Duocarmycin analogs

文档序号：39086 发布日期：2021-09-24 浏览：23次中文

阅读说明：本技术 倍癌霉素类似物 (Duocarmycin analogs ) 是由李和发莫阿娜·特塞尔于 2020-01-28 设计创作，主要内容包括：本发明涉及式I的2-甲基苯并噁唑化合物,其为倍癌霉素的DNA烷基化亚单位的类似物。式I化合物可用于合成DNA烷基化剂和抗体-药物缀合物以及相关化合物。式I的2-甲基苯并噁唑单元在结合高细胞毒性、低亲脂性和异常的水稳定性方面具有有利的性质,所有这些性质对于在有效的抗体-药物缀合物中作为有效负荷的应用是合乎需要的。(The present invention relates to 2-methylbenzoxazole compounds of formula I which are analogs of the DNA alkylated subunit of duocarmycin.)

1. A compound of formula I or a pharmaceutically acceptable salt, hydrate or solvate thereof

Wherein:

LG is a leaving group;

x is a group selected from hydroxy, protected hydroxy, prodrug hydroxy, amino, and protected amino; wherein amino is-NH₂or-NH (C)₁-C₆) An alkyl group; and is

Y is an N-protecting group.

2. The compound of claim 1, wherein Y is selected from Boc, COCF₃N-protecting groups for Fmoc, Alloc, Cbz, Teoc and Troc.

3. A compound of formula II or

Pharmaceutically acceptable salts, hydrates or solvates thereof

Wherein:

LG is a leaving group;

x is a group selected from hydroxy, protected hydroxy, prodrug hydroxy, amino, and protected amino; wherein amino is-NH₂or-NH (C)₁-C₆) An alkyl group; and is

DB is DNA minor groove binding unit.

4. The compound of claim 3, wherein DB is optionally substituted aryl or optionally substituted heteroaryl, linked directly or through an alkenyl.

5. The compound of claim 4, wherein DB is an optionally substituted indole, azaindole, benzene, benzofuran, pyridine, pyrimidine, pyrrole, imidazole, thiophene, thiazole, oxazole, pyrazole, triazole, pyrazine or pyridazinyl.

6. The compound of any one of claims 1-5, wherein X is selected from the group consisting of: -OH, -OBn, -OTf, -OMOM, -OMEM, -OBOM, -OTBDMS, -OPMB, -OSEM, piperazine-1-carboxylate, wherein N in position 4 is (C)₁-C₁₀) Alkyl, -OP (O) (OH)₂、-OP(O)(OR²)₂、-NH₂、-N＝C(Ph)₂、-NHZ、NH(C₁-C₁₀) Alkyl and-N-Z (C)₁-C₁₀) Alkyl substitution;

wherein R is²Is t-Bu, Bn or allyl; z is selected from Boc and COCF₃Fmoc, Alloc, Cbz, Teoc and Troc.

7. The compound according to any one of claims 1-6, wherein LG is selected from the group consisting of: chloride, bromide, iodide and-OSO₂R¹(ii) a Wherein R is¹Is selected from (C)₁-C₁₀) Alkyl, (C)₁-C₁₀) Heteroalkyl group, (C)₁-C₁₀) Aryl or (C)₁-C₁₀) A heteroaryl group.

8. A compound of formula III or a pharmaceutically acceptable salt, hydrate or solvate thereof

Wherein:

LG is a leaving group;

x is a group selected from hydroxy, protected hydroxy, prodrug hydroxy, amino, and protected amino; wherein the amino group is-NH₂or-NH (C)₁-C₆) An alkyl group; and is

Y is selected from:

(a) an N-protecting group;

(b)-C(O)-Ar¹

(c)-C(O)-Ar¹-NH-C(O)-Ar²

(d)-C(O)-Ar¹-NH-C(O)-CH＝CH-Ar³(ii) a Or

(e)-C(O)-CH＝CH-Ar³

Wherein Ar is¹、Ar²And Ar³Each independently selected from heteroaryl or aryl, wherein the heteroaryl or aryl is optionally substituted with one or more of: - (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -OH, -O- (C)₁-C₆) Alkyl, -NH₂、-NH(C₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl and-NHC (O) - (C)₁-C₆) An alkyl group;

in each case of- (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -O- (C)₁-C₆) Alkyl, -NH (C)₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl and-NHC (O) - (C)₁-C₆) Alkyl is independently optionally substituted by-NMe₂、-NHMe、-NH₂-OH, morpholine and-SH.

9. The compound of claim 8, wherein Y is selected from Boc, COCF₃N-protecting groups for Fmoc, Alloc, Cbz, Teoc and Troc.

10. The compound of claim 8, wherein Y is selected from the group consisting of:

(b)-C(O)-Ar¹

(c)-C(O)-Ar¹-NH-C(O)-Ar²

(d)-C(O)-Ar¹-NH-C(O)-CH＝CH-Ar³(ii) a And

(e)-C(O)-CH＝CH-Ar³

wherein

Wherein Ar is¹、Ar²And Ar³Independently selected from the group consisting of

WhereinRepresents a point of attachment, and each aryl or heteroaryl group may be substituted at the numbered position with up to three substituents selected from: - (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -OH, -O- (C)₁-C₆) Alkyl, -NH₂、-NH(C₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl and-NHC (O) - (C)₁-C₆) Alkyl radicals, and in each case- (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -O- (C)₁-C₆) Alkyl, -NH (C)₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl and-NHC (O) - (C)₁-C₆) Alkyl is independently optionally substituted by-NMe₂、-NHMe、-NH₂-OH, morpholine and-SH.

11. According to claim 10, wherein Ar¹Is heteroaryl, preferably indole, azaindole, benzofuran or benzothiophenyl, which is linked to the DNA alkylation unit at the 2-position of the heteroaryl.

12. The compound of claim 10 or 11, wherein Ar²Selected from the group consisting of: indole, azaindole, benzene, benzofuran, pyridine, pyrimidine, pyrrole, imidazole, thiophene, thiazole, oxazole, pyrazole, triazole, pyrazine or pyridazine, preferably indole, azaindole, benzene, benzofuran, pyrrole or imidazole.

13. The compound of any one of claims 10-12, wherein Ar³Selected from benzene, pyridine, pyrimidine and pyridazine, preferably benzene or pyridine, more preferably benzene.

14. The compound of claim 10, wherein Y is-c (o) -Ar¹-NH-C(O)-Ar²；

Ar¹Is an indole, azaindole, benzofuran or benzothiophenyl group attached to the DNA alkylation unit at the 2-position of the heteroaryl group, and Ar²Selected from indole, azaindole, benzene, benzofuran, pyridine, pyrimidine, pyrrole, imidazole, thiophene, thiazole, oxazole, pyrazole, triazole, pyrazine or pyridazine.

15. The compound of claim 10, wherein Y is-c (o) -Ar¹-NH-C(O)-CH＝CH-Ar³；Ar¹Is an indole, azaindole, benzofuran or benzothiophenyl group attached to the DNA alkylation unit at the 2-position of the heteroaryl group, and Ar³Selected from benzene, pyridine, pyrimidine and pyridazine.

16. The compound of claim 14 or claim 15, wherein Ar¹The point of attachment is at the 5-position of the indole, azaindole, benzofuran, or benzothiophene group.

17The compound of any one of claims 8-16, wherein X is selected from the group consisting of: -OH, -OBn, -OTf, -OMOM, -OMEM, -OBOM, -OTBDMS, -OPMB, -OSEM, piperazine-1-carboxylate, wherein N in position 4 is (C)₁-C₁₀) Alkyl, -OP (O) (OH)₂、-OP(O)(OR²)₂、-NH₂、-N＝C(Ph)₂、-NHZ、NH(C₁-C₁₀) Alkyl or-N-Z (C)₁-C₁₀) Alkyl substitution;

wherein R is²Is t-Bu, Bn or allyl; z is selected from Boc and COCF₃Fmoc, Alloc, Cbz, Teoc and Troc.

18. The compound according to any one of claims 8-17, wherein LG is selected from the group consisting of: chloride, bromide, iodide and-OSO₂R¹(ii) a Wherein R is¹Is selected from (C)₁-C₁₀) Alkyl, (C)₁-C₁₀) Heteroalkyl group, (C)₁-C₁₀) Aryl or (C)₁-C₁₀) A heteroaryl group.

19. The compound according to any one of claims 8-17, wherein LG is halogen, preferably chloro, and the configuration of the chiral carbon to which LG is attached is (S).

20. A compound selected from the group consisting of:

21. a pharmaceutical composition comprising a compound according to any one of claims 1-20 and a pharmaceutically acceptable carrier.

1. Field of the invention

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

2. Background of the invention

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

The mechanism of binding action involves the addition of adenine to the cyclopropyl ring of an alkylated subunit, the general structure of which is used is illustrated in scheme 1 below. The reaction is fast and selective for DNA, but orders of magnitude slower for other nucleophiles such as water, so that in the absence of DNA, the alkylated subunits persist in aqueous buffers under physiological conditions for very long periods of time.

Scheme 1

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 is so facile under physiological conditions that both forms (ring opening and cyclopropyl) exhibit essentially the same cytotoxicity. However, if the phenol of the ring-opened alkylated subunit is in a chemical form that prevents cyclization, cytotoxicity is greatly reduced.

The natural product, such as duocarmycin SA, was isolated as a single enantiomer as shown. However, both enantiomers can alkylate DNA, although the unnatural enantiomer is generally less cytotoxic.

Many synthetic forms of the duocarmycin alkylated subunit have been reported. These typically vary in the nature of the loop fusions indicated by the dashed lines in the structures in scheme 1 above. The nature of these loop fusions can have a strong effect on cytotoxicity.

For example, CBI (cyclopropylbenzindole) alkylated subunits (illustrated below in ring-opened form in combination with TMI (trimethoxyindole) side chain found in duocarmycin SA) produced duocarmycin analogs (j.org.chem. (1990)55,5823) with similar cytotoxic potency as the natural product.

In contrast, compounds comprising a combination of alternative COI (cyclopropyl oxazoloindole) alkylated subunits with TMI were several hundred times less cytotoxic (bioorg. med. chem. lett. (2010)20,1854).

Other synthetic variations have also been reported. For example, amino ring opening-CBI is known, wherein an amine group replaces the hydroxyl group of a phenol. These variants undergo the same spiro cyclization and DNA alkylation reactions as their phenolic analogs and have equal cytotoxic potency (chem biochem (2014)15,1998).

Further variations include linking two alkylated subunits together in a manner that allows interchain crosslinking of two adenines in the DNA (J.Am.chem.Soc. (1989)111,6428; Angew.chem. (2010)49,7336). These dimers may even be more cytotoxic than the corresponding monoalkylating agents, although the activity of the dimer need not be predicted by the activity of its component monoalkylation units.

Dimers of the amino seco-duo-oncomycin and alkylated subunits are shown below.

Several analogs of duocarmycin 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.

Recently, duocarmycin analogs have found application as payloads of antibody-drug conjugates (ADCs). ADCs are most commonly used in cancer therapy, where a small cytotoxic molecule (payload) is linked via a linker to an antibody that recognizes a tumor-associated antigen (nat. rev. drug disorders. (2017)16,315; pharmacol. rev. (2016)68, 3).

ADCs are constructed by chemically linking the payload to a suitable linker, which is itself conjugated to the desired antibody. ADCs are designed to be stable in the circulation, but typically release a payload at a predetermined target site following receptor binding and internalization into the target cell. In this way, the cytotoxic effects of the payload are specifically directed to the site where injury is desired, i.e., the tumor. Several such ADCs have been approved as anti-cancer therapies.

The ADC concept is a clever means of directing the payload specifically to its target cells, thereby minimizing the adverse effects associated with conventional non-specific systemic delivery of toxic compounds in vivo. Unfortunately, major technical challenges limit the successful application of the ADC concept.

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

Due to their high cytotoxicity, duocarmycin analogs have been investigated as ADCs payloads, as monoalkylating agents, or as components that can crosslink dimers of DNA (see, e.g., mol. pharm. (2015)12,1813; WO 2011/133039; biopharmam. 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).

In the vast majority of these examples, the alkylating subunit is a highly toxic ring-opened-CBI, as shown below.

Although the use of such alkylated subunits would result in ADCs with the desired cytotoxicity, the ring-opened-CBI has significant drawbacks in terms of high lipophilicity. Lipophilic payloads and derivatives thereof 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. The necessity of removing ADC aggregates prior to using the ADC adds complexity and cost to producing a clinically useful ADC.

Lipophilic payloads have also been associated with faster clearance of ADCs from the bloodstream, which reduces their overall exposure and thus their anti-tumor efficacy (nat. biotechnol. (2015)33,733-.

Lipophilic payloads, particularly those that cause aggregation, can also produce ADCs that elicit an immune response in vivo, resulting in unexpected toxicity or reduced therapeutic efficacy.

However, despite the problems associated with lipophilicity, the high cytotoxicity of ring-opened-CBI compounds continues to make them attractive payloads for anticancer ADCs, employing various strategies to mitigate the disadvantages.

For example, to counteract the high lipophilicity of ring-opened-CBI, some researchers have resorted to modifying other component moieties 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).

However, solving the payload lipophilicity problem by modifying other components of the ADC may have disadvantages in itself. In addition to making the synthesis of payload-linker components more complex and time consuming, this technique may also limit the choice of linker.

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 adequate stability in circulation but lyses in vivo when needed. The ADC design includes consideration of the physiological conditions under which the payload is released so that appropriate linkers can be used. For example, linkers containing hydrazone moieties are pH sensitive and will cleave in lower pH environments such as endosomes and lysosomes. The disulfide linker releases the payload in a reducing environment (e.g., an intracellular environment).

Some linker modifications to mitigate the undesirable lipophilicity of the alkylated subunits may reduce the efficacy of ADCs using them, such that the resulting ADCs may not be the most efficient products that can be prepared from a given payload and antibody.

Other solutions to the lipophilicity problem include the ADC Bio patent lock-release technology (www.adcbio.com) Which immobilizes the antibody on a solid support prior to conjugation to the payload-linker component. Aggregation is prevented by physically separating the ADC molecules from each other during their manufacture. This allows a wider range of linkers to be used, but adds additional cost to the conjugation process without actually reducing the lipophilicity of the final ADC product.

Thus, the lipophilicity of highly toxic alkylated subunits is a problem that hinders the development of new ADCs.

Another potential drawback of known duocarmycin analogs as ADCs payloads is the high stability of the cyclopropyl form of the alkylated subunit. The stable payload released from the ADC into the circulation may last long enough to cause undesirable systemic toxicity. Unfortunately, high stability is a characteristic of nearly all synthetic analogs of CBI alkylated subunits that retain the desired potent 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).

Thus, there is a need for alternative DNA-alkylating subunits that promote efficient synthesis of a range of ADCs while demonstrating the cytotoxic and stability properties required for efficacy.

It is therefore an object of the present invention to address this need in at least some way; and/or at least to provide the public with a useful choice. Other objects of the invention will become apparent from the following description which is given by way of example only.

In this specification, reference has been made to external sources of information, including patent specifications and other documents, which are generally intended to provide 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

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

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

Accordingly, in a first aspect, the present invention provides a compound of formula I or a pharmaceutically acceptable salt, hydrate or solvate thereof

Wherein:

LG is a leaving group;

x is a group selected from the group consisting of hydroxy, protected hydroxy, prodrug hydroxy, amine, and protected amine; wherein the amine is-NH₂or-NH (C)₁-C₆) An alkyl group; and is

Y is an N-protecting group.

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

Wherein:

LG is a leaving group;

x is a group selected from the group consisting of hydroxy, protected hydroxy, prodrug hydroxy, amine, and protected amine; wherein the amine is-NH₂or-NH (C)₁-C₆) An alkyl group; and is

DB is DNA minor groove binding unit.

