 gives the payload-linker compound 32.

[0313] Scheme 8 outlines the preparation of compounds of the invention wherein the linker is attached via a carbamate.

[0314]

[0315] Solution 8

[0316] Compound 33 reacts with a known dithiochloroformate reagent 34 (ACS Med. Chem. Lett. (2016) 7, 988) in the presence of pyridine as a base to give compound 35 of the present invention.

[0317] These embodiments are also used to illustrate that the linker should contain specific functional groups for selective metabolism or reaction in vivo. Examples include dipeptides recognized by lysosomal enzymes (e.g., valine-citrulline or valine-alanine dipeptides that are recognition cleavage sites for cathepsins) or disulfide bonds that are cleaved when exposed to high levels of intracellular reducing agents such as glutathione. The linker should also contain functional groups that allow connection to antibodies, such as maleimides that selectively react with thiols, such as those produced by reducing disulfide bonds within antibody chains, or those on cysteine ​​residues that are modified in site-selective modified antibodies.

[0318] An alternative site for attaching the payload to the linker is at a functional group within the side chain Y. This approach can be used when X = OH or X = prodrug OH. In the latter case, the prodrug is typically introduced into the alkylated subunit prior to attachment of the side chain, as shown in Scheme 9 below.

[0319]

[0320] Solution 9

[0321] Scheme 9 outlines the preparation of compounds of the invention wherein the linker is attached to the side chain via a carbamate. In this scheme, the phenol of the alkylated subunit is protected as a carbamate prodrug. Compound 18 is reacted with 4-methylpiperazinecarbonyl chloride in the presence of DMAP to give compound 36. The Boc protecting group is removed using HCl and the intermediate is reacted with 5-(2-(methylamino)ethoxy)-1H-indole-2-carboxylic acid using EDCI as a coupling agent to give compound 37. Reaction with the activated linker 25 described above gives drug-linker 38.

[0322] When the prodrug is a phosphate ester OP(O)(OH)2, the synthesis preferably uses the phosphate intermediate OP(O)(OR 4 )2 is carried out until the last step of phosphate deprotection. When connected to the side chain Y, there is a lot of flexibility about how the linker is connected to the side chain. The connection can be traceless, as shown in the above scheme, for which suitable side chain functional groups include alcohols (for carbamate connection), primary and secondary amines (for carbamate connection), tertiary amines (for quaternary salt connection) and thiols (for disulfide connection). These functional groups can be connected to the side chain at any determined substituent position. The connection can also be a traceless connection, that is, after cleavage from ADC, the effective load still contains a fragment of the linker. This method is suitable when the structure and position of the linker fragment do not interfere with the alkylation of the effective load to DNA, that is, do not adversely affect the cytotoxicity of the released substance. Within this limitation, non-traceless linkers can be connected to any type of side chain reactive part RM that has been defined.

[0323] 5.4 Uses of the compounds of the present invention

[0324] The compounds of the present invention comprise highly cytotoxic payloads or payload components useful for preparing ADCs and other biologically active compounds.

[0325] The compounds of the present invention wherein the 2-methylbenzoxazole subunit is linked to a DNA minor groove binding unit can be converted into ADCs by linking an antibody or other ligand binding group via a linker. The linker can be directly linked to the DNA alkylation subunit via a hydroxyl or amino group X, or indirectly linked to the DNA alkylation subunit via a DNA minor groove binding unit.

[0326] The binding ligand (e.g., antibody) and appropriate linker can be selected for the specific clinical application for which the ADC is intended. The nature of the linker can affect the pharmacokinetic properties of the conjugate and should therefore be selected for compatibility with the binding ligand to be used and the pharmacological requirements of the conjugate as a whole. The linker may include stretching units, spacer units, and moieties that increase solubility.

[0327] Examples of such linker groups, stretcher units, and spacer units include, but are not limited to, those described in US Pat. No. 7,964,566 B2 and US 2017 / 0232108 A1, which are incorporated herein by reference in their entirety.

[0328] In one aspect, the invention provides the use of a compound of Formula I, Ia, II, IIa, III or IIIa in the preparation of an ADC.

[0329] In another aspect, the invention provides a method of preparing an ADC or ADC component comprising reacting a compound of Formula I, Ia, II, Ha, III or IIIa with a linker or linker-antibody moiety.

[0330] The new 2-methylbenzoxazole DNA alkylating unit is much less lipophilic than the widely used CBI alkylating unit present in many duocarmycin analogs, as shown in Examples 6 and 24.

[0331] This will make the corresponding ADC easier to manufacture, because the effective load component will be more soluble in the aqueous solvent used to conjugate the antibody-linker component. The less lipophilic effective load will also cause the ADC to aggregate less, which will further simplify manufacturing. Reduced aggregation will reduce the risk of immune response in vivo, and the less lipophilic ADC will have a longer clearance from the bloodstream and therefore have a greater overall exposure. In short, the effective load based on 2-methylbenzoxazole will produce an ADC that is easier to prepare, safer and more effective.

[0332] DNA alkylating agents based on 2-methylbenzoxazole analogs of duocarmycin are highly cytotoxic compounds. This is demonstrated in Examples 7 and 25, which describe cytotoxicity tests comparing the compounds of the present invention with open-CBI-TMI. The latter is widely considered to have sufficient cytotoxicity to be used as an ADC effective load. The closest comparison between open-CBI-TMI and the compounds of the present invention is performed with compound 23. Because compound 23 shares the same minor groove binding side chain (i.e., TMI), direct head-to-head comparisons can be performed on these two compounds, which demonstrates equal cytotoxicity. The high cytotoxicity of the compounds of the present invention is very surprising. 2-methylbenzoxazole analogs are structurally closely related to known COI duocarmycin analogs (Bioorg. Med. Chem. Lett. (2010) 20, 1854). As shown in open-COI-TMI, the only difference between these structures is the orientation of the oxazole ring fusion and the nature of the 2-substituent (Me relative to CO2Me). However, the cytotoxicity of COI analogs is hundreds of times lower than that of CBI analogs, making COI analogs unsuitable for use as ADC payloads.

[0333]

[0334] Another advantage of the 2-methoxybenzoxazole duocarmycin analogs of the present invention is that they undergo rapid hydrolysis in aqueous buffers under physiological conditions to form inactive products, as shown in Examples 8 and 26.

[0335] This means that they are unlikely to persist in the circulation for very long periods of time and therefore are unlikely to cause relevant off-mechanism toxicity if systemic release from the ADC occurs. This stability is also very surprising, since the cytotoxicity and aqueous stability of duocarmycin analogs have been found to be correlated. In other words, all previously reported analogs with the highest cytotoxicity (including CBI) are also the most stable, and therefore all previously reported analogs have the potential to cause undesirable side effects if released into the systemic circulation.

[0336] Although the compounds of the present invention are primarily used as payloads to be incorporated into ADCs, in some embodiments, they can be used as therapeutic agents in their own right. Accordingly, the present invention also relates to pharmaceutical compositions comprising a compound of Formula I, Ia, II, IIa, III or IIIa and a pharmaceutically acceptable carrier.

[0337] The term "pharmaceutically acceptable carrier" refers to a carrier (e.g., an adjuvant or vehicle) that can be administered to a subject together with a compound of Formula I, Ia, II, IIa, III or IIIa, which is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes carriers suitable for veterinary as well as human pharmaceutical use.

[0338] Pharmaceutically acceptable carriers that can be used in the composition include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) (e.g., tocopherol polyethylene glycol 1000 succinate), surfactants for pharmaceutical dosage forms (e.g., Tween or other similar polymer delivery matrices), serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin. Cyclodextrins such as α-, β-, and γ-cyclodextrins, or chemically modified derivatives such as hydroxyalkyl cyclodextrins, including 2- and 3-hydroxypropyl-3-cyclodextrins, or other soluble derivatives can also be advantageously used to enhance delivery. The oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and / or suspensions.

[0339] The pharmaceutical composition of the present invention can be used as a single dose or in a multiple dose regimen as a single therapeutic agent or in combination with one or more other therapeutic agents simultaneously, sequentially or separately. The one or more other therapeutic agents will depend on the disease to be treated or the therapeutic benefit of other expectations. As known to those skilled in the art, one or more other therapeutic agents can be used for the therapeutic amount indicated or approved for a particular agent.

[0340] The pharmaceutical composition is formulated to allow administration to a subject by any selected approach, including but not limited to oral or parenteral (including topical, subcutaneous, intramuscular and intravenous) administration. In some embodiments, the composition is formulated for oral, intravenous, subcutaneous, intramuscular, percutaneous, intraperitoneal or other pharmacologically acceptable approach administration. For example, the composition can be prepared together with a suitable pharmaceutically acceptable carrier (including excipient, diluent, adjuvant and combination thereof) selected according to the expected route of administration and standard pharmaceutical practice. For example, the composition can be used as powder, liquid, tablet or capsule oral administration, or as ointment, cream or lotion topical application. Suitable preparations can contain other reagents as required, including emulsifier, antioxidant, flavoring or coloring agent, and can be suitable for immediate release, delayed release, modified release, sustained release, pulse release or controlled release.

[0341] The composition can be administered by a parenteral route. Examples of parenteral dosage forms include aqueous solutions, isotonic saline or 5% glucose of the active agent, or other well-known pharmaceutically acceptable excipients. For example, cyclodextrin or other solubilizing agents well known to those skilled in the art can be used as pharmaceutical excipients for delivering therapeutic agents.

[0342] Examples of dosage forms suitable for oral administration include, but are not limited to, tablets, capsules, lozenges, or any liquid form of the composition that can provide a therapeutically effective amount, such as syrups, aqueous solutions, emulsions, etc. The capsule can contain any standard pharmaceutically acceptable material, such as gelatin or cellulose. Tablets can be prepared according to conventional procedures by compressing a mixture of active ingredients with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. The active ingredient can also be used in the form of a hard shell tablet or capsule containing a binder (e.g., lactose or mannitol), a conventional filler, and a compressed tablet.

[0343] Examples of dosage forms suitable for transdermal administration include, but are not limited to, transdermal patches, transdermal bandages, and the like.

[0344] Examples of dosage forms suitable for topical administration of the composition include any lotion, stick, spray, ointment, paste, cream, gel, etc., whether applied directly to the skin or via a vehicle such as a pad, patch, etc.

[0345] Examples of dosage forms suitable for suppository administration of the composition include any solid dosage form for insertion into a body orifice, particularly those inserted rectally, vaginally, and urethrally.

[0346] Examples of dosage forms suitable for injectable compositions include single or multiple administrations by bolus delivery, such as by intravenous injection, subcutaneous, subdermal and intramuscular administration, or oral administration.

[0347] Examples of dosage forms suitable for depot administration of the composition include pills or solid forms in which the active agent is entrapped in a matrix of a biodegradable polymer, microemulsions, liposomes, or is microencapsulated.

[0348] Examples of infusion devices for the composition include infusion pumps for providing the desired number of doses or steady-state administration, and include implantable drug pumps. Examples of implantable infusion devices for the composition include any solid form in which the active substance is encapsulated or dispersed throughout a biodegradable polymer or synthetic polymer such as silicone, silicone rubber, silicone rubber or similar polymers.

[0349] Examples of dosage forms suitable for transmucosal delivery of compositions include storage solutions for enemas, vaginal suppositories, tampons, creams, gels, pastes, foams, atomized solutions, powders and similar preparations, which contain suitable carriers known in the art in addition to active ingredients. Such dosage forms include forms suitable for inhalation or insufflation of compositions, including compositions of solutions and / or suspensions contained in pharmaceutically acceptable aqueous or organic solvents or their mixtures and / or powders. Transmucosal administration of compositions can utilize any mucosa, but typically utilizes nose, cheek, vagina and rectal tissues. Preparations suitable for nasal administration of compositions can be applied in liquid form, such as nasal sprays, nasal drops, or by aerosol application via a sprayer, including aqueous or oily solutions of polymer particles. Preparations can be prepared into aqueous solutions, such as solutions in saline, solutions using benzyl alcohol or other suitable preservatives, absorption promoters, fluorocarbons and / or other solubilizing or dispersants known in the art that enhance bioavailability.

[0350] Examples of dosage forms suitable for buccal or sublingual administration of the composition include lozenges, tablets, etc. Examples of dosage forms suitable for ophthalmic administration of the composition include inserts and / or compositions comprising solutions and / or suspensions in pharmaceutically acceptable aqueous or organic solvents.

