WO2009060208A1

WO2009060208A1 - Pyrrolobenzodiazepines

Info

Publication number: WO2009060208A1
Application number: PCT/GB2008/003757
Authority: WO
Inventors: Philip Howard; Luke Masterson; John Roffey
Original assignee: Spirogen Limited
Priority date: 2007-11-09
Filing date: 2008-11-07
Publication date: 2009-05-14
Also published as: GB0722088D0

Abstract

A compound of formula I: wherein: the dotted line indicates the optional presence of a double bond between C2 and C3; R2 is selected from -H, -OH, =O, =CH2, -CN, -R, OR, halo, =CH-R, O-SO2-R, CO2R and COR; R7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halo; where R and R' are independently selected from optionally substituted C1-7 alkyl, C3-20 heterocyclyl and C5-20 aryl groups; R10 and R11 either together form a double bond, or are selected from H and YRY, where Y is selected from O, S and NH and RY is H or C1-7 alkyl or H and SOxM, where x is 2 or 3, and M is a monovalent pharmaceutically acceptable cation; each X is independently a C5-6 heteroarylene group; n is from 1 to 6.

Description

PYRROLOBENZODIAZEPINES

The present invention relates to pyrrolobenzodiazepines (PBDs), and in particular pyrrolobenzodiazepines bearing a polyamide chain at the C8 position, and their use in downregulation of target sequences and consequently in the treatment of varipus diseases.

Background to the invention

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu. The first PBD antitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc, 87, 5793-5795 (1965); Leimgruber, et al., J. Am. Chem. Soc, 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogues (Thurston, et al., Chem. Rev. 1994, 433-465 (1994)). Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chicamycin (Konishi, etal., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem. Brit, 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41, 1366- 1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc, 110, 2992-2993 (1988)) and tomamycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of the general structure:

They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N=C), a carbinolamine(NH-CH(OH)), or a carbinolamine methyl ether (NH-CH(OMe)) at the N10-C11 position which is the electrophilic centre responsible for alkylating DNA. All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA₁ leading to a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Ace. Chem. Res., 19, 230-237 (1986)). Their ability to form an adduct in the minor groove, enables them to interfere with DNA processing, hence their use as antitumour agents.

A number of conjugates of PBD with pyrroles and imidazoles have been reported.

Lown has reported (Damayanthi, Y., etal., Journal of Organic Chemistry, 64(1), 290-292 (1999)) the synthesis of PBD conjugates (named PBD-lexitropsin conjugates):

, wherein n = 1-3 with a propyl linker and an N-dimethylaminopropyl terminus. Although these are stated to be lexitropsin derivatives with the n = 2 and 3 analogues mimicking the 2 and 3 N-methylpyrrole rings of netropsin and distamycin, respectively, the N- dimethylaminopropyl tail differs significantly from the guanidine methyl or guanidine ethyl tails of netropsin or distamycin, respectively. The compounds, which were produced in an overall yield of 28-30% as a mixture of the N10-C11 imine and carbinolamine methyl ether forms. They were found to be highly polar as the imines and only soluble in a mixture of chloroform and methanol, and were insufficiently soluble in either solvent to allow production of pure imine or methyl ether forms. The compounds with n=2 and 3 were reported (Reddy, B.S.P., etal., Anti-Cancer Drug Design, 15(3), 225-238 (2000)) to have modest cytotoxicity with values ranging from 7.5-86.5μM (for n=2) and 0.9-93μM (for n=3).

Lown has also reported (Kumar, R. and Lown, J.W. Oncology Research, 13(4), 221-233 (2003)); Kumar, R., etal., Heterocyclic Communications, 8(1), 19-26 (2002)) the synthesis of three equivalent imidazole analogues:

, wherein n=1-3 and two mixed pyrrole-imidazole analogues:

, wherein n=1-2. These compounds, obtained in overall yields of 35-40% were also produced as mixtures of imines and carbinolamine methyl ethers in a 1 :1 ratio, and had similar solubility characteristics. In the NCI 60 panel screen, the mixed compound (n=1) was not active in any cell lines, and the other compounds had mean Gl₅₀, TGI and LC₅₀ values in the range 16.22-95.50μM.

In an effort to enhance water solubility, Lown and co-workers more recently reported

(Kumar, R., etal., Organic & Biomolecular Chemistry, 1(19), 3327-3342 (2003)) a set of sixteen PBD-heterocycle conjugates containing varying numbers of pyrrole and imidazole units but with heterocycles glycosylated on their ring nitrogens. In eight of these molecules the hydroxyl moieties of the glycosyl units were fully acetylated. The other eight were similar in structure except that all acetyl groups had been removed. Data from the NCI panel indicate that these compounds are significantly less cytotoxic than the corresponding compounds with glycosyl units. Also, although the group of compounds which are deacetylated are designed to be more water soluble than those which are acetylated, the average IC₅₀ values show only a marginal improvement which in fact appears to be due to just one compound which shows a marked improvement (0.588 μM) over its non-deacetylated equivalent (93.3μM). No DNA-binding data has been reported for any of these conjugates.

Baraldi and co-workers have reported (Baraldi, P. G., et al., Bioorganic & Medicinal Chemistry Letters, 8(21), 3019-3024 (1998)) the synthesis of a conjugate of a PBD and distamycin:

where n = 3, which differs from the conjugates synthesized by Lown in having a two- methylene linker between the PBD and first heterocycle rather than three, and in possessing a guanidine terminus as found in the natural products distamycin and netropsin rather than the dimethylaminopropyl terminus of the Lown compounds.

Interestingly, this compound appeared to be more cytotoxic than the Lown conjugates with an IC₅₀ in K562 cells of 0.2 μM. The DNA binding of the conjugate was assessed in a PCR-based assay against DNA sequences that were GC-rich (Ha-ras oncogene) or AT-rich (oestrogen, ER) receptor. Unlike Distamycin A which inhibited PCR in only the AT-rich sequence, the compound was equipotent in inhibiting PCR in both the GC-rich and AT-rich DNAs at a level 6 x more potent than Distamycin in the AT-rich sequence. Baraldi also reported (Baraldi, P.G., et a/., Journal of Medicinal Chemistry, 42(25), 5131- 5141 (1999)) an extended set of the compound above containing one to four pyrrole units (n = 1 - 4). Cytotoxicity evaluations in K562 (>100 μM [n=1] to 0.04 μM [n=4]) and Jurkat (80 μM [for n=1] to 0.07 μM [for n=4]) cell lines showed that increase in the length of the polypyrrole backbone led to an increase of in vitro activity. Only the n = 3 and 4 compounds were more potent than either the PBD fragment alone or the relevant tetrapyrrole devoid of PBD. To investigate sequence selectivity and stability of the drug/DNA complexes, DNase I footprinting and arrested polymerase chain reaction (PCR) were performed on fragments of the human c-myc oncogene and human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) (both GC-rich), and the estrogen receptor gene (AT-rich). It was found that the ability of the compounds to arrest PCR of the c-myc gene (IC₅₀ = 2-6 μM) and HIV gene (IC₅₀ = 0.8-2.0 μM) was higher than distamycin A (25 μM for C-myc; 50 μM for HIV), suggesting that the presence of the PBD might be favouring a shift to GC-recognition. Interestingly, for the ER gene, compounds with n = 1 or 2 were similar (IC₅₀ = 3.0 μM, 2.0 μM, respectively) to distamycin (IC₅₀ = 5μM), whereas compounds with n= 3 or 4 were marginally more active (0.8 μM, 0.2 μM, respectively) suggesting that there was a more profound effect on raising GC-selectivity. Analysis of arrest sites of ER PCR suggested that the compound where n=1 arrests at 5'-AGTTTAAA-3', whereas the compounds where n=2-4 cause arrest at the same site and in addition at δ'-CATATATGTGTG-S'. Footprinting experiments suggested that comparing these compounds, similar footprints were obtained suggesting that changes in the number of pyrrole rings did not produce significant changes in sequence recognition. However, it was noted that the footprints generated by the compound where n=4 were larger than that generated by distamycin. Finally, using a PCR-based dialysis experiment, it was demonstrated that these hybrid compounds exhibit different DNA-binding activity with respect to both distamycin and the parent PBD. In addition, a direct relationship was found between number of pyrrole rings present in the hybrids and stability of drug/DNA complexes. Confirming the previous studies of Baraldi, Gambari and co-workers reported (Borgatti, M., et al., Drug Development Research, 60(3), 173-185 (2003)) the effects of these compounds on the interaction between purified NF-κB and [³²P]-labelled oligomers mimicking the NF-κB HIV-1 LTR binding sites using both gel retardation (EMSA) and filter binding assays. The results showed that the conjugates were effective in inhibiting NF-κB p52/ NF-κB DNA interactions according to the EMSA assay but only compounds where n = 2-4 were active according to the filter assay. Similarly, conjugates where n = 2-4 (but not n=1) were shown to efficiently inhibit HIV-1 LTR driven transcription in vitro whereas the PBD fragment alone was not. Baraldi and co-workers (Baraldi, P. G., et al., Nucleosides Nucleotides & Nucleic Acids, 19(8), 1219-1229 (2000)) also reported that the compound where n = 3 inhibits binding of the transcription factor Sp1 , a protein important for the control of transcription of cellular and viral genes, to its cognate DNA sequence. Nuclear proteins were isolated from K562 cells and immobilised on a filter after electrophoresis. The ability of the compound where n = 3 to inhibit the binding of [³²P]-labelled Sp1 oligomer to the filter was then studied. Although the PBD fragment or distamycin A failed to inhibit binding at concentrations of up to 50μM, the compound where n=3 completely blocked the Sp1/DNA binding interaction at 10μM, a result which was confirmed by gel shift experiments.

Finally, Gambari and co-workers have reported (Mischiati, C, et al., Biochemical Pharmacology, 67(3), 401-410 (2004)) that these compounds bind to TAR-RNA and inhibit TAR/protein(s) interaction, and also interact with structured TAR-RNA of HIV-1. The authors studied the effects of these compounds on protein/TAR-RNA interactions in vitro by both EMSA and filter binding experiments using nuclear extracts and Tat, and ex vivo using the HL3T1 cell line as a cellular system to study Tat-induced HIV-1 LTR driven transcription. The compounds bind TAR-RNA since they slow down migration of radiolabeled HIV-1 TAR-RNA, whereas distamycin A and the PBD fragment are inactive. In the EMSA experiments, binding of the compounds to either the structured AU-rich or GC-rich RNA was less efficient than to wild type TAR-RNA. Again the PBD fragment and distamycin A were inactive. Denaturing experiments suggested that the compounds where n=1 and 2 might be binding non-covalently to the DNA whereas the compounds where n=3 and 4 might be binding covalently. They also reported IC₅₀ values in HL3T1 cells (72 hours) confirming earlier observed trends of cytotoxicity increasing with increasing numbers of pyrrole units attached.

Androgens are important growth factors for the normal prostate. Androgen signalling is mediated by the Androgen Receptor (AR) which is localised in the cytoplasm of stromal and epithelial cells. AR is a DNA binding transcription factor that becomes active when androgens bind to it. Ligand binding induces a conformational change in the AR protein which allows it to escape its cytoplasmic chaperone and translocate to the nucleus. Once in the nucleus AR homodimerises and binds to response elements containing palindromes of a 6-base pair core sequence (5'-AGAACA-3^J), which are separated by a 3 nucleotide spacer, in the promoters of target genes. Proliferative genes such as

Cyclins A, D1-3, D3 and E along with CDKs 1 , 2, 4 and 6 are transcriptionally activated by AR. On the other hand anti-proliferative and tumour suppressor genes such as p16 (ink4) and Rb are repressed.

Tumour proliferation in early stage prostate cancers is driven by androgens and androgen deprivation (either chemical or surgical) causes tumour regression in most patients. However, in almost all cases, the disease progresses, and the prostate cells acquire additional mutations which result in them becoming androgen independent. The AR gene may be amplified, raising cellular levels of AR and rendering the prostate cell hypersensitive to even very low levels of androgens. Other mutations can alter the ligand specificity of AR allowing it to bind promiscuously to alternative steroids, such as estrogen, alosterone and cortisone, allowing the inappropriate activation of target genes even in the absence of androgens. Finally, AR can be activated by alternative pathways involving growth factors, receptor tyrosine kinases or Akt. Although these changes in the cancer cells allows them to proliferate in the absence of androgens themselves, it is important to note that they still rely on active signalling through the androgen receptor (AR), which continues to activate its transcriptional targets independently of androgen binding.

One approach to treating prostate cancer in both its androgen dependent and independent stages is to block production of the androgen receptor itself. The present inventors have discovered that the DNA binding agents heterocycle-PBD conjugates can bind in the minor groove of DNA inhibiting transcription of the AR gene and production of the AR protein. This approach should continue to work whether the AR gene is amplified or the AR protein looses androgen specificity or is activated by an alternative signalling pathway.

Disclosure of the invention

Accordingly, a first aspect of the present invention provides a compound of formula I:

(or a salt or solvate thereof), wherein: the dotted line indicates the optional presence of a double bond between C2 and C3;

R² is selected from -H, -OH, =0, =CH₂, -CN, -R, OR₁ halo, =CH-R, 0-SO₂-R, CO₂R and

COR; R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR', nitro, Me₃Sn and halo; where R and R' are independently selected from optionally substituted Ci_-7 alkyl, C_3-20 heterocyclyl and C_5-2O aryl groups;

R¹⁰ and R¹¹ either together form a double bond, or are selected from H and YR^Y, where

Y is selected from O, S and NH and R^γ is H or C_1-7 alkyl or H and SO_xM, where x is 2 or 3, and M is a monovalent pharmaceutically acceptable cation; each X is independently a C_5-6 heteroarylene group; n is from 1 to 6.

A second aspect of the present invention provides a precursor to compounds of formula I, where R¹⁰ and R¹¹ together form a double bond, which are of formula II:

wherein R^10A is a nitrogen protecting group and R^11A is OH or OProt⁰, where Prot⁰ is a hydroxy protecting group. A third aspect provides a pharmaceutical composition comprising a compound of the first aspect and a pharmaceutically acceptable carrier or diluent, as well as a compound of the first aspect for use in a method of medical treatment.

A fourth aspect provides the use of a compound according to the first aspect in the manufacture of a medicament for treating a disease or condition ameliorated by the down-regulation of the Androgen Receptor, as well as a compound of the first aspect for use in a method of treatment of a disease or condition ameliorated by the down- regulation of the Androgen Receptor.

A fifth aspect provides a method of treatment of a disease or condition ameliorated by the down-regulation of the Androgen Receptor, comprising administering to a subject in need of treatment a therapeutically effective amount of a compound according to the first aspect, or a pharmaceutical composition of the third aspect.

In the fourth and fifth aspects of the invention, the disease or condition ameliorated by the down-regulation of the Androgen Receptor is prostate cancer.

Figures Figure 1 shows the effect of compounds of the invention over time at 1 μM on Androgen

Receptor expression in LNCaP-FGC cells.

Figure 2 shows the effect of compounds of the invention over time at 10 μM on

Androgen Receptor expression in LNCaP-FGC cells.

Figure 3 shows the effect a single compound of the invention over time at varying concentrations on Androgen Receptor expression in LNCaP-FGC cells.

Figure 4 shows the effect a different compound of the invention over time at varying concentrations on Androgen Receptor expression in LNCaP-FGC cells.

Definitions Points of connection

When used in structures shown herein, asterisks (*) represent the positions of bonds which connect to further chemical moieties or groups. Cs-6 heteroarylene

The term "C_5-6 heteroarylene", as used herein, pertains to a divalent moiety obtained by removing two hydrogen atoms from aromatic ring atoms of a heteroaromatic compound having a single ring comprising 5 or 6 ring atoms.

Examples of C_5-6 heteroarylene groups include, but are not limited to, those derived from:

Ni : pyrrole (azole) (Cs), pyridine (azine) (C₆);

O₁: furan (oxole) (C₅); S₁: thiophene (thiole) (C₅);

NiO₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂O₁: oxadiazole (furazan) (C₅);

N₃O-_I : oxatriazole (C₅);

NiS₁: thiazole (C₅), isothiazole (C₅); N₂: imidazole (1 ,3-diazole) (C₅), pyrazole (1 ,2-diazole) (C₅), pyridazine (1 ,2-diazine) (C₆), pyrimidine (1 ,3-diazine) (C₆) (e.g., cytosine, thymine, uracil), pyrazine (1 ,4-diazine) (C₆);

N₃: triazole (C₅), triazine (C₆); and,

N₄: tetrazole (C₅).

Building blocks

In the compounds of the present invention, some example chains are composed of numerous standard chemical blocks. For ease of reference, these are referred to in abbreviated form. Several of these standard blocks form fragments of the formula I. The abbreviations used herein are Dp, β, Py, Im and 5M4Tz. These abbreviations represent the following structures when used herein:

Dp Py

Im 5M4Tz Pharmaceutically acceptable cations

Examples of pharmaceutically acceptable monovalent and divalent cations are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), which is incorporated herein by reference.

The pharmaceutically acceptable cation may be inorganic or organic.

Examples of pharmaceutically acceptable monovalent inorganic cations include, but are not limited to, alkali metal ions such as Na⁺ and K⁺. Examples of pharmaceutically acceptable divalent inorganic cations include, but are not limited to, alkaline earth cations such as Ca²⁺ and Mg²⁺. Examples of pharmaceutically acceptable organic cations include, but are not limited to, ammonium ion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

Nitrogen protecting groups

Nitrogen protecting groups are well known in the art. Preferred nitrogen protecting groups are carbamate protecting groups that have the general formula:

R'¹⁰-O^O

wherein R'¹⁰ is an optionally substituted alkyl (e.g. Ci_-20 alkyl), aryl (e.g. C₅.₂₀ aryl) or heteroaryl (e.g. C_3-2O heterocyclyl) group.