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

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

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

Wherein:

LG is a leaving group;

x is a group selected from the group consisting of hydroxy, protected hydroxy, prodrug hydroxy, amine, and protected amine; wherein the amine is-NH 2 or-NH (C)₁-C₆) An alkyl group; and is

Y is selected from:

(a) an N-protecting group;

(b)-C(O)-Ar¹

(c)-C(O)-Ar¹-NH-C(O)-Ar²

(d)-C(O)-Ar¹-NH-C(O)-CH＝CH-Ar³(ii) a Or

(e)-C(O)-CH＝CH-Ar³

Wherein Ar is¹、Ar²And Ar³Each independently selected from heteroaryl or aryl, wherein each said heteroaryl or aryl is optionally substituted with one or more of: - (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkane (I) and its preparation methodRadical, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -OH, -O- (C)₁-C₆) Alkyl, -NH₂、-NH(C₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl and-NHC (O) - (C)₁-C₆) An alkyl group;

In one embodiment, Ar¹、Ar²And Ar³Independently selected from the group consisting of

WhereinRepresents 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: - (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -OH, -O- (C)₁-C₆) Alkyl, -NH₂、-NH(C₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl and-NHC (O) - (C)₁-C₆) An alkyl group;

wherein in each case a substituent-(C₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -O- (C)₁-C₆) Alkyl, -NH (C)₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl and-NHC (O) - (C)₁-C₆) The alkyl groups may independently optionally be substituted by-NMe₂、-NHMe、-NH₂-OH, morpholine and-SH.

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

Wherein V is Y or DB and X, Y and DB have the same meaning as defined for the compounds of formulae I, II and III and X' is X with the loss of H.

The compounds of formula IV can be formed by in vitro or in vivo rearrangement with concomitant elimination of H-LG from the corresponding ring-opened compounds of formulae I, II and III. All embodiments of the invention described herein for compounds of formula I, II or III, or any portion thereof, are also expressly contemplated as part of the inventive aspects directed to compounds of formula IV, unless the context dictates otherwise.

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.

In one embodiment, LG is halogen, preferably chloro, and the configuration on the chiral carbon to which LG is attached is (S).

In one embodiment, X is selected from the group consisting of: -OH, -OBn, -OTf, -OMOM, -OMEM, -OBOM, -OTBDMS, -OPMB, -OSEM, piperazine-1-carboxylate, wherein N in position 4 is (C)₁-C₁₀) Alkyl, -OP (O) (OH)₂、-OP(O)(OR²)₂、-NH₂、-N＝C(Ph)₂、-NHZ、NH(C₁-C₁₀) Alkyl and-N-Z (C)₁-C₁₀) Alkyl substitution;

wherein R is²Is t-Bu, Bn or allyl; z is selected from Boc and COCF₃Fmoc, Alloc, Cbz, Teoc and Troc.

In one embodiment, Y is selected from Boc, COCF₃N-protecting groups for Fmoc, Alloc, Cbz, Teoc and Troc.

A numerical range disclosed herein (e.g., 1 to 10) also includes 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) and 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 therefore, all subranges of all ranges explicitly disclosed herein are hereby explicitly disclosed. These are only examples of what is specifically intended, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

While the invention is broadly defined above, those skilled in the art will appreciate that the invention is not so limited and that the invention also includes embodiments exemplified by the following description.

4. Description of the drawings

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

FIG. 1 shows LCMS analysis of the stability of Ring-opened-CBI-TMI in neutral aqueous buffer. The upper panel shows an example of a chromatogram (after an incubation time of 20 minutes). The lower panel summarizes the change in mixture composition over total incubation time >300 min.

Figure 2 shows LCMS analysis of the stability of compound 23 in neutral aqueous buffer. The upper panel shows an example of a chromatogram (after an incubation time of 200 minutes). The lower panel summarizes the change in composition of the mixture over a total incubation time of 500 min.

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

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

Figure 5 shows HPLC analysis of the stability of compound 66 in neutral aqueous buffer. The experiment was monitored every hour for a total of 8 hours.

5. Detailed description of the preferred embodiments

5.1 definition

The term "comprising" as used in the present specification and claims means "consisting at least in part of …". When interpreting each expression in this specification and claims that includes the term "comprising", features other than, or prefaced by, that term can also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same way.

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

As used herein, "s" following a noun means the plural and/or singular form of the noun.

Asymmetric centers may be present in the compounds described herein. The asymmetric center may be designated as (R) or (S), depending on the configuration of the substituent in three-dimensional space at the chiral carbon atom. Unless a particular stereochemistry or isomeric form is indicated, all chiral, diastereomeric and racemic forms of a structure are intended. All stereochemically isomeric forms of the compounds, including diastereomeric, enantiomeric and epimeric forms, and cis and trans isomers, and mixtures thereof, including mixtures of both enantiomerically and diastereomerically enriched stereochemically isomeric forms, are intended to be within the scope of the invention.

The individual enantiomers can be prepared synthetically from commercially available, enantiomerically pure starting materials, orPrepared by preparing a mixture of enantiomers and resolving the mixture into the individual enantiomers. Resolution methods include (a) separation of enantiomeric mixtures by chromatography on a chiral stationary phase and (b) conversion of the enantiomeric mixtures to mixtures of diastereomers and separation of diastereomers by, for example, recrystallization or chromatography, and any other suitable method known in the art. Starting materials with defined stereochemistry may be commercially available or prepared and, if desired, resolved by techniques well known in the art. Preferably at the chiral carbon (with CH in the ring-opened form)₂The carbon of the LG moiety) has the "natural" configuration.

The compounds described herein may also exist as conformational or geometric isomers, including cis (cis), trans (trans), cis (syn), trans (anti), entgegen (e), and zusammen (z) isomers. All such isomers and any mixtures thereof are within the scope of the present invention.

Any tautomer of the compound or mixture thereof is also within the scope of the invention. As will be understood by those skilled in the art, a variety of functional groups and other structures may exhibit tautomerism. Examples include, but are not limited to, keto/enol, imine/enamine, and thione/enethiol tautomerism.

The compounds described herein may also exist as isotopic isomers and isotopic equivalents, wherein one or more atoms in the compound are replaced by a different isotope. Suitable isotopes include, for example¹H、²H(D)、³H(T)、¹²C、¹³C、¹⁴C、¹⁶O and¹⁸and O. Procedures for incorporating such isotopes into the compounds described herein will be apparent to those skilled in the art. Isotopic isomers and isotopic equivalents of the compounds described herein are also within the scope of the present invention.

Salts, including pharmaceutically acceptable salts, of the compounds described herein are also within the scope of the invention. Such salts include acid addition salts, base addition salts, and quaternary salts of basic nitrogen-containing groups. Acid addition salts may be prepared by reacting the free base form of the compound with an inorganic or 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 free acid form of the compound 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 (e.g., dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and diamyl sulfate), and the like.

The compounds described herein may form solvates with or exist as solvates. If the solvent is water, the solvate may be referred to as a hydrate, e.g. a monohydrate, dihydrate or trihydrate. All solvated and unsolvated forms of the compounds described herein are intended to be within the scope of the present invention.

General chemical terms used herein have their ordinary meanings.

Standard abbreviations for chemical groups are well known in the art and take their usual meaning, for example Me ═ methyl, Et ═ ethyl, Bu ═ butyl, t-Bu ═ tert-butyl, Ph ═ phenyl, Bn ═ benzyl, Ac ═ acetyl, Boc ═ tert-butoxycarbonyl, Fmoc ═ 9-fluorenylmethoxycarbonyl, Tf ═ triflate, OMOM ═ methoxymethyl ether, ome ═ methoxyethoxymethyl ether, OBOM ═ benzyloxymethyl ether, otms ═ tert-butyldimethylsilyl ether, DPPA ═ diphenylphosphoryl azide, nbds ═ N-bromosuccinimide, NIS ═ N-iodosuccinimide, OPMB ═ 4-methoxybenzyl ether, EDCI ═ 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, HOBt ═ hydroxybenzotriazole, OSEM ═ 2- (trimethylethoxy) methylsilyl ] carbodiimide, alloc ═ allyloxycarbonyl, Cbz ═ benzyloxycarbonyl, Teoc ═ 2- (trimethylsilyl) ethoxycarbonyl, TEMPO ═ 2,2,6, 6-tetramethyl-1-piperidinyloxy, Troc ═ 2,2, 2-trichloroethylcarbonyl, and the like.

These abbreviations apply to all examples below unless otherwise indicated.

The term "alkyl" as used herein, alone or in combination with other terms, refers to a straight or branched chain saturated or unsaturated acyclic hydrocarbon group, unless otherwise specified. 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-dimethylbutyl, 2, 3-dimethylbutyl, 2-dimethylpentyl, 2, 3-dimethylpentyl, 3-dimethylpentyl, 2,3, 4-trimethylpentyl, 2-dimethylpentyl, 3-dimethylpentyl, 2,3, 4-trimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-tetradecyl, n-pentadecyl, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, -neopentyl, 2-methylpentyl, 3-methylpentyl, 2, 4-methylpentyl, 2, 3-methylpentyl, and so-pentyl, 3-methylhexyl, 2-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 3, 5-dimethylhexyl, 2, 4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, n-heptyl, isoheptyl, isooctyl, isononyl, isodecyl, isoundecyl, isododecyl, isotridecyl, isotetradecyl, and isopentadecyl, and the like. 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, methyl-2-butenyl, and methyl-2-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. Prefix "C_x-C_y", wherein x and y are each an integer, when used in combination with the term" alkyl ", refers to the number of carbon atoms in the alkyl group. In some embodiments, an "alkyl" group may be substituted with one or more optional substituents as described herein.

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, unless otherwise specified. Aryl includes monocyclic, bicyclic and tricyclic ring systems. Examples of aryl groups include, but are not limited to, phenyl, azulenyl, heptenyl, biphenyl, fluorenyl, phenanthryl, anthracyl, indenyl, indanyl, pentalenyl, and naphthyl. In some embodiments, the aryl group 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. Prefix "C_x-C_y", wherein x and y are each an integer, when used in combination with the term" aryl "refers to the number of ring carbon atoms in the aryl group. In some embodiments, an "aryl" group may be substituted with one or more optional substituents as described herein.

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 is a heteroatom, unless otherwise specified. In some embodiments, the heteroatom is nitrogen, oxygen, or sulfur. Heteroaryl groups are various heterocyclic groups having an aromatic electronic structure. In some embodiments, heteroaryl includes mono-, di-, 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 groups include, but are not limited to, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridyl), indazolyl, benzimidazolyl, pyrazolopyridyl, triazolopyridyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, imidazopyridinyl, imidazolyl, isoxazolopyridinyl xanthyl, amidino (guaninyl), quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl. Heteroaryl groups include fused ring systems, wherein all rings are aromatic, e.g., indolyl, and fused ring systems, wherein only one ring is aromatic, e.g., 2, 3-indolinyl. When used in combination with the term "heteroaryl," the prefix "x-y membered" refers to the number of ring atoms in the heteroaryl, where x and y are each integers. In some embodiments, a "heteroaryl" group may be substituted with one or more optional substituents as described herein.

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

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.

As used herein, the term "substituted" is intended to mean that one or more hydrogen atoms in the indicated group is replaced by one or more independently selected suitable substituents, provided that the normal valency of each atom attached to the substituent is not exceeded, and that the substitution results in a stable compound. In various embodiments, suitable optional substituents in the compounds described herein include, but are not limited to, halogen, - (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -OH, -O- (C)₁-C₆) Alkyl, -NH₂、-NH(C₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl and-NHC (O) - (C)₁-C₆) Alkyl, -NMe₂、-NHMe、-NH₂-OH, morpholine and-SH. Unless otherwise indicated, the term "stable" herein means having sufficient stability to permit manufacture and maintaining its integrity sufficient for use hereinA compound for a period of time for the purposes described herein.

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 that contains an antigen binding site that immunospecifically binds to an antigen or portion thereof of a target of interest, including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The term "antibody" includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so 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: variable and constant regions. The variable region at the end of the Y arm binds to and interacts with the target antigen. The variable region includes Complementarity Determining Regions (CDRs) that recognize and bind to specific binding sites on a particular antigen. The constant region located at the tail of Y is recognized and interacts with the immune system (Janeway, c., Travers, p., Walport, m., shmchik (2001) Immuno Biology,5th ed., Garland Publishing, New York). Target antigens typically have a number of binding sites, also referred to as epitopes, that are recognized by CDRs on various antibodies. Each antibody that specifically binds a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody.

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 prior functionalization. The reaction between the reactive moiety and the second functional group requires only the application of heat, pressure, catalysts, acids and/or bases. Examples of reactive moieties include carbamoyl halides, acyl halides, active esters, anhydrides, α -haloacetyl, α -haloacetamide, maleimide, isocyanates, isothiocyanates, disulfides, thiols, hydrazines, hydrazides, sulfonyl chlorides, aldehydes, methyl ketones, vinyl sulfones, halomethyl, and methyl sulfonates. The second functional group is typically a linker or a ligand.

As used herein, the term "ligand" means a ligand that binds to or reactively associates or complexes with a receptor, antigen, or other receptive moiety associated with a given target cell population. The ligands are typically bound by intermolecular forces such as hydrogen bonds, ionic bonds, and van der waals forces. The ligand typically delivers a payload to a target cell population to which the ligand binds by binding to cells expressing a particular antigen or cell surface receptor. For example, the ligand may bind a cell surface receptor or surface protein that is overexpressed in diseased cells, such as cancer cells.

Examples of ligands for use in the 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.

The term "pharmaceutically acceptable salt" as used herein, unless otherwise indicated, refers to pharmaceutically acceptable organic or inorganic salts of the compounds described herein. For example, the compounds 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, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1, 1' -methylenebis (2-hydroxy-3-naphthoic acid)). Pharmaceutically acceptable salts may involve inclusion of another molecule, such as an acetate, succinate, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. The plurality of charged atoms may be part of a pharmaceutically acceptable salt, and may have a plurality of counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

The term "pharmaceutically acceptable solvate" or "solvate" as used herein, unless otherwise indicated, 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.

5.2 Compounds of the invention

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 efficient ADCs using conventional techniques.

Specifically, the compounds of the present invention comprise:

(a) n-protected 2-methylbenzoxazole (2-methylbenzoxaxole) DNA alkylated unit (where Y is an N-protecting group) and

(b) a 2-methylbenzoxazole DNA alkylation unit linked to a DNA minor groove binding unit.

The N-protected 2-methylbenzoxazole DNA alkylating units of the present invention can be readily linked to DNA minor groove binding units to provide highly cytotoxic duocarmycin analogs. They may also be conjugated with other chemical moieties to form novel bioactive compounds, including ADCs.

Compounds of the invention in which a DNA alkylating subunit is linked to a DNA minor groove binding unit can be converted into ADCs by linking the antibody or other ligand via a linker. The linker may be directly linked to the DNA alkylating subunit through a hydroxyl or amine group X, or indirectly linked to the DNA alkylating subunit through a DNA minor groove binding unit at the Y position.

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

Wherein:

LG is a leaving group;

x is a group selected from hydroxy, protected hydroxy, prodrug hydroxy, amino, and protected amino; wherein amino is-NH₂or-NH (C)₁-C₆) An alkyl group; and is

Y is an N-protecting group.

The compounds of formula I are useful in the synthesis of compounds of formulae II and III, as well as in ADCs and precursors thereof.

The invention also relates to compounds of formula Ia,

wherein LG, X and Y are as defined for formula I.

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

Wherein:

LG is a leaving group;

x is a group selected from hydroxy, protected hydroxy, prodrug hydroxy, amino, and protected amino; wherein amino is-NH₂or-NH (C)₁-C₆) An alkyl group; and is

DB is DNA minor groove binding unit.

The invention also relates to compounds of the formula IIa,

wherein LG, X and DB are as defined for formula II.

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

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

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 patterns and strengths. Thus, one skilled in the art can readily determine whether a particular chemical entity constitutes a DNA minor groove binding unit.

In one embodiment, DB comprises a reactive moiety RM compatible with a linker group or complementary reactive sites on a component of a linker group, wherein the linker group is attached to a ligand, or is adapted to be attached to a ligand, such as an antibody.

In one embodiment, the RM is a reactive moiety selected from the group consisting of: azides, alkynes, carbamoyl halides, acyl halides, active esters, anhydrides, α -haloacetyl, α -haloacetamide, maleimide, isocyanates, isothiocyanates, disulfides, thiols, hydrazines, hydrazides, sulfonyl chlorides, aldehydes, methyl ketones, vinyl sulfones, halomethyl and methylsulfonates.