[0351] Examples of composition formulations can be found, for example, in Sweetman, SC (Ed.). Martindale. The Complete Drug Reference, 33rd Edition, Pharmaceutical Press, Chicago, 2002, 2483 pp.; Aulton, ME (Ed.) Pharmaceutics. The Science of Dosage Form Design. Churchill Livingstone, Edinburgh, 2000, 734 pp.; and Ansel, HC, Allen, LV and Popovich, NG Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott 1999, 676 pp. Excipients for making drug delivery systems are described in various publications known to those skilled in the art, including, for example, Kibbe, EH Handbook of Pharmaceutical Excipients, 3rd Ed., American Pharmaceutical Association, Washington, 2000, 665 pp. USP also provides examples of sustained-release oral dosage forms, including those formulated as tablets or capsules. See, e.g., The United States Pharmacopeia 23 / National Formulary 18, The United States Pharmacopeial Convention, Inc., Rockville MD, 1995 (hereinafter referred to as "USP"), which also describes specific tests for determining the drug release capacity of extended release and delayed release tablets and capsules. The USP test for drug release of extended release and delayed release products is based on the dissolution of the drug from the dosage unit relative to the test time passed. Descriptions of various test devices and procedures can be found in the USP. FDA provides further guidance on the analysis of sustained-release dosage forms.(See Guidance for Industry. Extended release oral dosage forms: development, evaluation, and application of in vitro / in vivo correlations. Rockville, MD: Center for Drug Evaluation and Research, Food and Drug Administration, 1997).

[0352] The dosage forms described herein may be in the form of physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect.

[0353] Dosage levels of the active ingredients in the pharmaceutical compositions may be varied to provide an amount of the active ingredient (effective amount) effective to achieve the desired therapeutic effect for a particular patient, composition, and mode of administration without being toxic to the patient.

[0354] The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition used, the route of administration, the time of administration, the excretion rate of the particular compound of the invention used, other drugs, compounds and / or substances used in combination with the particular composition used, the age, sex, weight, condition, the general health and previous medical history of the patient being treated, and similar factors well known in the medical field. Typically, the daily amount or regimen should be in the range of about 0.01 mg to about 2000 mg of the compound of the invention per kilogram (kg) of body weight.

[0355] 6. Examples

[0356] General methods and materials

[0357] All reagents were purchased at reagent grade and used without further purification. The solvents used for the reaction were distilled according to standard procedures before use. Petroleum ether refers to the fraction with a boiling point of 40-60° C., and the solvent was dried over anhydrous Na2SO4 or MgSO4 according to standard procedures. Column chromatography was performed on silica gel (Merck 230-400 mesh).

[0358] NMR spectra were recorded on a Bruker Avance 400 MHz instrument. 1 H and 100MHz 13 C spectra chemical shifts are reported in parts per million (ppm) and are 1 The H spectra were calibrated to tetramethylsilane (0 ppm) as an internal standard. 13C spectra were calibrated to residual solvent. Multiplicities were reported as follows: br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, dt = doublet of triplet, ddd = doublet of doublets. High resolution mass spectra (HRMS) were obtained using a Bruker microTOF-Q II or Agilent 6530BAccurate Mass Q-TOF mass spectrometer. LRMS was performed using a SurveyorMSQ mass spectrometer.

[0359] The above general materials and methods apply to all examples below unless alternative general methods and materials are given.

[0360] Example 1. 6-methyl-4-oxo-8,8a-dihydro-1H-cyclopropane[c]oxazolo[4,5-e]indole-2(4H)-carboxylic acid tert-butyl ester (50)

[0361]

[0362] Solution 10

[0363] A solution of NaH2PO4·2H2O (4.29 g, 27.5 mmol) in H2O (10 mL) was added to a solution of 3,4-dihydroxy-5-nitrobenzaldehyde 39 (5.03 g, 27.5 mmol) in DMSO (25 mL). To the stirred mixture was added a solution of NaClO2 (80%, 4.35 g, 38.5 mmol) in H2O (25 mL) dropwise over 50 min, keeping the internal temperature below 45°C. The dark red-brown mixture was stirred at room temperature for 16 h and then poured into aqueous NaHCO3 solution (5%, 80 mL). The mixture was washed with CH2Cl2 (×2) and the aqueous phase was acidified with c.HCl to obtain pH ~1. The mixture was extracted with EtOAc (×3) and the combined extracts were washed with brine, then dried and evaporated to give 3,4-dihydroxy-5-nitrobenzoic acid (40) as a tan solid (5.12 g, 94%); 1 HNMR (d6-DMSO) δ13.02 (br s, 1H), 10.80 (br s, 2H), 7.87 (d, J = 2.0 Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H).

[0364] A mixture of 40 (3.99 g, 20.0 mmol) in MeOH (60 mL) and c.H2SO4 (1.5 mL) was stirred at reflux for 19 h, then cooled and evaporated. The residue was partitioned between EtOAc and brine (×2). The EtOAc extract was dried and evaporated, and the resulting solid was recrystallized from H2O to give methyl 3,4-dihydroxy-5-nitrobenzoate (41) as an orange-brown crystalline solid (3.30 g, 77%); mp 137°C to 139°C; 1 H NMR (d6-DMSO) δ10.84 (br s, 2H), 7.89 (d, J = 2.1 Hz, 1H), 7.57 (d, J = 2.1 Hz, 1H), 3.83 (s, 3H).

[0365] HCl (4M, 2.94 mL, 11.8 mmol) and Pd / C (10%, 0.25 g) in dioxane were added to a solution of 41 (2.51 g, 11.8 mmol) in EtOH (35 mL). The mixture was degassed with nitrogen, flushed with hydrogen, and then stirred under a hydrogen balloon until the reduction was complete (~8 h). The mixture was filtered through celite, washed with EtOH, and the filtrate was evaporated to give methyl 3-amino-4,5-dihydroxybenzoate hydrochloride (42) as a light yellow solid (3.0 g), which was used directly in the next step. Anal. (C8H 10 NO4) is calculated as [M+H] + 184.1; measured value 184.1.

[0366] Trimethyl orthoacetate (13.6 mL, 107 mmol) was added to a portion of 42 (2.71 g) prepared as described above. The suspension was stirred at reflux for 1 h, then cooled and diluted with petroleum ether. The precipitate was filtered off and dried to give methyl 7-hydroxy-2-methylbenzo[d]oxazole-5-carboxylate (43) as a light brown solid (2.02 g, 91% from 41); 1 H NMR(d6-DMSO) δ10.76(s,1H),7.66(d,J=1.5Hz,1H),7.43(d,J=1.5Hz,1H),3.85(s,3H),2.62(s,3H); 13 CNMR(d6-DMSO)δ166.1,165.0,143.0,142.2,141.9,126.5,112.1,111.1,52.2,14.1. Anal.(C 10 H9NO4) is calculated as [M+H] + 208.06043; measured value 208.06061.

[0367] Benzyl bromide (98%, 1.24 mL, 10.2 mmol) and K2CO3 (1.48 g, 10.7 mmol) were added to a solution of 43 (2.02 g, 9.75 mmol) in DMF (20 mL), and the mixture was stirred at room temperature for 18 h. The mixture was diluted with H2O, the solid was filtered off and dried to give methyl 7-(benzyloxy)-2-methylbenzo[d]oxazole-5-carboxylate (44) (2.81 g, 97%) as a light brown solid; 1 H NMR(d6-DMSO) δ7.83(d,J=1.3Hz,1H),7.63(d,J=1.3Hz,1H),7.54-7.49(m,2H),7.46-7.34(m,3H),5.36(s,2H),3.87(s,3H),2.63(s,3H); 13 C NMR(d6-DMSO)δ165.9,165.4,143.0,142.8,142.6,136.1,128.5,128.2,128.0,126.7,113.2,109.5,70.5,52.4,14.1. Anal.(C 17 H 15 NO4) is calculated as [M+H] + 298.10738; measured value 298.10834.

[0368] A solution of KOH (1.62 g, 29.1 mmol) in H2O (15 mL) was added to a suspension of 44 (2.79 g, 9.38 mmol) in MeOH (60 mL) and the mixture was stirred at 70°C for 40 min. The MeOH was evaporated and the aqueous residue was acidified with aqueous HCl (2N, 12 mL) at 0°C. The precipitated solid was filtered off and dried to give 7-(benzyloxy)-2-methylbenzo[d]oxazole-5-carboxylic acid (45) as an off-white solid (2.62 g, 98%); 1 H NMR(d6-DMSO) δ13.06(br s,1H),7.81(d,J=1.3Hz,1H),7.62(d,J=1.3Hz,1H),7.53-7.48(m,2H),7.46-7.34(m,3H),5.36(s,2H),2.63(s,3H). Anal.(C 16 H 13 NO4) is calculated as [M+H] + 284.09173; measured value 284.09164.

[0369] Et3N (1.54 mL, 11.1 mmol) and DPPA (97%, 2.25 mL, 10.1 mmol) were added to a suspension of 45 (2.61 g, 9.21 mmol) in anhydrous tert-butyl alcohol (80 mL), and the mixture was stirred at reflux for 3 h. The solvent was evaporated and the residue was purified by column chromatography (petroleum ether:EtOAc 9:1, then 4:1) to give tert-butyl (7-(benzyloxy)-2-methylbenzo[d]oxazol-5-yl)carbamate (46) as a cream solid (2.44 g, 75%); 1 H NMR(d6-DMSO)δ9.37(s,1H),7.52-7.47(m,2H),7.44-7.33(m,4H),7.21(s,1H),5.21(s,2H),2.56(s,3H),1.48(s,9H); 13 C NMR (d6-DMSO) δ164.1,152.8,142.8,142.4,136.8,136.3,135.1,128.5,128.2,128.1,100.72,100.67,79.1,70.3,28.1,14.1. Anal.(C 20 H 22 N2O4) is calculated as [M+H] + 355.1652; found 355.1668. A sample was recrystallized from EtOAc / petroleum ether to give white needles, mp 150-152 °C.

[0370] NBS (1.16 g, 6.52 mmol) was added portionwise to a solution of 46 (2.31 g, 6.52 mmol) in CH3CN (100 mL) at 0°C over 10 minutes. The mixture was stirred at room temperature for 3 h, and then the solvent was evaporated. The residue was dissolved in EtOAc, and the solution was washed with H2O (×2) and then with brine, then dried and evaporated. The resulting milky white solid was recrystallized from EtOAc / petroleum ether to give tert-butyl (7-(benzyloxy)-4-bromo-2-methylbenzo[d]oxazol-5-yl)carbamate (47) as a white solid (2.15 g, 76%); mp 154-156°C; 1 H NMR(CDCl3)δ8.01(s,1H),7.53-7.47(m,2H),7.43-7.32(m,3H),7.03(s,1H),5.27(s,2H),2.65(s,3H),1.55(s,9H); 13C NMR (CDCl3) δ165.0,152.9,143.0,142.0,136.4,136.1,133.9,128.8,128.6,128.3,102.2,92.4,81.4,71.6,28.5,14.8. Anal.(C 20 H 21 79 BrN2O4) calculated as [M+H] + 433.0757; measured value 433.0762; (C 20 H 21 81 BrN2O4) calculated as [M+H] + 435.0740; found 435.0745. The mother liquor was evaporated and the residue was purified by column chromatography (petroleum ether: EtOAc 4: 1) to give more 47 (0.60 g, 21%).

[0371] 1,3-Dichloropropene (mixed isomers, 90%, 1.92 mL, 18.6 mmol) and K2CO3 (4.3 g, 31 mmol) were added to a solution of 47 (2.69 g, 6.21 mmol) in DMF (12 mL) and the mixture was stirred at 80°C for 7 h. DMF was evaporated and the residue was partitioned between EtOAc and H2O. The aqueous layer was extracted again with EtOAc (×2) and the combined EtOAc layers were washed with brine (×3), then dried and evaporated to give crude tert-butyl (7-(benzyloxy)-4-bromo-2-methylbenzo[d]oxazol-5-yl)(3-chloroallyl)carbamate (48) as a light brown gum (3.21 g, 100%); 1 H NMR (CDCl3) (mixture of E and Z isomers and Boc rotamers) δ 7.50-7.32 (m, 5H), 6.85-6.66 (m, 1H), 6.05-5.88 (m, 2H), 5.34-5.15 (m, 2H), 4.52-4.44, 4.42-4.26, 4.06-4.03, 3.89-3.80 (4×m, 2H), 2.69 (s, 3H), 1.56 (s, 9H). Anal. (C 23 H 24 79 BrClN2O4) is calculated as [M+H] + 507.06807; measured value 507.06909.