A large number of possible carbamate nitrogen protecting groups are listed on pages 503 to 549 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.

Particularly preferred protecting groups include Alloc, Troc, Teoc, BOC₁ Doc, Hoc, TcBOC, Fmoc, 1-Adoc and 2-Adoc. Also suitable for use in the present invention are nitrogen protecting groups which can be removed in vivo (e.g. enzymatically, using light) as described in WO 00/12507, which is incorporated herein by reference. Examples of these protecting groups include:

, which is nitroreductase labile (e.g. using ADEPT/GDEPT);

, which are photolabile; and

which is glutathione labile (e.g. using NPEPT).

Hydroxyl protecting groups

Hydroxyl protecting groups are well known in the art. A large number of suitable groups are described on pages 23 to 200 of Greene, T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^rd Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein by reference.

Classes of particular interest include silyl ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, benzoates, carbonates, and sulfonates.

Substituents

The phrase "optionally substituted" as used herein, pertains to a parent group which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term "substituted" as used herein, pertains to a parent group which bears one or more substituents. The term "substituent" is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.

Examples of substituents are described in more detail below.

C_1-7 alkyl: The term "Ci_-7 alkyl" as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 7 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term "alkyl" includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.

Examples of saturated alkyl groups include, but are not limited to, methyl (C-i), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl (C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limited to, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl (amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃), iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), and neo-pentyl (C₅).

C_2-7 Alkenyl: The term "C_2-7 alkenyl" as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, - CH=CH₂), 1-propenyl (-CH=CH-CH₃), 2-propenyl (allyl, -CH-CH=CH₂), isopropenyl (1- methylvinyl, -C(CH₃)=CH₂), butenyl (C₄), pentenyl (C₅), and hexenyl (C₆).

C_2-7 alkynyl: The term "C_2-7 alkynyl" as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (ethinyl, - C=CH) and 2-propynyl (propargyl, -CH₂-C=CH).

C_3-7 cycloalkyl: The term "C_3-7 cycloalkyl" as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, those derived from: saturated monocyclic hydrocarbon compounds: cyclopropane (G₃), cyclobutane (C₄), cyclopentane (C₅), cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄), dimethylcyclopropane (C₅), methylcyclobutane (C₅), dimethylcyclobutane (C₆), methylcyclopentane (C₆), dimethylcyclopentane (C₇) and methylcyclohexane (C₇); unsaturated monocyclic hydrocarbon compounds: cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅), cyclohexene (C₆), methylcyclopropene (C₄), dimethylcyclopropene (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆), methylcyclopentene (C₆), dimethylcyclopentene (C₇) and methylcyclohexene (C₇); and saturated polycyclic hydrocarbon compounds: norcarane (C₇), norpinane (C₇), norbornane (C₇).

C_3-20 heterocyclyl: The term "C_3-20 heterocyclyl" as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.

In this context, the prefixes (e.g. C_3-20, C_3-7, C_5-5, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C_5-6 heterocyclyl", as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole) (C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆), dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇); O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole (dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆), pyran (C₆), oxepin (C₇); Si : thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅), thiane (tetrahydrothiopyran) (C₆), thiepane (C7); O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇); O₃: trioxane (C₆); N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline (C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);

N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole (C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆), dihydrooxazine (C₆), oxazine (C₆); N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆); N₂O₁: oxadiazine (C₆);

O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and, N₁OiSi: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C₅), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.

C_5-20 aryl: The term "C_5-20 aryl", as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C_3-20, C_5-7, C_5-6, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C_5-6 aryl" as used herein, pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in "carboaryl groups".

Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C₆), naphthalene (Ci₀), azulene (Ci₀), anthracene (Ci₄), phenanthrene (Ci₄), naphthacene (Ci₈), and pyrene (Ci₆).

Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2, 3-d i hydro- 1H- indene) (C₉), indene (C₉), isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C-io), acenaphthene (Ci₂), fluorene (Ci₃), phenalene (Ci₃), acephenanthrene (C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in "heteroaryl groups". Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:

N₁: : pyrrole (azole) (C₅), pyridine (azine) (C₆);

Cy. furan (oxole) (C₅); S₁ : thiophene (thiole) (C₅);

N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂Oi-. oxadiazole (furazan) (C₅);

N₃O₁: oxatriazole (C₅);

N₁S-,: thiazole (C₅), isothiazole (C₅); N₂: imidazole (1 ,3-diazole) (C₅), pyrazole (1 ,2-diazole) (C₅), pyridazine (1 ,2-diazine) (C₆), pyrimidine (1 ,3-diazine) (C₆) (e.g., cytosine, thymine, uracil), pyrazine (1 ,4-diazine) (C₆);

N₃: triazole (C₅), triazine (C₆); and,

N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are not limited to:

C₉ (with 2 fused rings) derived from benzofuran (Ot), isobenzofuran (Oi), indole (Ni), isoindole (N-i), indolizine (N₁), indoline (N-i), isoindoline (N₁), purine (N₄) (e.g., adenine, guanine), benzimidazole (N₂), indazole (N₂), benzoxazole (N1O1), benzisoxazole (N₁Oi), benzodioxole (O₂), benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (Si), benzothiazoie (N₁S1), benzothiadiazole (N₂S);

C₁₀ (with 2 fused rings) derived from chromene (O-i), isochromene (Oi), chroman (Oi), isochroman (Oi), benzodioxan (O₂), quinoline (N₁), isoquinoline (N₁), quinolizine (Ni), benzoxazine (Niθi), benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂), quinazoline (N₂), cinnoline (N₂), phthalazine (N₂), naphthyridine (N₂), pteridine (N₄); Ci₁ (with 2 fused rings) derived from benzodiazepine (N₂);

C₁₃ (with 3 fused rings) derived from carbazole (Ni), dibenzofuran (d), dibenzothiophene (Si), carboline (N₂), perimidine (N₂), pyridoindole (N₂); and,

C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene (O₁), thioxanthene (Si), oxanthrene (O₂), phenoxathiin (O₁Si), phenoxazine (N1O1), phenothiazine (N₁S₁), thianthrene (S₂), phenanthridine (Ni), phenanthroline (N₂), phenazine (N₂). C_2-7 Alkenylene: The term "C_2-7 alkenylene" as used herein, pertains to an alkylene group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenylene groups include, but are not limited to, ethenylene (vinylene, -CH=CH₂), 1-propenylene (-CH=CH-CH₃), 2-propenylene (allylene,

-CH-CH=CH₂), isopropenylene (1-methylvinylene, -C(CH₃)=CH₂), butenylene (C₄), pentenylene (C₅), and hexenylene (C₆).

C_2-7 alkynylene: The term "C_2-7 alkynylene" as used herein, pertains to an alkylene group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynylene groups include, but are not limited to, ethynylene (ethinylene, -C=C-) and 2-propynylene (propargylene, -CH₂-C=C-).

C_3-7 cycloalkylene: The term "C_3-7 cycloalkylene" as used herein, pertains to an alkylene group which is also a cyclyl group; that is, a monovalent moiety obtained by removing two hydrogen atoms from alicyclic ring atoms of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkylene groups include, but are not limited to, those derived from: saturated monocyclic hydrocarbon compounds: cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅), cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄), dimethylcyclopropane (C₅), methylcyclobutane (C₅), dimethylcyclobutane (C₆), methylcyclopentane (C₆), dimethylcyclopentane (C₇) and methylcyclohexane (C₇); unsaturated monocyclic hydrocarbon compounds: cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅), cyclohexene (C₆), methylcyclopropene (C₄), dimethylcyclopropene (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆), methylcyclopentene (C₆), dimethylcyclopentene (C₇) and methylcyclohexene (C₇); and saturated polycyclic hydrocarbon compounds: norcarane (C₇), norpinane (C₇), norbornane (C₇).

C_3-20 heterocyclylene: The term "C_3-20 heterocyclylene" as used herein, pertains to a monovalent moiety obtained by removing two hydrogen atoms from ring atoms of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.

In this context, the prefixes (e.g. C_3-20, C_3-7, C_5-5, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C_5-6 heterocyclylene", as used herein, pertains to a heterocyclylene group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclylene groups include, but are not limited to, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole) (C₅), pyrroline (e.g.,

3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆), dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇);

O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole (dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆), pyran (C₆), oxepin (C₇);

Si : thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅), thiane

(tetrahydrothiopyran) (C₆), thiepane (C₇);

O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);

O₃: trioxane (C₆); N₂: imidazoline (C₅), pyrazolidine (diazolidine) (C₅), imidazoline (C₅), pyrazoline

(dihydropyrazole) (C₅), piperazine (C₆);

N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole (C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆), dihydrooxazine (C₆), oxazine (C₆); NiS₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);

N₂Oi: oxadiazine (C₆);

OiSi: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,

N₁OiSi: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclylene groups include those derived from saccharides, in cyclic form, for example, furanoses (C₅), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose. C_5-2O arylene: The term "C_5-20 arylene", as used herein, pertains to a monovalent moiety obtained by removing two hydrogen atoms from aromatic ring atoms of an aromatic compound, which moiety has from 3 to 20 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C_3-20, C_5-7, C_5-e, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term "C_5-6 arylene" as used herein, pertains to an arylene group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in "carboarylene groups". Examples of carboarylene groups include, but are not limited to, those derived from benzene (i.e. phenylene) (C₆), naphthalene (C-io), azulene (C₁₀), anthracene (Ci₄), phenanthrene (C₁₄), naphthacene (Ci₈), and pyrene (Ci₆).

Examples of arylene groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3- dihydro-1H-indene) (C₉), indene (C₉), isoindene (C₉), tetraline (1 ,2,3,4-tetrahydronaphthalene (Ci₀), acenaphthene (Ci₂), fluorene (C₁3), phenalene (C-₁3), acephenanthrene (Ci₅), and aceanthrene (Ci₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in "heteroarylene groups". Examples of monocyclic heteroarylene groups include, but are not limited to, those derived from: N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);

Cv furan (oxole) (C₅);

S₁: thiophene (thiole) (C₅);

N₁Oi: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂O₁: oxadiazole (furazan) (C₅); N₃Oi: oxatriazole (C₅);

N₁Si: thiazole (C₅), isothiazole (C₅);

N₂: imidazole (1 ,3-diazole) (C₅), pyrazole (1 ,2-diazole) (C₅), pyridazine (1 ,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);

N₃: triazole (C₅), triazine (C₆); and, N₄: tetrazole (C₅). Examples of heteroarylene which comprise fused rings, include, but are not limited to:

Cg (with 2 fused rings) derived from benzofuran (O-i), isobenzofuran (Oi), indole (N₁), isoindole (N₁), indolizine (N₁), indoline (Ni), isoindoline (N₁), purine (N₄) (e.g., adenine, guanine), benzimidazole (N₂), indazole (N₂), benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (O₂), benzofurazan (N₂Oi), benzotriazole (N₃), benzothiofuran (S-i), benzothiazole (NiS₁), benzothiadiazole (N₂S);

C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene (Oi), chroman (Oi), isochroman (O₁), benzodioxan (O₂), quinoline (N-i), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁Oi), benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂), quinazoline (N₂), cinnoline (N₂), phthalazine (N₂), naphthyridine (N₂), pteridine (N₄);

C₁-_I (with 2 fused rings) derived from benzodiazepine (N₂);

C₁₃ (with 3 fused rings) derived from carbazole (N-i), dibenzofuran (O-i), dibenzothiophene (S-i), carboline (N₂), perimidine (N₂), pyridoindole (N₂); and,

C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene (O₁), thioxanthene (S₁), oxanthrene (O₂), phenoxathiin (0₁S₁), phenazine (N₂), phenoxazine (NiO₁), phenothiazine (N₁S₁), thianthrene (S₂), phenanthridine (Ni), phenanthroline (N₂), phenazine (N₂).

The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from the groups listed above and the additional substituents listed below.

Halo: -F, -Cl, -Br, and -I.

Hydroxy: -OH.

Ether: -OR, wherein R is an ether substituent, for example, a C_1-7 alkyl group (also referred to as a Ci_-7 alkoxy group, discussed below), a C_3-20 heterocyclyl group (also referred to as a C_3-20 heterocyclyloxy group), or a C_5-20 aryl group (also referred to as a C_5-20 aryloxy group), preferably a Ci_-7alkyl group.

Alkoxy: -OR, wherein R is an alkyl group, for example, a C_1-7 alkyl group. Examples of Ci_-7 alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), - O(nPr) (n-propoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy). Acetal: -CH(OR¹)(OR²), wherein R¹ and R² are independently acetal substituents, for example, a C_1-7 alkyl group, a C_3-2O heterocyclyl group, or a C_5-2O aryl group, preferably a C_1-7 alkyl group, or, in the case of a "cyclic" acetal group, R¹ and R², taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, -CH(OMe)₂, -CH(OEt)₂, and -CH(OMe)(OEt).

Hemiacetal: -CH(OH)(OR¹), wherein R¹ is a hemiacetal substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, -CH(OH)(OMe) and - CH(OH)(OEt).

Ketal: -CR(OR¹)(OR²), where R¹ and R² are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a Ci_-7 alkyl group. Examples ketal groups include, but are not limited to, -C(Me)(OMe)₂, -C(Me)(OEt)₂, -C(Me)(OMe)(OEt), -C(Et)(OMe)₂, - C(Et)(OEt)₂, and -C(Et)(OMe)(OEt).

Hemiketal: -CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a Ci_-7 alkyl group. Examples of hemiacetal groups include, but are not limited to, -C(Me)(OH)(OMe), -C(Et)(OH)(OMe), -C(Me)(OH)(OEt), and -C(Et)(OH)(OEt).

Oxo (keto, -one): =0.

Thione (thioketone): =S.

lmino (imine): =NR, wherein R is an imino substituent, for example, hydrogen, Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a Cs_-20 aryl group, preferably hydrogen or a Ci_-7 alkyl group. Examples of ester groups include, but are not limited to, =NH, =NMe, =NEt, and =NPh.

Formyl (carbaldehyde, carboxaldehyde): -C(=0)H. Acyl (keto): -C(=O)R, wherein R is an acyl substituent, for example, a C_halky! group (also referred to as Ci_-7 alkylacyl or Ci_-7 alkanoyl), a C_3-2O heterocyclyl group (also referred to as C_3-2O heterocyclylacyl), or a C_5-2O aryl group (also referred to as C_5-20 arylacyl), preferably a Ci_-7 alkyl group. Examples of acyl groups include, but are not limited to, -C(=O)CH₃ (acetyl), -C(=O)CH₂CH₃ (propionyl), -C(=O)C(CH₃)₃ (t-butyryl), and -C(=O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): -C(=O)OH.

Thiocarboxy (tricarboxylic acid): -C(=S)SH.

Thiolocarboxy (thiolocarboxylic acid): -C(=O)SH.

Thionocarboxy (thionocarboxylic acid): -C(=S)OH.

lmidic acid: -C(=NH)OH.

Hydroxamic acid: -C(=NOH)OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): -C(=O)OR, wherein R is an ester substituent, for example, a C₁-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-2O aryl group, preferably a Ci_-7 alkyl group. Examples of ester groups include, but are not limited to, -C(=O)OCH₃, -C(=O)OCH₂CH₃, -C(=O)OC(CH₃)₃, and -C(=O)OPh.

Acyloxy (reverse ester): -OC(=O)R, wherein R is an acyloxy substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a Ci_-7 alkyl group. Examples of acyloxy groups include, but are not limited to, -OC(=O)CH₃ (acetoxy), -OC(=O)CH₂CH₃, -OC(=O)C(CH₃)₃, -OC(=O)Ph, and -OC(=O)CH₂Ph.

Oxycarboyloxy: -OC(=O)OR, wherein R is an ester substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a Ci_-7 alkyl group. Examples of ester groups include, but are not limited to, -OC(=O)OCH₃, -OC(=O)OCH₂CH₃, -OC(=O)OC(CH₃)₃, and -OC(=O)OPh.

Amino: -NR¹R², wherein R¹ and R² are independently amino substituents, for example, hydrogen, a Ci_-7 alkyl group (also referred to as Ci_-7 alkylamino or di-Ci_-7 alkylamino), a C_3-2o heterocyclyl group, or a C_5-20 aryl group, preferably H or a Ci_-7 alkyl group, or, in the case of a "cyclic" amino group, R¹ and R², taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Amino groups may be primary (-NH₂), secondary (-NHR¹), or tertiary (-NHR¹R²), and in cationic form, may be quaternary (-1MR¹R²R³). Examples of amino groups include, but are not limited to, -NH₂, -NHCH₃, -NHC(CHa)₂, -N(CH₃)₂, -N(CH₂CH₃)₂, and -NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): -C(=O)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=O)NH₂, -C(=O)NHCH₃, -C(=O)N(CH₃)₂, -C(=O)NHCH₂CH₃, and -C(=O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.

Thioamido (thiocarbamyl): -C(=S)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=S)NH₂, -C(=S)NHCH₃, -C(=S)N(CH₃)₂, and -C(=S)NHCH₂CH₃.