In some embodiments of the invention, for example compounds of formula II, the 2-methylbenzoxazole DNA alkylating subunit is bound to a DNA minor groove binding unit (DB). Suitable DNA binding moieties for use in the present invention have an affinity for binding in the minor groove of double stranded DNA. There is a wealth of information available on the Methods of designing and synthesizing these molecules and determining their DNA binding patterns and strengths, for example, as described in Drug-Nucleic Acid Interactions (drugs-Nucleic Acid Interactions) edited by j.b. charies and m.j.waring (Methods in Enzymology Vol 340, Academic Press, 2001); molecular Recognition of DNA by Small Molecules (Molecular Recognition of DNA by Small Molecules) (bioorg.med.chem. (2001)9,2215); a Structure-Based DNA Targeting strategy using Small Molecule Ligands for Drug Discovery (Structure-Based DNA-Targeting molecules with Small Molecule Ligands for Drug Discovery) (med. res. rev. (2013)33,1119); and a Fluorescent Intercalator Displacement Assay (A Fluorescent Intercalator Displacement Assay for assessing DNA Binding Selectivity and Affinity) (chem. Rev. (2004)37,61) for Establishing DNA Binding Selectivity and Affinity.

The DNA binding moiety for attachment to the DNA-alkylating unit is a heteroaryl or aryl group that may be substituted with other functional groups. Generally, planar aryl and heterocyclic systems have the appropriate physicochemical properties for binding within the minor groove, which are typically driven by the combination of H-bonding and van der waals interactions with the DNA components of the walls and bottom of the groove. Aryl and heteroaryl ring substituents that enhance these interactions increase the strength of the binding.

Joining is further facilitated by joining two or more ring systems together to create a longer and more extensive interaction with the sulcus, so long as the overall structure maintains the correct curvature and twist to match that of the sulcus into which it is incorporated. This is most advantageously achieved when there is minimal distortion of the small molecule ligand or DNA; i.e. when there is a high degree of shape complementarity. The use of amide linkages to link the ring system is an advantageous motif because the amide itself can participate in hydrogen bonding interactions with DNA, while also providing sufficient flexibility to accommodate the desired distortion and curvature.

Another factor to consider, particularly for extended length DNA minor groove binders, is to maintain proper alignment or positioning between the H-bond donor and acceptor on the ligand and on the DNA. In some cases, this may be achieved by changing the nature of the substituents on the aryl or heteroaryl ring or changing the position of the substituents. A large number of DNA minor groove Binding ligand libraries have been constructed and their Binding affinities determined (e.g., total synthesis of distamycin A and 2640 analogs: a Solution-Phase Combinatorial Approach for Discovery of novel biologically active DNA Binding Agents and Development of Rapid, High Throughput screens to determine Relative DNA Binding affinities or DNA Binding Sequence selectivities (A Solution-Phase Binding Affinity to the Discovery of New, biological DNA Binding Agents and Development of a Rapid, High-Throughput screening for Determining sensitivity or DNA Binding 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 CC-1065and duocarmycin (+) -1,2,9,9a-tetrahydro [ C ] indole [4 ] indole-4-one analogs, it defines the role of the DNA Binding Domain (Parallel Synthesis and Evaluation of 132(+) -1,2,9,9 a-tetracylopa [ c ] benz [ e ] indole-4-one (CBI) antigens of CC-1065and the Duocarmycins refining the conjugation of the DNA-Binding Domain, J.org.chem. (2001)66,6654).

In a particular embodiment of the compounds of formulae II and IIa, the DNA minor groove binding unit comprises a heteroaryl group that can be linked to a second heteroaryl or aryl group through an amide linkage.

In other embodiments of the compounds of formula II and IIa, the DNA minor groove binding unit comprises a single aryl or heteroaryl group linked to the DNA alkylating unit through an alkenyl (-CH ═ CH-) group.

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

Wherein:

LG is a leaving group;

x is a group selected from hydroxy, protected hydroxy, prodrug hydroxy, amino, and protected amino; wherein amino is-NH₂or-NH (C)₁-C₆) An alkyl group; and is

Y is selected from:

(a) an N-protecting group;

(b)-C(O)-Ar¹

(c)-C(O)-Ar¹-NH-C(O)-Ar²

(d)-C(O)-Ar¹-NH-C(O)-CH＝CH-Ar³(ii) a Or

(e)-C(O)-CH＝CH-Ar³

Wherein Ar is¹、Ar²And Ar³Each independently selected from heteroaryl or aryl, wherein each said heteroaryl or aryl is optionally substituted with one or more of: - (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -OH, -O- (C)₁-C₆) Alkyl, -NH₂、-NH(C₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl and-NHC (O) - (C)₁-C₆) An alkyl group;

The invention also relates to compounds of formula IIIa,

wherein LG, X and DB are as defined for formula III.

The following embodiments apply to compounds of formulae I, Ia, II, IIa, III and IIIa.

The term "leaving group" as used herein refers to a group that is removed from the carbon center in a substitution reaction. Typically such groups are stable in the 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 (C)₁-C₆) Alkanesulfonates (e.g., methanesulfonate, trifluoromethanesulfonate and trifluoroethanesulfonate) and optionally substituted benzenesulfonates.

In one embodiment, LG is selected from the group consisting of: chloride, bromide, iodide and-OSO₂R¹(ii) a Wherein R is¹Is selected from (C)₁-C₁₀) Alkyl, (C)₁-C₁₀) Heteroalkyl group, (C)₁-C₁₀) Aryl or (C)₁-C₁₀) A heteroaryl group. In one embodiment, LG is a halogen group, preferably chloro. Scheme 2 below shows the synthesis of DNA alkylated subunits containing different leaving groups.

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

The term "prodrug hydroxy" 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 the "prodrug Design (Design of produgs)" edited by h. Examples are well known in the art and include, but are not limited to, phosphate, carbamate, and glycoside.

Compounds of the invention in which X is a prodrug hydroxyl group are useful in ADCs in which the linker will be attached to the DNA minor groove binding unit rather than through X. This will yield a prodrug form of the ADC. Those skilled in the art, designing ADCs that incorporate the compounds of the invention, will be able to select the appropriate position of the tethered antibody component and determine the desirability of a particular prodrug form for the intended application of the ADC.

As used herein, the term "protected hydroxyl group" refers to a hydroxyl group that has been protected from undesirable reactions during synthetic procedures. Protected hydroxyl groups are readily converted to free hydroxyl groups when the hydroxyl groups are no longer needed and/or allowed to react. Hydroxy protecting groups are described in protecting groups in organic synthesis edited by t.w. greene et al. (John Wiley & Sons, 1999). As used herein, the term "protected hydroxyl" also includes groups such as OTf which contain a good leaving group and which are useful in the synthesis of derivatives in which the OH group is replaced by a replacement group. Examples of hydroxy protecting groups useful in compounds of the invention include-OBn, -OTf, -OMOM, -OMEM, -OBOM, -OTBDMS, -OPMB, -OSEM.

The term "protected amino group" as used herein refers to an amino group that has been protected from undesired reactions during the synthetic procedure. Protected amino groups are readily converted to free amino groups when the amino groups are no longer needed and/or allowed to react. In the compounds of the invention, the amino group in the X position is selected from-NH₂and-NH (C)₁-C₆) An alkyl group. Amino protecting groups are described in the organic synthesis edited by t.w. greene et al (John Wiley)&Sons,1999) and the 'amino acid-protecting group' edited by Fernando Albericio (compare with Albert Isidro-Llobet and Mercedes Alvarez) Chemical Reviews 2009(109) 2455-2504.

Examples of amino protecting groups that may 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-nitrophenyloxyacetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, picolinoyl, aminocaproyl, benzoyl, methoxy-carbonyl, 9-fluorenylmethoxycarbonyl, 2,2, 2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-carbonyl, t-butoxycarbonyl, benzyloxycarbonyl, p-nitrophenoxycarbonyl, 2, 4-dichloro-benzyloxycarbonyl, and the like. Further examples include nitrobenzenesulfonyl (o-or p-nitrobenzenesulfonyl), Bpoc (2- (4-biphenylyl) isopropoxycarbonyl) and Dde (1- (4, 4-dimethyl-2, 6-dioxohexylidene) ethyl).

wherein R is²Is t-Bu, Bn or allyl; z is selected from Boc and COCF₃Fmoc, Alloc, Cbz, Teoc and Troc.

In one embodiment, X is-OH or-NH₂。

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

In one embodiment, X is protected-NH₂。

In one embodiment, X is selected from the group consisting of-OBn, -OTf, -OMOM and-OMEM.

A process for preparing compounds of the present invention wherein X is hydroxy, protected hydroxy or prodrug hydroxy is shown in scheme 2 below. A process for preparing compounds of the present invention wherein X is amino or protected amino is shown in scheme 3 below.

The compounds of the present invention wherein Y is an N-protecting group provide 2-methylbenzoxazole DNA-alkylating units that can be readily linked to DNA minor groove binding units to provide highly cytotoxic duocarmycin analogs.

The term "N-protecting group" as used herein refers to a group that can be easily removed to provide free N and protect the N atom from undesired reactions during synthetic procedures. Such protecting groups are described in the organic synthesis edited by T.W. Greene et al (John Wiley & Sons,1999) and the 'amino acid-protecting group' edited by Fernando Albericio (with Albert Isidro-Llobet and Mercedes Alvarez) Chemical Reviews 2009(109) 2455-4. Examples include, but are not limited to, acyl and acyloxy groups such as acetyl, chloroacetyl, trichloroacetyl, o-nitrophenylacetyl, o-nitrophenyloxyacetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, picolinoyl, aminocaproyl, benzoyl, methoxy-carbonyl, 9-fluorenylmethoxycarbonyl, 2,2, 2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxy-carbonyl, t-butoxycarbonyl, benzyloxycarbonyl, p-nitrophenoxycarbonyl, 2, 4-dichloro-benzyloxycarbonyl, and the like. Further examples include nitrobenzenesulfonyl (o-or p-nitrobenzenesulfonyl), Bpoc (2- (4-biphenylyl) isopropoxycarbonyl) and Dde (1- (4, 4-dimethyl-2, 6-dioxohexylidene) ethyl).

In one embodiment, Y is selected from Boc, COCF₃N-protecting groups for Fmoc, Alloc, Cbz, Teoc and Troc.

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

In compounds 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-.

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

In one embodiment, Ar¹、Ar²And Ar³Independently selected from the group consisting of

As understood by those skilled in the art, 5-azaindole, 6-azaindole, 7-azaindole, imidazole, thiazole, oxazole and pyrazole groups can be substituted with up to two groups and a triazole, only one.

When Ar is¹Attached to Ar by NH-C (O)²When Ar is¹Linked to Ar by-NH-C (O) -CH ═ CH³When is, Ar¹The connection point on may be any one of the numbered positions. As will be appreciated by those skilled in the art, such a connection will reduce Ar¹One of the number of possible substituents above.

In each case a substituent- (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -O- (C)₁-C₆) Alkyl, -NH (C)₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl and-NHC (O) - (C)₁-C₆) The alkyl groups may independently optionally be substituted by-NMe₂、-NHMe、-NH₂-OH, morpholine and-SH.

In one embodiment, Ar¹Is heteroaryl. In one embodiment, heteroaryl is indole, azaindole, benzofuran, or benzothienyl, which is linked to the DNA alkylation unit at the 2-position of the heteroaryl via-c (o) or c (o) -CH ═ CH-.

In one embodiment, Ar¹Attached to Ar by NH-C (O)²Or Ar¹Linked to Ar by-NH-C (O) -CH ═ CH³. In one embodiment, Ar¹The point of attachment is at the 5-position of the indole, azaindole, benzofuran, or benzothiophene group.

In one embodiment, Ar²Selected from the group consisting of: indole, azaindole, benzene, benzofuran, pyridine, pyrimidine, pyrrole, imidazole, thiophene, thiazole, oxazole, pyrazole, triazole, pyrazine or pyridazine.

In one embodiment, Ar²Selected from indole, azaindole, benzene, benzofuran, pyrrole or imidazole.

In one embodiment, Ar³Selected from benzene, pyridine, pyrimidine and pyridazine. In one embodiment, Ar³Is benzene or pyridine, preferably benzene.

In one embodiment Y is-C (O) -Ar¹Wherein Ar is¹Is heteroaryl or aryl, optionally substituted with one or more of: - (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON(C₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -OH, -O- (C)₁-C₆) Alkyl, -NH₂、-NH(C₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl or-NHC (O) - (C)₁-C₆) An alkyl group, a carboxyl group,

In one embodiment, Ar¹Substituted at one position of the heteroaryl or aryl ring.

In one embodiment, Ar¹Is heteroaryl.

In one embodiment, heteroaryl is indole, azaindole, benzofuran, or benzothienyl, which is linked to the DNA alkylation unit at the 2-position of the heteroaryl via-c (o) or c (o) -CH ═ CH-.

In one embodiment, Ar¹Is composed of

Wherein A is NH, O or S, and

R¹⁰、R¹¹and R¹²Independently selected from H, - (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -OH, -O- (C)₁-C₆) Alkyl, -NH₂、-NH(C₁-C₆) Alkyl radical、-N(C₁-C₆) Alkyl radical (C)₁-C₆) Alkyl and-NHC (O) - (C)₁-C₆) An alkyl group, a carboxyl group,

in each case of- (C)₁-C₆) Alkyl, -O- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl and-NH-C (O) - (C)₁-C₆) Alkyl is independently optionally substituted by-NMe₂、-NHMe、-NH₂-OH, morpholine and-SH.

In one embodiment, a is NH.

In one embodiment, R¹⁰、R¹¹And R¹²Is OMe.

In one embodiment, R¹⁰is-O- (C)₁-C₆) Alkyl, optionally substituted by-NMe₂、-NHMe、-NH₂-one or more of OH, morpholine and-SH; r¹¹And R¹²Are all H.

In one embodiment, R¹⁰is-NHC (O) - (C)₁-C₆) Alkyl, optionally substituted by-NMe₂、-NHMe、-NH₂-one or more of OH, morpholine and-SH; r¹¹And R¹²Are all H.

In one embodiment, R¹⁰is-NH-C (O) -Ar²Wherein Ar is²Is optionally substituted indole, azaindole, benzene, benzofuran, pyrrole or imidazole; r¹¹And R¹²Are all H.

In one embodiment, R¹⁰is-NH-C (O) -Ar²Wherein Ar is²Is an optionally substituted indolyl group; r¹¹And R¹²Are all H. In one embodiment, the indole group is substituted with-O- (C)₁-C₆) Alkyl substituted, optionally by-NMe₂、-NHMe、-NH₂-OH, morpholine and-SH.

In one embodiment, R¹⁰is-NH-C (O) -Ar²Wherein Ar is²Is optionally substitutedPhenyl of (a); r¹¹And R¹²Are all H. In one embodiment, the phenyl group is substituted with-OH, -NH₂or-O- (C)₁-C₆) Alkyl substitution of which-O- (C)₁-C₆) Alkyl is optionally substituted by-NMe₂And (4) substitution.

In one embodiment, R¹⁰is-NH-C (O) -CH ═ CH-Ar³Wherein Ar is³Is optionally substituted phenyl, pyrimidinyl or pyrrolyl; r¹¹And R¹²Are all H. In one embodiment, the benzene, pyrimidinyl or pyrrolyl group is substituted with-O- (C)₁-C₆) Alkyl, -NH₂or-NHC (O) - (C)₁-C₆) Alkyl substitution of which-O- (C)₁-C₆) Alkyl is optionally substituted with morpholine.

In some embodiments, Ar¹Selected from the group consisting of:

wherein R is¹⁰、R¹¹And R¹²(when present) is independently selected from H, - (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -OH, -O- (C)₁-C₆) Alkyl, -NH₂、-NH(C₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl and-NHC (O) - (C)₁-C₆) An alkyl group, a carboxyl group,

In one embodiment, R¹⁰、R¹¹And R¹²Is OMe.

In one embodiment, R¹⁰、R¹¹And R¹²One of them is-O- (C)₁-C₆) Alkyl, optionally substituted by-NMe₂、-NHMe、-NH₂-OH, morpholine and-SH.

In one embodiment, R¹⁰、R¹¹And R¹²One of them is-NHC (O) - (C)₁-C₆) Alkyl, optionally substituted by-NMe₂、-NHMe、-NH₂-OH, morpholine and-SH.

In one embodiment, R¹⁰、R¹¹And R¹²One of them is-NH-C (O) -Ar²Wherein Ar is²Is optionally substituted indole, azaindole, benzene, benzofuran, pyrrole or imidazole.

In one embodiment, R¹⁰、R¹¹And R¹²One of them is-NH-C (O) -Ar²Wherein Ar is²Is an optionally substituted indolyl group. In one embodiment, the indolyl group is substituted with-O- (C)₁-C₆) Alkyl substituted, optionally by-NMe₂、-NHMe、-NH₂-OH, morpholine and-SH.