[0372] Under nitrogen, Bu3SnH (97%, 1.09 mL, 3.92 mmol) and AIBN (64 mg, 0.39 mmol) were added to a solution of 48 (995 mg, 1.96 mmol) in anhydrous toluene (15 mL), and the mixture was stirred under reflux. Additional portions of Bu3SnH (97%, 0.54 mL, 2.0 mmol) and AIBN (32 mg, 0.2 mmol) were added after 1.5 and 3 hours. After 4 hours, toluene was evaporated and the residue was distributed between CH3CN and petroleum ether. The petroleum ether layer was extracted again with CH3CN (×2), the combined extracts were washed with petroleum ether (×2), and then evaporated. The residue was purified by column chromatography (petroleum ether: EtOAc 9:1, then 6:1) to give a crude product as white foam. This was stirred with petroleum ether (8 mL) to give tert-butyl 4-(benzyloxy)-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indole-6-carboxylate (49) as a white solid (668 mg, 80%); mp 142-143 °C; 1 H NMR(CDCl3)δ7.78(br s,1H),7.52-7.45(m,2H),7.43-7.31(m,3H),5.26(s,2H),4.21(dd,J=11.5,9 .7Hz,1H),4.15-3.96(m,3H),3.65(t,J=9.9Hz,1H),2.62(s,3H),1.57(s,9H); 13 C NMR (CDCl3) δ165.0,152.6,143.7,141.4,139.3,136.9,136.4,128.8,128.4,128.1,111.6,97.6,81.2,71.4,53.2,46.8,40.9,28.7,14.8. Anal.(C 23 H 25 ClN2O4) calculated as [M+H] + 429.15756; measured value 429.15735.

[0373] Under nitrogen at 0 ℃, NH4HCO2 aqueous solution (25%, 3.9 mL, 15.4 mmol) and Pd / C (10%, 53% H2O moistening, 0.13 g) were added to a THF (10 mL) solution of 48 (660 mg, 1.54 mmol), and the mixture was stirred vigorously at this temperature. After 3 hours, additional portions of NH4HCO2 aqueous solution (25%, 3.9 mL, 15.4 mmol) and Pd / C (10%, 53% H2O moistening, 0.13 g) were added. After 7 hours, the mixture was filtered through diatomaceous earth and washed with EtOAc. The EtOAc layer from the filtrate was washed with brine, then dried and evaporated. The residue was recrystallized from EtOAc / petroleum ether to give tert-butyl 8-(chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indole-6-carboxylate (18) as a white solid (444 mg, 85%); mp 234-238°C; 1 H NMR(CDCl3)δ7.66(br s,1H),7.63(br s,1H),4.18(dd,J=11.5,9.6Hz,1H),4.11-3.95(m,3H),3.63(apparent t,J=9.9Hz,1H),2.62(s,3H),1.56(s,9H); 13 C NMR(CDCl3)δ165.2,153.0,141.1,140.9,139.2,136.1,111.1,99.9,81.7,53.3,46.9,40.8,28.7,14.8. Anal.(C 16 H 19 ClN2O4) calculated as [M+H] + 339.11061; measured value 339.11149.

[0374] K2CO3 (41 mg, 0.3 mmol) was added to a solution of 18 (67 mg, 0.2 mmol) in DMF (1 mL), and the mixture was stirred at room temperature for 3.5 h, then diluted with EtOAc and H2O. The EtOAc layer was washed with brine (×3), then dried and evaporated. The residue was purified by column chromatography (petroleum ether: EtOAc 2:3, then 1:4) to give 50 as a colorless oil (22 mg, 37%); 1H NMR(CDCl3)δ6.72(s,1H),4.05(d,J=11.4Hz,1H),3.99(dd,J=11.4,5.0Hz,1H),2.94-2.88 (m,1H),2.57(s,3H),2.01(dd,J=7.8,4.0Hz,1H),1.55(s,9H),1.46(dd,J=4.9,4.4Hz,1H); 13 C NMR (CDCl3) δ175.1,165.5,158.6,151.7,148.1,145.8,109.8,83.6,53.7,33.1,28.3,24.9,23.4,14.6. Anal.(C 16 H 18 N2O4) is calculated as [M+H] + 303.13393; measured value 303.13458.

[0375] Example 2. (8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-6-yl)(5,6,7-trimethoxy-1H-indol-2-yl)methanone (23)

[0376]

[0377] Solution 11

[0378] A mixture of 49 (51 mg, 0.12 mmol) and HCl in dioxane (4 M, 1 mL) was stirred at room temperature for 3 h and then evaporated. The residue was suspended in DMA (1.5 mL) and 5,6,7-trimethoxy-1H-indole-2-carboxylic acid (31.4 mg, 0.13 mmol) and EDCI.HCl (46 mg, 0.24 mmol) were added. The mixture was stirred at room temperature for 1 h and then dilute aqueous NaHCO3 solution was added. The precipitated solid was filtered off, washed with H2O, and dried. The crude product was recrystallized from EtOAc to give (4-(benzyloxy)-8-(chloromethyl)-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-6-yl)(5,6,7-trimethoxy-1H-indol-2-yl)methanone (51) as an off-white solid (34 mg, 51%); mp 225-227°C; 1H NMR(d6-DMSO)δ11.44(d,J=1.4Hz,1H),8.04(br s,1H),7.53-7.47(m,2H),7.45-7.33(m,3H),7.03(d,J=2.0Hz,1H),6.97(s,1H),5.27(s,2H),4.74(t,J=10.2Hz,1H),4.40(dd,J=10. 9,5.3Hz,1H),4.25-4.16(m,1H),4.13(dd,J=10.9,3.1Hz,1H),4.06-4.00(m,1H),3.93(s,3H),3.83(s,3H),3.78(s,3H),2.64(s,3H); 13 C NMR(d6-DMSO)δ165.2,160.1,149.2,142.3,141.5,139.9,139.1,138.5,136.7,136.3,130.8,1 28.6,128.2,127.9,125.3,123.2,113.1,106.1,70.5,61.1,61.0,56.0,54.6,46.8,40.7,14.2. Anal.(C 30 H 28 ClN3O6) calculated as [M+H] + 562.17394; measured value 562.17311.

[0379] Aqueous NH4HCO2 (25%, 0.14 mL, 0.55 mmol) and Pd / C (10%, wetted with 53% H2O, 34 mg) were added to a solution of 51 (31 mg, 0.055 mmol) in THF (20 mL) at 0°C under nitrogen, and the mixture was vigorously stirred at this temperature. After 1 hour, more aqueous NH4HCO2 (25%, 0.28 mL, 1.1 mmol) and Pd / C (10%, wetted with 53% H2O, 35 mg) were added. After 2 hours, the mixture was filtered through celite, washed with THF, and the filtrate was evaporated to dryness at 30°C. The residue was triturated with H2O, the solid was filtered off and dried. Further trituration with EtOAc gave 23 as a white solid (21 mg, 81%); mp 285°C (dec); 1H NMR(d6-DMSO) δ11.39(d,J=1.6Hz,1H),10.45(s,1H),7.84(s,1H),7.01(d,J=2.1Hz,1H),6.96(s,1H),4.71(t,J=10.1Hz,1H),4. 36(dd,J=10.9,5.1Hz,1H),4.17-4.09(m,2H),3.97(dd,J=11.2,7.8Hz,1H),3.94(s,3H),3.82(s,3H),3.78(s,3H),2.61(s,3H); 13 C NMR(d6-DMSO)δ164.8,156.0,149.2,141.3,141.2,139.8,139.1,138.6,136.2,131. 0,125.2,123.2,111.0,105.9,101.4,98.0,61.1,61.0,56.0,54.7,46.9,40.7,14.2. Anal.(C 23 H 22 ClN3O6) calculated as [M+H] + 472.12699; measured value 472.12694.

[0380] Example 3. (8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-6-yl)(5-(2-(dimethylamino)ethoxy)-1H-indol-2-yl)methanone (52)

[0381]

[0382] Solution 13

[0383] A mixture of 18 (33 mg, 0.097 mmol) and HCl in dioxane (4 M, 1 mL) was stirred at room temperature for 4 h and then evaporated. The residue was suspended in DMA (1.0 mL) and 5-(2-(dimethylamino)ethoxy)-1H-indole-2-carboxylic acid hydrochloride (34 mg, 0.12 mmol), EDCI.HCl (65 mg, 0.34 mmol) and anhydrous toluenesulfonic acid (3.3 mg, 0.019 mmol) were added. The mixture was stirred at room temperature for 1.5 h and then cooled in an ice bath. EtOAc (50 mL) and H2O (10 mL) were added and the pH was adjusted to 8-9 with saturated aqueous NaHCO3. The EtOAc layer was separated, washed with water, then dried and evaporated. The residue was purified by column chromatography (CH2Cl2:MeOH 10:1) to give 52 (34 mg, 75%); mp 206-210°C (dec); 1HNMR(d6-DMSO)δ11.59(br s,1H),10.46(br s,1H),7.91(s,1H),7.38(d,J=8.9Hz,1H),7.17(d,J=2.3Hz,1H),7.05(d,J=1.7Hz,1H),6.92(dd,J=8.9,2.4H z,1H),4.76(t,J=10.2Hz,1H),4.48-4.40(m,1H),4.22-4.06(m,4H),4.04-3.95(m,1H),2.80(poorlyresolved t,J=5.3Hz,2H),2.61(s,3H),2.34(s,6H); 13 C NMR(d6-DMSO)δ164.7,159.8,152.8,141.28,141.25,138.6,136.2,131.6,131.0,127. 5,115.7,113.1,111.0,105.1,103.3,101.6,65.6,57.4,54.5,46.9,45.1,40.8,14.2. Anal(C 24 H 25 ClN4O4) is calculated as [M+H] + 469.1637; measured value 469.1642.

[0384] Example 4. N-(2-(8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indole-6-carbonyl)-1H-indol-5-yl)-5-(2-(dimethylamino)ethoxy)-1H-indole-2-carboxamide (53)

[0385]

[0386] Solution 14

[0387] A mixture of 18 (33 mg, 0.097 mmol) and a solution of HCl in dioxane (4 M, 1 mL) was stirred at room temperature for 4 h and then evaporated. The residue was suspended in DMA (1.0 mL) and 5-(5-(2-(dimethylamino)ethoxy)-1H-indole-2-formamido)-1H-indole-2-carboxylic acid hydrochloride (43 mg, 0.097 mmol), EDCI.HCl (65 mg, 0.34 mmol) and anhydrous toluenesulfonic acid (3.3 mg, 0.019 mmol) were added. The mixture was stirred at room temperature for 1.5 h and then cooled in an ice bath. EtOAc (150 mL) and H2O (50 mL) were added and the pH was adjusted to 8-9 with saturated NaHCO3 aqueous solution. The EtOAc layer was separated, washed with water, then dried and evaporated. The residue was purified by column chromatography (CH2Cl2:MeOH 10:1 then 5:1) to give 53 (46 mg, 75%); mp 231°C (dec); 1 HNMR(d6-DMSO)δ11.70(br s,1H),11.59(br s,1H),10.47(br s,1H),10.13(s,1H),8.21(d,J=1.5Hz,1H),7.93(s,1H),7.57(dd,J=8.9,1.9Hz,1H),7.46(d,J=8.8Hz,1H),7.41-7.27(m,2 H),7.22-7.11(m,2H),6.87(dd,J=8.9,2.4Hz,1H),4.80(t,J=10.2,1H),4.53-4.43(m,1H),4.24-3.98(m,5H),2.77(poorly resolved t,J=5.4Hz,2H),2.62(s,3H),2.34(s,6H); 13 C NMR(d6-DMSO)δ164.8,159.8,159.5,152.8,141.3,141.2,138.6,138.6,136.3,133.2,132.3,132.1,131.3,127.4,127. 1,119.3,115.1,113.2,112.9,112.2,111.1,105.6,103.2,103.1,101.6,65.6,57.5,54.5,46.9,45.2,40.8,39.5,14.2. Anal(C 33 H 31 ClN6O5) is calculated as [M+H] + 627.2117; measured value 627.2119.