Acylamido (acylamino): -NR¹C(=O)R², wherein R¹ is an amide substituent, for example, hydrogen, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably hydrogen or a Ci_-7 alkyl group, and R² is an acyl substituent, for example hydrogen, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20aryl group, preferably hydrogen or a C_1-7 alkyl group. Examples of acylamide groups include, but are not limited to, -NHC(=O)CH₃ , -NHC(=O)CH₂CH₃, and -NHC(=O)Ph. R¹ and R² may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:

succinimidyl maleimidyl

Aminocarbonyloxy: -OC(=O)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, -0C(=0)NH₂, -0C(=0)NHMe, -0C(=0)NMe₂, and -OC(=O)NEt₂.

Ureido: -N(R¹)CONR²R³ wherein R² and R³ are independently amino substituents, as defined for amino groups, and R¹ is a ureido substituent, for example, hydrogen, a C_1-7 alkyl group, a C_3-2O heterocyclyl group, or a C_5-2O aryl group, preferably hydrogen or a C_1-7 alkyl group. Examples of ureido groups include, but are not limited to, -NHCONH₂, - NHCONHMe, -NHCONHEt, -NHCONMe₂, -NHCONEt₂, -NMeCONH₂, -NMeCONHMe, -NMeCONHEt, -NMeCONMe₂, and -NMeCONEt₂.

Guanidino: -NH-C(=NH)NH₂.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms and one carbon atom,

Imino: =NR, wherein R is an imino substituent, for example, for example, hydrogen, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C₅,₂o aryl group, preferably H or a C_1-7alkyl group. Examples of imino groups include, but are not limited to, =NH, =NMe, and =NEt.

Amidine (amidino): -C(=NR)NR_2> wherein each R is an amidine substituent, for example, hydrogen, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably H or a Ci_-7 alkyl group. Examples of amidine groups include, but are not limited to, -C(=NH)NH₂, -C(=NH)NMe₂, and -C(=NMe)NMe₂.

Nitro: -NO₂.

Nitroso: -NO.

Azido: -N₃.

Cyano (nitrile, carbonitrile): -CN. Isocyano: -NC.

Cyanato: -OCN.

Isocyanato: -NCO.

Thiocyano (thiocyanato): -SCN.

lsothiocyano (isothiocyanato): -NCS.

Sulfhydryl (thiol, mercapto): -SH.

Thioether (sulfide): -SR, wherein R is a thioether substituent, for example, a C_1-7 alkyl group (also referred to as a Ci_-7alkylthio group), a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl group. Examples of Ci_-7 alkylthio groups include, but are not limited to, -SCH₃ and -SCH₂CH₃.

Disulfide: -SS-R, wherein R is a disulfide substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a Ci_-7 alkyl group (also referred to herein as C_1-7 alkyl disulfide). Examples of C_1-7 alkyl disulfide groups include, but are not limited to, -SSCH₃ and -SSCH₂CH₃.

Sulfine (sulfinyl, sulfoxide): -S(=O)R, wherein R is a sulfine substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a d-₇ alkyl group. Examples of sulfine groups include, but are not limited to, -S(=O)CH₃ and -S(=O)CH₂CH₃.

Sulfone (sulfonyl): -S(=O)₂R, wherein R is a sulfone substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a Ci_-7 alkyl group, including, for example, a fluorinated or perfluorinated C_1-7 alkyl group. Examples of sulfone groups include, but are not limited to, -S(=O)₂CH₃ (methanesulfonyl, mesyl), -S(=O)₂CF₃ (triflyl), -S(=O)₂CH₂CH₃ (esyl), -S(=O)₂C₄F₉ (nonaflyl), -S(=O)₂CH₂CF₃ (tresyl), -S(=O)₂CH₂CH₂NH₂ (tauryl), -S(=O)₂Ph (phenylsulfonyl, besyl), A- methylphenylsulfonyl (tosyl), 4-chlorophenylsulfony! (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenylsulfonyl (nosyl), 2-naphthalenesulfonate (napsyl), and 5-dimethylamino-naphthalen-1 -ylsulfonate (dansyl). Sulfinic acid (sulfino): -S(=O)OH, -SO₂H.

Sulfonic acid (sulfo): -S(=O)₂OH, -SO₃H.

Sulfinate (sulfinic acid ester): -S(=O)OR; wherein R is a sulfinate substituent, for example, a C_1-7 alkyl group, a C_3-2O heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl group. Examples of sulfinate groups include, but are not limited to, -S(=O)OCH₃ (methoxysulfinyl; methyl sulfinate) and -S(=O)OCH₂CH₃ (ethoxysulfinyl; ethyl sulfinate).

Sulfonate (sulfonic acid ester): -S(=O)₂OR, wherein R is a sulfonate substituent, for example, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl group. Examples of sulfonate groups include, but are not limited to, -S(=O)₂OCH₃ (methoxysulfonyl; methyl sulfonate) and -S(=O)₂OCH₂CH₃ (ethoxysulfonyl; ethyl sulfonate).

Sulfinyloxy: -OS(=O)R, wherein R is a sulfinyloxy substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl group. Examples of sulfinyloxy groups include, but are not limited to, -OS(=O)CH₃ and -OS(=O)CH₂CH₃.

Sulfonyloxy: -OS(=O)₂R, wherein R is a sulfonyloxy substituent, for example, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl group. Examples of sulfonyloxy groups include, but are not limited to, -OS(=O)₂CH₃ (mesylate) and -OS(=O)₂CH₂CH₃ (esylate).

Sulfate: -OS(=O)₂OR; wherein R is a sulfate substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a Cs_-20 aryl group, preferably a C_1-7 alkyl group. Examples of sulfate groups include, but are not limited to, -OS(=O)₂OCH₃ and -SO(=O)₂OCH₂CH₃.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): -S(=O)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, -S(=O)NH₂, -S(=O)NH(CH₃), -S(=O)N(CH₃)₂, -S(=O)NH(CH₂CH₃), -S(=O)N(CH₂CH₃)₂, and -S(=O)NHPh. Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): -S(=O)₂NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of sulfonamido groups include, but are not limited to, -S(=O)₂NH₂, -S(=O)₂NH(CH₃), -S(=O)₂N(CH₃)₂, -S(=O)₂NH(CH₂CH₃), -S(=O)₂N(CH₂CH₃)₂, and -S(=O)₂NHPh.

Sulfamino: -NR¹S(=O)₂OH, wherein R¹ is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, -NHS(=O)₂OH and -N(CH₃)S(=O)₂OH.

Sulfonamino: -NR¹S(=O)₂R, wherein R¹ is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-2O aryl group, preferably a C_1-7 alkyl group. Examples of sulfonamino groups include, but are not limited to, -NHS(=O)₂CH₃ and -N(CH₃)SC=O)₂C₆H₅.

Sulfinamino: -NR¹S(=O)R, wherein R¹ is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a C_1-7 alkyl group. Examples of sulfinamino groups include, but are not limited to, -NHS(=O)CH₃ and -N(CH₃)S(=O)C₆H₅.

Phosphino (phosphine): -PR₂, wherein R is a phosphino substituent, for example, -H, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably -H₁ a C_1-7 alkyl group, or a C_5-20 aryl group. Examples of phosphino groups include, but are not limited to, -PH₂, -P(CHs)₂, -P(CH₂CHa)₂, -P(t-Bu)₂, and -P(Ph)₂.

Phospho: -P(=O)₂.

Phosphinyl (phosphine oxide): -P(=0)R₂, wherein R is a phosphinyl substituent, for example, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably a Ci_-7 alkyl group or a C_5-20 aryl group. Examples of phosphinyl groups include, but are not limited to, -P(=O)(CH₃)₂, -P(=O)(CH₂CH₃)₂, -P(=O)(t-Bu)₂, and -P(=O)(Ph)₂.

Phosphonic acid (phosphono): -P(=O)(OH)₂.

Phosphonate (phosphono ester): -P(=O)(OR)₂, where R is a phosphonate substituent, for example, -H, a C₁._? alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably -H, a Ci_-7 alkyl group, or a C₅.₂o aryl group. Examples of phosphonate groups include, but are not limited to, -P(=O)(OCH₃)₂, -P(=O)(OCH₂CH₃)₂, -P(=O)(O-t-Bu)₂, and -P(=O)(OPh)₂.

Phosphoric acid (phosphonooxy): -OP(=O)(OH)₂.

Phosphate (phosphonooxy ester): -OP(=O)(OR)₂, where R is a phosphate substituent, for example, -H, a C_1-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably -H, a Ci_-7 alkyl group, or a C_5-2O aryl group. Examples of phosphate groups include, but are not limited to, -OP(=O)(OCH₃)₂, -OP(=O)(OCH₂CH₃)₂, -OP(=O)(O-t-Bu)₂, and -OP(=O)(OPh)₂.

Phosphorous acid: -OP(OH)₂.

Phosphite: -OP(OR)₂, where R is a phosphite substituent, for example, -H, a Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably -H, a C_1-7 alkyl group, or a C_5-2O aryl group. Examples of phosphite groups include, but are not limited to, -OP(OCH₃)₂, -OP(OCH₂CHs)₂, -OP(O-t-Bu)₂, and -OP(OPh)₂.

Phosphoramidite: -OP(OR¹)-NR² ₂, where R¹ and R² are phosphoramidite substituents, for example, -H, a (optionally substituted) Ci_-7 alkyl group, a C_3-2O heterocyclyl group, or a C_5-20 aryl group, preferably -H, a C_1-7 alkyl group, or a C_5-20 aryl group. Examples of phosphoramidite groups include, but are not limited to, -OP(OCH₂CH₃)-N(CH₃)₂, -OP(OCH₂CH₃)-N(i-Pr)₂, and -OP(OCH₂CH₂CN)-N(J-Pr)₂.

Phosphoramidate: -OP(=O)(OR¹)-NR² ₂, where R¹ and R² are phosphoramidate substituents, for example, -H, a (optionally substituted) Ci_-7 alkyl group, a C_3-20 heterocyclyl group, or a C_5-20 aryl group, preferably -H, a Ci_-7 alkyl group, or a C_5-2O aryl group. Examples of phosphoramidate groups include, but are not limited to, -OP(=O)(OCH₂CH₃)-N(CH₃)₂, -OP(=O)(OCH₂CH₃)-N(i-Pr)₂, and -OP^O)(OCH₂CH₂CN)- N(i-Pr)₂.

Methods of Treatment

As described above, the present invention provides as a third aspect the use of a compound in a method of therapy. Also provided as a fifth aspect is a method of treatment, comprising administering to a subject in need of treatment a therapeutically- effective amount of a compound of the first aspect or a pharmaceutical composition of the second aspect, preferably in the form of a pharmaceutical composition, which is the seventh aspect of the present invention. The term "therapeutically effective amount" is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.

A compound may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs); surgery; and radiation therapy.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a compound of the first aspect, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection, Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Includes Other Forms Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-COO^"), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N⁺HR¹R²), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (-0^"), a salt or solvate thereof, as well as conventional protected forms.

Isomers, Salts and Solvates Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomer^, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and I- forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers", as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, -OCH₃, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH₂OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. C_1-7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

keto enol enolate

Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including ¹H, ²H (D), and ³H (T); C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopic form, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, and protected forms of thereof, for example, as discussed below.

It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al., J. Pharm. ScL, 66, 1-19 (1977).

For example, if the compound is anionic, or has a functional group which may be anionic (e.g. -COOH may be -COO^"), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CHa)₄ ⁺.

If the compound is cationic, or has a functional group which may be cationic (e.g. -NH₂ may be -NH₃ ⁺), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term "solvate" is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Solvates of particular relevance to the present invention are those where the solvent adds across the imine bond present in the PBD moieties of formula I, which is illustrated below where the solvent is water or an alcohol (R^AOH, where R^A is an ether substituent as described above):

These forms can be called the carbinolamine and carbinolamine ether forms of the PBD. The balance of these equilibria depends on the conditions in which the compounds are found, as well as the nature of the moiety itself.

In general any nucleophilic solvent is capable of forming such solvates as illustrated above for hydroxylic solvents. Other nucleophilic solvents include thiols and amines.

These solvates may be isolated in solid form, for example, by lyophilisation.

Chemical Abbreviations

In this application several abbreviations are used when referring to well known chemical fragments and reagents.

Chemical abbreviations Alloc - allyloxycarbonyl

DMSO - dimethylsulphoxide ft

DMAP - 4-dimethylaminopyridine

EDCI - i-ethyl-Sβ'-dimethylaminopropyQ-carbodiimide

HOBt - 1-hydroxybenzotriazole

Su - succinimide

TFA - trifluoroacetic acid

F _FΛ— CO₂H THF - tetrahydrofuran

Boc: tertiary-butyl carbamate

Ala: alanine, or a derivative fragment thereof

DCM: dichloromethane

CI-^^CI DMF: N,N-dimethylformamide

DIPEA: N,N-diisopropylethylamine

DlC: 5-(3,3-dimethyl-1-triazenyl)-imidazo!e-4-carboxamide

HOBt: 1-hydroxybenzotriazole

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

DTT: dithiothreitol QH

,SH

HS^'

OH EDTA: ethylenediaminetetraacetic acid

APS: ammonium persulfate

TEMED: tetramethylethylenediamine

Further Preferences

The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.

X

Each X is independently a C_3-6 heteroarylene group. This group is preferably an optionally substituted C₅ heteroarylene group.

The adjoining carbonyl and amino groups may be attached to the heteroarylene group (X) at any two of the heteroarylene atoms, and preferably at two separate carbon atoms in the heteroarylene ring.

Where the X group is a six membered heteroarylene group, the carbonyl and amino groups are preferably attached at the 2,6, 2,5, 3,6 or 3,5 positions. Where the X group is a five membered heteroarylene group, the carbonyl and amino groups are preferably attached at the 2,5, 2,4 or 3,5 positions.

The group preferably has one or two ring heteroatoms, which are preferably selected from N, S and O. If the heteroarylene group is substituted, it is preferably substituted with an optionally substituted C_1-7 alkyl group, most preferably with a methyl group.

If the heteroarylene group is substituted with a methyl group, the substitution is preferably on the atom β to that which is bonded to the acyl carbon of amino residue represented by (-NH-E-C(=O)-).

Heteroarylene groups that are particularly preferred are those derived from: N₁: pyrrole (azole) (C₅); O₁: furan (oxole) (C₅); N₁S-_I.¹ thiazole (C₅); and

N₂: imidazole (1 ,3-diazole).

The fragments

may be preferably selected from:

n

In some embodiments, n is 1 , 2 or 3, and most preferably n = 2.

R¹O and R^

R-O and RH preferably together form a double bond between N10 and C11. R⁷

R⁷ is preferably selected from H, OH, OR, SH, SR, NH₂, NHR, NRR', and halo, and more preferably independently selected from H, OH and OR, where R is preferably selected from optionally substituted C_1-7 alkyl, C_3-I0 heterocyclyl and C_S-io aryl groups. Particularly preferred substituents at the 7- position are OMe and OCH₂Ph.

R²

In some embodiments, R² is H.

In some embodiments, R (and particularly R²) is an optionally substituted C_5-2o aryl group. It may be selected from: an optionally substituted C_5-7 aryl group, for example phenyl; an optionally substituted C_9-12 aryl group, for example for example naphthyl (e.g napthy-1-yl, napth-2-yl) and quinolinyl (e.g. quinolin-2-yl, quinolin-3-yl,quinolin-6-yl); an optionally substituted Cs_-7 heteroaryl group, for example furanyl (e.g. furan-2-yl, furan-3- yl), thiophenyl (e.g. thiophen-2-yl, thiophen-3-yl) and pyridyl (e.g. pyrid-2-yl, pyrid-3-yl).

The C_5-20 aryl group may bear any substituent group. It may bear from 1 to 3, 1 to 2 or 1 substituent groups. C_5-20 aryl substituents, particularly for phenyl, include, but are not limited to; halo (e.g. F, Cl, Br); C_1-7 alkoxy (e.g. methoxy, ethoxy); Ci_-7 alkyl (e.g. methyl, trifluoromethyl, ethyl, propyl, t-butyl); bis-oxy-alkylene (e.g. bis-oxy-methylene, -0-CH₂- O-).

C_5-2O aryl groups of particular interest, include, but are not limited to, phenyl, 4-methyl- phenyl, 4-methoxy-phenyl, 3-methoxyphenyl, 4-fluoro-phenyl, 3,4-bisoxymethylene- phenyl, 4-triflouoromethylphenyl, 4-methylthiophenyl, 4-cyanophenyl, 4-phenoxyphenyl, thiophen-2-yl, napth-2-yl, quinolin-3-yl and quinolin-6-yl.

In some embodiments, R² is =CH₂.

In some embodiments, R² is =CH-R. In these embodiments, R may be C_1-7 alkyl (e.g. methyl, ethyl). The C_1-7 alkyl group may contain one or more unsaturated bonds conjugated to the double bond bound to the C-ring. Thus, in this case R² may be, for example, =CH-CH=CH₂, =CH-CH=CH-CH₃, =CH-C≡CH, =CH-CH=CH-CH=CH₂. Alternatively in these embodiments, R may be C₅-₂₀ aryl, in particular C_5-6 aryl (e.g. phenyl, pyridyl, thiophenyl, furanyl). In some embodiments where there is a double bond between the C2 and C3 positions of the C-ring, R² may be C_1-7 alkyl containing one or more unsaturated bonds conjugated to the double bond in the C-ring. Thus, in this case R² may be, for example, -CH=CH₂, - CH=CH-CH₃, -C=CH, -CH=CH-CH=CH₂.

In some embodiments, R⁶ and R⁹ are H, R⁷ is OMe or OCH₂Ph, and R² is selected from the options set out above, in particular, when there is no double bond between C2 and C3: H, optionally substituted, C_5-7 aryl, =CH_Z, -CH-CH₃, =CH-CH=CH₂, and when there is a double bond between C2 and C3: -CH=CH₂, -CH=CH-CH₃ and -C=CH.