In one embodiment, R¹⁰、R¹¹And R¹²One of them is-NH-C (O) -Ar²Wherein Ar is²Is optionally substituted phenyl. In one embodiment, the phenyl group is substituted with-OH, -NH₂or-O- (C)₁-C₆) Alkyl substitution of which-O- (C)₁-C₆) Alkyl is optionally substituted by-NMe₂And (4) substitution.

In one embodiment, R¹⁰、R¹¹And R¹²One of them is-NH-C (O) -CH ═ CH-Ar³Wherein Ar is³Is an optionally substituted benzene, pyrimidinyl or pyrrolyl group. In one embodiment of the process of the present invention,phenyl, pyrimidinyl or pyrrolyl-O- (C)₁-C₆) Alkyl, -NH₂or-NHC (O) - (C)₁-C₆) Alkyl substitution of which-O- (C)₁-C₆) Alkyl is optionally substituted with morpholine.

In one embodiment Y is-C (O) -Ar¹-NH-C(O)-Ar²Wherein Ar is¹And Ar²Is heteroaryl or aryl, optionally substituted with one or more of: - (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -OH, -O- (C)₁-C₆) Alkyl, -NH₂、-NH(C₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl or-NHC (O) - (C)₁-C₆) An alkyl group, a carboxyl group,

In one embodiment, Ar¹Is heteroaryl and Ar²Is heteroaryl or aryl.

In one embodiment, Ar¹Is an indole, azaindole, benzofuran or benzothiophenyl group attached to the DNA alkylation unit at the 2-position of the heteroaryl group.

At one endIn one embodiment, Ar²Selected from indole, azaindole, benzene, benzofuran, pyrrole or imidazole.

In one embodiment, Ar¹Is an indole, azaindole, benzofuran or benzothiophenyl group attached to the DNA alkylation unit at the 2-position of the heteroaryl group, and Ar²Selected from indole, azaindole, benzene, benzofuran, pyridine, pyrimidine, pyrrole, imidazole, thiophene, thiazole, oxazole, pyrazole, triazole, pyrazine or pyridazine.

In one embodiment, Ar¹The point of attachment is at the 5-position of the indole, azaindole, benzofuran, or benzothiophene group.

In one embodiment Y is-C (O) -Ar¹-NH-C(O)-CH＝CH-Ar³Wherein Ar is²And Ar³Is heteroaryl or aryl, optionally substituted with one or more of: - (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -OH, -O- (C)₁-C₆) Alkyl, -NH₂、-NH(C₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl or-NHC (O) - (C)₁-C₆) An alkyl group, a carboxyl group,

In one embodiment, Ar¹Is heteroaryl and Ar³Is heteroaryl or aryl.

In one embodiment, Ar¹Is an indole, azaindole, benzofuran or benzothiophenyl group attached to the DNA alkylation unit at the 2-position of the heteroaryl group.

In one embodiment, Ar³Selected from benzene, pyridine, pyrimidine and pyridazine. In one embodiment, Ar³Is benzene or pyridine, preferably benzene.

In one embodiment, Ar¹Is an indole, azaindole, benzofuran or benzothiophenyl group attached to the DNA alkylation unit at the 2-position of the heteroaryl group, and Ar³Selected from benzene, pyridine, pyrimidine and pyridazine.

In one embodiment, the point of attachment on Ar1 is at the 5-position of the indole, azaindole, benzofuran, or benzothiophene group.

In one embodiment Y is-c (o) -CH ═ CH-Ar³Wherein Ar is³Is heteroaryl or aryl, optionally substituted with one or more of: - (C)₁-C₆) Alkyl, -CO- (C)₁-C₆) Alkyl, -CONH (C)₁-C₆) Alkyl, -CON (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl, -OH, -O- (C)₁-C₆) Alkyl, -NH₂、-NH(C₁-C₆) Alkyl, -N (C)₁-C₆) Alkyl radical (C)₁-C₆) Alkyl or-NHC (O) - (C)₁-C₆) An alkyl group, a carboxyl group,

In one embodiment, Ar³Selected from benzene, pyridine, pyrimidine and pyridazine. In one embodiment, Ar³Is benzene or pyridine, preferably benzene.

In one embodiment, Ar³Is optionally substituted by-O- (C)₁-C₆) Alkyl-substituted phenyl optionally substituted by-NMe₂、-NHMe、-NH₂-OH, morpholine and-SH.

Compounds of formulae I, II and III are ring-opened precursors of compounds of formula IV, which are considered active agents in vivo.

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

Wherein V is Y or DB and X, Y and DB have the same meaning as defined for the compounds of formulae I, II and III and X' is X with the loss of H.

The compounds of formulae I, II and III contain a Leaving Group (LG) that promotes cyclopropyl ring formation under physiological conditions to form compounds of formula IV.

In one aspect, the present 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.

In one embodiment, the present 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.

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

in one embodiment, the present invention provides a compound selected from the group consisting of:

5.3 Synthesis of Compounds of the invention

The compounds of the invention can be prepared using the methods and procedures described herein or similar methods and procedures thereto. Other suitable methods of preparing the compounds of the invention will be apparent to those skilled in the art.

It is to be understood that where typical or preferred process conditions are indicated (e.g., reaction temperature, time, molar ratios of reactants, solvents, pressures, etc.), other process conditions may also be used unless otherwise indicated. The optimum reaction conditions may vary depending on the particular reactants used.

The starting materials for this method and reaction are commercially available or can be prepared by known procedures or modifications thereof, such as, for example, those described in standard references, 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).

The various starting materials, intermediates and compounds may be isolated and purified as appropriate using conventional techniques such as precipitation, filtration, crystallization, evaporation, distillation and chromatography. Characterization of the compounds can be performed using conventional methods, e.g., by melting point, mass spectrometry, nuclear magnetic resonance, and various other spectroscopic analyses.

Conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesirable reactions. The need for protection and deprotection, and the choice of an appropriate protecting group, are readily determined by those skilled in the art. Suitable Protecting Groups for various functional Groups and suitable conditions for Protecting and deprotecting particular functional Groups are well known in the art (see, e.g., t.w.green and g.m.wuts, Protecting Groups in Organic Synthesis, third edition, Wiley, New York, 1999).

The individual enantiomers of the payload containing the alkylated subunit of 2-methylbenzoxazole can be prepared using chiral HPLC resolution of a suitable intermediate (e.g., compound 49 or 18). Suitable columns include those used for the resolution of the relevant alkylated subunits 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). The resolved intermediates can be converted into payload and drug-linker conjugates by methods similar to those described for the racemates.

A general method for preparing 2-methylbenzoxazole alkylated subunits where X ═ protected OH is shown in scheme 2. Compounds 5and 7 in this scheme are readily converted to 2-methylbenzoxazole alkylated subunits where X ═ free OH and X ═ prodrug OH.

Scheme 2

In this scheme, 3, 4-dihydroxy-5-nitrobenzoate 1 was used as starting material. These can be prepared, for example, by oxidation of 3, 4-dihydroxy-5-nitrobenzaldehyde to the corresponding acid, followed by esterification with the alcohol ROH. Alternatively, 1 may be prepared by nitration of 4-hydroxy-3-methoxybenzoic acid followed by the use of a reagent such as HBr or BBr₃Dealkylation of the methoxy group followed by esterification with the alcohol ROH.

The nitro group of 1 is reduced to the corresponding amine by exposure to suitable 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 under acidic conditions with a trialkyl orthoacetate (e.g. trimethyl orthoacetate) to induce cyclization to substituted 2-methylbenzoxazole 2.

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

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 may conveniently be carried out in a single pot using Diphenylphosphorylazide (DPPA) reagents 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, Y ═ Cbz was obtained using benzyl alcohol, Y ═ Teoc was obtained using 2- (trimethylsilyl) ethanol, and Y ═ Troc was obtained using 2,2,2- (trichloro) ethanol. Alternatively, the intermediate isocyanate may be reacted with water instead of an alcohol toThe corresponding unprotected amine is produced. It may then be converted to the protected form under standard conditions, for example reaction with trifluoroacetic anhydride will give Y ═ COCF₃And reaction with benzyl chloroformate will yield Y ═ Cbz.

Compound 4 may be selectively halogenated at the 4-position by reaction with a suitable reagent, for example NBS introduces bromide at the 4-position and NIS introduces iodide at the 4-position. These reactions are preferably carried out with a single equivalent of halogenating agent to minimize the dihalide in the 4-and 6-positions. To prepare the alkylated subunit with the leaving group chlorine, i.e., 5, the halogenated intermediate is then reacted with 1, 3-dichloropropene in a suitable base such as NaH or K₂CO₃The base serves to deprotonate the NHY group and direct the chloroallylation to that position. This intermediate is then treated with a suitable reagent such as tributyltin hydride or tris (trimethylsilyl) silane to extract the halogen atom and initiate free radical mediated cyclization on the pendant chloroallyl group, which yields product 5.

Alkylated subunits in which the leaving group is halide or sulfonate may be prepared via intermediate 6. Compound 6 can be prepared from 4 by modifying the above procedure. Allyl bromide was used instead of 1, 3-dichloropropene and the radical-mediated cyclization was carried out in the presence of spin trap TEMPO (2,2,6, 6-tetramethyl-1-piperidinyloxy). This results in a product containing N-O bonds that is cleaved by exposure to Zn and an acid such as acetic acid to yield 6. Conversion of primary alcohols of 6 to LG ═ halides or sulfonates by using standard reagents, such as bromine and triphenylphosphine, can be used to prepare LG ═ Br, while methanesulfonyl chloride and triethylamine can be used to prepare LG ═ OSO₂Me。

The protecting groups for X and Y can be selectively removed and replaced at compound 5and 7 stages. This may be the preferred method for its introduction for several Y protecting groups that may be less stable to the conditions used in the post-synthesis step of compound 4. For example, Y ═ COCF₃May be unstable to the basic conditions used in the allylation reaction, but may be introduced by deprotecting 5 or 7 (where, for example, Y ═ Boc), followed by reaction with trifluoroacetic anhydride. Similarly, Y ═ Fmoc may also be unstable under the same basic conditions, howeverY ═ Alloc may interfere with free radical mediated cyclization. However, these alternative protecting groups may be very useful in subsequent reactions of 5and 7 where stability to different reaction conditions is required. This is stated below as NH in which X ═ is protected₂The synthesis of alkylated subunits of (a).

For 5and 7, the group X ═ protected OH can also be converted to the free OH (i.e. compound 8 below), and thus to the prodrug OH. The latter transformation is carried out via well-established synthetic methods. For example, reaction of 8 with 4-alkylpiperazine carbonyl chloride affords 5and 7, where X ═ piperazine-1-carboxylic acid esters, in which the N in the 4-position is substituted by alkyl. In another example, the free OH of 8 is associated with the general structure R₂NP(OR²)₂Wherein R is lower alkyl, e.g. ethyl or isopropyl, R⁴Is t-Bu, Bn or allyl. The intermediate product is then oxidized with a suitable reagent, such as hydrogen peroxide OR an organic peracid, to give 5and 7, wherein X ═ op (o) (OR)²)₂。

A general method for preparing 2-methylbenzoxazole alkylated subunits is shown in scheme 3, where X ═ NH₂Protected NH₂、NHR¹Or protected NHR¹Wherein R is¹Is (C)₁–C₆) An alkyl group.

Scheme 3

Compound 8, containing a free OH group, is reacted with triflic anhydride and a suitable base, such as triethylamine, to give compound 9. Using suitable catalysts, e.g. Pd (OAc)₂And a ligand such as BINAP to react the compound with benzophenone imine to effect amination and produce compound 10. The benzophenone imine is cleaved under acidic conditions to yield compound 11. The acidic conditions required mean that some protecting groups Y are more suitable than others. For example, Y ═ COCF₃More suitable than Y ═ Boc, so a convenient route requires the exchange of Y ═ Boc to COCF at the stage of compound 7₃. Of protecting groupsCombinations thereof are also possible and may provide different synthetic advantages, depending on the particular compound of interest.

Compound 11 can be further converted to alkylated subunit 12 with NHR at the X position¹(wherein R is¹Is (C)₁–C₆) Alkyl) substituents. For example, 11 can be reacted with acetic formic anhydride to produce a formylated intermediate, which is reduced with borane to produce 12, where R¹Me. Alternatively, 11 may be reacted with aldehyde R¹CHO condensation, wherein R¹Is (C)₁-C₆) Alkyl to give an imine, reduction with a reagent such as sodium borohydride or sodium cyanoborohydride to give 12, wherein R¹＝(C₁-C₆) An alkyl group. Compounds 11 and 12 can be converted to protected analogues 13 and 14 using standard conditions as described above, for example reaction with trifluoroacetic anhydride will yield X ═ NHZ and NZR¹Wherein Z is COCF₃And R is¹Is (C)₁–C₆) Alkyl, and reaction with fluorenylmethoxycarbonyl chloride will yield X ═ NHZ and NZR¹Wherein Z ═ Fmoc and R¹Is (C)₁–C₆) An alkyl group.

A general method for preparing DNA alkylating agents incorporating 2-methylbenzoxazole alkylating subunits and a DNA minor groove binding moiety (DB) is shown in scheme 4.

Scheme 4

In the case where 15 contains X ═ OH, the protecting group Y is removed by an appropriate method (for example, by treatment with HCl for Y ═ Boc), and the resulting intermediate is allowed to bind to the DNA minor groove binding unit Ar¹CO₂H or Ar³CH＝CHCO₂H is reacted in the presence of a suitable amide coupling reagent such as EDCI or HOBt. Other activated forms of DNA minor groove binding units, such as acid chlorides, may also be used. This process directly produces 17 where X ═ OH.

Containing X ═ protected OH or protected NH at 15₂Or protected NHR¹In the case of (3), the synthesis proceeds in 2 steps via intermediate 16. It will be clear to the skilled person that the protecting groups in X and Y should be chosen appropriately to allow deprotection of Y 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 acid sensitive, such as NHTeoc or NRTeoc. Suitable combinations of orthogonal protecting groups are readily available by following the general scheme shown above. Amide coupling to form compound 16 in the same general manner as described above, wherein Y ═ c (o) Ar¹or-C (O) -CH-Ar³And 16 is converted to 17 by removal of the protecting group in X. Containing X ═ NH in compound 17₂In the case of (a), benzophenone imine may then be used as a suitable protected precursor, i.e. 15 wherein X ═ N ═ c (ph)₂。

The same method can be used to prepare payloads where the side chain contains two attached aromatic rings by substituting the appropriate side chain acid, i.e. by using Ar²C(O)NHAr¹CO₂H or Ar³CH＝CHC(O)NHAr¹CO₂H。

Scheme 5 shows the synthesis wherein X is NH₂Are also contemplated.

Scheme 5

In the synthesis presented in scheme 5, the alkylated subunit of 2-methylbenzoxazole containing a free amino group (general structure 22) can be prepared by a method similar to that reported for the ring-opened-CBI compound (bioorg. med. chem. (2016)24,6075). In particular, compound 18 was converted to triflate 19 using triflic anhydride and triethylamine. Use of benzophenone imine and Pd (OAc)₂Catalyst and BINAP ligand amination compound 19 affords 20. The Boc protecting group was selectively cleaved using HCl in methanol or TFA in dichloromethane, and the resulting intermediate was reacted with the appropriate side chain acid using EDCI as a coupling agent to afford amide 21. Removal of benzophenone protecting groups using aqueous acetic acidTo yield the desired product 22. The process can also be applied to the R and S enantiomers of 18 to give the corresponding R and S enantiomers of 22.

Many DNA minor groove binding unit acids Ar with suitable substituents¹CO₂H or Ar³CH＝CHCO₂H is a commercially available compound or can be readily prepared from commercially available compounds by simple functional group changes, for example 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 and/or metathesis reactions to obtain the various desired substituents. In addition, many other aryl and heteroaryl compounds useful for preparing suitable DNA minor groove binding monobasic 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 a.r.kattritzky, c.a.ramsden, e.f.v.scriven and r.j.k.taylor, Elsevier, 2008. For example, the synthesis of indole is reviewed in section 3.03 (Vol 3, pp 269-. There are many other reviews on 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.

More specifically, for use as DNA minor groove binding monobasic acid, methods are known for producing heteroaryl compounds having a carboxylate substituent at the desired 2-position. Many of these are included in the comments cited above, but specific examples are also given here: for indole-2-carboxylate org.lett. (2016)18,3586; org.lett. (2004)6,2953; for azaindole-2-carboxylic acid esters WO 2009/030725; for benzofuran-2-carboxylate org.lett. (2013)15,3876; chem.soc., Perkin trans.2(1998) 1063; and org.lett. (2013)15,3876 for benzothiophene-2-carboxylate; leg (2012)14,5334.