[0388] Example 5. N-(2-(8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indole-6-carbonyl)imidazo[1,2-a]pyridin-6-yl)-4-hydroxybenzamide (57)

[0389]

[0390] Solution 15

[0391] A solution of 6-aminoimidazo[1,2-a]pyridine-2-carboxylic acid ethyl ester (54) (50 mg, 0.24 mmol), 4-hydroxybenzoic acid (68 mg, 0.49 mmol), EDCI.HCl (163 mg, 0.85 mmol) and anhydrous toluenesulfonic acid (8.7 mg, 0.05 mmol) in DMA (1 mL) was stirred at room temperature for 2 h. The mixture was diluted with H2O and extracted with EtOAc. The EtOAc layer was dried and evaporated, and the residue was purified by column chromatography (EtOAc only, then EtOAc:MeOH 10:1) to give 6-(4-hydroxybenzamido)imidazo[1,2-a]pyridine-2-carboxylic acid ethyl ester (55) as a green-amber solid (41 mg, 52%); mp 263-266 °C; 1 H NMR (d6-DMSO) δ 10.17, 10.16 (partial overlap s, 2H), 9.40-9.35 (m, 1H), 8.64 (s, 1H), 7.93-7.84 (m, 2H), 7.62 (d, J = 9.7 Hz, 1H), 7.55 (dd, J = 9.8, 1.9 Hz, 1H), 6.93-6.84 (m, 2H), 4.30 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H); 13 C NMR(d6-DMSO)δ165.3,162.7,160.8,142.3,135.6,129.8,127.6,124.5,123.5,118.9,117.5,117.2,115.0,60.1,14.3. Anal(C 17 H 15 N3O4) is calculated as [M+H] + 326.1135; measured value 326.1125.

[0392] Solid KOH (171 mg, 3.05 mmol) was added to a solution of 55 (198 mg, 0.61 mmol) in THF (6 mL), methanol (6 mL) and H2O (3 mL), and the mixture was stirred at room temperature for 1 h and then evaporated. The residue was diluted with water (10 mL) and acidified with aqueous HCl (5 M) to pH < 1. The solid was filtered off, washed with H2O and dried to give 6-(4-hydroxybenzamido)imidazo[1,2-a]pyridine-2-carboxylic acid (56) as a yellow solid (143 mg, 79%), mp 268-271 °C; 1 H NMR(d6-DMSO)δ10.17(s,2H),9.40(s,1H),8.59(s,1H),7.95-7.84(m,2H),7.63(d,J=9.7Hz,1H),7.56(dd,J=9.7,1.9Hz,1H),6.96-6.83(m,2H),1H not observed; 13 CNMR(d6-DMSO)δ165.3,163.9,160.8,142.0,136.1,129.8,127.6,124.6,123.5,118.8,117.3,117.2,115.0. Anal(C 15 H 11 N3O4) is calculated as [M+H] + 298.0822; measured value 298.0818.

[0393] A mixture of 18 (38 mg, 0.11 mmol) and HCl in dioxane (4 M, 1 mL) was stirred at room temperature for 3 h 15 min and then evaporated. The residue was suspended in DMA (1.0 mL) and 56 (28 mg, 0.093 mmol), EDCI.HCl (63 mg, 0.33 mmol) and anhydrous toluenesulfonic acid (3.3 mg, 0.019 mmol) were added. The mixture was stirred at room temperature for 1.5 h. EtOAc (150 mL) and H2O (50 mL) were added, the EtOAc layer was separated, washed with H2O (×2), then dried and evaporated. The residue was triturated with MeOH to give 57 (30 mg, 63%); mp 232-235°C; 1H NMR(d6-DMSO)δ10.41(s,1H),10.17(s,2H),9.42(br s,1H),8.62(s,1H),7.97(br s,1H),7.88(d,J=8.7Hz,2H),7.69(d,J=9.7Hz,1H),7.56(dd,J=9.7,2.0Hz,1H),6.90(d,J=8.7Hz, 2H), 4.93 (t, J = 10.7Hz, 1H), 4.73-4.62 (m, 1H), 4.18-4.07 (m, 2H), 4.03-3.92 (m, 1H), 2.61 (s, 3H); 13 C NMR(d6-DMSO)δ165.3,164.7,161.4,160.8,141.4,141.3,141.2,140.9,138.6,136.2,129 .8,127.5,124.6,123.0,118.6,117.4,117.1,115.0,111.1,101.5,54.8,46.9,40.7,14.2. Anal(C 26 H 20 ClN5O5) is calculated as [M+H] + 518.1226; measured value 518.1223.

[0394] Example 6: Lipophilicity of the compounds of the present invention

[0395] The lipophilicity of representative compounds of the invention was calculated using the ChemDraw Professional v.17.0.0 software package (Perkin Elmer Informatics Inc). The results are provided in Figure 1 middle.

[0396]

[0397] The compounds of the present invention carry multiple DNA minor groove binding side chains: side chain A is a 5,6,7-trimethoxyindole structure found in the natural product of duocarmycin; side chain B has been used as the payload in ADC BMS-936561 (Biopharm. Drug Disp. (2016) 37, 93); and side chain C has been used as the payload open-DUBA in ADC SYD985 (Mol. Pharm. (2015) 12, 1813).

[0398] In each case, compounds containing the 2-methylbenzoxazolyl alkylated subunit had significantly lower calculated logP (approximately 1.5 units) than those incorporating the ring-opening-CBI alkylated subunit, and this applied whether the alkylated subunit was in the form of a phenol (X=OH) or an amine (X=NH2).

[0399] These substantial reductions in payload lipophilicity are likely responsible for the favorable properties of drug-linkers and ADCs incorporating 2-methylbenzoxazolidinylated subunits.

[0400] Table 1: Lipophilicity comparison

[0401]

[0402] Example 7: Cytotoxicity

[0403] The cytotoxicity of the payloads of the invention was determined by measuring the proliferation inhibition of two human tumor cell lines, cervical cancer SiHa and ovarian cancer SKOV3. Logarithmic phase monolayers were continuously exposed to the payloads for 5 days in 96-well plates and then stained with sulforhodamine B. 50 Determined by interpolation, as the drug concentration required to inhibit cell density to 50% of the untreated control on the same plate. Each plate contained the reference compound open-CBI-TMI as an internal control.

[0404] The data presented in Table 2 show that DNA alkylating agents containing a 2-methylbenzoxazolyl alkylating subunit are highly cytotoxic compounds with an IC 50 In the nM or sub-nM range, ie, in the range considered suitable for ADC applications.

[0405] Importantly, compound 23 and serotype-CBI-TMI have the same TMI minor groove binding side chain, allowing a head-to-head comparison of the effects of the alkylated subunits on cytotoxicity. In this comparison, the 2-methylbenzoxazolyl alkylated subunit produced the same cytotoxicity as serotype-CBI, which is noteworthy because the latter is the most potent of all known variants of duocarmycin-type alkylated subunits (J. Med. Chem. (2009) 52, 5771).

[0406] The significant cytotoxicity of 23 is surprising, since the closely related compound, s-COI-TMI, which differs only in the orientation of the oxazole ring fusion and the nature of the 2-substituent, is several hundred times less cytotoxic than a related reference compound (Bioorg. Med. Chem. Lett. (2010) 20, 1854).

[0407] Table 2: Comparison of cytotoxicity

[0408]

[0409] Example 8: Stability

[0410] The aqueous stability of 23 and the reference compound sec-CBI-TMI was investigated by LC-MS analysis. Samples containing the payload at a concentration of 4 μM in Tris buffer (pH 7.4) containing 10% DMF at 37°C were monitored at regular intervals over 300-500 min.

[0411] like Figure 1 As shown, the open-CBI-TMI undergoes a clean conversion to the cyclopropyl form (CBI-TMI), which is stable under these conditions. These observations are consistent with the reported behavior (ChemBioChem (2014) 15, 1998; J. Am. Chem. Soc. (1994) 116, 7996).

[0412] On the contrary, Figure 2 As shown, although 23 also undergoes conversion to the corresponding cyclopropyl form (identified by characteristic UV-visible absorption spectrum and mass spectrum), the compound is unstable under these conditions and quickly converted into various products. The UV-visible absorption spectrum and mass spectrum of the main product are consistent with hydrolysis, i.e., cyclopropane is opened by adding water. Such products cannot alkylate DNA and can be considered non-toxic. The instability of the cyclopropyl intermediate derived from the alkylated subunit of 2-methylbenzoxazol is very surprising, because there is a well-defined correlation between solvent decomposition stability and cytotoxicity, and it is reported that all other known alkylated subunits with cytotoxicity like CBI are stable to hydrolysis at neutral pH (J.Med.Chem. (2009) 52,5771). The instability of the payload containing the alkylated subunit of 2-methylbenzoxazol is advantageous in ADC applications by being used as a detoxification mechanism for any systemically released payload.

[0413] Example 9. (E)-1-(8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-6-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (58)

[0414]

[0415] Scheme 16

[0416] A mixture of 18 (31.3 mg, 0.092 mmol) and HCl in dioxane (4 M, 1 mL) was stirred at room temperature for 4 h and then evaporated. The residue was suspended in DMA (1.5 mL) and 4-methoxycinnamic acid (18 mg, 0.10 mmol) and EDCI.HCl (53 mg, 0.28 mmol) were added. The mixture was stirred at room temperature for 19 h. Water was slowly added and the precipitated solid was filtered off and dried to give 58 as a light brown solid (10 mg, 27%); 1 H NMR(d6-DMSO)δ10.39(s,1H),7.96(s,1H),7.80-7.70(m,2H),7.60(d,J=15.3Hz,1H),7.07-6.97(m,3H),4.6 3-4.54(m,1H),4.32-4.25(m,1H),4.17-4.08(m,2H),3.98(dd,J=10.8,7.5Hz,1H),3.31(s,3H),2.60(s,3H). Anal(C 21 H 19 ClN2O4) calculated as [M+H] + 399.1106; measured value 399.1108.

[0417] Example 10. (E)-1-(8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-6-yl)-3-(3-hydroxy-4-methoxyphenyl)prop-2-en-1-one (59)

[0418]

[0419] Solution 17

[0420] A mixture of 18 (30.1 mg, 0.089 mmol) and HCl in dioxane (4M, 1 mL) was stirred at room temperature for 4 h and then evaporated. The residue was suspended in DMA (1.0 mL) and 3-hydroxy-4-methoxycinnamic acid (19 mg, 0.10 mmol) and EDCI.HCl (51 mg, 0.27 mmol) were added. The mixture was stirred at room temperature for 3 h, then diluted with water and extracted with EtOAc (×2). The combined extracts were washed with water (×3) and brine, then dried and evaporated. The residue was purified with a small amount of EtOAc (ca.1 mL) to give 59 as a very light green solid (7.7 mg, 21%); 1H NMR(d6-DMSO)δ10.41(s,1H),9.14(s,1H),7.96(s,1H),7.51(d,J=15.3Hz,1H),7.24(d,J=2.0Hz,1H),7.16(dd,J=8.4,2.0Hz,1H),6.96(d,J=8.4 Hz,1H),6.91(d,J=15.3Hz,1H),4.63-4.54(m,1H),4.31-4.24(m,1H),4. 16-4.06(m,2H),3.95(dd,J=11.0,7.8Hz,1H),3.83(s,3H),2.60(s,3H); 13 C NMR(d6-DMSO)δ164.6,163.5,149.6,146.6,142.4,141.3,141.2,138.6,136.0, 127.8,121.2,117.2,114.2,111.9,110.7,101.1,55.6,52.8,47.0,40.3,14.2. Anal(C 21 H 19 ClN2O5) is calculated as [M+H] + 415.1055; measured value 415.1060.

[0421] Example 11. (E)-N-(2-(8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indole-6-carbonyl)-1H-indol-5-yl)-3-(4-methoxyphenyl)acrylamide (63)

[0422]

[0423] Scheme 18

[0424] A mixture of ethyl 5-aminoindole-2-carboxylate (60) (354 mg, 1.73 mmol), 4-methoxycinnamic acid (309 mg, 1.73 mmol) and EDCI.HCl (0.67 g, 3.46 mmol) in DMA (4 mL) was stirred at room temperature for 18 hours. Water was added and the precipitated solid was filtered off, washed with water and dried. The crude product was stirred with hot EtOH (40 mL) and the suspension was cooled. The solid was filtered off and dried to give (E)-ethyl 5-(3-(4-methoxyphenyl)acrylamido)-1H-indole-2-carboxylate (61) as a cream solid (506 mg, 80%); mp 244-247°C; 1H NMR(d6-DMSO)δ11.82(s,1H),10.03(s,1H),8.15(s,1H),7.58(d,J=8.6Hz,2H),7.53(d,J=15.6Hz,1H),7.48-7.37(m,2H ), 7.12 (s, 1H), 7.02 (d, J = 8.6Hz, 2H), 6.70 (d, J = 15.6Hz, 1H), 4.34 (q, J = 7.1Hz, 2H), 3.80 (s, 3H), 1.35 (t, J = 7.1Hz, 3H). Anal(C 21 H 20 N2O4·1 / 4H2O) calculated as C, 68.37; H, 5.60; N, 7.59%; found C, 68.56; H, 5.53; N, 7.66%.