Precursor

R^10A is preferably selected from boc, Troc or alloc. R^11A is preferably THP or a silyl oxygen protecting group (for example TBS) and is most preferably THP.

Bisulphite preferences

In the group SO_ZM, z is preferably 3. M is preferably Na⁺.

Compounds

Particularly preferred compounds include: 23, 31 , 48, 49 and 57.

Examples

General Synthetic Routes

Pyrrolobenzodiazepine moieties

The syntheses of pyrrolobenzodiazepine moieties are described in WO 00/12506. Protection at the C11 position can be readily introduced, as for example described in

WO 2007/039752.

The synthesis of the polyamide chains is described below.

General methods for ester hydrolysis

Hydrolysis Method A (Alloc or Boc protected compounds)

An 0.5M solution of NaOH (2 eq) was added to a solution of the ester in 1,4-dioxane.

The reaction mixture was allowed to stir at room temperature until reaction was complete. The 1 ,4-dioxane was evaporated and the residue, diluted with water as necessary, was acidified to pH 3 with either cone. HCI or 1M citric acid. The product was collected by filtration, purified as necessary and dried. Hydrolysis Method B (Boc protected compounds)

NaOH solution (excess) was added to a solution of the ester in MeOH. The reaction mixture was allowed to stir at 50-60⁰C until complete. The MeOH was evaporated and the residue, diluted with water as necessary was acidified to pH 3 with 1 M citric acid solution. The product was collected by filtration, purified as necessary and dried.

General methods for heterocvcle couplings

Heterocyclic Coupling Method A A solution of the acid, amine, EDCI and DMAP in DCM was stirred at room temperature until reaction was complete. The solution was washed with either 0.1 M HCI (x 2) or 1M citric acid (x 2), sat. NaHCO₃ (x 2), H₂O (x 1), brine (x 1), dried (MgSO₄) and evaporated.

The product was either used without further purification or was purified by standard techniques.

Heterocyclic Coupling Method B

A solution of the acid, amine, EDCI and DMAP in DMF was stirred at room temperature until reaction was complete. The reaction mixture was poured onto ice and extracted with DCM (x 3). The solution was washed with either 0.1 M HCI (x 2) or 1M citric acid (x 2), sat. NaHCO₃ (x 2), H₂O (x 1), brine (x 1), dried (MgSO₄) and evaporated. The product was either used without further purification or was purified by standard techniques.

Compounds 1(a & b), 15(b), 20(a & b), 23, 24 37, 43 and 47 are available from commercial sources.

Synthesis of Alloc Building Blocks

Ia X = CH₁ R = Me 2a X = CH, R = Me 3a X = CH Ib X = N, R = Et 2bX = N, R = Et 3b X = N

4a X = CH 5a X = CH 4b X = N 5b X = N

SCHEME 1

Scheme 1 (i) ^AIIyloxycarbonylamino-i-methyl-IH-pyrrole-Σ-carboxylic acid methyl ester (2a)

AIIyI chloroformate (39.1 g, 34.4 ml, 0.33 mol, 1.5 eq) was added dropwise to a solution of 4-amino-1-methyl-1H-pyrrole-2-carboxylic acid methyl ester 1a (33.5 g, 0.22 mol, 1 eq) and DIPEA (84.22 g, 114 ml, 0.65 mol, 3 eq) in EtOAc (400 ml) at -5°C. The reaction mixture was allowed to stir at 0⁰C for 1 hour then overnight at room temperature. Sat. NaHCO₃ (200 ml) was added and the mixture stirred for 30 minutes. The NaHCO₃ portion was separated and washed with EtOAc (200 ml). The combined EtOAc portions were washed with 2M HCI (3 x 200 ml), saturated NaHCO₃ (2 x 200 ml), H₂O (200 ml), brine (200 ml),dried (MgSO₄) and evaporated to give a pale brown oil which solidified on standing overnight. Trituration with n-hexane gave the product as a white solid (50.2g, 97%). ¹H NMR (CDCI₃) δ 7.09 (bs, 1H), 6.656 (d, J = 2 Hz, 1H), 6.47 (s, 1H), 6.0 - 5.90 (m, 1H), 5.37 -5.23 (m, 2H), 4.64 (m, 2H), 3.88 (s, 2H), 3.79 (s, 3H); MS (ES⁺) mlz 239.01 ([M + H]⁺, 50). 4-Allyloxycarbonylamino-1-methyl-1H-imidazole-2-carboxylic acid ethyl ester (2b) A solution of allyl chloroformate (3.88 g, 3.4 ml, 32.2 mmol, 1.1 eq) in anhydrous DCM (15 ml) was added dropwise to a suspension of 4-amino-1-methyl-1H-imidazoIe-2- carboxylic acid ethyl ester 1b (4.95 g, 29.3 mmol, 1 eq) and pyridine (5.32 g, 5.44 ml, 67.3 mmol, 2.3 eq) in anhydrous DCM (85 ml) at -10⁰C. The reaction mixture was allowed to stir at -10⁰C for 30 minutes then at room temperature for 3 hours. The solution was washed with sat. CuSO₄ (2 x 100 ml), H₂O (200 ml), brine (200 ml), dried (MgSO₄) and evaporated to give the product as a pale brown solid (β.6 g, 89%). ¹H NMR (CDCI₃) δ 7.24 (m, 2H), 6.0 - 5.90 (m, 1H), 5.36 - 5.24 (m, 2H), 4.65 (m, 2H), 4.4 (q, J = 7.07 Hz, 2H), 3.98 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H); MS (ES⁺) mlz 254.02 ([M + H]⁺, 100).

(H) 4-Allyloxycarbonylamino-1-methyl-1H-pyrrole-2-carboxylic acid (3a) The ester 2a (10.1 g. 42 mmol, 1 eq) in 1,4-dioxane (100 ml) was hydrolysed (Hydrolysis Method A) to give the product as a white solid (8.65 g, 92%). ¹H NMR (DMSO-cfe) δ 12.21 (bs, 1H), 9.47 (s, 1H), 7.08 (d, J = 1.24 Hz, 1H), 6.65 (d, J = 1.6 Hz, 1H), 6.04 -5.94 (m, 1H), 5.34 - 5.23 (m, 2H), 4.59 (m, 2H), 3.82 (s, 3H); MS (ES⁺) mlz 224.93 ([M + H]⁺, 100).

4-Allyloxycarbonylamino- 1 -methyl- 1 H-imidazole-2-carboxylic acid (3b) The ester 2b (6.56 g, 26 mmol, 1 eq) in 1 ,4-dioxane (50 ml) was hydrolysed (Hydrolysis Method A) to give the product as a pale pink solid (3.72 g, 64%). ¹H NMR (DMSO-Cf₆) δ 10.11 (s, 1H), 7.29 (s, 1H), 6.0 - 5.91 (m, 1H), 5.4 - 5.21 (m, 2H), 4.59 (m, 2H), 3.9 (s, 3H); MS (ES⁺) mlz 226.00 ([M + H]⁺, 100).

(Ui) 4-[(4-Allyloxycarbonylamino- 1-methyl-1H-pyrrole~2-carbonyl)-aminoJ- 1 -methyl- 1 H- pyrrole-2-carboxylic acid methyl ester (4a)

A solution of the acid 3a (7.53 g, 33.6 mmol, 1.2 eq), amine 1a HCI salt (5.33 g, 27.99 mmol, 1 eq), EDCI (10.73 g, 55.97 mmol, 2 eq) and DMAP (8.55 g, 69.97 mmol, 2.5 eq) in DMF (100 ml) was stirred for 18 hours. Work up (Heterocyclic Coupling Method B) gave the product as a yellow foam (9.4 g, 93%). ¹H NMR (CDCI₃) δ 7.61 (s, 1H), 7.40 (s, 1 H), 6.87 (S, 1 H), 6.74 (s, 1 H), 6.56 (s, 1 H), 6.50 (s, 1 H), 6.02 - 5.92 (m, 1 H), 5.4 - 5.24 (m, 2H), 4.65 (m, 2H), 3.96 (s, 3H), 3.89 (s, 3H), 3.81 (s, 3H); MS (ES⁺) mlz 361.01 ([M + H]⁺, 100). 4-[(4-Allyloxycarbonylamino-1-methyl- 1H-imidazole-2-carbonyl)-amino]-1 -methyl- 1H- pyrrole-∑carboxylic acid methyl ester (4b)

A solution of the acid 3b (15.0 g, 67 mmol, 1.2 eq), amine 1a HCI salt (10.58 g, 56 mmol, 1 eq), EDCI (21.28 g, 111 mmol, 2 eq) and DMAP (16.95 g, 139 mmol, 2.6 eq) in DMF (200 ml) was stirred for 18 hours. Work up (Heterocyclic Coupling Method B) gave the product as a brown foam (16.5 g, 82%), MS (ES⁺) mlz 362.04 ([M + H]⁺, 100).

(iv) 4-[(4-Allyloxycarbonylamino-1-methyl-1H-pyrrole-2-carbonyl)-aminoJ-1-methyl-1H- pyrrole-2-carboxylic acid (5a) The ester 4a (9.36 g, 26 mmol, 1 eq) in 1 ,4-dioxane (100 ml) was hydrolysed

(Hydrolysis Method A) to give the product as an off white solid (8.75 g, 97%). ¹H NMR

(DMSO-de) δ 11.98 (bs, 1H), 9.85 (s, 1H), 9.45 (s, 1H), 7.424 (d, J = 1.4 Hz, 1H), 6.94

(s, 1H), 6.85 (2s, 2H), 6.03 - 5.94 (m, 1H), 5.37 - 5.22 (m, 2H), 4.59 (m, 2H), 3.84 (s,

6H); MS (ES⁺) mlz 346.94 ([M + H]⁺, 100).

4-[(4-Allyioxycarbonylamino-1-methyl-1H-imidazole-2-carbonyl)-amino]-1-methyl-1H- pyrrole-2carboxylic acid (5b)

The ester 4b (16.5 g, 46 mmol, 1 eq) in 1,4 dioxane (150 ml) was hydrolysed (Hydrolysis

Method A) to give the product as a brown solid (14.3 g, 90%). ¹H NMR (DMSO-Cf₆) δ 12.01 (bs, 1H) 10.06 (s, 1H), 9.74 (bs, 1H), 7.46 (d, J = 1.92 Hz, 1H), 7.23 (s, 1H), 6.96

(d, J = 1.96 Hz, 1 H), 6.01 - 5.91 (m, 1 H), 5.36 - 5.32 (m, 2H), 4.60 (m, 2H), 3.95 (s, 3H),

3.83 (s, 3H); MS (ES⁺) mlz 347.92 ([M + H]⁺, 100).

6a X = CH 7a X = CH 6b X = N 7b X = N SCHEME 2 Scheme 2

(j) ^[ft-Allyloxycarbonylamino-i-methyl-IH-pyrrole-Σ-carbonyty-arninoJ-i-methyl-IH- imidazole-2-carboxylic acid ethyl ester (6a)

A solution of the acid 3a (15 g, 66.9 mmol, 1.2 eq), amine 1b (9.4 g, 55.75 mmol, 1 eq), EDCI (16.03 g, 83.7 mmol, 1.5 eq) and DMAP (8.2 g, 66.9 mmol, 1.2 eq) in DMF (120 ml) was stirred for 18h. Work up (Heterocyclic Coupling Method B) gave a yellow solid.

Trituration with diethyl ether gave the product as an off white solid (17.12 g, 82%). ¹H

NMR (CDCI₃) δ 8.75 (S₁ 1H), 8.47 (s, 1H), 7.3 (s, 1H), 6.80 (s, 1H)₁ 6.47 (s, 1H), 5.76 -

5.66 (m, 1 H), 5.12 - 4.97 (m, 2H), 4.38 (m, 2H), 4.15 (q, J = 4.78 Hz, 2H), 3.76 (s, 3H), 3.67 (s, 3H), 1.16 (t, J = 7.12 Hz, 3H); MS (ES⁺) m/z 376.03 ([M + H]⁺, 100).

Ψtft-Allyloxycarbonylamino-i-methyl-IH-imidazole-Σ-carbonyQ-aminoJ-i-methyl-IH- imidazole-2-carboxylic acid ethyl ester (Qb)

A solution of the acid 3b (13.1 g, 58 mmol, 1.2 eq), amine 1b (8.2 g, 48.5 mmol, 1 eq), EDCI (13.95 g, 73 mmol, 1.5 eq) and DMAP (7.1 g, 58 mmol, 1.2 eq) in DMF (100 ml) was stirred for 18 hours. Work up (Heterocyclic Coupling Method B) gave the product as a yellow solid. (18.24 g, 78%). ¹H NMR (DMSO-c/₆) δ 9.998 (bs, 1 H), 9.7 (s, 1H), 7.67 (s, 1H), 7.28 (s, 1H), 6.02 - 5.92 (m, 1H), 5.34 - 5.21 (m, 2H), 4.61 (m, 2H), 4.29 (q, J = 7.12 Hz, 2H), 3.96 (s, 3H), 3.95 (s, 3H), 1.33 (t, J = 7.12 Hz, 3H); MS (ES⁺) m/z 376.96 ([M + H]⁺, 100).

(H) ^[ft-Allyloxycarbonylamino-i-methyl-IH-pyrrole-Σ-carbonyiyaminoJ-i-methyl-IH- imidazole-2-carboxylic acid (7a)

The ester 6a (15.79 g, 42.1 mmol, 1 eq) in 1,4 dioxane (200 ml) was hydrolysed (Hydrolysis Method A) to give the product as a tan solid (12.7 g, 87%). ¹H NMR (DMSO- Cf₆) δ 10.64 (s, 1H), 9.46 (s, 1H), 7.62 (s, 1H), 7.02 (s, 1H), 6.97 (d, J = 1.72 Hz, 1H), 6.02 - 5.93 (m, 1H), 5.32 - 5.21 (m, 2H), 4.58 (m, 2H), 4.5 - 3.5 (bs, 1H), 3.93 (s, 3H), 3.84 (S, 3H); MS (ES⁺) m/z 347.96 ([M + H]⁺, 100).

4-[(4-Allyloxycarbonylamino-1-methyl- 1H-imidazole-2-carbonyl)-amino]-1-methyl- 1 H- imidazole-2-carboxylic acid (7b)

The ester 6b (14.26 g, 38 mmol, 1 eq) in 1 ,4 dioxane (150 ml) was hydrolysed

(Hydrolysis Method A) to give the product as a brown solid (12.44 g, 94%). ¹H NMR (DMSO-cy₆) δ 10.04 (bs, 1 H), 9.63 (s, 1 H), 7.63 (s, 1 H), 7.29 (s, 1 H), 6.0 - 5.93 (m, 1 H), 5.37 - 5.21 (m, 2H), 4.6 (m, 2H), 3.96 (s, 3H), 3.93 (s, 3H); MS (ES^f) m/z 348.97 ([M + H]⁺, 100).

8a X = CH, R = Me 9a X = CH 8b X = N, R = Et 9b X = N

E

1Oa X = CH Ha X = CH 1Ob X = N 11b X = N

SCHEME 3

Scheme 3

(i) 4-(3-Allyloxycarbonylamino-propionylamino)-1-methyl- 1 H-pyrrole-2-carboxylic acid methyl ester (8a)

A solution of Alloc-β-Alanine (21.8 g, 0.126 mol, 1,2 eq), amine 1a HCI salt (20.0 g,

0.105 mol, 1 eq), EDCI (40.23 g, 0.21 mol, 2 eq) and DMAP (32.04 g, 0.262 mol, 2.5 eq) in DCM (600 ml) was stirred for 18 hours. Work up (Heterocyclic Coupling Method A) followed by trituration with diethyl ether gave the product as an off white solid (28.26 g,

87%). ¹H NMR (CDCI₃) 5 7.75 (s, 1H), 7.333 (d, J = 1.92 Hz, 1H), 6.681 (O₁ J = 1.6 Hz,

1H), 5.94 - 5.84 (m, 1 H), 5.47 (m, 1H), 5.31 - 5.18 (m, 2H), 4.55 (m, 2H), 3.88 (s, 3H),

3.8 (s, 3H), 3.54 (dt, J = 6.12 Hz, 5.6 Hz, 2H), 2.57 (t, J = 5.9 Hz, 2H); MS (ES⁺) m/z

310.04 ([M + H]⁺, 100). 4-(3-Allyloxycarbonylamino-propionylamino)-1-methyl-1H-imidazole-2-carboxylic acid ethyl ester (8b)

A solution of Alloc-β-Alanine (5.00 g, 28.9 mmol, 1.2 eq), amine 1b (4.07 g, 24.1 mmol, 1 eq), EDCI (9.23 g, 48.2 mmol, 2 eq) and DMAP (7.36 g, 60.2 mmol, 2.5 eq) in DCM (100 ml) was stirred for 18 hours. Work up (Heterocyclic Coupling Method A) gave the product as a tan solid (4.76 g, 61%). ¹H NMR (DMSO-Cf₆) δ 10.71 (s, 1H), 7.56 (s, 1H), 7.28 (t, J = 5.6 Hz, 1H)₁ 5.97 - 5.87 (m, 1H), 5.35 - 5.17 (m, 2H), 4.48 (m, 2H), 4.29 (q, J = 7.1 Hz₁ 2H)₁ 3.94 (dt, J = 6.12 Hz, 6.9 Hz, 2H), 2.49 (t, J = 7.1 Hz₁ 2H), 1.32 (t, J = 7.1 Hz, 3H); MS (ES⁺) m/z 100 (, M+H).