Many of these methods have been used to synthesize specific DNA minor groove binding monobasic acids for the preparation of duocarmycin analogs. For example, the synthesis of 72 different substituted indole-2-carboxylic acid esters and their coupling to ring-opened CBI alkylation subunits is described in bioorg.med.chem. (2003)11,3815. Other examples of a series of substituted indole-2-carboxylic acid esters useful for preparing duocarmycin analogs are described in j.med.chem. (2017)60,5834; ChemMedChem (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. Wherein Ar has also been reported¹To aryl or heteroaryl Ar³Synthesis of DNA minor groove binding monobasic acids of the type described, for example, in WO 2011/133039; med chem. (2003)46,634; and j. org. chem. (2001)66,6654 (which describes the synthesis of such side chains 132 that bind together indole, benzofuran, benzothiophene, pyrrole, thiophene, imidazole, thiazole, and like rings).

Those skilled in the art will recognize that Ar¹And Ar²Or Ar³Many other side chains attached can be attached to Ar containing an amino substituent by using standard methods and coupling reagents¹Units and Ar containing carboxylic acid substituents²Or Ar³Amide bonds are formed between the units and prepared using appropriate protecting groups appropriate for the other substituents referred to in the specific examples. Thus, there is a wealth of information and precedent describing the preparation of the acid Ar which reacts with the DNA alkylation unit in the synthesis of the compounds of the invention¹CO₂H or Ar³CH＝CHCO₂H or Ar²CONHAr¹CO₂H or Ar³CH＝CHCONHAr¹CO₂General and specific methods for H.

DNA alkylating agents incorporating 2-methylbenzoxazole alkylated subunits and minor groove-binding side chains can be used as the payload of ADCs. To do this, a linker must be used to link the payload to the antibody. The connector can be attached to the payload in several different ways. For example, the linker may be attached at the X position, which may be via a carbamate (-OC (O) NHR or-)OC(O)NR₂OR-NHC (O) OR OR-NRC (O) OR') OR via an ether (-OR) functional group. These types of linkers require fragmentation after ADC metabolism to release X ═ OH or NH₂Or NHR, one type of linkage is referred to as a 'traceless' linker. Several examples of suitable linker types are known, which typically incorporate a self-immolative spacer, such as p-aminobenzyl ether or p-aminobenzyl carbamate, which may be further substituted in a manner that affects its cleavage rate, or linked to an additional cleavable spacer, such as N, N-dialkyl-1, 2-diaminoethane.

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

Scheme 6 outlines the preparation of compounds of the invention, where the linker is attached via a phenol carbamate.

Scheme 6

In this scheme, compound 23 was reacted with 4-nitrophenyl chloroformate, followed by reaction of the product with mono-Boc protected N, N' -dimethyl-1, 2-diaminoethane to afford 24. Deprotection of the Boc group of 24 with TFA gave the corresponding TFA salt. Activated maleimide-valine-citrulline-PABA compound 25 was prepared as described (mol. pharm. (2015)12,1813). The TFA salt and 25 were reacted under slightly basic conditions to give the drug-linker 26.

Scheme 7 outlines the preparation of compounds of the invention where the linker is attached via a phenol ether.

Scheme 7

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 afford compound 28. By treatment with TFARemoval of the two Boc protecting groups followed by the use of DIPEA in the presence of (Boc)₂O treatment instead of Boc protecting group on aliphatic amine gave compound 29. This compound was reacted with 5- (2- (dimethylamino) ethoxy) -1H-indole-2-carboxylic acid using EDCI as a coupling agent to give 30. Removal of the Boc protecting group with TFA and reaction of the amine with a commercially available NHS ester 31 provided the payload-linker compound 32.

Scheme 8 outlines the preparation of compounds of the invention, where the linker is attached via carbamate.

Scheme 8

Compound 33 is reacted 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.

These examples also serve 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 upon exposure to high levels of intracellular reducing agents such as glutathione. The linker should also contain functional groups that allow attachment to the antibody, such as maleimides that selectively react with thiols, such as those generated by reducing intra-antibody chain disulfide bonds, or on cysteine residues engineered in the site-selectively modified antibody.

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.

Scheme 9

Scheme 9 outlines the preparation of compounds of the invention, where 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. Removal of the Boc protecting group using HCl and reaction of the intermediate with 5- (2- (methylamino) ethoxy) -1H-indole-2-carboxylic acid using EDCI as coupling reagent gave compound 37. Reaction with the activated linker 25 as described above yields the drug-linker 38.

When the prodrug is phosphate OP (O) (OH)₂In this case, the synthesis preferably uses a phosphate ester intermediate OP (O) (OR)⁴)₂Proceeding to the final step of phosphate deprotection. When attached to side chain Y, there is a lot of flexibility as to how the linker is attached to the side chain. The linkage may be traceless, as shown in the above scheme, for which suitable pendant functional groups include alcohols (for carbamate linkages), primary and secondary amines (for carbamate linkages), tertiary amines (for quaternary salt linkages), and thiols (for disulfide linkages). These functional groups may be attached to the side chain at any defined substituent position. The ligation may also be a ligation without traces, i.e. after cleavage from the ADC, the payload still contains fragments of the linker. This approach is suitable when the structure and position of the linker fragment does not interfere with the alkylation of the payload with DNA, i.e., does not adversely affect the cytotoxicity of the released material. Within this limitation, the non-traceless linker may be attached to any type of side-chain reactive moiety RM already defined.

5.4 use of the Compounds of the invention

The compounds of the invention comprise highly cytotoxic payloads or payload components useful in the preparation of ADCs and other biologically active compounds.

Compounds of the invention in which a 2-methylbenzoxazole subunit is linked to a DNA minor groove binding unit can be converted to ADCs by linking the antibody or other ligand binding group via a linker. The linker may be directly linked to the DNA alkylating subunit through a hydroxyl or amino group X, or indirectly linked to the DNA alkylating subunit through a DNA minor groove binding unit.

The binding ligand (e.g., antibody) and appropriate linker may be selected for the particular clinical application for which the ADC is intended. The nature of the linker may influence 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 comprise an extension unit, a spacer unit and a solubility-enhancing moiety.

Examples of such linker groups, extension units, and spacer units include, but are not limited to, those described in US7,964,566B2 and US2017/0232108a1, which are incorporated herein by reference in their entirety.

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.

In another aspect, the invention provides a method of making an ADC or ADC component comprising reacting a compound of formula I, Ia, II, IIa, III or IIIa with a linker or linker-antibody moiety.

The lipophilicity of the novel 2-methylbenzoxazole DNA alkylation unit is much less than the widely used CBI alkylation unit present in many duocarmycin analogs, as shown in examples 6 and 24.

This will make the corresponding ADC easier to manufacture, as the payload component will be more soluble in the aqueous solvent used for conjugating the antibody-linker component. A less lipophilic payload will also result in less ADC aggregation, which will further simplify manufacturing. Reduced aggregation will reduce the risk of immune responses in vivo, and less lipophilic ADCs will have longer clearance from the bloodstream and thus greater overall exposure. In conclusion, a payload based on 2-methylbenzoxazole would result in an ADC that is easier to prepare, safer and more efficient.

DNA alkylating agents based on 2-methylbenzoxazole analogues of duocarmycin are highly cytotoxic compounds. This is demonstrated in examples 7 and 25, examples 7 and 25 describe cytotoxicity assays comparing compounds of the invention with seco-CBI-TMI. The latter is widely recognized as having sufficient cytotoxic potency to be used as an ADC payload. Ring opening with Compound 23-CBI-TMI and protocolClosest comparison between the clear compounds. Since compound 23 shares the same minor groove binding side chain (i.e., TMI), a direct head-to-head comparison of the two compounds could be made, demonstrating equal cytotoxic potency. The high cytotoxicity of the compounds of the invention is very surprising. 2-methylbenzoxazole analogs are structurally closely related to the known COI duocarmycin analog (bioorg.Med.chem.Lett. (2010)20,1854). The only difference between these structures, as shown by ring opening-COI-TMI, is the orientation of the oxazole ring fusion and the nature of the 2-substituent (Me vs. CO)₂Me). However, the cytotoxicity of COI-fold oncomycin analogs is hundreds of fold lower than CBI analogs, making COI analogs unsuitable for use as ADC payloads.

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.

This means that they are unlikely to last a very long time in the circulation and therefore if systemic release from the ADC occurs, they are unlikely to cause associated off-mechanism toxicity. This stability is also very surprising, since it has been found that the cytotoxicity and the aqueous stability of duocarmycin analogs are correlated. In other words, all previously reported analogs that are most cytotoxic (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.

While the compounds of the present invention are primarily useful as payloads to be incorporated into ADCs, in some embodiments they may be used as therapeutic agents in their own right. Thus, the invention also relates to a pharmaceutical composition comprising a compound of formula I, Ia, II, IIa, III or IIIa and a pharmaceutically acceptable carrier.

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

Pharmaceutically acceptable carriers that may 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., tweens or other similar polymeric 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, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol-polyoxyethylene-block polymers, polyethylene glycol-sodium stearate, sodium, Polyethylene glycol and lanolin. Cyclodextrins, such as alpha-, beta-, and gamma-cyclodextrins, or chemically modified derivatives such as hydroxyalkyl cyclodextrins, including 2-and 3-hydroxypropyl-3-cyclodextrin, or other solubilized derivatives may also be advantageously used to enhance delivery. The oil solution or suspension may also contain a long chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents, which are commonly used to formulate pharmaceutically acceptable dosage forms, such as emulsions and/or suspensions.

The pharmaceutical compositions of the present invention may be administered simultaneously, sequentially or separately as a single dose or in a multiple dose regimen, as the sole therapeutic agent or in combination with one or more additional therapeutic agents. The additional therapeutic agent or agents will depend on the disease or condition to be treated or other desired therapeutic benefit. As known to those skilled in the art, one or more additional therapeutic agents may be used in therapeutic amounts indicated or approved for the particular agent.

The pharmaceutical composition is formulated to allow administration to a subject by any selected route, including but not limited to oral or parenteral (including topical, subcutaneous, intramuscular, and intravenous) administration. In some embodiments, the compositions are formulated for oral, intravenous, subcutaneous, intramuscular, transdermal, intraperitoneal, or other pharmacologically acceptable routes of administration. For example, the compositions may be formulated with suitable pharmaceutically acceptable carriers (including excipients, diluents, adjuvants and combinations thereof) selected with respect to the intended route of administration and standard pharmaceutical practice. For example, the composition may be administered orally as a powder, liquid, tablet or capsule, or topically as an ointment, cream or lotion. Suitable formulations may contain additional agents as desired, including emulsifiers, antioxidants, flavoring or coloring agents, and may be adapted for immediate release, delayed release, modified release, sustained release, pulsed release or controlled release.

The compositions may be administered by parenteral routes. Examples of parenteral dosage forms include aqueous solutions, isotonic saline or 5% dextrose of the active agent, or other well known pharmaceutically acceptable excipients. For example, cyclodextrins or other solubilizing agents well known to those skilled in the art may be used as pharmaceutical excipients for the delivery of therapeutic agents.

Examples of dosage forms suitable for oral administration include, but are not limited to, tablets, capsules, lozenges, and the like, or any liquid form capable of providing a therapeutically effective amount of the composition such as syrups, aqueous solutions, emulsions, and the like. The capsules may contain any standard pharmaceutically acceptable material, such as gelatin or cellulose. Tablets may be formulated according to conventional procedures by compressing a mixture of the active ingredient with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. The active ingredient may also be administered in the form of hard shell tablets or capsules containing binders such as lactose or mannitol, conventional fillers and tableting agents.

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

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

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

Examples of dosage forms suitable for injecting the composition include single or multiple administrations delivered by bolus injection, e.g., by intravenous injection, subcutaneous, subdermal, and intramuscular administration, or oral administration.

Examples of dosage forms suitable for depot administration of the composition include pellets or solid forms in which the active agent is entrapped in a matrix of biodegradable polymers, microemulsions, liposomes or is microencapsulated.

Examples of infusion devices for the composition include infusion pumps for providing the required number of doses or steady state administration, and include implantable drug pumps. Examples of implantable infusion devices for use in the compositions include any solid form in which the active substance is encapsulated or dispersed throughout a biodegradable or synthetic polymer such as silicone, silicone rubber, or the like.

Examples of dosage forms suitable for transmucosal delivery of the compositions include depot solutions for enemas, pessaries, tampons, creams, gels, pastes, foams, nebulized solutions, powders, and similar formulations, containing in addition to the active ingredient such carriers as are known in the art to be appropriate. Such dosage forms include forms suitable for inhalation or insufflation compositions, including compositions comprising solutions and/or suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures and/or powders thereof. Transmucosal administration of the compositions can utilize any mucosal membrane, but typically utilizes nasal, buccal, vaginal and rectal tissue. Formulations suitable for nasal administration of the compositions may be administered in liquid form, e.g., nasal spray, nasal drops, or by aerosol administration via a nebulizer, including aqueous or oily solutions of the polymer particles. The formulations may be prepared as aqueous solutions, for example, in saline, in solution with benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

Examples of dosage forms suitable for buccal or sublingual administration of the compositions include lozenges, tablets and the like. 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.

Examples of composition formulations can be found, for example, in sweet man, s.c. (Ed.). martindale the Complete Drug Reference,33rd Edition, Pharmaceutical Press, Chicago,2002,2483 pp.; aulton, M.E. (Ed.) pharmaceuticals, the Science of the document Form design, Churchill Livingstone, Edinburgh,2000,734 pp.; and Ansel, H.C, Allen, l.v. and popivich, n.g. pharmaceutical Dosage Forms and Drug Delivery Systems,7th ed., Lippincott 1999,676 pp. Excipients for the manufacture of drug delivery systems are described in various publications known to those skilled in the art, including, for example, Kibbe, e.h. 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, for example, The United States Pharmacopeia 23/National Formulary 18, The United States Pharmacopeia Convention, Inc., Rockville MD,1995 (hereinafter "USP"), which also describes specific tests to determine The drug release capacity of extended release and delayed release tablets and capsules. USP testing for drug release of extended release and delayed release preparations is based on dissolution of the drug from the dosage unit versus elapsed test time. Descriptions of various testing devices and procedures can be found in the USP. Further guidance regarding sustained release dosage form analysis is provided. (see guide for industry extended release oral procedures for: depth, Evaluation, and application of in vitro/in vivo correlation. Rockville, MD: Center for Drug Evaluation and Research, Food and Drug Administration, 1997).

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.

The dosage level of the active ingredient in the pharmaceutical composition can be varied to provide an amount (effective amount) of the active ingredient which is effective to achieve the desired therapeutic effect for a particular patient, composition, and mode of administration, without toxicity to the patient.

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

6. Examples of the embodiments

General methods and materials

All reagents were purchased at reagent grade and used without further purification. The solvent used for the reaction was distilled according to standard procedures prior to use. Petroleum ether refers to a fraction with a boiling point of 40-60 ℃ according to standard procedures, i.e. by anhydrous Na₂SO₄Or MgSO 2₄The solvent is dried. Column chromatography was performed on silica gel (Merck 230-.

NMR spectra were recorded on a Bruker Avance 400MHz instrument¹H and 100MHz¹³Chemical shifts of the C spectrum are reported in parts per million (ppm) and are reported in¹Calibration in the H spectrum to tetramethylsilane (0ppm) as internal standard¹³C spectrum calibrated to residual solvent. Multiplicity is reported as follows: br is broad, s is singlet, d is doublet, t is triplet, q is quartet, m is multiplet, dd is doublet, dt is doublet, ddd is doublet of doublet. High Resolution Mass Spectra (HRMS) were obtained using a Bruker microTOF-Q II or Agilent 6530B Accurate Mass Q-TOF Mass spectrometer. LRMS was performed with a Surveyor MSQ mass spectrometer.

Unless alternative general methods and materials are given, the above general materials and methods apply to all examples below.