[0425] A solution of KOH (0.40 g, 7.1 mmol) in water (6 mL) was added to a suspension of 61 (480 mg, 1.32 mmol) in ethanol (15 mL), and the mixture was heated under reflux for 5 minutes. The solution was cooled to room temperature and acidified with aqueous HCl (2N, 5 mL). The resulting suspension was diluted with water, and the solid was filtered off, washed with water, and dried to give (E)-5-(3-(4-methoxyphenyl)acrylamido)-1H-indole-2-carboxylic acid (62) as a light brown solid (414 mg, 93%); mp 276-279°C; 1 H NMR(d6-DMSO)δ12.90(br s,1H),11.68(s,1H),10.01(s,1H),8.14(s,1H),7.57(d,J=8.8Hz,2H),7.53(d,J=15.6Hz,1H),7. 45-7.35(m,2H),7.06(d,J=1.8Hz,1H),7.01(d,J=8.8Hz,2H),7.21(d,J=15.6Hz,1H),3.80(s,3H). Anal(C 19 H 16 N2O4) calculated as C, 67.85; H, 4.79; N, 8.33%; found C, 68.09; H, 4.77; N, 8.39%.

[0426] A mixture of 18 (30.0 mg, 0.089 mmol) and HCl in dioxane (4 M, 1 mL) was stirred at room temperature for 4 h and then evaporated. The residue was suspended in DMA (1.0 mL) and 62 (33 mg, 0.10 mmol) and EDCI.HCl (51 mg, 0.27 mmol) were added. The mixture was stirred at room temperature for 3 h. Water was slowly added, and the precipitated solid was filtered off and dried. The crude product was triturated with EtOAc to give 63 as a light brown solid (24 mg, 49%); 1 H NMR(d6-DMSO)δ11.67(s,1H),10.46(s,1H),10.04(s,1H),8.20(s,1H),7.91(s,1H) ,7.58(d,J=8.8Hz,2H),7.54(d,J=15.6Hz,1H),7.43-7.36(m,2H),7.15(d,J=2.1Hz, 1H),7.02(d,J=8.8Hz,2H),6.72(d,J=15.6Hz,1H),4.80(t,J=10.2Hz,1H),4.47(dd, J=10.9,5.1Hz,1H),4.23-4.10(m,2H),4.06-3.99(m,1H),3.80(s,3H),2.62(s,3H); 13 C NMR(d6-DMSO)δ164.8,163.5,160.5,159.8,141.3,141.2,139.1,138.6,136.3,133.0,132.3,131.3,129 .2,127.5,127.2,120.2,118.1,114.5,112.3,111.5,111.1,105.6,101.6,55.3,54.5,46.9,40.8,14.2. Anal(C 30 H 25 ClN4O5) is calculated as [M+H] + 557.1586; measured value 557.1593.

[0427] Example 12. (8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-6-yl)(5-methoxybenzofuran-2-yl)methanone (64)

[0428]

[0429] Compound 64 was prepared from 18 by the same general procedure as described in Examples 3 and 4 and isolated as a grey solid; 1H NMR(d6-DMSO)δ10.50(s,1H),7.85(br s,1H),7.65(d,J=9.2Hz,1H),7.63(s,1H),7.30(d,J=2.6Hz,1H),7.10(dd,J=9.0,2.6Hz,1H),4.79(t,J=10.4H z,1H),4.46(dd,J=11.2,5.1Hz,1H),4.20-4.09(m,2H),4.02(dd,J=10.7,7.2Hz,1H),3.33(s,3H),2.62(s,3H). Anal(C 21 H 17 ClN2O5) is calculated as [M+H] + 413.0899; measured value 413.0899.

[0430] Example 13. (8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-6-yl)(5-methoxybenzo[b]thiophen-2-yl)methanone (65)

[0431]

[0432] Solution 19

[0433] A mixture of 18 (40.0 mg, 0.12 mmol) and HCl in dioxane (4 M, 2 mL) was stirred at room temperature for 1.5 h and then evaporated. The residue was suspended in DMA (1.5 mL) and 5-methoxybenzo[b]thiophene-2-carboxylic acid (25 mg, 0.12 mmol), EDCI.HCl (68 mg, 0.36 mmol) and toluenesulfonic acid (4 mg, 0.024 mmol) were added. The mixture was stirred at room temperature for 3 h. Water was slowly added and the precipitated solid was filtered off and dried. The crude product was triturated with EtOAc to give 65 as a cream solid (35 mg, 69%); 1 H NMR(d6-DMSO)δ10.49(s,1H),8.02(s,1H),7.94(d,J=8.9Hz,1H),7.79(brs,1H),7.53(d,J=2.5Hz,1H),7.14(dd,J=8.9,2.5H z,1H),4.74(t,J=10.0Hz,1H),4.40(dd,J=10.8,5.2Hz,1H),4.20-4.08(m,2H),4.05-3.98(m,1H),3.85(s,3H),2.62(s,3H); 13C NMR(d6-DMSO)δ164.9,160.7,157.4,141.3,140.8,140.21,140.17,138.6,136.5 ,132.2,126.5,123.3,117.1,111.4,106.9,101.5,55.3,55.1,46.7,40.8,14.2. Anal(C 21 H 17 ClN2O4S) is calculated as [M+H] + 429.0670; measured value 429.0677.

[0434] Example 14. (8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-6-yl)(5-(2-methoxyethoxy)-1H-indol-2-yl)methanone (66)

[0435]

[0436] Solution 20

[0437] A mixture of 18 (36 mg, 0.106 mmol) and HCl in dioxane (4 M, 1 mL) was stirred at room temperature for 4 h and then evaporated. The residue was suspended in DMA (1.0 mL) and 5-(2-methoxyethoxy)indole-2-carboxylic acid (27.5 mg, 0.12 mmol), EDCI.HCl (61 mg, 0.32 mmol) and toluenesulfonic acid (3.7 mg, 0.021 mmol) were added. The mixture was stirred at room temperature for 2 h. Water was slowly added, and the precipitated solid was filtered off, washed with water and dried. The crude product was triturated with EtOAc to give 66 as a gray solid (28 mg, 58%); 1 H NMR (d6-DMSO) δ11.58 (d, J=1.5 Hz, 1H), 10.45 (s, 1H), 7.92 (s, 1H), 7.39 (d, J=8.9 Hz, 1H), 7.16 (d, J=2.3 Hz, 1H), 7.04 (d, J=1.7 Hz, 1H), 6.90 (dd, J=8.9, 2.4 Hz, 1H), 4.78 (t, J=10.1 Hz, 1H), 4.45 (dd, J=10.8, 5.1 Hz, 1H), 4.21-4.06 (m, 2H), 4.04-3.97 (m, 1H), 3.71-3.66 (m, 2H), 2.62 (s, 3H) (one 3H s is obscured by the water peak at δ3.3); 13C NMR(d6-DMSO)δ164.8,159.9,152.9,141.3,141.2,138.6,136.3,131.6,131.0,127.5 ,115.7,113.2,111.0,105.2,103.1,101.6,70.6,67.2,58.2,54.6,46.9,40.9,14.2. Anal(C 23 H 22 ClN3O5) is calculated as [M+H] + 456.1321; measured value 456.1323.

[0438] Example 15. N-(2-(8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indole-6-carbonyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)acetamide (70)

[0439]

[0440] Solution 21

[0441] Acetyl chloride (0.12 mL, 1.7 mmol) was added to a mixture of 5-amino-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid methyl ester (67) (165 mg, 0.86 mmol) and Et3N (0.36 mL, 2.6 mmol) in CH2Cl2 (8 mL) and THF (10 mL) at 0°C. The ice bath was removed and the mixture was stirred for 1 h and then diluted with water. The organic solvent was evaporated to leave an aqueous suspension. The solid was filtered off and dried to give 5-acetamido-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid methyl ester (68) as a white solid (178 mg, 89%); 1 H NMR(d6-DMSO) δ12.42(s,1H),10.07(s,1H),8.42(s,2H),7.15(s,1H),3.87(s,3H),2.07(s,3H). Anal(C 11 H 11 N3O3) is calculated as [M+H] + 234.0873; measured value 234.0868.

[0442] A solution of KOH (205 mg, 3.6 mmol) in water (3 mL) was added to a suspension of 68 (167 mg, 0.72 mmol) in MeOH (6 mL), and the mixture was heated under reflux for 2 min and then cooled to room temperature. Aqueous HCl (2N, 1.6 mL) was added, and the precipitated solid was filtered off and dried to give 5-acetamido-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (69) as a cream solid (134 mg, 85%); 1 H NMR (d6-DMSO) δ13.08 (br s, 1H), 12.21 (s, 1H), 10.04 (s, 1H), 8.40 (d, J = 2.3Hz, 1H), 8.38 (d, J = 2.3Hz, 1H), 7.06 (d, J = 2.0Hz, 1H), 2.07 (s, 3H). Anal(C 10 H9N3O3) is calculated as [M+H] + 220.0717; measured value 220.0712.

[0443] A mixture of 18 (39 mg, 0.12 mmol) and HCl in dioxane (4 M, 2 mL) was stirred at room temperature for 1.5 h and then evaporated. The residue was suspended in DMA (1.0 mL) and 69 (25 mg, 0.12 mmol), EDCI.HCl (66 mg, 0.36 mmol) and toluenesulfonic acid (15 mg, 0.09 mmol) were added. The mixture was stirred at room temperature for 3 h. Water was slowly added and the precipitated solid was filtered off, washed with water and dried. The crude product was triturated with EtOAc to give 70 as a brown solid (31 mg, 61%); 1 H NMR(d6-DMSO)δ12.15(s,1H),10.47(s,1H),10.06(s,1H),8.42(d,J=2.4Hz,1H),8.40(d,J=2.3Hz,1H),7.87(br s,1H),7.11(d,J=2.1Hz,1H),4.74(t,J=10.1Hz,1H),4.40(dd,J=10.9,5. 1Hz,1H),4.20-4.08(m,2H),4.05-3.96(m,1H),2.62(s,3H),2.08(s,3H); 13 C NMR(d6-DMSO)δ168.4,164.8,159.7,144.7,141.3,141.0,139.5,138.6,136.3, 132.0,129.8,120.1,118.7,111.2,103.8,101.5,54.6,46.8,40.7,23.7,14.2. Anal(C21 H 18 ClN5O4) calculated as [M+H] + 440.1120; measured value 440.1123.

[0444] Example 16. N-(2-(8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indole-6-carbonyl)imidazo[1,2-a]pyridin-6-yl)acetamide (74)

[0445]

[0446] Solution 22

[0447] Acetyl chloride (0.11 mL, 1.6 mmol) was added to a mixture of ethyl 6-aminoimidazo[1,2-a]pyridine-2-carboxylate (71) (161 mg, 0.78 mmol) and Et3N (0.33 mL, 2.3 mmol) in CH2Cl2 (10 mL) at room temperature. After stirring for 5 minutes, the mixture was diluted with water and the organic solvent was evaporated to leave an aqueous suspension. The solid was filtered off and dried, then triturated with EtOAc to give ethyl 6-acetamidoimidazo[1,2-a]pyridine-2-carboxylate (72) as a light green solid (99 mg, 51%); 1 H NMR(d6-DMSO)δ10.14(s,1H),9.25(s,1H),8.62(s,1H),7.58(d,J=9.7Hz,1H),7.2 4 (dd, J = 9.7, 2.0 Hz, 1H), 4.29 (q, J = 7.1 Hz, 2H), 2.08 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H). Anal(C 12 H 13 N3O3) is calculated as [M+H] + 248.1030; measured value 248.1021.

[0448] A solution of KOH (122 mg, 2.2 mmol) in water (3 mL) was added to a suspension of 72 (96 mg, 0.39 mmol) in MeOH (5 mL) and the mixture was stirred at room temperature for 4 h. MeOH was evaporated and the aqueous residue was acidified with HCl (2N, 1.0 mL). The precipitated solid was filtered off and dried to give 6-acetamidoimidazo[1,2-a]pyridine-2-carboxylic acid (73) as a light brown solid (71 mg, 83%); 1HNMR (d6-DMSO) δ10.13 (s, 1H), 9.25 (s, 1H), 8.55 (s, 1H), 7.58 (d, J = 9.7 Hz, 1H), 7.23 (dd, J = 9.7, 2.0 Hz, 1H), 2.09 (s, 3H) (CO2H protons not observed). Anal (C 10 H9N3O3) is calculated as [M+H] + 220.0717; measured value 220.0709.