(H) 4-(3~Allyloxycarbonylamino-propionylamino)-1 -methyl- 1 H-pyrrole-2-carboxylic acid (9a)

The ester 8a (28.26 g, 91.4 mmol, 1 eq) in 1,4-dioxane (350 ml) was hydrolysed (Hydrolysis Method A) to give the product as a white solid (26.98 g, 97%). ¹H NMR

(DMSO-Cf₆) δ 12.13 (bs, 1H), 9.85 (s, 1H), 7.3 (d, J = 1.2 Hz, 1H), 7.245 (m, 1 H), 6.675 (d, J = 1.6 Hz₁ 1H)₁ 5.96 - 5.86 (m, 1H)₁ 5.3 - 5.16 (m, 2H)₁ 4.48 (m, 2H), 3.81 (s, 3H), 3.26 (m, 2H), 2.42 (t, J = 6.8 Hz, 2H); MS (ES⁺) mlz 295.94 ([M + H]⁺, 100).

4-(3-Allyloxycarbonylamino-propionylamino)-1-methyl-1H-imidazole-2-carboxylic acid (9b)

The ester 8b ( 5.05 g, 15.6 mmol, 1 eq), in 1 ,4-dioxane (50 ml) was hydrolysed (Hydrolysis Method A) to give the product as an off white solid (3.28 g, 71%). ¹H NMR (DMSO-d₆) δ 10.59 (s, 1 H), 7.49 (s, 1H), 7.26 (t, J = 5.2 Hz, 1H), 5.95 - 5.86, (m, 1H), 5.3 - 5.16 (m, 2H), 4.465 (m, 2H), 3.91 (s, 3H), 3.26 (dt, J = 6.4 Hz, 6 Hz, 2H), 2.47 (t, J = 6.8 Hz, 2H); MS (ES⁺) mlz 100 (, M+H).

(Hi) 4-{[4-(3-Allyloxycarbonylamino-propionylamino)-1-methyl-1H-pyrrole-2-carbonyl]- amino}-1-methyl-1 H-pyrrole-2-carboxylic acid methyl ester (10a) A solution of the acid 9a (18.61 g, 63.01 mmol,^' 1.2 eq) amine 1a HCI salt (10.01 g, 52.51 mmol, 1 eq), EDCI (20.13 g, 105.0 mmol, 2 eq) and DMAP (16.04 g, 131.3 mmol, 2.5 eq) in DMF (200 ml) was stirred for 72 hours. Work up (Heterocyclic Coupling Method B) gave the product as a white solid (18.22 g, 80%). ¹H NMR (DMSO-Cf₆) δ 9.94 (s, 1H)₁ 9.92 (s, 1 H), 7.48 (s, 1H)₁ 7.27 (t, J = 5 Hz, 1H), 7.19 (s, 1H)₁ 6.93 (s, 1H), 6.90 (s, 1H)₁ 6.0 - 5.88 (m, 1H)₁ 5.32 - 5.18 (m, 2H)₁ 4.50 (m, 2H)₁ 3.86 (s, 3H), 3.85 (s, 3H), 3.77 (s, 3H)₁ 3.30 (dt, J = 6.4 Hz, 4 Hz, 2H), 2.48 (t, J = 7.1 Hz, 2H); MS (ES⁺) mlz 431.97 ([M + H]⁺, 100).

4-{[4-(3-Allyloxycarbonylamino-propionylamino)-1-methyl-1H-imidazole-2-carbonylJ- amino}- i-methyl-IH-pyrrole-2-carboxylic acid methyl ester (1 Ob)

A solution of the acid 9b (3.08 g, 10.4 mmol, 1 eq), amine 1a HCl salt (2.4 g, 12.5 mmol, 1.2 eq), EDCI (3.98 g, 20.8 mmol, 2.0 eq) and DMAP (3.17 g, 26.0 mmol, 2.5 eq) in DCM (150 ml) was stirred for 18 hours. Work up (Heterocyclic Coupling Method A) gave the product as a yellow foam (3.67 g, 81%). ¹H NMR (DMSO-Cf₆) δ 10.11 (s, 1H), 9.95 (s, 1H), 7.35 (d, J = 1.92 Hz, 1H), 7.28 (s, 1 H), 7.09 (t, J = 5.6 Hz, 1H)₁ 6.82 (d, J = 1.29 Hz, 1H), 5.77 - 5.67 (m, 1H), 5.12 - 4.98 (m, 2H), 4.29 (m, 2H), 3.77 (s, 3H), 3.67 (s, 3H), 3.57 (S, 3H), 3.10 (dt, J = 6.1 Hz, 6.6 Hz₁ 2H)₁ 2.33 (m, 2H); MS (ES⁺) mlz 433.16 ([M + H]⁺, 100).

(iv) 4-{[4-(3-Allyloxycarbonylamino-propionylamino)-1-methyl-1H-pyrrole-2-carbonyl]- amino}~1-methyl-1H-pyrrole-2-carboxylic acid (11a)

The ester 10a (12.62 g, 29.26 mmol, 1 eq) in 1,4-dioxane (300 ml) was hydrolysed (Hydrolysis Method A) to give the product as an off white solid (10.96 g, 90%). ¹H NMR (DMSO-de) δ 12.18 (bs, 1H), 9.89 (s, 1H), 7.42 (d, J = 1.84 Hz, 1H), 7.28 (t, J = 5.6 Hz, 1 H), 7.17 (d, J = 1.64 Hz, 1 H), 6.86 (d, J = 1.72 Hz₁ 1 H), 6.83 (d, J = 1.92 Hz, 1 H), 5.95 - 5.85 (m, 1 H), 5.29 - 5.15 (m, 2H), 4.46 (m, 2H), 3.84 (2s, 6H), 3.26 (dt, J = 6.4 Hz, 6.8 Hz, 2H), 2.43 (t, J =7.1 Hz, 2H); MS (ES⁺) mlz 417.90 ([M + H]⁺, 100).

4-{[4-(3~Allyloxycarbonylamino-propionylamino)-1-methyl-1H-imidazole-2~carbonyl]- amino}-1 -methyl- 1 H-pyrrole-2-carboxylic acid (11b)

The ester 10b (3.67 g, 8.5 mmol, 1 eq) in 1 ,4-dioxane (50 ml) was hydrolysed (Hydrolysis Method A) to give the product as an off white solid (2.9 g, 82%). ¹H NMR (DMSO-cΕ) δ 12.1 (bs, 1 H), 10.25 (s, 1H), 10.00 (s, 1H), 7.45 (m, 2H), 7.22 (m, 1H), 6-94 (s, 1H), 5.93 - 5.86 (m, 1H), 5.29 - 5.15 (m, 2H), 4.47 (m, 2H), 3.95 (s, 3H)₁ 3.84 (s, 3H), 3.28 (m, 2H), 2.52 (m, 2H); MS (ES⁺) mlz 419.24 ([M + H]⁺, 100).

12a X = CH 13a X = CH 12b X = N 13b X = N

SCHEME 4

Scheme 4

(i) 4-{[4-(3-Allyloxycarbonylamino-propionylamino)- 1 -methyl- 1 H-pyrrole-2-carbonyl]- amino}-1-methyl-1H~imidazole-2-carboxylic acid ethyl ester (12a) A solution of the acid 9a (18.26 g, 61.84 mmol, 1.2 eq), amine 1b (8.72 g, 51.53 mmol, 1 eq), EDCI (14.82 g, 77.3 mmol, 1.5 eq), DMAP (7.55 g, 61.84 mmol, 1.2 eq) in DMF (150 ml) was stirred for 18 hours. Work up (Heterocyclic Coupling Method A) gave the product as a brown foam (15.8 g, 69%). ¹H NMR (CDCI₃) δ 8.31 (s, 1H), 8.11 (s, 1H), 7.54 (s, 1H), 7.30 (S₁ 2H), 6.44 (s, 1H), 5.90 - 5.45 (m, 1H), 5.50 (m, 1H), 5.30 - 5.15 (m, 2H), 4.55 (m, 2H), 4.38 (q, J = 7.07 Hz, 2H), 4.00 (s, 3H), 3.92 (s, 3H), 3.54 (dt, J = 5.8 Hz, 6.07 Hz, 2H), 2.58 (t, J =5.8 Hz); MS (ES⁺) m/z 447.03 ([M + H]⁺, 100).

4-{[4-(3-Allyloxycarbonylamino-propionylamino)-1-methyl-1H-imidazole-2-carbonyl]- amino}-1-methyl-1H-imidazole-2-carboxylic acid ethyl ester (12b) A solution of the acid 9b (3.38 g, 11.41 mmol, 1.3 eq), amine 1b (1.61 g, 9.5 mmol, 1 eq), EDCI (3.64 g, 19.0 mmol, 2.0 eq) and DMAP (2.9 g, 23.8 mmol, 2.5 eq) in DCM (250 ml) was stirred for 18 hours. Work up (Heterocyclic Coupling Method A) and purification by flash column chromatography [DCM/EtOH/NH₃, 150/8/1] gave the product as a yellow foam (3.5 g, 69%). ¹H NMR (DMSO-cfe) δ 10.43 (s, 1H), 9.83 (s, 1 H), 7.7 (s, 1H), 7.51 (s, 1H), 7.27 (t, J = 5.6 Hz, 1H), 6.0 - 5.9 (m, 1H), 5.30 - 5.16 (m, 2H), 4.47 (m, 2H), 4.29 (q, J = 7.1 Hz, 2H), 3.96 (s, 3H), 3.95 (s, 3H), 3.20 (dt, J = 6.12 Hz, 6.84 Hz, 2H), 2.53 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H); MS (ES⁺) m/z 448.14 ([M + H]⁺, 100). (H) 4-{[4-(3-Allyloxycarbonylaminoφropionylamino)-1-methyl-1Hφyrrole-2-carbonyl]- amino}-1-methyl-1H~imidazole-2~carboxylic acid (13a)

The ester 12a (15.8 g, 35.4 mmol, 1 eq) in 1,4-dioxane (150 ml) was hydrolysed (Hydrolysis Method A) to give the product as a yellow solid (12.7 g, 86%). ¹H NMR (DMSO-tfe) δ 10.68 (s, 1H), 9.95 (s, 1H), 7.45 (s, 1H), 7.3 (m, 2H), 6.92 (s, 1H), 5.98 - 5.83 (m, 1H), 5.52 - 5.13 (m, 2H), 4.53 (m, 2H), 3.97 (s, 3H), 3.86 (s, 3H), 3.29 (dt, J = 6.4 Hz, 6.5 Hz₁ 2H), 2.47 (t, J = 7.1 Hz, 2H); MS (ES⁺) mlz 419.00 ([M + H]⁺, 100).

4-{[4-(3~Allyloxycarbonylamino-propionylamino)-1-methyl-1H-imidazole-2-carbonyl]- amino}-1~methyl-1H~imidazoie-2-carboxylic acid (13b)

The ester 12b (3.34 g, 7.5 mmol, 1 eq) in 1,4-dioxane (60 ml) was hydrolysed (Hydrolysis Method A) to give the product as a white solid (2.4 g, 76%). ¹H NMR (DMSO-de) δ 10.43 (s, 1H), 9.64 (s, 1H), 7.64 (s, 1H), 7.51 (s, 1H), 7.23 (m, 1H), 5.94 - 5.87 (m, 1H), 5.31 - 5.16 (m, 2H)₁ 4.48 (m, 2H), 3.98 (s, 3H), 3.95 (s, 3H), 3.29 (m, 2H), 2,52 (m, 2H); MS (ES⁺) mlz 420.24 ([M + H]⁺, 100).

Synthesis of Boc Building Blocks

14a X = CH, R = Me 15a X = CH 14b X = N, R = Et 15b X = N

16a X = CH 17a X = CH 16b X = N 17b X = N

SCHEME 5 Scheme 5

(i) 4-(3-tert-Butoxycarbonylamino-propionylamino)- 1 -methyl- 1 H-pyrrole-2-carboxylic acid methyl ester (14a)

Triethylamine (15.99 g, 22 ml, 158.4 mmol, 1.1 eq) was added to a solution of amine 1a HCI salt (27.44 g, 144 mmol, 1 eq) and Boc Ala succinimide (45.4 g, 158.4 mmol, 1.1 eq) in DCM (400 ml). The solution was stirred at room temperature for 1.5 hours. The reaction mixture was washed with H₂O (2 x 250 ml), 1 M citric acid (2 x 200 ml), saturated NaHCO₃ (2 x 200 ml), H₂O (250 ml) and brine (250 ml). Dried (MgSO₄) and evaporated to give the product as an off white solid (46.8 g, 100%). ¹H NMR (DMSO-Cf₆) δ 9.87 (s, 1H), 7.345 (d, J = 1.6 Hz, 1H), 6.81 (m, 1H), 6.725 (d, J = 2.0 Hz, 1H), 3.83 (s, 3H), 3.74 (S₁ 3H), 3.2 (dt, J = 6.8 Hz, 6 Hz, 2H), 2.4 (t, J = 7.2 Hz, 2H)₁ 1.39 (s, 9H); MS (ES-) m/z 326.37 ([M + H]^", 40).

Compound 14b was synthesised according to the following literature procedure: Seio, K. et al., Journal of Organic Chemistry, 70(26), 10311-10322 (2005)

(H) 4-(3-tert-Butoxycarbonylamino-propionylamino)-1-methyl-1 H-pyrrole-2-carboxylic acid

(15a)

4-(3-fert-Butoxycarbonylamino-propionylamino)-1 -methyl-1 H-pyrrole-2-carboxylic acid 14a (46.8 g, 144 mmol, 1 eq) in MeOH (500 ml) was hydrolysed (Hydrolysis Method B) to give the product as a white solid (39.96 g, 89%).¹H NMR (DMSO-c/₆) δ 12.14 (bs, 1H), 9.83 (s, 1H), 7.3 (d, J = 1.6 Hz, 1H), 6.81 (t, J= 5.06 Hz, 1 H), 6.67 (d, J = 1.96 Hz, 1H), 3.81 (S, 3H), 3.2 (dt, J = 7.2 Hz, 6 Hz, 2H), 2.39 (t, J = 7.2 Hz, 2H), 1.39 (s, 9H); MS (ES^" ) m/z 310.35 ([M - H]-, 100).

(Hi) 4-{[4-(3-tert-Butoxycarbonylamino-propionylamino)-1~methyl-1H-pyrrole-2-carbonyl]- amino}-1 -methyl-1 H-pyrrole-2-carboxylic acid methyl ester (16a) A solution of the acid 15a (8.0 g, 25.7 mmol, 1.2 eq), amine 1a HCI salt (4.08 g, 21.4 mmol, 1 eq), EDCI (8.2 g, 42.8 mmol, 2.0 eq) and DMAP (6.5 g, 53.5 mmol, 2.5 eq) in DCM (150 ml) was stirred for 72 hours. Work up (Heterocyclic Coupling Method A) gave the product as a yellow foam (8.98 g, 93%).¹H NMR (DMSO-Cf₆) δ 9.89 (s, 1 H), 9.86 (s, 1H), 7.46 (d, J = 1.92 Hz, 1H), 7.167 (d, J = 1.8 Hz, 1H), 6.913 (d, J =1.96 Hz, 1H), 6.89 (d, J = 1.88 Hz, 1H), 6.81 (t, J = 5.14 Hz, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.75 (s, 3H), 3.21 (dt, J = 7.2 Hz, 6 Hz, 2H)₁ 2.42 (t, J = 7.3 Hz, 2H), 1.34 (s, 9H); MS (ES⁺) mlz 470.37 ([M + Na]⁺, 100). 4-{[4-(3-tert~Butoxycarbonylaminoφropionylamino)-1-methyl-1H-imidazole-2-carbonyI]~ aminoJ-i-methyl-IH-pyrrole^-carboxylic acid methyl ester (16b) A solution of the acid 15b (4.93 g, 15.6 mrnol, 1 eq), amine 1a HCI salt (3.61 g, 18.94 mmol, 1.2 eq), EDCI (6.05 g, 31.6 mmol, 2.0 eq) and DMAP (4.82 g, 39.5 mmol, 2.5 eq) in DMF (150 ml) was stirred for 18 hours. Work up (Heterocyclic Coupling Method B) gave the product as an off white solid (5.92 g, 84%).¹H NMR (DMSO-Cf₆) δ 10.23 (s, 1H), 10.09 (s, 1H)₁ 7.53 (d, J = 2 Hz, 1H), 7.46 (s, 1H), 7.01 (d, J = 2 Hz, 1H), 6.795 (m, 1H), 3.96 (S, 3H), 3.86 (s, 3H), 3.76 (s, 3H), 3.21 (dt, J = 7.2 Hz, 6 Hz, 2H), 2.49 (t, J = 7.2 Hz, 2H), 1.39 (s, 9H); MS (ES⁺) mlz 449.38 ([M + H]⁺, 50).

(iv) 4-{[4-(3-tert-Butoxycarbonylamino-propionylamino)-1-methyl-1H-pyrrole-2-carbonyl]~ amino}-1-methyl-1H-pyrrole-2-carboxylic (17a)

The ester 16a (8.75 g, 19.55 mmol, 1 eq) in MeOH (200 ml) was hydrolysed (Hydrolysis Method B) to give the product as a pale yellow solid (7.73 g, 91 %).¹ H NMR (DMSO-cf_s) δ 12.05 (bs, 1H), 9.86 (S, 2H), 7.42 (d, J = 1.8 Hz, 1H), 7.17 (d, J = 1.5 Hz, 1H), 6.88 (d, J = 1.7 Hz, 1H), 6.85 (d, J = 1.8 Hz, 1H), 6.81 (m, 1H), 3.84 (s, 6H), 3.21 (dt, J = 6.8 Hz, 6.4 Hz, 2H), 2.42 (t, J = 7.3 Hz, 2H), 1.4 (s, 9H); MS (ES^") mlz 432.40 ([M ~- H]^', 50).