Example 1.6-methyl-4-oxo-8, 8 a-dihydro-1H-cyclopropa [ c ] oxazolo [4,5-e ] indole-2 (4H) -carboxylic acid tert-butyl ester (50)

Scheme 10

Reacting NaH with₂PO₄·2H₂O (4.29g, 27.5mmol) in H₂A solution of O (10mL) was added to a solution of 3, 4-dihydroxy-5-nitrobenzaldehyde 39(5.03g, 27.5mmol) in DMSO (25 mL). NaClO was added dropwise to the stirred mixture over 50 minutes₂(80%, 4.35g, 38.5mmol) of H₂O (25mL) solution, maintaining the internal temperature below 45 ℃. The dark red-brown mixture was stirred at room temperature for 16h, then NaHCO was poured in₃Aqueous solution (5%, 80 mL). Subjecting the mixture to CH₂Cl₂(. times.2) wash and acidify the aqueous phase with c.HCl to give a pH of 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 (5.12g, 94%);¹H NMR(d₆-DMSO)δ13.02(br s,1H),10.80(br s,2H),7.87(d,J＝2.0Hz,1H),7.57(d,J＝2.0Hz,1H)。

40(3.99g, 20.0mmol) in MeOH (60mL) and c.H₂SO₄The mixture in (1.5mL) was stirred at reflux for 19h, 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 isolated from H₂Recrystallization from O gave methyl 3, 4-dihydroxy-5-nitrobenzoate (41) as an orange-brown crystalline solid (3.30g, 77%); mp 137 ℃ to 139 ℃;¹H NMR(d₆-DMSO)δ10.84(br s,2H),7.89(d,J＝2.1Hz,1H),7.57(d,J＝2.1Hz,1H),3.83(s,3H)。

HCl in dioxane (4M, 2.94mL, 11.8mmol) and Pd/C (10%, 0.25g) were added to a solution of 41(2.51g, 11.8mmol) in EtOH (35 mL). The mixture was degassed with nitrogen, flushed with hydrogen, and then stirred under a hydrogen balloon until reduction was complete (-8 h). The mixture was filtered through celite, washed with EtOH and filtered by evaporationAs a liquid, methyl 3-amino-4, 5-dihydroxybenzoate hydrochloride (42) was obtained as a pale yellow solid (3.0g), which was used directly in the next step. Anal. (C)₈H₁₀NO₄) Is calculated as [ M + H]⁺184.1 of the total weight of the alloy; found 184.1.

Trimethyl orthoacetate (13.6mL, 107mmol) was added to a portion of 42(2.71g) prepared as described above. The suspension was stirred at reflux for 1h, then cooled and diluted with petroleum ether. The precipitate is filtered off and dried to give 7-hydroxy-2-methylbenzo [ d ]]Oxazole-5-carboxylic acid methyl ester (43) as a light brown solid (2.02g, 91%, from 41);¹H 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)；¹³C NMR(d₆-DMSO)δ166.1,165.0,143.0,142.2,141.9,126.5,112.1,111.1,52.2,14.1。Anal.(C₁₀H₉NO₄) Is calculated as [ M + H]⁺208.06043, respectively; found 208.06061.

Benzyl bromide (98%, 1.24mL, 10.2mmol) and K₂CO₃(1.48g, 10.7mmol) was added to a solution of 43(2.02g, 9.75mmol) in DMF (20mL) and the mixture was stirred at room temperature for 18 h. Subjecting the mixture to hydrogenation with H₂O dilution, filtration of the solid and drying to give 7- (benzyloxy) -2-methylbenzo [ d ]]Oxazole-5-carboxylic acid methyl ester (44) (2.81g, 97%) as a light brown solid;¹H 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)；¹³C 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.(C₁₇H₁₅NO₄) Is calculated as [ M + H]⁺298.10738, respectively; found 298.10834.

KOH (1.62g, 29.1mmol) in H₂A solution of O (15mL) was added to a suspension of 44(2.79g, 9.38mmol) in MeOH (60mL) and the mixture was stirred at 70 ℃ for 40 min. The MeOH was evaporated and the aqueous residue was acidified with aqueous HCl (2N, 12mL) at 0 deg.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 (2.62g, 98%);¹H 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.(C₁₆H₁₃NO₄) Is calculated as [ M + H]⁺284.09173, respectively; found 284.09164.

Adding Et₃N (1.54mL, 11.1mmol) and DPPA (97%, 2.25mL, 10.1mmol) were added to a suspension of 45(2.61g, 9.21mmol) of dry tert-butanol (80mL) and the mixture was stirred at reflux for 3 hours. The solvent was evaporated and the residue was purified by column chromatography (petroleum ether: EtOAc 9:1, then 4: 1) to give (7- (benzyloxy) -2-methylbenzo [ d ]]Oxazol-5-yl) carbamic acid tert-butyl ester (46) as a cream solid (2.44g, 75%);¹H 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)；¹³C 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.(C₂₀H₂₂N₂O₄) Is calculated as [ M + H]⁺355.1652, respectively; found 355.1668. The sample was recrystallized from EtOAc/petroleum ether to give white needles, mp 150-.

NBS (1.16g, 6.52mmol) was added portionwise to 46(2.31g, 6.52mmol) of CH at 0 ℃ over 10min₃CN (100 mL). The mixture was stirred at room temperature for 3h, then the solvent was evaporated. The residue was dissolved in EtOAc and taken up with H₂O (× 2), the solution was then washed with brine, then dried and evaporated. The resulting milky white solid was recrystallized from EtOAc/petroleum ether to give (7- (benzyloxy) -4-bromo-2-methylbenzo [ d ]]Oxazol-5-yl) carbamic acid tert-butyl ester (47) as a white solid (2.15g, 76%); mp 154-156 ℃;¹H 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.(C₂₀H₂₁ ⁷⁹BrN₂O₄) Is calculated as [ M + H]⁺433.0757, respectively; found 433.0762; (C)₂₀H₂₁ ⁸¹BrN₂O₄) Is calculated as [ M + H]⁺435.0740, respectively; 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(0.60g, 21%).

1, 3-dichloropropene (mixed isomer, 90%, 1.92mL, 18.6mmol) and K₂CO₃(4.3g, 31mmol) was added to a solution of 47(2.69g, 6.21mmol) in DMF (12mL) and the mixture was stirred at 80 ℃ for 7 h. DMF was evaporated and the residue was in EtOAc and H₂And (4) distributing among the O. The aqueous layer was re-extracted with EtOAc (. times.2) and the combined EtOAc layers were washed with brine (. times.3) then dried and evaporated to give crude (7- (benzyloxy) -4-bromo-2-methylbenzo [ d ]]Oxazol-5-yl) (3-chloroallyl) carbamic acid tert-butyl ester (48) as a light brown gum (3.21g, 100%);¹H NMR(CDCl₃) (mixture of E and Z isomers with Boc rotamers) delta 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 xm, 2H),2.69(s,3H),1.56(s, 9H). Anal. (C)₂₃H₂₄ ⁷⁹BrClN₂O₄) Is calculated as [ M + H]⁺507.06807, respectively; found 507.06909.

Under nitrogen, Bu₃SnH (97%, 1.09mL, 3.92mmol) and AIBN (64mg, 0.39mmol) were added to a solution of 48(995mg, 1.96mmol) in dry toluene (15mL) and the mixture was stirred at reflux. After 1.5 hours and 3 hours, additional portions of Bu were added₃SnH (97%, 0.54mL, 2.0mmol) and AIBN (32mg, 0.2 mmol). After 4 hours, the toluene was evaporated and the residue was in CH₃And (3) dividing between CN and petroleum ether. By CH₃CN (× 2) re-extracts the petroleum ether layer and 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 the crude product as a white foam. This was stirred with petroleum ether (8mL) to give 4- (benzyloxy) -8- (chloromethyl) -2-methyl-7, 8-dihydro-6H-oxazolo [4,5-e]Indole-6-carboxylic acid tert-butyl ester (49) as a white solid (668mg, 80%); mp 142-143 ℃;¹H 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)；¹³C 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.(C₂₃H₂₅ClN₂O₄) Is calculated as [ M + H]⁺429.15756, respectively; found 429.15735.

NH at 0 ℃ under nitrogen₄HCO₂Aqueous solution (25%, 3.9mL, 15.4mmol) and Pd/C (10%, with 53% H)₂O wet, 0.13g) was added to a solution of 48(660mg, 1.54mmol) in THF (10mL) and the mixture was stirred vigorously at this temperature. After 3 hours, a further portion of NH was added₄HCO₂Aqueous solution (25%, 3.9mL, 15.4mmol) and Pd/C (10%, with 53% H)₂O wet, 0.13 g). After 7 hours, the mixture was filtered through celite, washing 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 8- (chloromethyl) -4-hydroxy-2-methyl-7, 8-dihydro-6H-oxazolo [4,5-e]Indole-6-carboxylic acid tert-butyl ester (18) as a white solid (444mg, 85%); mp 234-;¹H NMR(CDCl₃) δ 7.66(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);¹³C 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.(C₁₆H₁₉ClN₂O₄) Is calculated as [ M + H]⁺339.11061, respectively; found 339.11149.

Will K₂CO₃(41mg, 0.3mmol) was added to a solution of 18(67mg, 0.2mmol) in DMF (1mL) and the mixture was stirred at room temperature for 3.5H, then EtOAc and H were used₂And (4) diluting with oxygen. 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 (22mg, 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)；¹³C 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.(C₁₆H₁₈N₂O₄) Is calculated as [ M + H]⁺303.13393, respectively; found 303.13458.

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)

Scheme 11

A mixture of 49(51mg, 0.12mmol) and HCl in dioxane (4M, 1mL) was stirred at room temperature for 3h, then evaporated. The residue was suspended in DMA (1.5mL) and 5,6, 7-trimethoxy-1H-indole-2-carboxylic acid (31.4mg, 0.13mmol) and EDCI.HCl (46mg, 0.24mmol) were added. The mixture was stirred at room temperature for 1h, then diluted NaHCO was added₃An aqueous solution. The precipitated solid is filtered off with H₂And O washing and drying. 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 (34mg, 51%); mp 225-;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)；¹³C NMR(d₆-DMSO)δ165.2,160.1,149.2,142.3,141.5,139.9,139.1,138.5,136.7,136.3,130.8,128.6,128.2,127.9,125.3,123.2,113.1,106.1,70.5,61.1,61.0,56.0,54.6,46.8,40.7,14.2。Anal.(C₃₀H₂₈ClN₃O₆) Is calculated as [ M + H]⁺562.17394, respectively; found 562.17311.

NH at 0 ℃ under nitrogen₄HCO₂Aqueous solution (25%, 0.14mL, 0.55mmol) and Pd/C (10%, using53％H₂O wet, 34mg) was added to a solution of 51(31mg, 0.055mmol) in THF (20mL) and the mixture was stirred vigorously at this temperature. More NH was added after 1 hour₄HCO₂Aqueous solution (25%, 0.28mL, 1.1mmol) and Pd/C (10%, with 53% H)₂O wet, 35 mg). After 2 hours, the mixture was filtered through celite, washed with THF and the filtrate was evaporated to dryness at 30 ℃. The residue is substituted by H₂Triturate, filter off the solid and dry. Further trituration with EtOAc afforded 23 as a white solid (21mg, 81%); mp 285 ℃ (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)；¹³C 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.(C₂₃H₂₂ClN₃O₆) Is calculated as [ M + H]⁺472.12699, respectively; found 472.12694.

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)

Scheme 13

A mixture of 18(33mg, 0.097mmol) and HCl in dioxane (4M, 1mL) was stirred at room temperature for 4h, then evaporated. The residue was suspended in DMA (1.0mL) and 5- (2- (dimethylamino) ethoxy) -1H-indole-2-carboxylic acid hydrochloride (34mg, 0.12mmol), EDCI.HCl (65mg, 0.34mmol) and anhydrous toluene sulfonic acid (3.3mg, 0.019mmol) were added. The mixture was stirred at room temperature for 1.5h, then cooled in an ice bath. EtOAc (50mL) and H were added₂O (10mL), with saturated NaHCO₃The aqueous solution is adjusted to pH 8-9. The EtOAc layer was separated, washed with water, then dried and evaporated. The residue is passed throughPurification by column Chromatography (CH)₂Cl₂MeOH 10:1) gave 52(34mg, 75%); mp 206-;¹H NMR(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＝8.9,2.4Hz,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(poorly resolved t,J＝5.3Hz,2H),2.61(s,3H),2.34(s,6H)；¹³C 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(C₂₄H₂₅ClN₄O₄) Is calculated as [ M + H]⁺469.1637, respectively; found 469.1642.

Example 4N- (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)

Scheme 14

A mixture of 18(33mg, 0.097mmol) and HCl in dioxane (4M, 1mL) was stirred at room temperature for 4h, then evaporated. The residue was suspended in DMA (1.0mL) and 5- (5- (2- (dimethylamino) ethoxy) -1H-indole-2-carboxamido) -1H-indole-2-carboxylic acid hydrochloride (43mg, 0.097mmol), EDCI.HCl (65mg, 0.34mmol) and anhydrous toluene sulfonic acid (3.3mg, 0.019mmol) were added. The mixture was stirred at room temperature for 1.5h, then cooled in an ice bath. EtOAc (150mL) and H were added₂O (50mL), with saturated NaHCO₃The aqueous solution is adjusted to pH 8-9. The EtOAc layer was separated, washed with water, then dried and evaporated. The residue was purified by column Chromatography (CH)₂Cl₂MeOH 10:1 then 5:1) gave 53(46mg, 75%); mp 231 ℃ (dec);¹H NMR(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,2H),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)；¹³C 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(C₃₃H₃₁ClN₆O₅) Is calculated as [ M + H]⁺627.2117, respectively; found 627.2119.

Example 5N- (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)

Scheme 15

Reacting 6-aminoimidazo [1,2-a ]]A solution of pyridine-2-carboxylic acid ethyl ester (54) (50mg, 0.24mmol), 4-hydroxybenzoic acid (68mg, 0.49mmol), EDCI.HCl (163mg, 0.85mmol) and anhydrous toluene sulfonic acid (8.7mg, 0.05mmol) in DMA (1mL) was stirred at room temperature for 2 hours. Subjecting the mixture to hydrogenation with H₂Dilute O and extract 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 (41mg, 52%); mp 263-266 deg.C;¹H 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 ═ 9.7Hz,1H),7.55(dd, J ═ 9.8,1.9Hz,1H),6.93-6.84(m,2H),4.30(q, J ═ 7.1Hz,2H),1.32(t, J ═ 7.1Hz, 3H);¹³C 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(C₁₇H₁₅N₃O₄) Is calculated as [ M + H]⁺326.1135, respectively; found 326.1125.

Solid KOH (171mg, 3.05mmol) was added 55(198mg, 0.61mmol) of THF (6mL), methanol (6mL) and H₂O (3mL) solution and the mixture was stirred at room temperature for 1h and then evaporated. The residue was diluted with water (10mL) and acidified to pH with aqueous HCl (5M)<1. The solid is filtered off and washed with H₂O washing and drying to obtain 6- (4-hydroxybenzoylamino) imidazo [1,2-a]Pyridine-2-carboxylic acid (56) as a yellow solid (143mg, 79%), mp 268-271 ℃;¹H 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；¹³C NMR(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(C₁₅H₁₁N₃O₄) Is calculated as [ M + H]⁺298.0822, respectively; found 298.0818.

A mixture of 18(38mg, 0.11mmol) and HCl in dioxane (4M, 1mL) was stirred at room temperature for 3h 15min, then evaporated. The residue was suspended in DMA (1.0mL) and 56(28mg, 0.093mmol), EDCI.HCl (63mg, 0.33mmol) and anhydrous toluene sulfonic acid (3.3mg, 0.019mmol) were added. The mixture was stirred at room temperature for 1.5 h. EtOAc (150mL) and H were added₂O (50mL), separate the EtOAc layer with H₂O (. times.2) washed, then dried and evaporated. The residue was triturated with MeOH to give 57(30mg, 63%); mp 232-235 ℃;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)；¹³C 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(C₂₆H₂₀ClN₅O₅) Is calculated as [ M + H]⁺518.1226, respectively; found 518.1223.

Example 6: lipophilicity of the Compounds of the invention

The lipophilicity of representative compounds of the invention was calculated using the ChemDraw Professional v.17.0.0 software package (Perkin Elmer information Inc). The results are provided in figure 1.

The compounds of the invention carry a variety of DNA minor groove binding side chains: the side chain A is a 5,6, 7-trimethoxy indole structure found in a natural product of the duocarmycin; side chain B has been used in the payload in ADC BMS-936561(biopharm. drug Disp. (2016)37, 93); and side chain C has been used for payload opening-DUBA in ADC SYD985(mol. pharm. (2015)12,1813).

In each case, compounds containing 2-methylbenzoxazole alkylated subunits have a calculated logP (about 1.5 units) that is significantly lower than those incorporating ring-opening-CBI alkylated subunits, and this applies to whether the alkylated subunit is phenol (X ═ OH) or an amine (X ═ NH)₂) In the form of (1).