[0449] A mixture of 18 (43 mg, 0.13 mmol) and HCl in dioxane (4 M, 1 mL) was stirred at room temperature for 3.5 h and then evaporated. The residue was suspended in DMA (1.0 mL) and 73 (27.8 mg, 0.13 mmol), EDCI.HCl (73 mg, 0.39 mmol) and toluenesulfonic acid (4.4 mg, 0.03 mmol) were added. The mixture was stirred at room temperature for 3 h. Water was added slowly to give an emulsion. Aqueous NaHCO3 solution was added and the mixture was extracted with EtOAc (×4). The combined extracts were washed with water and brine, then dried and evaporated. The residue was triturated with EtOAc to give 74 as an off-white solid (25.5 mg, 46%); 1 H NMR(d6-DMSO)δ10.41(s,1H),10.15(s,1H),9.29(d,J=1.0Hz,1H),8.58(s,1H),7.95(br s,1H),7.67(d,J=9.7Hz,1H),7.25(dd,J=9.7,2.0Hz,1H),4.96-4.87(m,1H),4.66(dd ,J=12.2,5.0Hz,1H),4.16-4.06(m,2H),4.01-3.93(m,1H),2.60(s,3H),2.10(s,3H); 13 C NMR (d6-DMSO) δ 168.6, 164.7, 161.4, 141.4, 141.2, 140.8, 138.6, 136.2, 127.2, 121.8, 118.6, 117.8, 116.0, 111.1, 101.5, 54.8, 46.9, 40.7, 23.7, 14.2 (one C was not observed). Anal (C 21 H 18 ClN5O4) calculated as [M+H] + 440.1120; measured value 440.1139.

[0450] Example 17. 4-Acetylamino-N-(2-(8-(chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indole-6-carbonyl)-1H-indol-5-yl)-1-methyl-1H-imidazole-2-carboxamide (79)

[0451]

[0452] Solution 23

[0453] Pd / C (10%, 90 mg) was added to a solution of 1-methyl-4-nitro-1H-pyrrole-2-carboxylic acid ethyl ester (75) (254 mg, 1.28 mmol) in EtOH (25 mL) and the mixture was hydrogenated at 50 psi for 2 h. The mixture was filtered through diatomaceous earth, washed with EtOAc, and the filtrate was evaporated. The residue was dissolved in CH2Cl2 (6 mL) and the solution was cooled in an ice bath. Et3N (0.54 mL, 3.8 mmol) and acetyl chloride (0.18 mL, 2.6 mmol) were added and the ice bath was removed. The mixture was stirred for 10 min, then diluted with water and extracted with CH2Cl2 (×2). The extract was washed with dilute aqueous HCl solution and water, then dried and evaporated. The residue was recrystallized from EtOH to give ethyl 4-acetamido-1-methyl-1H-pyrrole-2-carboxylate (76) as a light yellow solid (65 mg, 24%); mp 165-168° C. The aqueous phase from the liquid-liquid extraction was basified with aqueous NaHCO3 and extracted with EtOAc (×4). The extract was dried, combined with the mother liquor from the recrystallization, and evaporated. The residue was purified by column chromatography (EtOAc: petroleum ether 1:1, then 4:1, then EtOAc alone) to give more 76 (139 mg, 52%); 1 H NMR (CDCl3) δ7.91 (brs, 1H), 7.47 (s, 1H), 4.41 (q, J = 7.1Hz, 2H), 3.99 (s, 3H), 2.14 (s, 3H), 1.42 (t, J = 7.1Hz, 3H). Anal(C9H 13 N3O3) is calculated as [M+H] + 212.1030; measured value 212.1026.

[0454] A solution of KOH (218 mg, 3.9 mmol) in water (2 mL) was added to a solution of 76 (185 mg, 0.88 mmol) in MeOH (4 mL), and the mixture was stirred at room temperature for 30 min. The MeOH was evaporated, the aqueous layer was neutralized with aqueous HCl (2N, 1.3 mL), and then evaporated to dryness. Aminoindole 60 (179 mg, 0.88 mmol), EDCI.HCl (0.50 g, 2.6 mmol) and DMA (2 mL) were added, and the mixture was stirred at room temperature for 29 h. The mixture was diluted with water and extracted with EtOAc (×2). The extract was washed with water (×3), then dried and evaporated. The residue was triturated with EtOAc to give ethyl 5-(4-acetylamino-1-methyl-1H-imidazole-2-carboxamido)-1H-indole-2-carboxylate (77) as a cream solid (59 mg, 18% over 2 steps); 1 H NMR(d6-DMSO)δ11.85(s,1H),10.36(s,1H),9.74(s,1H),8.13(d,J=1.7Hz,1H),7.49(dd,J=8.9,2.0Hz,1H),7.47(s,1H ), 7.41 (d, J = 8.9Hz, 1H), 7.12 (d, J = 1.3Hz, 1H), 4.33 (q, J = 7.1Hz, 2H), 3.97 (s, 3H), 2.03 (s, 3H), 1.34 (t, J = 7.1Hz, 3H). Anal(C 18 H 19 N5O4) is calculated as [M+H] + 370.1510; measured value 270.1505.

[0455] A solution of KOH (89 mg, 1.6 mmol) in water (1 mL) was added to a suspension of 77 (54 mg, 0.15 mmol) in MeOH (8 mL) and the mixture was stirred at 50 °C for 3 h and then at reflux for 1.5 h. The mixture was cooled, the MeOH was evaporated, and the aqueous residue was acidified with aqueous HCl (2N, 0.8 mL). The precipitated solid was filtered off and dried to give 5-(4-acetamido-1-methyl-1H-imidazole-2-carboxamido)-1H-indole-2-carboxylic acid (78) as a grey solid (46 mg, 92%); 1HNMR(d6-DMSO)δ12.95(br s,1H),11.71(s,1H),10.37(s,1H),9.72(s,1H),8.11(s,1H),7.50-7.43(m, 2H), 7.39 (d, J = 8.9 Hz, 1H), 7.06 (d, J = 1.6 Hz, 1H), 3.97 (s, 3H), 2.02 (s, 3H). Anal(C 16 H 15 N5O4) is calculated as [M+H] + 342.1197; measured value 342.1204.

[0456] A mixture of 18 (39.4 mg, 0.12 mmol) and HCl in dioxane (4 M, 1 mL) was stirred at room temperature for 3.5 h and then evaporated. The residue was suspended in DMA (1.0 mL) and 78 (39.7 mg, 0.12 mmol), EDCI.HCl (67 mg, 0.36 mmol) and toluenesulfonic acid (4 mg, 0.02 mmol) were added. The mixture was stirred at room temperature for 5 h. Water was slowly added and the precipitated solid was filtered off, washed with water and dried. The crude product was triturated with EtOAc to give 79 as an off-white solid (36 mg, 55%); 1 HNMR(d6-DMSO)δ11.71(s,1H),10.46(s,1H),10.37(s,1H),9.75(s,1H),8.12 (d,J=1.7Hz,1H),7.92(s,1H),7.54(dd,J=8.9,2.0Hz,1H),7.48-7.43(m,2H) ,7.15(d,J=1.7Hz,1H),4.80(t,J=10.2Hz,1H),4.46(dd,J=10.9,5.1Hz,1H), 4.22-4.10(m,2H),4.05-3.99(m,1H),3.97(s,3H),2.63(s,3H),2.04(s,3H); 13 C NMR(d6-DMSO)δ167.2,164.8,159.8,156.8,141.3,141.2,138.6,136.3,134.0,133.3,131.4,13 1.0,127.1,118.8,114.0,112.4,112.3,111.1,105.5,101.6,54.5,46.9,40.8,35.0,22.7,14.2. Anal(C 27 H 24 ClN7O5) is calculated as [M+H] + 562.1600; measured value 562.1579.

[0457] Example 18. (S)-(8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-6-yl)(5,6,7-trimethoxy-1H-indol-2-yl)methanone (82) and (R)-(8-(Chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-6-yl)(5,6,7-trimethoxy-1H-indol-2-yl)methanone (83)

[0458]

[0459] Solution 24

[0460] Compound 18 (334 mg) was separated by preparative chiral HPLC (Daicel Chiralpak IA 250×21 mm column, EtOH:hexane 15:85 eluent, 6 mL / min flow rate, 1.43) to obtain baseline separation of the two enantiomers. The fractions containing the faster eluting enantiomer (R T 9.4 min) and evaporated to give (S)-8-(chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indole-6-carboxylate (80) as a white solid (131 mg, 39%); [] D = -31° (c 0.584, CH2Cl2); 1 H NMR (CDCl3) was the same as described for 18. Fractions containing the slower eluting enantiomer (R T 13.4 min) and evaporated to give (R)-8-(chloromethyl)-4-hydroxy-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indole-6-carboxylic acid tert-butyl ester (81) as a white solid (130 mg, 39%); [] D = +24° (c 0.586, CH2Cl2); 1 HNMR (CDCl3) was the same as described for 18. Reanalysis on an analytical column (Daicel Chiralpak IA 150 x 4.6 mm column) confirmed 100% ee for each enantiomer.

[0461] Compound 80 was converted to 82 by the general method described to give a white solid. 23 H 22 ClN3O6) calculated as [M+H] + 472.12699; found 472.12721. Compound 81 was converted to 83 by the general method described to give a white solid; 1H NMR (d6-DMSO) was the same as described for 23. Anal (C 23 H 22 ClN3O6) calculated as [M+H] + 472.12699; measured value 472.12719.

[0462] Example 19. 8-(Chloromethyl)-2-methyl-6-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-7,8-dihydro-6H-oxazolo[4,5-e]indol-4-yl(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoylamino)-3-methylbutanoylamino)-5-ureidopentanamido)benzyl)ethane-1,2-diylbis(methylcarbamate) (26)

[0463]

[0464] Solution 25

[0465] Phenyl p-nitrochloroformate (97%, 20 mg, 0.10 mmol) and Et3N (34 μL, 0.25 mmol) were added to a solution of 23 (23 mg, 0.049 mmol) in anhydrous THF (5 mL) and DMF (1.5 mL) at 0°C. After 1.5 h, phenyl p-nitrochloroformate (97%, 10 mg, 0.05 mmol) and Et3N (17 μL, 0.12 mmol) were added, and after another 50 min, tert-butyl methyl (2-(methylamino)ethyl)carbamate (95%, 40 mg, 0.21 mmol) was added. The ice bath was removed and the mixture was stirred for another 20 h before being evaporated to dryness. The residue was purified by column chromatography (EtOAc:petroleum ether 1:4, then 1:3, 1:1, 3:1) to give tert-butyl(8-(chloromethyl)-2-methyl-6-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-7,8-dihydro-6H-oxazolo[4,5-e]indol-4-yl)ethane-1,2-diylbis(methylcarbamate) (24) as a colorless oil (31 mg, 94%); 1HNMR (CDCl3) (some signals are split due to rotational isomers) δ 9.37 (s, 1H), 8.24 and 8.23 ​​(2s, 1H), 6.97 (d, J = 2.3 Hz, 1H), 6.87 (s, 1H), 4.77 (t, J = 9.6 Hz, 1H), 4.61 (dd, J = 10.7, 4.6 Hz, 1H), 4.29-4.21 (m, 2H), 4.091 and 4.090 (2s, 3H), 3.95 (s, 3H), 3.92 (s, 3H), 3.76-3.34 (m, 5H), 3.21 and 3.08 (2s, 3H), 3.01-2.84 (m, 3H), 2.65 (s, 3H), 1.53-1.42 (m, 9H). Anal (C 33 H 40 ClN5O9) is calculated as [M+H] + 686.2587; measured value 686.2573.