4-{[4-(3-tert-Butoxycarbonylamino-propionylamino)-1-methyl-1H-imidazole-2-carbonyl]- amino}- 1-methyl-1H~pyrrole~2-carboxylic acid (17b)

The ester 16b (13.95 g, 31.1 mmol, 1 eq) in 1 ,4-dioxane (300 ml) was hydrolysed (Hydrolysis Method A) to give the product as a white solid (12.84 g, 95 %). MS (ES⁺) m/z 457.27 ([M + Na]⁺, 50).

18 19

SCHEME 6 Scheme 6

(i) 4-{[4-(3~tert-Butoxycarbonylamino-propionylamino)- 1 -methyl- 1 H-pyrrole-2-carbonyl]- amino}-1-methyl-1H-imidazole-2-carboxylic acid ethyl ester (18) A solution of the acid 15a (8.0 g, 25.7 mmol, 1.2 eq), amine 1b (3.62 g, 21.4 mmol, 1 eq), EDCI (8.2 g, 42.8 mmol, 2.0 eq) and DMAP (6.5 g, 53.5 mmol, 2.5 eq) in DCM (150 ml) was stirred for 72 hours. Work up (Heterocyclic Coupling Method A) gave the product as a yellow foam (9.04 g, 91%). ¹H NMR (DMSO-Cf₆) δ 10.7 (s, 1H), 9.87 (s, 1H), 7.67 (s, 1H)₁ 7.308 (d, J = 1.7 Hz, 1H), 6.98 (d, J= 1.8 Hz, 1H), 6.80 (t, J = 5.0 Hz, 1H), 4.3 (q, J = 7.1 Hz, 2H), 3.95 (s, 3H), 3.85 (S₁ 3H), 3.20 (dt, J = 6.8 Hz, 6.4 Hz, 2H), 2.42 (t, J = 7.3 Hz, 2H), 1.4 (s, 9H), 1.32 (t, J = 7.1 Hz, 3H); MS (ES⁺) m/z 463.29 ([M + H]⁺, 100).

(H) ^{ft-fi-tert-Butoxycarbonylamino-propionylaminoj-i-methyl-IH-pyrrole-Σ-carbonyl]- amino}-1-methyl-1H-imidazole-2-carboxylic acid (19) The ester 18 (11.65 g, 25.2 mmol, 1 eq) in 1,4-dioxane (100 ml) was hydrolysed

(Hydrolysis Method A) to give the product as a tan solid (8.89 g, 81%).¹H NMR (DMSO- Cf₆) δ 10.56 (bs, 1H), 9.87 (bs, 1H)₁ 7.54 (bs, 1H), 7.28 (s, 1H), 6.96 (s, 1H), 6.81 (m, 1H), 4.5 - 3.8 (bs 1H), 3.95 (s, 3H), 3.85 (s, 3H), 3.21 (dt, J = 6.9 Hz, 6.3 Hz, 2H), 2.42 (t, J = 7.2 Hz, 2H), 1.4 (s, 9H); MS (ES^") mlz 433.4 ([M - H]^', 100).

R¹ = Me CO. ,Me

SCHEME 7

Scheme 7

(i) 2-{[4-(3-tert~Butoxycarbonylamino-propionylamino)- 1 -methyl- 1 H-pyrrole-2-carbonyl]- amino}-5-methyl-thiazole-4-carboxylic acid methyl ester (21a) A solution of the acid 15a (11.5 g, 36.94 mmol, 1.2 eq), 2-amino-5-methyl-thiazole-4- carboxylic acid methyl ester 20a (5.3 g, 30.78 mmol, 1.0 eq), EDCI (11.8 g, 61.56 mmol, 2.0 eq) and DMAP (9.79 g, 76.95 mmol, 2.5 eq) in DMF (300 ml) was stirred for 72 hours. Work up (Heterocyclic Coupling Method B) gave a yellow oil. Trituration with Et₂O gave the product as a white solid (7.96 g, 55%). MS (ES^") m/z 464.45 ([M - Hf₁ 100).

2-{[4-(3-tert-Butoxycarbonylamino-propionylamino)-1-methyl-1H-pyrrole-2-carbonyl]- amino}-4-methy/-thiazole-5-carboxylic acid methyl ester (21b) A solution of the acid 15a (12.56 g, 40.34 mmol, 1.2 eq), 2-amino-4-methyl-thiazole-5- carboxylic acid methyl ester 20b (5.79 g, 33.62 mmol, 1.0 eq), EDCI (12.9 g, 67.24 mmol, 2.0 eq) and DMAP (10.7 g, 84.05 mmol, 2.5 eq) in DMF (300 ml) was stirred for 96 hours. Work up (Heterocyclic Coupling Method B) gave an off white solid. Trituration with Et₂O gave the product as a white solid (9.6 g, 61%).¹H NMR (DMSO-d₆) δ 12.55 (s, 1H), 9.97 (s, 1H), 7.454 (d, J = 1.56 Hz, 1H), 7.247 (d, J = 1.4 Hz, 1H), 6.82 (t, J = 5.2 Hz, 1H), 3.89 (s, 3H), 3.8 (s, 3H), 3.21 (dt, J = 7.2 Hz, 6 Hz, 2H), 2.59 (s, 3H), 2.43 (t, J = 7.2 Hz, 2H), 1.4 (s, 9H); MS (ES^") m/z 464.43 ([M - H]^", 100).

(H) 2-{[4-(3-tert-Butoxycarbonylamino-propionylamino)-1-methyl-1H-pyrrole-2-carbonyl]- aminoJS-methyl-thiazole^-carboxylic acid (22a)

The ester 21a (3.9 g, 8.4 mmol, 1 eq) in MeOH (100 ml) was hydrolysed (Hydrolysis Method B) at room temperature to give the product as an off white solid (2.7 g, 71%).¹H NMR (DMSO-d₆) δ 12.7 (bs, 1H), 12.5 (bs, 1H), 10.03 (s, 1H)₁ 7.51 (s, 1H), 7.3 (s, 1H), 6.9 (S, 1H), 3.98 (s, 3H), 3.3 (m, 2H), 2.72 (s, 3H), 2.61 (m, 2H), 1.49 (s, 9H); MS (ES^") m/z 450.61 ([M - H]^", 100).

2-{[4-(3-tert-Butoxycarbonylamino-propionyIamino)-1-methyl-1H-pyrrole-2-carbonyl]- amino}~4-methyl-thiazole-5-carboxylic acid (22b)

The ester 21b (9.6 g, 20.62 mmol, 1 eq) in MeOH (250 ml) was hydrolysed (Hydrolysis Method B) at room temperature to give a yellow solid. Trituration with Et₂O gave the product as a white solid (9.3 g, 83%).¹H NMR (DMSO-d₆) δ 12.46 (bs, 2H), 9.96 (s, 1 H),

7.44 (d, J= 1.24 Hz, 1H)₁ 7.22 (d, J = 1.4 Hz, 1H), 6.82 (m, 1H), 3.89 (s, 3H), 3.05 (dt, J

= 7.2 Hz, 6 Hz, 2H), 2.57 (s, 3H), 2.43 (t, J = 7.2 Hz, 2H) 1.4 (s, 9H); MS (ES^") m/z

450.54 ([M - H]^", 100).

SCHEME 8

Scheme 8

(i) δ-tfi-Methyl-^nitro-IH-pyrrOle-Σ-carbonyiyaminoJ-furan-Σ-carboxylic acid methyl ester (25)

Oxalyl Chloride (6.88 g, 4.73 mmol, 54.2 mmol, 1.2 eq) was added to a suspension of the nitro acid 23 (7.68 g, 45.2 mmol, 1 eq) in anhydrous DCM (150 ml). DMF (3 drops) was added and the suspension was allowed to stir at room temperature under a nitrogen atmosphere for 18 hours. The solvent was evaporated under reduced pressure and the residue was redissolved in anhydrous THF (150 ml). The solution was added dropwise to a solution of 5-amino-furan-2-carboxylic acid methyl ester 24 (7.65 g, 54.2 mmol, 1.2 eq) and triethylamine (11.4 g, 15.7 ml, 113 mmol, 2.5 eq) in anhydrous THF (150 ml) at 0°C.The resultant solution was allowed to stir at room temperature for 18hours. The reaction mixture was filtered, the filtrate was evaporated under reduced pressure and the residue triturated with DCM to give the solid product (2.81 g, 21 %). This was used without further purification. ¹H NMR (DMSO-c/₆) δ 11.84 (s, 1H), 8.26 (s, 1 H), 7.88 (s, 1H), 7.365 (d, J = 3.2 Hz, 1H), 6.53 (d, J = 2.8 Hz, 1H), 3.975 (s, 3H), 3.82 (s, 3H); MS (ES^") mlz 292.367 ([M - H]^", 100).

(//) δ-W-β-tert-Butoxycarbonylamino-propionylaminoyi-methyl-IH-pyrrole-Σ-carbonyl]- amino}-furan-2-carboxylic acid (26) A solution of the nitro derivative 25 (4.11 g, 14.02 mmol, 1 eq) in DMF (250 ml) was hydrogenated over 10% Palladium on Carbon (0.8 g, 20% by wt) at 45 psi for 1 hour. The reaction mixture was filtered through celite washing with DMF. Boc-β-Alanine succinimide ester (4.41 g, 15.4 mmol, 1.1 eq) was added and the solution was stirred at room temperature for 1 hour. The reaction mixture was poured onto ice and extracted with DCM (3 x 150 ml), washed with 1M citric acid (2 x 100 ml), sat. NaHCO₃ (2 x 100 ml), H₂O (100 ml), brine (100 ml), dried (MgSO₄) and evaporated to give a brown solid. 1M sodium hydroxide solution (68 ml, 68 mmol, 4 eq) was added to a solution of the brown solid in MeOH (100 ml). This was stirred at room temperature for 72h, the MeOH was evaporated under reduced pressure and the residue acidified to pH3 with 2M citric acid. The resultant suspension was extracted with EtOAc (3 x 100 ml) the combined extracts were dried (MgSO₄) and evaporated to give the product as a brown foam (3.5 g, 59%).¹H NMR (DMSO-CZ₆) δ 12.72 (bs, 1 H), 11.37 (s, 1H)₁ 9.91 (s, 1H), 7.34 (d, J = 1.3 Hz, 1H), 7.246 (d, J= 3.5 Hz, 1H), 7.11 (d, J= 1.5 Hz, 1H), 6.81 (m, 1H), 6.46 (d, J = 3.5 Hz, 1H), 3.85 (s, 3H), 3.21 (dt, J = 6.8 Hz, 6.4 Hz, 2H), 2.43 (t, J = 7.2 Hz, 2H), 1.4 (s, 9H); MS (ES) mlz 419.44 ([M - H]^", 100).

SCHEME 9

Scheme 9

(i) & (ii) Compounds 27 and 28 were synthesised according to the following literature procedure: Marques, MA, et al., Helvetica Chimica Acta, 85(12), 4485-4517 (2002).

(Hi) 4-{[5-(3-tert-Butoxycarbonylaminoφropionylamino)-furan-2-carbonyl]-amino}-1- methyl-1H-pyrrole-2-carboxylic acid methyl ester (29) A solution of 5-(3-tert-butoxycarbonylamino-propionylamino)-furan-2carboxylic acid 28 (2.34 g, 7.84 mmol, 1.25 eq), amine 1a HCI salt (1.20 g, 6.28 mmol, 1.0 eq), EDCI (1.81 g, 9.41 mmol, 1.5 eq) and DMAP (1.92 g, 15.69 mmol, 2.5 eq) in DCM (50 ml) was stirred for 18 hours. Workup (Heterocyclic Coupling Method A) followed by trituration with hexane/ether gave the product as a brown solid (1.53 g, 56 %). MS (ES^") m/z 433.54 ([M - H]-, 100).

(7VJ ^{[δ-β-tert-Butoxycarbonylaminoφmpionylaminoj-furan-Σ-carbonylJ-aminoJ-i- methyl-1 H-pyrrole-2-carboxylic acid (30)

The ester 29 (1.53 g, 3.53 mmol, 1 eq) in MeOH (7 ml) was hydrolysed (Hydrolysis Method B) to give the product as an off-white solid (847 mg, 57 %). ¹H NMR (DMSO-d⁶) δ 12.22 (bs, 1H), 11.32 (bs, 1H), 10.02 (s, 1H), 7.40 (d, 1 H, J = 1.92 Hz), 7.30 (d, 1H, J = 2.04 Hz), 7.29 (d, 1H, J = 3.52 Hz), 6.84 (m, 2H), 6.36 (d, 1H, J = 3.52 Hz), 3.85 (s, 3H), 3.23 (m, 2H), 2.51 (m, 2H), 1.40 (s, 9H). MS (ES^") m/z 419.59 ([M - H]^", 100). NaOH

20

31

32

33

34 SCHEME 10

Scheme 10

(i) 2-(3-tert-Butoxycarbonylaminoφropionylamino)-5-methyl-thiazole-4-carboxylic acid methyl ester (31)

1,3-Diisopropylcarbodiimide (15.14 g, 18.6 ml, 120 mmol, 2 eq) was added to a solution of Boc-β-Alanine (45.4 g, 240 mmol, 4 eq) in DCM (350 ml). The reaction mixture was allowed to stir at room temperature for 30 minutes. 2-Amino-5-methyl-thiazole-4- carboxylic acid methyl ester 20a (10.33 g, 60 mmol, 1 eq) was added portionwise followed by DIPEA (7.75 g, 9.73 ml, 60 mmol, 1 eq), and DMAP (2.2 g, 18 mmol, 0.3 eq) and the reaction mixture was allowed to stir at room temperature for 18 hours. The mixture was filtered and extracted with 1M citric acid (3 x 200 ml), sat. NaHCO₃ (3 x 200 ml), water (200 ml), brine (200 ml), dried (MgSO₄) and evaporated under reduced pressure to give a yellow foam. The product was contaminated with dicyclohexylcarbodiimide and was used without further purification assuming 100% yield.

(H) 2-(3-tert-Butoxycarbonylamino-propionylamino)-5-methyl-thiazole-4-carboxylic acid (32)

The ester 31 (20.6 g, 60 mmol, 1 eq) in 1 ,4 dioxane (250 ml) was hydrolysed (Hydrolysis Method A) to give the product as an off white solid (15.83 g, 88%). ¹H NMR (DMSO-Cf₆) δ 12.3 (bs, 2H), 6.87 (m, 1H), 3.24 (dt, J = 6.8 Hz, 6 Hz, 2H), 2.61 (s, 3H), 2.56 (t, J = 6.8 Hz, 2H), 1.38 (s, 9H); MS (ES^") mlz 328.24 ([M - HV, 30), 284.31 ([M - CO₂]^", 100).

(Hi) 4-{[2-(3-tert-Butoxycarbonylamino-propionylamino)-5-methyl-thiazole-4-carbonyl]- amino}~1-methyl-1H-pyrrole-2-carboxylic acid methyl ester (33) A solution of the acid 32 (10.74 g, 32.61 mmol, 1.0 eq), amine 1a HCI salt (7.46 g, 39.13 mmol, 1.2 eq), EDCI (12.5 g, 65.21 mmol, 2.0 eq) and DMAP (9.96 g, 88.15 mmol, 2.5 eq) in DMF (100 ml) was stirred for 18 hours. Work up (Heterocyclic Coupling Method B) followed by recrystallisation (EtOAc) gave the product as a white solid (7.89 g). Evaporation of the EtOAC and flash column chromatography of the residue (EtOAc) gave a white solid (2.21 g). Yield (10.1 g, 66%). ¹H NMR (DMSO-c/₆) δ 12.11 (s, 1H), 9.78 (s, 1H), 7.56 (s, 1H), 6.962 (6, J = 1.8 Hz, 1H), 6.92 (t, J = 5Hz, 1H), 3.86 (s, 3H), 3.75 (s, 3H), 3.25 (dt, J - 6.4 Hz, 6 Hz, 2H), 2.66 (s, 3H), 2.62 (t, J = 6.8 Hz, 2H), 1.38 (s, 9H); MS (ES⁺) mlz 466.42 ([M + H]⁺, 30), 488.38 ([M + Na]⁺, 80).

(M 4-{[2-(3-tert-Butoxycarbonylamino-propionylamino)-5-methyl-thiazole-4-carbonylJ- amino}-1-methyl-1H-pyrrole-2-carboxylic acid (34) The ester 33 (5.075 g, 10.9 mmol, 1.0 eq) in 1,4-dioxane (100 ml) was hydrolysed (Hydrolysis Method A) to give the product as a white solid (4.34 g, 88%).¹H NMR (DMSO-cfe) δ 12.1 (bs, 2H), 9.67 (s, 1H), 7.482 (d, J = 1.4 Hz, 1H), 6.88 (m, 2H), 3.85 (s, 3H), 3.26 (dt, J = 6.4 Hz, 6 Hz 2H), 2.66 (s, 3H), 2.62 (t, J = 6.8 Hz, 2H), 1.39 (s, 9H); MS (ES-) mlz 450.49 ([M - H]^", 100).