These substantial reductions in payload lipophilicity are very likely to result in the advantageous properties of drug-linkers and ADCs that bind 2-methylbenzoxazole alkylated subunits.

Table 1: comparison of lipophilicity

Example 7: cytotoxicity

The cytotoxicity of the payloads of the present invention was determined by measuring the inhibition of proliferation of two human tumor cell lines, cervical cancer SiHa and ovarian cancer SKOV 3. Log phase monolayers were exposed to payload for 5 consecutive days in 96-well plates and then stained with sulforhodamine B. IC (integrated circuit)₅₀Drug concentration required to suppress cell density to 50% of untreated controls on the same plate was determined by interpolation. Each plate contained the reference compound Ring opened-CBI-TMI as an internal control.

The data presented in Table 2 show that DNA alkylating agents containing 2-methylbenzoxazole alkylating subunits are highly cytotoxic compounds, IC₅₀In the nM or sub-nM range, i.e. where it is considered appropriate for ADCWithin the range of use.

Importantly, compound 23 and seco-CBI-TMI have identical TMI minor groove binding side chains, allowing head-to-head comparison of the effect of the alkylated subunit on cytotoxicity. In this comparison, the 2-methylbenzoxazole alkylating subunit produces the same cytotoxicity as seco-CBI, which is noteworthy in that the latter is the most effective one of all known variants of duocarmycin-type alkylating subunits (j.med.chem. (2009)52,5771).

The significant cytotoxicity of 23 is surprising, since the cytotoxicity of the closely related compound ring-opening-COI-TMI (which differs only in the orientation of the oxazole ring fusion and the nature of the 2-substituent) is hundreds of times lower than that of the related reference compound (bioorg.med.chem.lett. (2010)20,1854).

Table 2: comparison of cytotoxicity

Example 8: stability of

The water stability of ring-opened-CBI-TMI was investigated 23 and the reference compound by LC-MS analysis. Samples containing payload at a concentration of 4 μ M in Tris buffer (pH7.4) containing 10% DMF at 37 ℃ were monitored at regular intervals over 300-500 min.

As shown in FIG. 1, Ring-opened-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).

In contrast, as shown in figure 2, while 23 also undergoes conversion to the corresponding cyclopropyl form (identified by characteristic UV-visible absorption spectroscopy and mass spectrometry), the compound is unstable and rapidly converted to a variety of products under these conditions. The UV-visible absorption spectrum and mass spectrum of the main product are consistent with hydrolysis, i.e. the cyclopropane is boiled by the addition of water. Such products do not alkylate DNA and can be considered non-toxic. The instability of the cyclopropyl intermediate derived from the alkylated subunit of 2-methylbenzoxazole is very surprising, since there is a well-defined correlation between solvolytic stability and cytotoxic efficacy, and all other known alkylated subunits that are as cytotoxic as CBI are reported to be stable to hydrolysis at neutral pH (j.med.chem. (2009)52,5771). The instability of payloads containing 2-methylbenzoxazole alkylated subunits is advantageous in ADC applications by acting as a detoxification mechanism for any systemically released payload.

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)

Scheme 16

A mixture of 18(31.3mg, 0.092mmol) and HCl in dioxane (4M, 1mL) was stirred at room temperature for 4h, then evaporated. The residue was suspended in DMA (1.5mL) and 4-methoxycinnamic acid (18mg, 0.10mmol) and EDCI.HCl (53mg, 0.28mmol) were added. The mixture was stirred at rt for 19 h. Water was slowly added and the precipitated solid was filtered off and dried to give 58 as a light brown solid (10mg, 27%);¹H 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),4.63-4.54(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(C₂₁H₁₉ClN₂O₄) Is calculated as [ M + H]⁺399.1106, respectively; found 399.1108.

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)

Scheme 17

18(30.1mg, 0.08)9mmol) and HCl in dioxane (4M, 1mL) were stirred at room temperature for 4h, then evaporated. The residue was suspended in DMA (1.0mL) and 3-hydroxy-4-methoxycinnamic acid (19mg, 0.10mmol) and EDCI.HCl (51mg, 0.27mmol) were added. The mixture was stirred at rt for 3h, 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.1ml) to give 59 as a very light green solid (7.7mg, 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＝8.4Hz,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)；¹³C 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(C₂₁H₁₉ClN₂O₅) Is calculated as [ M + H]⁺415.1055, respectively; found 415.1060.

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)

Scheme 18

A mixture of ethyl 5-aminoindole-2-carboxylate (60) (354mg, 1.73mmol), 4-methoxycinnamic acid (309mg, 1.73mmol) and EDCI.HCl (0.67g, 3.46mmol) in DMA (4mL) was stirred at room temperature for 18 h. Water was added and the precipitated solid was filtered off, washed with water and dried. The crude product was stirred with hot EtOH (40mL) 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 an off-white solid (506mg, 80%); mp 244-247 deg.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(C₂₁H₂₀N₂O₄·1/4H₂o) is calculated as C, 68.37; h, 5.60; n, 7.59%; found C, 68.56; h, 5.53; and N,7.66 percent.

A solution of KOH (0.40g, 7.1mmol) in water (6mL) was added to a suspension of 61(480mg, 1.32mmol) in ethanol (15mL), and the mixture was heated at reflux for 5 min. 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 pale brown solid (414mg, 93%); mp 276-279 ℃;¹H 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(C₁₉H₁₆N₂O₄) Calculated as C, 67.85; h, 4.79; n, 8.33%; found C, 68.09; h, 4.77; n,8.39 percent.

A mixture of 18(30.0mg, 0.089mmol) and HCl in dioxane (4M, 1mL) was stirred at room temperature for 4h, then evaporated. The residue was suspended in DMA (1.0mL) and 62(33mg, 0.10mmol) and EDCI.HCl (51mg, 0.27mmol) 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 (24mg, 49%);¹H 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)；¹³C 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(C₃₀H₂₅ClN₄O₅) Is calculated as [ M + H]⁺557.1586, respectively; found 557.1593.

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)

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＝10.4Hz,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(C₂₁H₁₇ClN₂O₅) Is calculated as [ M + H]⁺413.0899, respectively; found 413.0899.

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)

Scheme 19

A mixture of 18(40.0mg, 0.12mmol) and HCl in dioxane (4M, 2mL) was stirred at room temperature for 1.5h, then evaporated. The residue was suspended in DMA (1.5mL) and 5-methoxybenzo [ b ] was added]Thiophene-2-carboxylic acid (25mg, 0.12mmol), EDCI.HCl (68mg, 0.36mmol) and toluene sulfonic acid (4mg, 0.024 mmol). The mixture was stirred at room temperature for 3 h. Water was slowly added and the precipitated solid was filtered off and dried. Trituration of the crude product with EtOAc afforded 65 as a milky white solid (35mg, 69%);¹H NMR(d₆-DMSO)δ10.49(s,1H),8.02(s,1H),7.94(d,J＝8.9Hz,1H),7.79(br s,1H),7.53(d,J＝2.5Hz,1H),7.14(dd,J＝8.9,2.5Hz,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(C₂₁H₁₇ClN₂O₄s) is calculated as [ M + H]⁺429.0670, respectively; found 429.0677.

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)

Scheme 20

A mixture of 18(36mg, 0.106mmol) and HCl in dioxane (4M, 1mL) was stirred at room temperature for 4h, then evaporated. The residue was suspended in DMA (1.0mL) and 5- (2-methoxyethoxy) indole-2-carboxylic acid (27.5mg, 0.12mmol), EDCI.HCl (61mg, 0.32mmol) and toluene sulfonic acid (3.7mg, 0.021mmol) 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 grey solid (28mg, 58%);¹H NMR(d₆-DMSO) δ 11.58(d, J ═ 1.5Hz,1H),10.45(s,1H),7.92(s,1H),7.39(d, J ═ 8.9Hz,1H),7.16(d, J ═ 2.3Hz,1H),7.04(d, J ═ 1.7Hz,1H),6.90(dd, J ═ 8.9,2.4Hz,1H),4.78(t, J ═ 10.1Hz,1H),4.45(dd, J ═ 10.8,5.1Hz,1H),4.21-4.06(m,2H),4.04-3.97(m,1H),3.71-3.66(m,2H),2.62(s,3H) (one H s δ is masked by water peak at 3.3);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(C₂₃H₂₂ClN₃O₅) Is calculated as [ M + H]⁺456.1321, respectively; found 456.1323.

Example 15N- (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)

Scheme 21

Acetyl chloride (0.12mL, 1.7mmol) was added to 5-amino-1H-pyrrolo [2,3-b ] at 0 deg.C]Pyridine-2-carboxylic acid methyl ester (67) (165mg, 0.86mmol) and Et₃N (0.36mL, 2.6mmol) in CH₂Cl₂(8mL) and THF (10 mL). The ice bath was removed and the mixture was stirred for 1h and then diluted with water. The organic solvent was evaporated, leaving an aqueous suspension. The solid is filtered off and dried to give 5-acetamido-1H-pyrrolo [2,3-b]Pyridine-2-carboxylic acid methyl ester (68) as a white solid (178mg, 89%);¹H 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(C₁₁H₁₁N₃O₃) Is calculated as [ M + H]⁺234.0873, respectively; found 234.0868.

A solution of KOH (205mg, 3.6mmol) in water (3mL) was added to a suspension of 68(167mg, 0.72mmol) in MeOH (6mL), and the mixture was heated at reflux for 2min, then cooled to room temperature. Aqueous HCl (2N, 1.6mL) 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 (134mg, 85%);¹H 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(C₁₀H₉N₃O₃) Is calculated as [ M + H]⁺220.0717, respectively; found 220.0712.

A mixture of 18(39mg, 0.12mmol) and HCl in dioxane (4M, 2mL) was stirred at room temperature for 1.5h, then evaporated. The residue was suspended in DMA (1.0mL) and 69(25mg, 0.12mmol), EDCI.HCl (66mg, 0.36mmol) and toluene sulfonic acid (15mg, 0.09mmol) 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 (31mg, 61%);¹H 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)；¹³C 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(C₂₁H₁₈ClN₅O₄) Is calculated as [ M + H]⁺440.1120, respectively; found 440.1123.

Example 16N- (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)

Scheme 22

Acetyl chloride (0.11mL, 1.6mmol) was added to 6-aminoimidazo [1,2-a ] at room temperature]Pyridine-2-carboxylic acid ethyl ester (71) (161mg, 0.78mmol) and Et₃N (0.33mL, 2.3mmol) CH₂Cl₂(10mL) of the solution. After stirring for 5 minutes, the mixture was diluted with water and the organic solvent was evaporated, leaving an aqueous suspension. The solid was filtered off and dried, then triturated with EtOAc to give 6-acetamidoimidazo [1,2-a ]]Pyridine-2-carboxylic acid ethyl ester (72) as a pale green solid (99mg, 51%);¹H NMR(d₆-DMSO)δ10.14(s,1H),9.25(s,1H),8.62(s,1H),7.58(d,J＝9.7Hz,1H),7.24(dd,J＝9.7,2.0Hz,1H),4.29(q,J＝7.1Hz,2H),2.08(s,3H),1.31(t,J＝7.1Hz,3H)。Anal(C₁₂H₁₃N₃O₃) Is calculated as [ M + H]⁺248.1030, respectively; found 248.1021.

A solution of KOH (122mg, 2.2mmol) in water (3mL) was added to a suspension of 72(96mg, 0.39mmol) in MeOH (5mL) 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 (71mg, 83%);¹HNMR(d₆-DMSO)δ10.13(s,1H),9.25(s,1H) 8.55(s,1H),7.58(d, J ═ 9.7Hz,1H),7.23(dd, J ═ 9.7,2.0Hz,1H),2.09(s,3H) (no CO observed)₂H protons). Anal (C)₁₀H₉N₃O₃) Is calculated as [ M + H]⁺220.0717, respectively; found 220.0709.

A mixture of 18(43mg, 0.13mmol) and HCl in dioxane (4M, 1mL) was stirred at room temperature for 3.5h, then evaporated. The residue was suspended in DMA (1.0mL) and 73(27.8mg, 0.13mmol), EDCI.HCl (73mg, 0.39mmol) and toluene sulfonic acid (4.4mg, 0.03mmol) were added. The mixture was stirred at room temperature for 3 h. Water was slowly added to obtain an emulsion. Adding NaHCO₃Aqueous solution, 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 (25.5mg, 46%);¹H 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)；¹³C 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 not observed). Anal (C)₂₁H₁₈ClN₅O₄) Is calculated as [ M + H]⁺440.1120, respectively; found 440.1139.

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)

Scheme 23

Pd/C (10%, 90mg) was added to a solution of ethyl 1-methyl-4-nitro-1H-pyrrole-2-carboxylate (75) (254mg, 1.28mmol) in EtOH (25mL) and the mixture was hydrogenated at 50psi for 2H. The mixture was filtered through celite, washing with EtOAc, and the filtrate was evaporated. Dissolving the residue in CH₂Cl₂(6mL) and the solution was cooled in an ice bath. Et was added₃N (0.54mL, 3.8mmol) and acetyl chloride (0.18mL, 2.6mmol), the ice bath was removed. The mixture was stirred for 10min, then diluted with water and CH₂Cl₂(. times.2) extraction. The extract was washed with dilute aqueous HCl and water, then dried and evaporated. The residue was recrystallized from EtOH to give 4-acetamido-1-methyl-1H-pyrrole-2-carboxylic acid ethyl ester (76) as a pale yellow solid (65mg, 24%); mp 165-168 ℃. The aqueous phase from the liquid-liquid extraction is treated with NaHCO₃The aqueous solution was basified 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 only) to afford more 76(139mg, 52%);¹H NMR(CDCl₃)δ7.91(br s,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(C₉H₁₃N₃O₃) Is calculated as [ M + H]⁺212.1030, respectively; found 212.1026.

A solution of KOH (218mg, 3.9mmol) in water (2mL) was added to a solution of 76(185mg, 0.88mmol) in MeOH (4mL), and the mixture was stirred at room temperature for 30 min. The MeOH was evaporated, the aqueous layer was neutralized with aqueous HCl (2N, 1.3mL) and then evaporated to dryness. Aminoindole 60(179mg, 0.88mmol), EDCI.HCl (0.50g, 2.6mmol) and DMA (2mL) 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. Trituration of the residue with EtOAc afforded 5- (4-acetylamino-1-methyl-1H-imidazole-2-carboxamido) -1H-indole-2-carboxylic acid ethyl ester (77) as an off-white solid (59mg, more than 2 steps 18%);¹H 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(C₁₈H₁₉N₅O₄) Is calculated as [ M + H]⁺370.1510, respectively; found 270.1505.

KOH (89mg, 1.6mmol) in water (1mL)) The solution was added to a suspension of 77(54mg, 0.15mmol) in MeOH (8mL) and the mixture was stirred at 50 ℃ for 3h and then at reflux for 1.5 h. The mixture was cooled, 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 (46mg, 92%);¹H NMR(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＝8.9Hz,1H),7.06(d,J＝1.6Hz,1H),3.97(s,3H),2.02(s,3H)。Anal(C₁₆H₁₅N₅O₄) Is calculated as [ M + H]⁺342.1197, respectively; found 342.1204.

A mixture of 18(39.4mg, 0.12mmol) and HCl in dioxane (4M, 1mL) was stirred at room temperature for 3.5h, then evaporated. The residue was suspended in DMA (1.0mL) and 78(39.7mg, 0.12mmol), EDCI.HCl (67mg, 0.36mmol) and toluene sulfonic acid (4mg, 0.02mmol) were added. The mixture was stirred at rt 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 (36mg, 55%);¹H NMR(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)；¹³C NMR(d₆-DMSO)δ167.2,164.8,159.8,156.8,141.3,141.2,138.6,136.3,134.0,133.3,131.4,131.0,127.1,118.8,114.0,112.4,112.3,111.1,105.5,101.6,54.5,46.9,40.8,35.0,22.7,14.2。Anal(C₂₇H₂₄ClN₇O₅) Is calculated as [ M + H]⁺562.1600, respectively; found 562.1579.