[0466] At 0 ° C, TFA (0.7 mL, 9.1 mmol) was added to a solution of 24 (31 mg, 0.045 mmol) in CH2Cl2 (0.7 mL). After 30 minutes, the mixture was evaporated to dryness at room temperature, and the residue was dissolved in CH2Cl2 and evaporated again. The residue was dissolved in DMF (0.5 mL) and the solution was cooled in an ice bath. 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoylamino)-3-methylbutanoylamino)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (25) (36.7 mg, 0.050 mmol) and Et3N (32 μL, 0.23 mmol) were added. The ice bath was removed and the mixture was stirred for 18 h, then evaporated to dryness at room temperature. The residue was purified by column chromatography (CH2Cl2, then CH2Cl2:MeOH 100:1, then 50:1, 30:1, 15:1) to afford recovered 23 (6 mg, 28%) and crude product. The latter was purified by column chromatography (EtOAc:MeOH 9:1) to afford 26 as a light yellow glass (22 mg, 41%); 1H NMR (d6-DMSO) (some signals are split due to rotamers) δ 11.48-11.37 (m, 1H), 9.99-9.90 (m, 1H), 8.08 and 8.06 (2s, 1H), 8.05-7.97 (m, 1H), 7.81 and 7.79 (2s, 1H), 7.60-7.46 (m, 2H), 7.36-7.16 (m, 2H), 7.07-7.02 (m, 1H), 7.00 (s, 2H), 6.96 (s, 1H), 5.96 (t, J = 5.5 Hz, 1H), 5.40 (s, 2H), 5.08-4.95 (m, 2H) ,4.81-4.72(m,1H),4.48-4.25(m,3H),4.21-4.06(m,3H),3.93(s,3H),3.32(s,3H),3.28(s,3H),3.67-3.44(m,4H),3.36(t,J=7.1Hz,2 H),3.14-2.85(m,8H),2.67-2.57(m,3H),2.23-2.06(m,2H),2.01-1.90(m,1H),1.74-1.31(m,8H),1.24-1.13(m,2H),0.87-0.76(m,6H). Anal(C 57 H 70 C1N 11 O 15 ) is calculated as [M+H] + 1184.4814; measured value 1184.4840.

[0467] Example 20. 8-(Chloromethyl)-2-methyl-6-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-7,8-dihydro-6H-oxazolo[4,5-e]indol-4-yl(2-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)propionamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(2-(2-hydroxyethoxy)ethyl)carbamate (87)

[0468]

[0469] Scheme 26

[0470] At 0°C, phenyl p-nitrochloroformate (97%, 39 mg, 0.18 mmol) and Et3N (64 μL, 0.46 mmol) were added to a solution of 23 (43.5 mg, 0.092 mmol) in DMF (4 mL). Additional portions of phenyl p-nitrochloroformate (97%, 19 mg, 0.18 mmol) were added after 45 min and 2 h, and more Et3N (32 μL, 0.28 mmol) was added after 70 min. After 2.5 h, a solution of tert-butyl (2-((2-(2-hydroxyethoxy)ethyl)amino)ethyl)(methyl)carbamate (84) (73 mg, 0.28 mmol) in DMF (1 mL) was added and the ice bath was removed. The mixture was stirred for 18 h and then evaporated to dryness. The residue was triturated with CH2Cl2, the solid was filtered off and dried to recover 23 (23 mg, 53%). The filtrate was evaporated and the residue was purified by column chromatography (EtOAc:petroleum ether 1:1, then 3:1, then EtOAc only, then EtOAc:MeOH 20:1) to give 8-(chloromethyl)-2-methyl-6-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-7,8-dihydro-6H-oxazolo[4,5-e]indol-4-yl (2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(2-(2-hydroxyethoxy)ethyl)carbamate (85) as a colorless oil (20 mg, 29%); 1 H NMR (CDCl3) (some signals split due to rotamers) δ 9.39 (s, 1H), 8.36, 8.34 and 8.29 (3s, 1H), 6.97 (d, J = 2.3 Hz, 1H), 6.87 (s, 1H), 4.82-4.74 (m, 1H), 4.64-4.57 (m, 1H), 4.29-4.20 (m, 2H), 4.09 (s, 3H), 3.95 (s, 3H), 3.92 (s, 3H), 3.85-3.44 (m, 14H), 2.99-2.86 (m, 3H), 2.66 (s, 3H), 1.54-1.42 (m, 9H). Anal (C 36 H 46 ClN5O 11 ) is calculated as [M+H] + 760.2955; measured value 760.2940.

[0471] Compound 85 (31 mg, 0.041 mmol) is treated with CH2Cl2 (0.7 mL) and a mixture of TFA (0.7 mL, 9.1 mmol) at 0 ° C for 15 min. The mixture is evaporated to dryness at room temperature and the residue is dissolved in CH2Cl2 and evaporated again. The residue is dissolved in DMF (0.5 mL) and the solution is cooled in an ice bath. ((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxobutyl-2-yl)carbamic acid tert-butyl ester (86) (23 mg, 0.041 mmol) and Et3N (28 μ L, 0.21 mmol) are added and the ice bath is removed. After 2.5 h, the mixture was evaporated and the residue was purified by column chromatography (EtOAc:petroleum ether 2:3, then 3:2, then EtOAc only, then EtOAc:MeOH 20:1) to afford recovered 23 (4.3 mg, 22%) and 87 as a colorless glass (24.8 mg, 56%); 1 H NMR (CDCl3) (some signals are split due to rotamers) δ 9.64-9.44 (m, 1H), 8.96-8.74 (m, 1H), 8.28-8.02 (m, 1H), 7.63-7.46 (m, 2H), 7.36-7.23 (m, 1H), 6.97 (s, 1H), 6.90-6.80 (m, 2H), 5.19-5.06 (m, 2H), 4.80-4.72 (m, 1H), 4.67 -4.56(m,2H),4.28-4.20(m,2H),4.08(s,3H),4.02-3.93(m,1H),3.94(s,3H),3.91(s,3H),3.84-3.4 1(m,13H),3.05-2.95(m,3H),2.66-2.61(m,3H),2.21-2.11(m,1H),1.44(s,12H),0.98-0.89(m,6H). Anal(C 52 H 67 ClN8O 15 ) is calculated as [M+H] + 1079.4487; measured value 1079.4489.

[0472] Example 21. 8-(Chloromethyl)-6-(5-(2-(dimethylamino)ethoxy)-1H-indole-2-carbonyl)-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-4-yl(2-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)propionamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(2-(2-hydroxyethoxy)ethyl)carbamate (89)

[0473]

[0474] Solution 27

[0475] At 0°C, phenyl p-nitrochloroformate (97%, 34.5 mg, 0.16 mmol) and Et3N (88 μL, 0.63 mmol) were added to a solution of 52 (59.3 mg, 0.126 mmol) in THF (6 mL) and DMF (1.5 mL). After 30 minutes, more phenyl p-nitrochloroformate (97%, 34.5 mg, 0.16 mmol) was added, and after 2.5 hours, a solution of 84 (66 mg, 0.32 mmol) in THF (1 mL) was added. The ice bath was removed and the mixture was stirred for 18 h and then evaporated to dryness. The residue was triturated with CH2Cl2, the solid was filtered off and dried to afford recovered 52 as the hydrochloride salt (21 mg, 32%). The filtrate was evaporated and the residue was purified by column chromatography (EtOAc only, then EtOAc:MeOH 10:1, then 5:1, then 3:2) to give 8-(chloromethyl)-6-(5-(2-(dimethylamino)ethoxy)-1H-indole-2-carbonyl)-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-4-yl(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(2-(2-hydroxyethoxy)ethyl)carbamate (88) as a colorless oil (30 mg, 31%); 1 H NMR (CDCl3) (some signals split due to rotational isomers) δ 9.52-9.41 (m, 1H), 8.26 and 8.23 ​​(2s, 1H), 7.34 (d, J = 8.9 Hz, 1H), 7.14 (d, J = 2.2 Hz, 1H), 7.03 (dd, J = 8.9, 2.3 Hz, 1H), 6.97 (d, J = 1.4 Hz, 1H), 4.81-4.74 (m, 1H), 4.64-4.57 (m, 1H), 4.28-4.16 (m, 4H), 3.83-3.41 (m, 14H), 2.97-2.85 (m, 5H), 2.65 (s, 3H), 2.45 (s, 6H), 1.52-1.42 (m, 9H). Anal (C 37 H49 ClN6O9) calculated as [M+H] + 757.3322; measured value 757.3332.

[0476] Compound 88 (30 mg, 0.040 mmol) was treated with a mixture of CH2Cl2 (0.7 mL) and TFA (0.7 mL, 9.1 mmol) at 0°C for 15 min. The mixture was evaporated to dryness at room temperature and the residue was dissolved in CH2Cl2 and evaporated again. The residue was dissolved in DMF (0.5 mL) and the solution was cooled in an ice bath. Compound 86 (22 mg, 0.040 mmol) and Et3N (33 μL, 0.24 mmol) were added and the ice bath was removed. After stirring for 2 hours, the mixture was evaporated and purified by column chromatography (CH2Cl2:MeOH 50:1, then 20:1, 10:1, 8:1; then eluted with a second column with EtOAc:MeOH 10:1, then 5:1, 3:1, 3:2) to give 89 as a colorless glass (7.8 mg, 18%); 1 H NMR (CDCl3) (some signals are split due to rotational isomers) δ 10.01-9.47 (m, 1H), 8.95-8.61 (m, 1H), 8.27-8.06 (m, 1H), 7.61-7.44 (m, 2H), 7.41-7.19 (m, 2H), 7.14 (d, J = 2.2 Hz, 1H), 7.06-6.95 (m, 2H), 5.19-4.99 (m, 2H), 4.83-4.73 (m, 1H), 4.71-4.57 ( m,2H),4.28-4.17(m,2H),4.14(t,J=5.7Hz,2H),4.02-3.92(m,1H),3.83-3.34(m,14H),3.05-2.93(m,3H),2. 80(t,J=5.7Hz,2H),2.67-2.61(m,3H),2.38(s,6H),2.23-2.14(m,1H),1.47-1.38(m,12H),1.00-0.90(m,6H). Anal(C 53 H 70 ClN9O 13 ) is calculated as [M+H] + 1076.4854; measured value 1076.4857.

[0477] Example 22. 8-(Chloromethyl)-6-(5-(2-methoxyethoxy)-1H-indole-2-carbonyl)-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-4-yl(2-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)propionamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(2-(2-hydroxyethoxy)ethyl)carbamate (91)

[0478]

[0479] Scheme 28

[0480] At 0°C, p-nitrophenyl chloroformate (96%, 160 mg, 0.76 mmol) and EtN (0.37 mL, 2.7 mmol) were added to a solution of 66 (242 mg, 0.53 mmol) in THF (8 mL). After 75 minutes, a solution of 84 (279 mg, 1.1 mmol) in THF (1.5 mL) was added. The ice bath was removed and the mixture was stirred for 5.5 h and then evaporated to dryness. The remaining tan foam was dissolved in a minimum amount of CHCl and the solution was left standing overnight at 4°C. The solid was filtered off, washed with CHCl and the filtrate was evaporated. The residue was purified by column chromatography (EtOAc:petroleum ether 1:1, then 3:1, then EtOAc alone, then EtOAc:MeOH 20:1) to give 8-(chloromethyl)-6-(5-(2-methoxyethoxy)-1H-indole-2-carbonyl)-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-4-yl(2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(2-(2-hydroxyethoxy)ethyl)carbamate (90) as a colorless oil (90 mg, 23%); 1 H NMR (CDCl3) (some signals split due to rotational isomers) δ 9.43 (s, 1H), 8.26 and 8.24 (2s, 1H), 7.34 (d, J = 8.9 Hz, 1H), 7.14 (d, J = 2.3 Hz, 1H), 7.05 (dd, J = 8.9, 2.4 Hz, 1H), 6.98 (d, J = 1.5 Hz, 1H), 4.81-4.74 (m, 1H), 4.65-4.57 (m, 1H), 4.28-4.13 (m, 4H), 3.85-3.51 (m, 15H), 3.48 (s, 3H), 2.98-2.85 (m, 3H), 2.65 (s, 3H), 1.53-1.42 (m, 9H). Anal (C 36 H 46 ClN5O 10 ) is calculated as [M+H]+ 744.3006; measured value 744.3014.

[0481] The solid insoluble in CH2Cl2 was combined with matching fractions from column chromatography to afford recovered 66 (142 mg, 59%).

[0482] TFA (0.7 mL, 9.1 mmol) was added to a solution of 90 (90 mg, 0.12 mmol) in CH2Cl2 (2.0 mL) at 0°C and the mixture was stirred at this temperature for 40 minutes. The mixture was evaporated to dryness at room temperature and the residue was dissolved in CH2Cl2 and evaporated again. The residue was dissolved in DMF (1 mL) and the solution was cooled in an ice bath. Compound 86 (67.5 mg, 0.12 mmol) and Et3N (84 μL, 0.6 mmol) were added and the ice bath was removed. After 3 hours, the mixture was evaporated and the residue was purified by column chromatography (EtOAc: petroleum ether 2:3, then 1:1, 3:1, then EtOAc only, then EtOAc: MeOH 50:1, then 25:1, 15:1) to give recovered 66 as a white solid (11 mg, 20%) and 91 as a white glass (58 mg, 45%); 1 H NMR (CDCl3) (some signals are split due to rotational isomers) δ 9.95-9.40 (m, 1H), 8.91-8.55 (m, 1H), 8.27-8.05 (m, 1H), 7.61-7.45 (m, 2H), 7.40-7.20 (m, 2H), 7.15 (d, J = 2.2 Hz, 1H), 7.06 (dd, J = 8.9, 2.2 Hz, 1H), 7.00 (s, 1H), 6.72-6.53 (m, 1H), 5.21-4 .94(m,3H),4.84-4.74(m,1H),4.72-4.56(m,2H),4.28-4.16(m,4H),4.01-3.92(m,1H),3.84-3.35(m,15H), 3.47(s,3H),3.06-2.94(m,3H),2.66-2.60(m,3H),2.24-2.13(m,1H),1.48-1.37(m,12H),1.01-0.90(m,6H). Anal(C 52 H 67 ClN8O 14 ) is calculated as [M+H] + 1063.4538; measured value 1063.4546.