SCHEME 11

Scheme 11

(i) 4-{[2-(3-tθrt-Butoxycarbonylamino~propionylamino)-4-methyl-thiazole-5-carbonyl]- amino}-1-methyl-1H-pyrrole-2-carboxylic acid methyl ester (35) 1.0 M 1 ,3-Diisopropylcarbodiimide in DCM (22.4 ml, 22.4 mmol, 2 eq) and Boc-β-Alanine (8.49 g, 44.8 mmol, 4 eq) in DCM (40 ml) were allowed to stir at room temperature for 15 minutes. Amine 49 (3.3 g, 11.2 mmol, 1 eq) was added portionwise followed by DIPEA (1.45 g, 1.82 ml, 11.2 mmol, 1 eq), and DMAP (0.41 g, 3.3 mmol, 0.3 eq) and the reaction mixture was allowed to stir at room temperature for 4 hours. The mixture was filtered and extracted with 1M citric acid (3 x 50 ml), sat. NaHCO₃ (3 x 50 ml), water (50 ml), brine (50 ml), dried (MgSO₄) and evaporated under reduced pressure to give a yellow foam. The product was contaminated with dicyclohexylcarbodiimide and was used without further purification assuming 100% yield.

(H) 4-{[2-(3-tert-Butoxycarbonylamino-propionylamino)-4-methyl-thiazole-5-carbonyl]- amino}-1-methyl-1H-pyrrole-2-carboxylic acid (36)

The ester 35 (5.22g 11.2 mmol 1 eq.) in 1 ,4-dioxane (70 ml) was hydrolysed (Hydrolysis Method A) to give the product as a white solid (4.9 g, 97% %). ¹H NMR (DMSO-Cf₆) δ 12.29 (bs, 1H), 9.93 (s, 1 H), 7.42 (d, J = 1.6 Hz, 1H), 6.87 (s, 1H), 6.865 (s, 1H), 3.83 (s, 3H), 3.26 (dt, J = 6.4 Hz, 6.4 Hz 2H), 2.608 (t, J = 6.8 Hz, 2H), 2.52 (s, 3H), 1.365 (s, 9H); MS (ES-) mlz 450.5 ([M - H]^", 100).

38a X = CH 39a X = CH 37b X = N 38b X = N 39b X = N

42a X = CH 42b X = N

SCHEME 12

Scheme 12

(i) Compounds 38a and 38b were synthesised according to the following literature procedure: Boger, D.L., et al., Journal of the American Chemical Society, 122(27), 6382-6394 (2000).

(ii) Compound 39a was synthesised according to the following literature procedure: Yeung, B.K.S. & Boger, D.L., Journal of Organic Chemistry, 68(13), 5249-5253 (2003).

Compound 39b was synthesised according to WO2005/085250. (iii) Compound 40 was synthesised according to WO 2007/039752

Compound 41a was synthesised according to the following literature procedure: Wells, G., et a\., Journal of Medicinal Chemistry, 49(18), 5442-5461 (2006).

(11S, 1 laSj-T-Methoxy-β-β-p-fδ-methoxycarbonyl-i-methyl-IH-pyriOl-S-ylcarhamoylj-i- methyl-1H-imidazol-4-ylcarbamoyl]-propoxy}-5-oxo- 11-(tetrhaydro-pyran-2-yloxy)- 2,3, 11,11a-tetrahydro-1H,5H-pyrrolo[2,1~c][1,4]benzodiazepine-10-carboxylic acid allyl ester (41b)

A solution of the acid 40 (4.54 g, 8.76 mmol, 1.0 eq), amine 39b HCI salt (3.30 g, 10.5 mmol, 1.2 eq), EDCI (3.36 g, 17.5 mmol, 2.0 eq) and DMAP (2.68 g, 21.9 mmol, 2.5 eq) in DMF (100 ml) was stirred at room temperature for 18 hours. Work up (Heterocyclic Coupling Method B) and purification by flash column chromatography (EtOAc) gave the product as a yellow foam (4.6 g, 56%). ¹H NMR (DMSO-CZ₆) (Mixture of diastereoisomers) δ 10.29 (s, 1H), 10.05 (s, 1H), 7.531 (d, J = 1.88 Hz, 1H), 7.45 (s, 1H), 7.11 (s, 1H), 7.007 (d, J = 1.9 Hz, 1H), 6.92 (bs, major diastereoisomer, 1H), 6.735 (s, minor diastereoisomer, 1H), 5.85 - 5.65 (m, 1H), 5.73 (d, J = 9.28 Hz, 1H), 5.15 - 4.9 (m, 3H), 4.7 - 4.35 (m, 2H), 4.1 - 4.0 (m, 2H), 3.96 (m, 3H), 3.87 - 3.82 (m, 7H), 3.76 (s, 3H), 3.55 - 3.45 (m, 2H), 3.45 - 3.35 (m, 2H),2.15 - 1.75 (m 8H), 1.7 - 1.3 (m, 7H); MS (ES⁺) mlz 778.51 ([M + H]⁺, 100).

(iv) (11S, 1 laSj-δ-β-lδ-fi-Carboxy-i-methyl-IH-pyrrol-S-ylcarbamoyO-i-methyl-IH- pyrrol-3-ylcarbamoyl]-propoxy}-7-methoxy-5-oxo~11-(tetrahydro-pyran-2-yloxy)- 2,3, 11, 11a-tetrahydro- 1H, 5H-pyrrolo[2, 1-c][1, 4]benzodiazepine- 10-carboxylic acid allyl ester (42a)

The ester 41a (1.73 g, 2.2 mmol, 1 eq.) in 1,4-dioxane (9 ml) was hydrolysed (Hydrolysis Method A) to give the product as a white solid (1.39 g, 81 %). ¹H (DMSO-d₆) δ 12.13 (bs, 1H), 9.86 (s, 1H), 7.41 (d, 1H₁ J = 1.41 Hz), 7.16 (m, 1H), 7.09 (m, 1H), 6.93 - 6.83 (m, 3H), 5.82 - 5.67 (m, 2H), 5.09 - 5.05 (m, 3H), 4.61 - 4.36 (m, 2H), 4.05 - 3.98 (m, 2H),

3.83 (bs, 9H), 3.49 - 3.48 (m, 2H), 3.41 - 3.36 (m, 2H), 3.29 (s, 1H)₁ 2.11 - 1.38 (m, 14H); MS (ES-) m/z 761.37 ([M - H]^', 100). (11S, 1 laSyδ-fS-fδ-fS-Carboxy-i-methyl-IH-pyrrol-S-ylcarbamoyO-i-methyl-IH-irnidazol- 4-ylcarbamoyl]-propoxy}-7-methoxy-5-oxo-11-(tetrahydro-pyran-2-yloxy)-2,3, 11,11a- tetrahydro-1H,5H-pyrfOlo[2, i-ctfi^Jbenzodiazepine-IO-carboxylic acid allyl ester (42b) The ester 41b (4.36 g, 5.6 mmol, 1.0 eq) in 1,4 dioxane (100 ml) was hydrolysed (Hydrolysis Method A) to give the product as a yellow solid (3.52 g, 82%). ¹H (DMSO-d⁶) δ 12.27 (bs, 1H), 10.37 (s, 1H), 10.06 (s, 1H), 7.54 (m, 1H), 7.52 (m, 1H), 6.99, 6.91 (2s, 1 H), 5.84 - 5.75 (m, 2H), 5.17 - 5.06 (m, 3H), 4.70 - 4.45 (m, 2H), 4.12 - 4.05 (m, 2H), 4.03 (m, 3H), 3.92 - 3.89 (m, 7H), 3.63 - 3.51 (m, 2H), 3.50 - 3.45 (m, 2H), 2.20 - 1.45 (m, 14H). MS (ES⁺) m/z 764.69 ([M + H]⁺, 100).

SCHEME 13

46a R - Me 46b R = H

Scheme 13 (i) 4-[(2-tert-Butoxycarbonylamino-5-methyl-thiazole-4-carbonyl)-amino]- 1-methyl-1H- pyrrole-2-carboxylic acid methyl ester (44)

A solution of Boc 5-methylthiazole acid 43 (4.40 g, 17.0 mmol, 1 eq), amine 1a HCI salt (3.25 g, 17.0 mmol, 1 eq), EDCI (6.53 g, 34.0 mmol, 2 eq) and DMAP (5.20 g, 42.5 mmol, 2.5 eq) in DCM (50 ml) was stirred for 18 hours at 26⁰C. Work up (Heterocyclic Coupling Method B) gave a crude residue which was purified by trituration with diethyl ether. The product was collected by filtration as a white solid (4.5 g, 67%). ¹H NMR

(DMSO-CZ₆; 400 MHz) δ 11.42 (bs, 1H), 9.60 (s, 1H), 7.51 (d, J = 1.93 Hz, 1H), 6.95 (d, J = 1.98 Hz, 1 H), 3.84 (s, 3H), 3.74 (s, 3H), 2.63 (s, 3H), 1.49 (s, 9H); MS (ES-) m/z 393.36 ([M - H]-, 100).

(H) 4-[(2-Amino-5-methyl-thiazole-4~carbonyl)-amino]-1-methyl-1H-pyrrole-2-carboxylic acid methyl ester (45)

A solution of Boc dirtier 44 (4.3 g, 10.9 mmol, 1 eq) in TFA/DCM (20/80, v/v) was allowed to stir at room temperature for 3 hours. Completion of the reaction was checked by LC/MS. The solution was added to a mixture of concentrated ammonium hydroxide/ice with vigorous stirring. The product was recovered by filtration, washed with water and dried (3.2 g, 100%). ¹H NMR (DMSO-c/₆; 400 MHz) δ 9.66 (s, 1H), 7.50 (d, J = 1.93 Hz, 1H), 6.97 (d, J = 1.98 Hz, 1H), 6.82 (s, 2H)₁ 3.83 (s, 3H), 3.73 (s, 3H), 2.54 (s, 3H). MS (ES⁺) m/z 295.17 ([M + H]⁺, 100).

(Hi) (11S, 11aS)-7-Methoxy-8-{3-[4-(5-methoxycarbonyl-1-methyl-1H-pyrrol~3- ylcarbamoylj-S-methyl-thiazol-Σ-ylcarbamoylJ-propoxyϊ-δ-oxo- 11 ~(tetrahydro-pyran-2- yloxy)-2,3, 11,11 a-tetrahydro-1 H, 5H-pyιrolo[2, 1-c][1,4]benzodiazepine-10-carhoxylic acid allyl ester (46a)

A solution of amino dimer 45 (1.71 g, 5.8 mmol, 1 eq), PBD acid 40 (3.62 g, 7.0 mmol, 1.2 eq), EDCI (2.23 g, 11.6 mmol, 2 eq) and DMAP (1.77 g, 14.5 mmol, 2.5 eq) in DMF (50 ml) was stirred for 18 hours at 25°C. Work up (Heterocyclic Coupling Method B) gave the crude product which was purified by flash chromatography (EtOAc) and trituration with diethyl ether. The product was collected by filtration to give a white solid (2.15 g, 46%). ¹H NMR (DMSO-d₆; 400 MHz) (Mixture of diastereoisomers in a 65/35 ratio) 5 12.15 (s, 1H), 9.75 (s, 1H), 7.54 (d, J = 1.91 Hz₁ 1H), 7.08 (m, 1H), 6.95 (d, J = 1.97 Hz, 1H), 6.91 (s, 0.65 H), 6.81 (s, 0.35 H), 6.14-5.54 (m, 2H), 5.49-4.78 (m, 3H),

4.75-4.20 (m, 2H), 4.15-3.89 (m, 2H), 3.89-3.60 (m, 10H), 3.60-3.43 (m, 2H), 2.73-2.59 (m, 5H), 2.19-1.74 (m, 3H), 1.72-1.56 (m, 2H), 1.55-1.23 (m, 4H); MS (ES^") m/z 793.87 ([M - H]-, 50).

(11S, 11aS)-8-{3-[4-(5-Carboxy-1-methyt-1H-pyrrol-3-ylcarbamoyl)-5-methyl-thiazol-2- ylcarbamoyl]-propoxy}-7-methoxy-5-oxo-11-(tetrahydro-pyran-2-yloxy)-2,3, 11, 11a- tetrahydro-1H,5H-pyrrolo[2, 1-c][1,4]benzodiazepm^' e-10-carboxyHc acid allyl ester (46b) Methyl ester 46a (1.9Og, 2.4 mmol) was hydrolysed (Hydrolysis Method A) to give the title acid (1.6 g, 86 %). ¹H NMR (DMSO-Cf₆; 400 MHz) (Mixture of diastereoisomers in a 65/35 ratio) δ 12.13 (s, 1H), 9.64 (s, 1H), 7.48 (d, J = 1.91 Hz, 1H), 7.09 (m,1H), 6.90 (s, 0.65 H), 6.88 (d, J = 1.97 Hz, 1 H), 6.82 (s, 0.35 H), 5.90-5.60 (m, 2H)₁ 5.18-4.88 (m, 3H), 4.75-4.24 (m, 2H)₁ 4.14-3.91 (m, 2H), 3.88-3.70 (m, 7H), 3.57-3.42 (m, 2H), 2.75-2.57 (m, 5H), 2.22-2.01 (m, 3H), 2.00-1.73 (m, 3H), 1.72-1.56 (m, 2H), 1.55-1.23 (m, 4H); MS (ES^") m/z 779.87 ([M - H]^", 100).

SCHEME 14

50a R = Me 50b R = H

Scheme 14

(i) 4-[(2-tert-Butoxycarbonylamino-4-methyl-thiazole-5-carbonyl)-amino]- 1 -methyl- 1H- pyrrole-2-carboxylic acid methyl ester (48) A solution of Boc 4-methylthiazole acid 47 (6.36 g, 24.5 mmol, 1 eq), amine 1a HCI salt (4.70 g, 24.6 mmol, 1 eq), EDCI (9.44 g, 49.2 mmol, 2 eq) and DMAP (7.51 g, mmol, 2.5 eq) in DCM (50 ml) was stirred for 18 hours at 26⁰C. Work up (Heterocyclic Coupling Method B) gave a crude residue which was purified by trituration with diethyl ether. The product was collected by filtration to give a white solid (4.5 g, 67%). ¹H NMR (DMSO-Cf₆; 400 MHz) δ 11.73 (bs, 1 H), 9.92 (s, 1 H), 7.46 (d, J = 1.92 Hz, 1 H), 6.90 (d, J = 1.96 Hz, 1H), 3.84 (s, 3H), 3.74 (s, 3H), 2.48 (s, 3H), 1.50 (s, 9H); MS (ES^") m/z 393.36 ([M - H]^", 100). (H) 4-[(2-Amino-4-methyl-thiazole-5-carbonyl)-amino]~1-methyl-1H-pyrrole-2-carboxylic acid methyl ester (49)

A solution of Boc dimer 48 (7.0 g, 17.8 mmol, 1 eq) in TFA/DCM (20/80, v/v) was allowed to stir at room temperature for 3 hours. Completion of the reaction was checked by LC/MS. The solution was added to a mixture of concentrated ammonium hydroxide/ice with vigorous stirring. The product was recovered by filtration, washed with water and dried (5.20 g, 100%). ¹H NMR (DMSO-cfe; 400 MHz) δ 9.44 (s, 1H), 7.45 (s, 2H), 7.41 (d, J = 1.90 Hz, 1H), 6.87 (d, J = 1.96 Hz, 1H), 3.82 (s, 3H), 3.73 (s, 3H), 2.37 (s, 3H); MS (ES⁺) m/z 295.22 ([M + H]⁺, 100).

(Hi) (11S, 11aS)-7-Methoxy-8-{3-[5-(5-methoxycarbonyl-1-methyl-1H-pyrrol-3- ylcarbamoyl)-4-methyl-thiazol-2-ylcarbamoyl]-propoxy}-5-oxo-11-(tetrahydro-pyran-2- yloxy)-2,3, 11,11 a-tetrahydro-1 H, 5H-pyrrolo[2, 1-c][1, 4]benzodiazepine- 10-carboxylic acid allyl ester (50a) A solution of amino dimer 49 (1.71 g, 5.8 mmol, 1 eq), protected PBD acid 40 (3.62 g, 7.0 mmol, 1.2 eq), EDCI (2.23 g, 11.6 mmol, 2 eq) and DMAP (1.77 g, 14.5 mmol, 2.5 eq) in DMF (50 ml) was stirred for 18 hours at 25°C. Work up (Heterocyclic Coupling Method B) gave the crude product which was purified by flash chromatography (EtOAc) (3.6 g, 78 %).

(11 S, 11aS)-8-{3-[5-(δ-Carboxy-1-methyi-1H-pyrrol-3-ylcarbamoyl)-4-methyi-th\azoi-2- ylcarbamoyl]-propoxy}-7-methoxy-5-oxo-11-(tetrahydro-pyran-2-yloxy)-2,3, 11, 11a- tetrahydro-1 H,5H-pyrrolo[2, 1-c][1 ,4]benzodiazepine-10-carboxylic acid allyl ester (50b) Methyl ester 50a (3.6g, 4.5 mmol) was hydrolysed (Hydrolysis Method A) to give the crude acid, which was purified by flash chromatography (MeOH/EtOAc, 10/90, v/v),(1.19 g, 86 %). ¹H NMR (DMSO-Cf₆; 400 MHz) (Mixture of diastereoisomers in a 65/35 ratio) δ 12.35 (s, 1H), 9.91 (m, 1H), 7.41 (d, J = 1.88 Hz, 1H), 7.08 (m,1H), 6.88 (s, 0.65 H), 6.85 (d, J = 1.92 Hz, 1H), 6.82 (s, 0.35 H), 5.99-5.57 (m, 2H), 5.21-4.88 (m, 3H), 4.70- 4.30 (m, 2H), 4.10-3.91 (m, 2H), 3.89-3.72 (m, 7H), 3.57-3.42 (m, 2H), 2.64 (t, J = 7.03 Hz, 2H), 2.52 (s, 3H, with DMSO signals), 2.17-2.01 (m, 3H), 1.97-1.74 (m, 3H), 1.74- 1.56 (m, 2H), 1.55-1.30 (m, 4H); MS (ES^") m/z 779.88 ([M - HY, 100).