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)

Scheme 24

Compound 18(334mg) was resolved by preparative chiral HPLC (Daicel Chiralpak IA 250X 21mm column, EtOH: Hexane 15: 85 eluent, flow rate 6mL/min, 1.43) to give baseline separation of the two enantiomers. Fractions containing the faster eluting enantiomer were combined (R)_T9.4min) 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 (131mg, 39%); []_D＝-31°(c 0.584,CH₂Cl₂)；¹H NMR(CDCl₃) As described in 18. Fractions containing the slower eluting enantiomer were pooled (R)_T13.4min) 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 (130mg, 39%); []_D＝+24°(c 0.586,CH₂Cl₂)；¹H NMR(CDCl₃) As described in 18. Reanalysis on an analytical column (Daicel Chiralpak IA 150X 4.6mm column) confirmed 100% ee for each enantiomer.

Compound 80 was converted to 82 by the general procedure described to give a white solid. Anal (C)₂₃H₂₂ClN₃O₆) Is calculated as [ M + H]⁺472.12699, respectively; found 472.12721. Compound 81 was converted to 83 by the general procedure described to give a white solid;¹H NMR(d₆-DMSO) is the same as described for 23. Anal (C)₂₃H₂₂ClN₃O₆) Is calculated as [ M + H]⁺472.12699, respectively; found 472.12719.

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-diyl bis (methylcarbamate) (26)

Scheme 25

Phenyl p-nitrochloroformate (97%, 20mg, 0.10mmol) and Et were reacted at 0 deg.C₃N (34. mu.L, 0.25mmol) was added to a solution of 23(23mg, 0.049mmol) in dry THF (5mL) and DMF (1.5 mL). After 1.5 hours, additional p-nitrophenylchloroformate (97%, 10mg, 0.05mmol) and Et were added₃N (17. mu.L, 0.12mmol) and after a further 50 minutes (2- (methylamino) ethyl) carbamic acid tert-butyl methyl ester (95%, 40mg, 0.21mmol) was added. The ice bath was removed and the mixture was stirred for a further 20h and then 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 (31mg, 94%);¹H NMR(CDCl₃) (some signals split due to rotamers) δ 9.37(s,1H),8.24 and 8.23(2s,1H),6.97(d, J ═ 2.3Hz,1H),6.87(s,1H),4.77(t, J ═ 9.6Hz,1H),4.61(dd, J ═ 10.7,4.6Hz,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)₃₃H₄₀ClN₅O₉) Is calculated as [ M + H]⁺686.2587, respectively; found 686.2573.

TFA (0.7mL, 9.1mmol) was added to 24(31mg, 0.045mmol) of CH at 0 deg.C₂Cl₂(0.7mL) in solution. After 30 minutes, the mixture was evaporated to dryness at room temperature and the residue was dissolved in CH₂Cl₂And evaporated again. The residue was dissolved in DMF (0.5mL) and the solution was cooled in an ice bath. 4- ((S) -2- ((S) -2- (6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanoylamino) -3-methylbutanoylamino) -5-ureidopentanamido) benzyl (4-nitrophenyl) carbonate (25) (36.7mg, 0.050mmol) and Et₃N (32. mu.L, 0.23 mmol). The ice bath was removed and the mixture was stirred for 18h and then at room temperatureWarm to evaporate to dryness. The residue was purified by column Chromatography (CH)₂Cl₂Then only CH₂Cl₂: MeOH 100: 1, then 50: 1. 30: 1. 15: 1) purification yielded 23(6mg, 28%) recovered and the crude product. The latter was purified by column chromatography (EtOAc: MeOH 9:1) to give 26 as a pale yellow glass (22mg, 41%);¹H NMR(d₆-DMSO) (some signals are split by rotamers) delta 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 ═ 5.5Hz,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), 3.85 (m,3H), 7.7.7.7.7 (s,1H), 7.7.60 (s,1H), 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)₅₇H₇₀ClN₁₁O₁₅) Is calculated as [ M + H]⁺1184.4814, respectively; found 1184.4840.

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)

Scheme 26

Phenyl p-nitrochloroformate (97%, 39mg, 0.18mmol) and Et were reacted at 0 deg.C₃N (64. mu.L, 0.46mmol) was added to a solution of 23(43.5mg, 0.092mmol) in DMF (4 mL). After 45 min and 2h, an additional portion of phenyl p-nitrochloroformate (97%, 19mg, 0.18mmol) was added, and after 70 min more Et was added₃N (32. mu.L, 0.28 mmol). After 2.5 h, a solution of tert-butyl (2- ((2- (2-hydroxyethoxy) ethyl) amino) ethyl) (methyl) carbamate (84) (73mg, 0.28mmol) in DMF (1mL) was added and the ice bath removed. Mixing the mixtureStirred for 18 hours and then evaporated to dryness. The residue is substituted by CH₂Cl₂Trituration, filtration of the solid and drying gave recovery 23(23mg, 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 (20mg, 29%);¹H 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 ═ 2.3Hz,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)₃₆H₄₆ClN₅O₁₁) Is calculated as [ M + H]⁺760.2955, respectively; found 760.2940.

Compound 85(31mg, 0.041mmol) was reacted with CH₂Cl₂A mixture of (0.7mL) and TFA (0.7mL, 9.1mmol) was treated at 0 ℃ for 15 min. The mixture was evaporated to dryness at room temperature and the residue was dissolved in CH₂Cl₂Neutralized and evaporated again. The residue was dissolved in DMF (0.5mL) and the solution was cooled in an ice bath. (S) -3-methyl-1- (((S) -1- ((4- ((((4-nitrophenoxy) carbonyl) oxy) methyl) phenyl) amino) -1-oxoprop-2-yl) amino) -1-oxobutan-2-yl) carbamic acid tert-butyl ester (86) (23mg, 0.041mmol) and Et₃N (28. mu.L, 0.21mmol) and the ice bath was 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 alone, then EtOAc: MeOH 20: 1) to give recovered 23(4.3mg, 22%) and 87 as colorless glasses (24.8mg, 56%);¹H NMR(CDCl₃) (some signals are split by 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), 3.8.44 (m,1H),8.96-8.74(m,1H), 8.7 (m,1H), 4.19-5.06 (m,2H), 4.80-4.5.5 (m,2H),4.80-4.72(m, 2H)4-3.41(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(C₅₂H₆₇ClN₈O₁₅) Is calculated as [ M + H]⁺1079.4487, respectively; found 1079.4489.

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) propanamido) benzyl) oxy) carbonyl) (methyl) amino) ethyl) (2- (2-hydroxyethoxy) ethyl) carbamate (89)

Scheme 27

Phenyl p-nitrochloroformate (97%, 34.5mg, 0.16mmol) and Et were reacted at 0 deg.C₃N (88. mu.L, 0.63mmol) was added to a solution of 52(59.3mg, 0.126mmol) in THF (6mL) and DMF (1.5 mL). After 30 minutes more p-nitrophenyl chloroformate (97%, 34.5mg, 0.16mmol) was added and after 2.5 hours a solution of 84(66mg, 0.32mmol) in THF (1mL) was added. The ice bath was removed and the mixture was stirred for 18h and then evaporated to dryness. The residue is substituted by CH₂Cl₂Trituration, filtration of the solid and drying afforded recovered 52 as the hydrochloride salt (21mg, 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 (30mg, 31%);¹H NMR(CDCl₃) (some signals split due to rotamers) δ 9.52-9.41(m,1H),8.26 and 8.23(2s,1H),7.34(d, J ═ 8.9Hz,1H),7.14(d, J ═ 2.2Hz,1H),7.03(dd, J ═ 8.9,2.3Hz,1H),6.97(d, J ═ 1.4Hz,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)₃₇H₄₉ClN₆O₉) Is calculated as [ M + H]⁺757.3322, respectively; found 757.3332.

Compound 88(30mg, 0.040mmol) was reacted with CH₂Cl₂A mixture of (0.7mL) and TFA (0.7mL, 9.1mmol) was treated at 0 ℃ for 15 min. The mixture was evaporated to dryness at room temperature and the residue was dissolved in CH₂Cl₂Neutralized and evaporated again. The residue was dissolved in DMF (0.5mL) and the solution was cooled in an ice bath. Add Compound 86(22mg, 0.040mmol) and Et₃N (33. mu.L, 0.24mmol), the ice bath was removed. After stirring for 2 hours, the mixture is evaporated and chromatographed on a Column (CH)₂Cl₂: MeOH 50: 1, then 20: 1. 10: 1. 8: 1; then eluted with a second column, where EtOAc: MeOH 10:1, then 5: 1. 3: 1. 3: 2) purification afforded 89 as a colorless glass (7.8mg, 18%);¹H NMR(CDCl₃) (some signals are split by rotamers) 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 ═ 2.2Hz,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, 7.7H), 2H),4.02-3.92(m,1H),3.83-3.34(m,14H),3.05-2.93(m, 2H), 2H, 1H, 23.47 (m,2H), 1.00-0.90(m, 6H). Anal (C)₅₃H₇₀ClN₉O₁₃) Is calculated as [ M + H]⁺1076.4854, respectively; found 1076.4857.

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)

Scheme 28

Phenyl p-nitrochloroformate (96%, 160mg, 0.76mmol) and Et were reacted at 0 deg.C₃N(0.37mL2.7mmol) was added to a solution of 66(242mg, 0.53mmol) in THF (8 mL). After 75 minutes, a solution of 84(279mg, 1.1mmol) in THF (1.5mL) was added. The ice bath was removed and the mixture was stirred for 5.5h and then evaporated to dryness. Dissolving the residual tan foam in a minimal amount of CH₂Cl₂And the solution was allowed to stand at 4 ℃ overnight. The solid is filtered off and washed with CH₂Cl₂Washed and the filtrate evaporated. 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) -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 (90mg, 23%);¹H NMR(CDCl₃) (some signals split due to rotamers) δ 9.43(s,1H),8.26 and 8.24(2s,1H),7.34(d, J ═ 8.9Hz,1H),7.14(d, J ═ 2.3Hz,1H),7.05(dd, J ═ 8.9,2.4Hz,1H),6.98(d, J ═ 1.5Hz,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)₃₆H₄₆ClN₅O₁₀) Is calculated as [ M + H]⁺744.3006, respectively; found 744.3014.

Will not be soluble in CH₂Cl₂Was combined with the matched fractions from column chromatography to give recovery 66(142mg, 59%).

TFA (0.7mL, 9.1mmol) was added to 90(90mg, 0.12mmol) of CH at 0 deg.C₂Cl₂(2.0mL) and the mixture stirred at this temperature for 40 minutes. The mixture was evaporated to dryness at room temperature and the residue was dissolved in CH₂Cl₂Neutralized and evaporated again. The residue was dissolved in DMF (1mL) and the solution was cooled in an ice bath. Add Compound 86(67.5mg, 0.12mmol) and Et₃N (84. mu.L, 0.6mmol), the ice bath was removed. After 3h, 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 white solids (11mg, 20%) and 91 as white solidsColored glass (58mg, 45%);¹H NMR(CDCl₃) (some signals are split by rotamers) 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 ═ 2.2Hz,1H),7.06(dd, J ═ 8.9,2.2Hz,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 (m, 3.47, 2H), 3.01-3.48 (m,1H), 3.06-4.48 (m,1H), 3.48 (m,1H), 6H) in that respect Anal (C)₅₂H₆₇ClN₈O₁₄) Is calculated as [ M + H]⁺1063.4538, respectively; found 1063.4546.

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

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-diazatridecanamido) benzyl) oxy) carbonyl) (methyl) amino) ethyl) (2- (2-hydroxyethoxy) ethyl) carbamate (93).

Scheme 29

TFA (0.2mL, 2.6mmol) was added to 91(14mg, 0.013mmol) of CH at 0 deg.C₂Cl₂(1.0mL) and the solution was held at that temperature for 3 h. The mixture was evaporated to dryness at room temperature and the residue was dissolved in CH₂Cl₂Neutralized and evaporated again. The residue was dissolved in DMF (0.5mL) 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) (4.6mg, 0.013mmol) and Et were added₃N (9. mu.L, 0.065mmol), the ice bath was removed. After 19h, 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 (8.3mg, 54%);¹H NMR(CDCl₃) (some signals are split due to rotamers, no 2 exchangeable protons are 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), 1.46-1.03 (m,1H), 1.90-6.0 (m, 3H). Anal (C)₅₆H₆₈ClN₉O₁₇) Is calculated as [ M + H]⁺1174.4494, respectively; found 1174.4516.

Example 24 additional lipophilicity data

The lipophilicity of small molecule compounds can be calculated in many different ways (reviewed in e.g. j. phase. sci. (2009)98,861) to supplement the data provided in example 6, the calculated lipophilicity of several compounds of the invention was compared to the lipophilicity of ring-opened-CBI analogues with the same minor groove binding side chain, obtained by swissanew (http:// swisssandame. et al.,/Swiss Institute of Bioinformatics), using four different software packages, namely ChemDraw Professional (Perkin Elmer information Inc), as in example 6, ACD/logp (advanced Chemistry development), XLOGP3 (shanghai Institute of organic Chemistry) and MLOGP (tall SRL, Milano, Italy). The structures of the compounds are shown (where DNA binding units A, D, E and F, when linked to the 2-methylbenzoxazole alkylating subunit, represent compounds 23, 59, 66 and 74, respectively) and the calculated logP values are collected in table 3.

It is clear from Table 3 that although the absolute logP value varies according to the calculation procedure used, there is a consistent and significant reduction in logP when comparing the 2-methylbenzoxazole alkylated subunit with the ring-opened-CBI alkylated subunit (. DELTA.logP in the range from 0.98 to 1.48) in each case. This applies to whether the computing program is property-based (e.g., MLOGP) or substructure-based (e.g., fragment-based, such as ACD, or atom-based, such as XLOG 3).

As already noted, these substantial reductions in payload lipophilicity are very likely to result in the advantageous properties of drug-linkers and ADCs that bind 2-methylbenzoxazole alkylated subunits.

Table 3: lipophilicity of the Compounds of the invention

Example 25 additional cytotoxicity data

Following the general procedure described in example 7, the cytotoxicity of the payloads of the present invention was determined by measuring the inhibition of proliferation of two human tumor cell lines, cervical cancer SiHa and ovarian cancer SKOV 3. In a new assay, a stock solution of payload in DMSO was serially diluted prior to addition to the cell-containing wells of a 96-well plate. The cytotoxicity assays were repeated several times (depending on compound and cell line, n-3-7) and the results were collected in table 4.

The new data reinforces the information presented in Table 2, showing that DNA alkylating agents containing 2-methylbenzoxazole alkylating subunits may be highly cytotoxic compounds, IC₅₀In the nM or sub-nM range, i.e. in the range considered suitable for ADC applications. They also show that appropriate selection of minor groove binding side chains can be used to modulate the cytotoxicity of payloads, with the examples in table 4 spanning an IC over the 100-fold range in the 2 cell lines examined₅₀. The data in table 4 further allow for a comparison between the two enantiomeric forms of the alkylated subunit of 2-methylbenzoxazole. Compounds 82 and 83 showed 93-fold differential cytotoxic potency in SiHa and 110-fold differential cytotoxic potency in SKOV 3. These observations are consistent with the behavior of other analogs of duocarmycin, since the natural enantiomers are usually cytotoxic due to alkylated DNAIs more cytotoxic.

Importantly, in IC₅₀Within the error of the measurement, compound 23 and seco-CBI-TMI sharing the same minor groove binding side chain were again found to have the same cytotoxic potency. This means that the 2-methylbenzoxazole alkylating subunit can be considered to be the most effective of all the known variants of the duocarmycin-type alkylating subunit, surprisingly different from the closely related ring-opening-COI alkylating subunit.

Table 4: cytotoxicity of Compounds of the invention

Example 26 additional stability data

The water 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 sample contained a payload at a concentration of 4. mu.M in Tris buffer (pH7.4) containing 10% DMF at 37 ℃. In this experiment, the solution was monitored at hourly intervals over 8 hours.

As shown in fig. 3-5, all three payload compounds were converted to intermediates identified as the corresponding cyclopropyl forms based on characteristic shifts of the uv-vis absorption spectra. In all three cases, the cyclopropyl intermediate is unstable to further reaction in aqueous buffer, producing multiple products, two of which predominate as hydrolysis products. This behavior closely matches the stability and product distribution observed for compound 23 in example 8 (see figure 2), but is in marked contrast to the prolonged stability of the reference compound ring-opened-CBI-TMI (see figure 1). Thus, it appears that the unusual instability is a general property of the alkylated subunit of 2-methylbenzoxazole. It does not significantly depend on the nature of the minor groove binding side chain of the DNA. In other words, the potential advantages of hydrolysis as a detoxification mechanism for any systemically released payload can reasonably be assumed to be properties common to all novel compounds of the present invention.

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