[0483] Another less pure portion of 91 was obtained from the column as a white glass (14 mg, crude yield 11%).

[0484] Example 23. 8-(Chloromethyl)-6-(5-(2-methoxyethoxy)-1H-indole-2-carbonyl)-2-methyl-7,8-dihydro-6H-oxazolo[4,5-e]indol-4-yl(2-((((4-((2S,5S)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7-dioxo-8,11-dioxa-3,6-diazatridecanoylamino)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(2-(2-hydroxyethoxy)ethyl)carbamate (93)

[0485]

[0486] Solution 29

[0487] TFA (0.2 mL, 2.6 mmol) was added to a solution of 91 (14 mg, 0.013 mmol) in CH2Cl2 (1.0 mL) at 0°C and the solution was kept at this temperature for 3 h. The mixture was evaporated to dryness at room temperature and the residue was dissolved in CH2Cl2 and evaporated again. The residue was dissolved in DMF (0.5 mL) and the solution was cooled in an ice bath. 2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl(4-nitrophenyl)carbonate (92) (4.6 mg, 0.013 mmol) and Et3N (9 μL, 0.065 mmol) were added and the ice bath was removed. After 19 h, the mixture was evaporated and the residue was purified by column chromatography (EtOAc only, then EtOAc:MeOH 20:1) to give 93 as a colorless oil (8.3 mg, 54%); 1 HNMR (CDCl3) (some signals split due to rotational isomers, 2 exchangeable protons not observed) δ 9.94-9.46 (m, 1H), 8.87-8.57 (m, 1H), 8.31-8.00 (m, 1H), 7.63-7.46 (m, 2H), 7.43-7.22 (m, 2H), 7.16-6.93 (m, 4H), 6.68 (s, 2H), 5.40-5.32 (m, 1H),5.16-5.04(m,2H),4.83-4.56(m,3H),4.35-3.98(m,7H),3.83-3.36(m,21H),3.47(s,3H), 3.05-2.94(m,3H),2.66-2.60(m,3H),2.30-2.20(m,1H),1.46-1.36(m,3H),1.03-0.90(m,6H). Anal(C 56 H 68 ClN9O17 ) is calculated as [M+H] + 1174.4494; measured value 1174.4516.

[0488] Example 24. Additional Lipophilicity Data

[0489] The lipophilicity of small molecule compounds can be calculated in many different ways (reviewed in, for example, J. Pharm. Sci. (2009) 98, 861). To supplement the data provided in Example 6, the calculated lipophilicity of several compounds of the present invention was compared with the lipophilicity of open-ring-CBI analogs with the same minor groove binding side chain using four different software packages, namely ChemDraw Professional (Perkin Elmer Informatics Inc), as in Example 6, ACD / logP (Advanced Chemistry Development), XLOGP3 (Shanghai Institute of Organic Chemistry) and MLOGP (Talete SRL, Milano, Italy), the latter three of which were obtained by SwissADME (http: / / swissadme.ch / , Swiss Institute of Bioinformatics). The structures of the compounds are shown (wherein DNA binding units A, D, E and F, when linked to the 2-methylbenzoxazolidinylating subunit, represent compounds 23, 59, 66 and 74, respectively) and the calculated logP values ​​are collected in Table 3.

[0490]

[0491] It is clear from Table 3 that, although the absolute logP values ​​vary depending on the computational program used, in each case there is a consistent and significant reduction in logP when comparing the 2-methylbenzoxazolyl alkylated subunit to the ring-opening-CBI alkylated subunit (ΔlogP in the range of 0.98-1.48). This applies whether the computational program is property-based (e.g., MLOGP) or substructure-based (fragment-based such as ACD or atom-based such as XLOG3).

[0492] As already noted, these substantial reductions in payload lipophilicity are likely responsible for the favorable properties of drug-linkers and ADCs incorporating 2-methylbenzoxazolidinylated subunits.

[0493] Table 3: Lipophilicity of compounds of the present invention

[0494]

[0495]

[0496] Example 25. Additional cytotoxicity data

[0497] The cytotoxicity of the payloads of the invention was determined by measuring the proliferation inhibition of two human tumor cell lines, cervical carcinoma SiHa and ovarian carcinoma SKOV3, following the general procedure described in Example 7. In the new assay, the stock solution of the payload in DMSO was serially diluted before addition to the wells containing cells in a 96-well plate. The cytotoxicity assay was repeated several times (n=3-7 depending on the compound and cell line) and the results are collected in Table 4.

[0498] The new data reinforce the information presented in Table 2 and show that DNA alkylating agents containing a 2-methylbenzoxazolyl alkylating subunit can be highly cytotoxic compounds with an IC 50 They also show that appropriate selection of minor groove binding side chains can be used to modulate the cytotoxicity of the payload, with the examples in Table 4 spanning more than a 100-fold range of IC in the two cell lines examined. 50 The data in Table 4 further allow comparison between the two enantiomeric forms of the alkylated subunit of 2-methylbenzoxazol. Compounds 82 and 83 showed a 93-fold differential cytotoxic potency in SiHa and a 110-fold differential cytotoxic potency in SKOV3. These observations are consistent with the behavior of other analogs of duocarmycin, in that although both enantiomers are cytotoxic due to alkylation of DNA, the natural enantiomer is generally more cytotoxic.

[0499] Importantly, in IC 50 Compound 23 and ring-open-CBI-TMI, which share the same minor groove binding side chain, were again found to have the same cytotoxic potency within the measured error. This means that the 2-methylbenzoxazolyl alkylating subunit can be considered the most potent of all known variants of the duocarmycin-type alkylating subunits, surprisingly different from the closely related ring-open-COI alkylating subunit.

[0500] Table 4: Cytotoxicity of compounds of the present invention

[0501]

[0502]

[0503] Example 26. Additional Stability Data

[0504] The aqueous stability of compounds 52, 59 and 66 was investigated by HPLC analysis. The conditions were the same as those described in Example 8, i.e., the samples contained a 4 μM concentration of the payload in Tris buffer (pH 7.4) containing 10% DMF at 37° C. In this experiment, the solution was monitored at hourly intervals over 8 hours.

[0505] like Figure 3-5 As shown, all three payload compounds were converted into intermediates identified as the corresponding cyclopropyl forms based on characteristic shifts in UV-visible absorption spectra. In all three cases, the cyclopropyl intermediates were unstable for further reaction in aqueous buffer, producing a variety of products, with two major hydrolysis products dominating. This behavior closely matches the stability and product distribution observed for compound 23 in Example 8 (see Figure 2 ), but in marked contrast to the extended stability of the reference compound open-CBI-TMI (see Figure 1 ). It therefore appears that the unusual instability is a general property of the 2-methylbenzoxazolidinyl subunit. It does not clearly depend on the properties of the DNA minor groove binding side chain. In other words, the potential advantage of hydrolysis as a detoxification mechanism for any systemically released payload can reasonably be assumed to be a property common to all novel compounds of the present invention.

Claims

1. A compound of formula III or a pharmaceutically acceptable salt thereof, in: LG is a leaving group selected from the group consisting of chloride, bromide, iodide and -OSO2R 1 ; where R 1 Selected from (C1-C 10 )alkyl, (C1-C 10 )heteroalkyl, (C1-C 10 )aryl or (C1-C 10 ) heteroaryl; X is a group selected from hydroxyl, protected hydroxyl, amino and protected amino; wherein the protected hydroxyl is -OBn, -OTf, -OMOM, -OMEM, -OBOM, -OTBDMS, -OPMB, -OSEM, piperazine-1-carboxylate, wherein the N at the 4th position is (C1-C 10 )alkyl, -OP(O)(OH)2, -OP(O)(OR 2 )2, -NH2, -N=C(Ph)2, -NHZ, NH(C1-C 10 )alkyl or -NZ(C1-C 10 ) alkyl substituted, wherein R 2 is t-Bu, Bn or allyl; and Z is selected from Boc, COCF3, Fmoc, Alloc, Cbz, Teoc and Troc; and wherein the amino group is -NH2, or -NH(C1-C6)alkyl; wherein the amino protecting group is selected from the group consisting of acetyl, chloroacetyl, trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxyacetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, pyridine formyl, aminocaproyl, benzoyl, methoxy-carbonyl, 9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-carbonyl, tert-butoxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2,4-dichloro-benzyloxycarbonyl, o-nitrobenzenesulfonyl, p-nitrobenzenesulfonyl, 2-(4-biphenylyl)isopropoxycarbonyl, and 1-(4,4-dimethyl-2,6-dioxohexylidene)ethyl; and Y is selected from: (a)-C(O)-On 1 (b)-C(O)-Ar 1 -NH-C(O)-Ar 2 (c)-C(O)-Ar 1 -NH-C(O)-CH=CH-Ar 3 ; or (d)-C(O)-CH=CH-Ar 3 Among them, Ar 1 is selected from indole, azaindole, benzofuran and benzothienyl; Ar 2 is selected from the group consisting of indole, azaindole, benzene, benzofuran, pyridine, pyrimidine, pyrrole, imidazole, thiophene, thiazole, oxazole, pyrazole, triazole, pyrazine or pyridazine; and Ar 3 is selected from benzene, pyridine, pyrimidine and pyridazine, wherein Ar 1 ,Ar 2 and Ar 3 each optionally substituted with one or more of: -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -OH, -O-(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl, and -NHC(O)-(C1-C6)alkyl; wherein in each case -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -O-(C1-C6)alkyl, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl are independently optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

2. The compound according to claim 1, wherein Y is selected from the group consisting of: (a)-C(O)-On 1 (b)-C(O)-Ar 1 -NH-C(O)-Ar 2 (c)-C(O)-Ar 1 -NH-C(O)-CH=CH-Ar 3 ; and (d)-C(O)-CH=CH-Ar 3 in Ar 1 Select from the group consisting of: Ar 2 Select from the group consisting of: as well as Ar 3 Select from the group consisting of: in represents the connection point, and Ar 1 ,Ar 2 and Ar 3 each may be substituted at the numbered positions with up to three substituents selected from the group consisting of -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -OH, -O-(C1-C6)alkyl, -NH2, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl, and -NHC(O)-(C1-C6)alkyl, and wherein in each instance -(C1-C6)alkyl, -CO-(C1-C6)alkyl, -CONH(C1-C6)alkyl, -CON(C1-C6)alkyl(C1-C6)alkyl, -O-(C1-C6)alkyl, -NH(C1-C6)alkyl, -N(C1-C6)alkyl(C1-C6)alkyl and -NHC(O)-(C1-C6)alkyl are independently optionally substituted with one or more of -NMe2, -NHMe, -NH2, -OH, morpholine and -SH.

3. The compound according to claim 2, wherein Y is -C(O)-Ar 1 -NH-C(O)-Ar 2 ; Ar 1 is indole, azaindole, benzofuran or benzothienyl, which is linked to the DNA alkylation unit at the 2-position of the heteroaryl group, and Ar 2 is selected from indole, azaindole, benzene, benzofuran, pyridine, pyrimidine, pyrrole, imidazole, thiophene, thiazole, oxazole, pyrazole, triazole, pyrazine or pyridazine.

4. The compound according to claim 2, wherein Y is -C(O)-Ar 1 -NH-C(O)-CH=CH-Ar 3 ; and Ar 1 is indole, azaindole, benzofuran or benzothienyl, which is linked to the DNA alkylation unit at the 2-position of the heteroaryl group, and Ar 3 Selected from benzene, pyridine, pyrimidine and pyridazine.

5. according to the compound described in claim 3 or claim 4, wherein Ar 1 The point of attachment is at the 5-position of the indole, azaindole, benzofuran or benzothiophenyl group.

6. The compound according to any one of claims 1 to 5, wherein LG is chloride, bromide or iodide, and the configuration of the chiral carbon to which -CH2-LG is attached is (S).

7. A compound selected from the group consisting of:

8. A pharmaceutical composition comprising the compound according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

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