51

54 SCHEME 15

Scheme 15

(i) (11S.1 laSyj-Methoxy-δ-β-fS-methoxycarbonyl-furan-Σ-ylcarbamoyQ-propoxyJ-δ-oxo-

11-(tetrahydro-pyran-2-yloxy)-2,3, 11,11a-tetrahydro-1H, 5H-pyrrolo[2, 1- c][1,4]benzodiazepine-10-carboxylic acid ally! ester (51)

A solution of the acid 40 (4.42 g, 8.52 mmol, 1.2 eq), 5-amiπo-furan-2-carboxylic acid methyl ester 24 (1.00 g, 7.1 mmol, 1 eq), EDCI (2.72 g, 14.2 mmol, 2.0 eq) and DMAP

(2.26 g, 17.75 mmol, 2.5 eq) in DMF (70 ml) was stirred at room temperature for 72 hours. Work up (Heterocyclic Coupling Method B) gave the product as a brown foam

(3.9 g, 71%). MS (ES⁺) m/z 664.78 ([M + Na]⁺, 100). (H) (11S, 1 laSl-δ-β-fδ-Carboxy-furan-Σ-ylcarbamoylJ-propoxyJ-y-methoxy-δ-oxo-H- (tetrahydro-pyran-2-yloxy)-2,3, 11, 11a-tetrahydro-1H,5H-pyrrolo[2, 1- c][1,4)benzodiazepine-10-carboxylic acid allyl ester (52)

The ester 51 (3.9 g, 6.1 mmol, 1 eq) in 1,4 dioxane (100 ml) was hydrolysed (Hydrolysis Method A) to give a brown gum. The product was dissolved in DCM (150 ml) and the solution was washed with brine (100 ml), dried (MgSO₄) and evaporated under reduced pressure to give the product as a brown foam which was used without further purification (3.04 g, 80%). MS (ES^") m/z 626.72 [(M - H]⁺, 100).

(Hi) (HSJIaSΪ-J-Methoxy-δ-β-lS-tS-methQxycarbonyl-i-methyl-IH-pyrrol-S- ylcarbamoyO-furan-Σ-ylcarbamoylJ-propoxyJ-δ-oxo-H-ftetrahydro-pyran-Σ-yloxy)- 2,3, 11, 11a-tetrahydro-1H,5H-pyrrolo[2, 1-c][1,4]benzodiazepine-10-carboxylic acid allyl ester (53) A solution of the acid 52 (3.0 g, 4.8 mmol, 1 eq), amine 1a HCI salt (1.11 g, 5.8 mmol, 1.2 eq), EDCI (1.86 g, 9.69 mmol, 2.0 eq) and DMAP (1.54 g, 12.11 mmol, 2.5 eq) in DMF (50 ml) was stirred at room temperature for 72 hours. Work up (Heterocyclic Coupling Method B) gave the product as a brown foam (3.4 g, 92%). MS (ES⁺) m/z 764.84 ([M + H]⁺, 30).

(iv) (11S, 11aS)-8-{3~[δ-(δ-methoxycarbonyl-1-methyl-1H-pyrrol-3-ylcarbamoyl)-furan-2- ylcarbamoyl]-propoxy}-7-methoxy-5-oxc~11-(tetrahydro-pyran-2-yloxy)-2,3, 11,11a- tetrahydro-1H,5H-pyrrolo[2, 1-c][1,4]benzodiazepine-10-carboxylic acid allyl ester (54) The ester 53 (3.4 g, 4.45 mmol, 1 eq) in 1 ,4 dioxane (70 ml) was hydrolysed (Hydrolysis Method A) to give an off white solid. The product was dissolved in DCM (150 ml) and the solution was washed with brine (100 ml), dried (MgSO₄) and evaporated under reduced pressure to give the product as an off white foam (1.9 g, 57%). ¹H NMR (DMSO-c/₆) (Mixture of diastereoisomers) δ 11.38 (bs, 1H), 10.01 (s, 1H), 7.376 (d, J = 1.64 Hz, 1H), 7.288 (s, 1H), 7.103 (d, J = 3.68 Hz, 1H), 6.93 (bs, major diastereomer, 1H), 6.84 (s, minor diastereomer, 1H), 6.827 (d, J = 1.64 Hz, 1H), 6.35 (d, J = 3.44 Hz, 1H), 5.85 - 5.65 (m, 1H), 5.73 (d, J = 9.28 Hz, 1H), 5.15 - 4.95 (m, 3H), 4.7 - 4.35 (m, 2H), 4.1 -

3.95 (m, 2H), 3.84 - 3.8 (m, 7H), 3.6 - 3.25 (m, 4H, with H₂O signals), 2.25 (m, 2H, with DMSO signals), 2.15 - 1.8 (m, 6H), 1.7 - 1.6 (m, 2H), 1.55 - 1.35 (m, 4H); MS (ES^") m/z 748.58 ([M - H]-, 70). General Coupling Methods General Coupling Method A (coupling only)

A 1 M solution of DIC (2 eq) was added to a solution of the acid (1.25 - 1.5 eq) in DMF. The solution was stirred/shaken for 10 minutes and a 1M solution of HOBt (2.5 eq) was added. After a further 10 minutes a solution of the amine (1 eq) in DMF was added. The reaction was stirred/shaken until complete by LCMS (typically 18 hours). The reaction mixture was loaded onto an lsolute SCX-2 acidic ion-exchange resin cartridge that had been pre-equilibrated with MeOH (1 vol). The cartridge was washed successively with volumes of DMF, MeOH, DMF and MeOH. The product was eluted with 2M NH₃ in MeOH (2 vol), evaporation under reduced pressure gave the product which was used without further purification.

General Coupling Method B (coupling and Boc deprotection)

The coupling reaction was carried out as for Method A. The cartridge was then washed further with AM HCI in 1,4 dioxane (2 x 0.5 vol) and MeOH several volumes. The product was eluted with 2M NH₃ in MeOH (2 vol), evaporation under reduced pressure gave the product which was used without further purification.

General Deprotection Methods Deprotection Method A (Alloc Deprotection)

Tetrakis(triphenylphosphine)palladium(0) (0.05 eq) was added to a solution of the Alloc protected compound (1 eq) and phenylsilane (5 eq) in either DCM or DMF. The solution was stirred/shaken at room temperature until the reaction was complete by LCMS (typically 1 hour). The reaction mixture was loaded onto an lsolute SCX-2 acidic ion- exchange resin cartridge that had been pre-equilibrated with DCM (1 vol). The cartridge was washed successively with volumes of DMF, MeOH, DMF and MeOH. The product was eluted with 2M NH₃ in MeOH (2 vol), evaporation under reduced pressure gave the product which was used without further purification.

Deprotection Method B (Boc Protected PBD Deprotection)

The Boc protected compound (1eq) was dissolved in anisole (1 ml) and 95% trifluoroacetic acid solution was added. The solution was stirred/shaken at room temperature for 50 minutes. The reaction mixture was diluted with DCM (5 ml) and loaded onto an lsolute SCX-2 acidic ion-exchange resin cartridge that had been pre- equilibrated with DCM (1 vol). The cartridge was washed successively with volumes of DCM, CH₃CN and DCM. The product was eluted with 2M NH₃ in MeOH (2 vol), evaporation under reduced pressure gave the product which was purified by preparative LCMS.

Deprotection Method C (Alloc Protected PBD Deprotection) Tetrakis(triphenylphosphine)palladium(0) (0.1 eq) was added to a solution of the Alloc protected compound (1 eq) and pyrrolidine (1.25 eq) in DCM and the minimum amount of DMF. The solution was stirred/shaken at room temperature until the reaction was complete by LCMS (typically 1 hour). The DCM was evaporated under reduced pressure and the residue was diluted with an excess of Et₂O. The precipitated product was collected by filtration, washing with Et₂O and air dried. The product was purified by preparative LCMS.

Example Compound Synthesis

Dimethylaminopropylamine was coupled to a building block using the above coupling methods. Successive coupling and deprotection steps were employed, using the above building blocks, to prepare polyamides of the appropriate length. The PBD building blocks were then coupled to the polyamides as above. Final deprotection of the PBD derivatives gave the target compounds

Example Compounds Key

Λ_^-_N

Dp

5MTz

LN4PBD

Dimethylaminopropylamine (Dp) β-Alanine (β) Pyrrole (Py) Imidazole (Im) Furan (Fr)

4-Methylthiazole-5-carboxylic acid (4WITz) δ-Methylthiazole^-carboxylic acid (5MTz) PBD acid capping unit (LN4PBD)

Compounds containing one β-alanine residue

Compounds containing two β-alanine residues

Compounds containing three β-alanine residues

Quantitative Real-Time Polymerase Chain Reaction

Ce// culture and drug treatment LNCaP-FGC prostate carcinoma cells were seeded into six-well plates at 8 x 10⁵ cells per well. Cells were left to adhere overnight and drug (or vehicle - DMSO) was added directly to the culture medium subsequently. For initial studies, the compounds to be tested were added at 1 μM and 10 μM final concentrations. Later, for dose-response relationship studies the compounds to be tested were added at multiple varying concentrations, whilst for time course studies a single concentration was used and the compound to be tested was removed following a variable length of incubation.

RNA extraction, quantitation and cDNA generation

RNA extraction was performed with QIAGEN's RNeasy Kit according to the manufacturer's instructions. Briefly, culture medium containing drug was removed and cells cleaned with PBS. 350 μl RLT Lysis Buffer was added to each well and each homogenised sample transferred to an individual QIAshredder spin column. Following centrifugation (2 min at 12000 r.p.m.) 350 μl of 70% ethanol was added to the flow- throughs. The mixtures were transferred to RNeasy spin columns and spun at 12000 r.p.m. for 15 seconds. 700 μl RW1 Wash Buffer was added to each column followed by centrifugation at 12000 r.p.m. for 15 seconds. 500 μL RPE Wash Buffer was added to each column followed by centrifugation at 12000 r.p.m. for 15 seconds. A further 500 μl RPE Wash Buffer was added to each column followed by centrifugation at 12000 r.p.m. for 2 minutes. Any remaining ethanol was allowed to evaporate. 40 μl of RNAse-free water was added to each column and left to stand for 1 minute. Columns were then spun at 12000 r.p.m. for 1 minute. Flow-throughs were collected and replaced in the columns to increase RNA yield. Again, the columns were spun at 12000 r.p.m. for 1 minute. RNA samples could then be stored at -80⁰C indefinitely.

RNA yield was quantified via the following method: RNA standards at 1 μg/ml, 0.5 μg/ml, 0.1 μg/ml and 0.02 μg/ml were prepared in Tris- EDTA buffer. 50 μl of each standard (in duplicate) were transferred to a fluorescence- compatible 96-well plate. 50 μl Tris-EDTA buffer was used as a blank. Each RNA sample was diluted 100-fold in Tris-EDTA buffer and 50 μl of each diluted RNA sample was also transferred to the 96-well plate. 50 μl RiboGreen was added to each well and the plate was shaken gently for 5 min. Fluorescence read-out from the 96-well plate was collected on an Envision 2101 Multilabel Reader (Perkin Elmer). GraphPad Prism software was used to create standard curve and then used to analyse sample data.

Complementary DNA (cDNA) was synthesised from isolated RNA using QIAGEN's Omniscript Kit according to the manufacturer's instructions. Briefly, for each sample 1.4 μg RNA was added diluted into 12 μl RNAse-free water, heated to 65 ⁰C for 10 min and immediately placed on ice. 8 μl pre-prepared Omniscript mix was added to each reaction and samples were incubated at 37⁰C for 1 hour to permit cDNA synthesis. Samples were stored at 4⁰C for up to one week.

Real-time PCR

Real time PCR reactions were performed in 25 μl reaction volumes in a fluorescence- compatible 96-well plate. Each 25 μl reaction comprised 12.5 μl Taqman Master Mix (ABI), 1.25 μl primer probe mix (either Taqman pre-designed mix (ABI) or self-designed primers at 18 μM and probe at 10 μM), 0.25 μl cDNA direct from reverse transcriptase step, and 11 μl RNAse-free water. Primers and probes were designed to amplify and detect cDNA corresponding to the following genes: Androgen Receptor; β-actin

'Home-made' probes were labelled with FAM and TAMRA.

All samples were run in triplicate and both an internal housekeeping gene (β-actin) and non-template controls were included. Reactions were run and data collected using an ABI 7500 real-time PCR machine. DNA was amplified via the following cycling program:

Initial denaturation at 95°C for 10 minutes Cyclical denaturation at 95⁰C for 15 seconds Cyclical annealing at 6O⁰C for 1 minute Cyclical elongation at 72⁰C for 1 minute

Final elongation at 72⁰C for 10 minutes

Forty cycles of amplification were performed and collected data were transferred to Microsoft Excel for analysis using the delta delta Ct method (ΔΔCt).

Relative cDNA Quantity (RQ) = 2^("MCt) Where:

AACt ΔCt value(s) of control samples

ΔCt = Cttarget - Ctj3_-aotin n₀ = Number of control samples

Ctta_rget = Cycle threshold(s) at midpoint of logarithmic phase for target gene samples Ctβ-actin = Cycle threshold(s) at midpoint of logarithmic phase for β-actin samples

RQ values were imported into GraphPad Prism software for graphical representation. Effects on β-actin were displayed as ΔCt values rather than RQ.

Tables of results:

These data tables show evidence of selective inhibition of androgen receptor (AR) mRNA transcription by particular compounds (23, 31 , 48, 49 and 57). In addition, the tables provide a clear indication of the lack of concomitant effects on β-actin mRNA transcription, emphasising that the activity of these compounds is selective.

By way of an example; at both 6 and 24 hour time points, compounds 23 and 31 exhibit the greatest magnitude of reduction in AR mRNA transcription. After 6 hours incubation with 1 μM compound, the fold expressions of AR mRNA are 0.0884 and 0.0466 for compounds 23 and 31 respectively compared to control. At 10 μM, these values are 0.0014 and 0.0043 respectively. The ΔCt values for β-actin expression ranged between - 1.9 and 0.86 within the same samples. (A value of n corresponds to a fold increase/decrease in mRNA levels of 2"; expression of 0.0014 corresponds to a ΔCt value of ~-9.5; and a ΔCt value of -1.9 corresponds to a fold expression of 0.267.) Compared to the expression level effects of compounds 23 and 31 , this fluctuation in β- actin levels is negligible and it can be assumed that drug effect on β-actin mRNA transcription is minimal. The lack of effect on β-actin is seen throughout the series of molecules described herein.

Additionally, other molecules show a selective inhibition of AR mRNA transcription. For example, after a 24 hour incubation with 1 μM compound 49, the expression on AR is only 0.0008-fold the level of the control samples. The concomitant change is the levels of β-actin is minimal (ΔCt value of -0.25). Similarly, the amount of AR mRNA detected after 24 hours in 1 μM of compound 57 is merely 2.39 x 10^"6-fold of the control. The β- actin ΔCt value under the same conditions is only -1.07.

In conclusion, several of the compounds described show selective activity in downregulating the transcription of androgen receptor mRNA when compared to the transcription of β-actin. The compounds highlighted (23, 31 , 48, 49, 57) show particularly high levels of activity in this context. When the effects of these drugs on the expression of other genes are measured, it is clear that there is a preferential reduction in AR levels compared to, say, TOPOIIα or EGFR (data not shown).

The effect of example compounds 3 to 38 (1 μM and 10 μM, 6 hour incubation) on Androgen Receptor in LNCaP-FGC cells

The effect of example compounds 3 to 38 (1 μM and 10 μM, 24 hour incubation) on Androgen Receptor in LNCaP-FGC cells.

The effect of example compounds at concentrations of 1 μM and lower (24 hour incubation) on Androgen Receptor in LNCaP-FGC cells:

β-Actin ΔCt

The effect of example compounds over time (1 μM and 10 μM) on Androgen Receptor in LNCaP-FGC cells.

μM (see fig. 1)

Timecourse for 1 μM β-Actin ΔCt

The effect of example compounds 39 to 59 (1μM, 24 hour incubation) on Androgen Receptor in LNCaP-FGC cells.

The effect of example compound 57 on Androgen Receptor in LNCaP-FGC cells over time at concentrations of 1 μM and lower (see fig. 3):

The effect of example compound 49 on Androgen Receptor in LNCaP-FGC cells over time at concentrations of 1 μM and lower (see fig. 4):

Effect of compounds in xenograft

The following compounds were tested for their effect against a human prostate tumour model (DIM 45) growing as a xenograft in male athymic nude mice: SG3018, SG3028, SG-2669 and SG-2677. The compounds were administered intravenously under varying dosing regimens, and showed evidence of tumour growth delay in every case.

Claims

1. A compound of formula I:

wherein: the dotted line indicates the optional presence of a double bond between C2 and C3;

R² is selected from -H, -OH, =0, =CH₂, -CN, -R, OR, halo, =CH-R, 0-SO₂-R, CO₂R and

COR;

R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR', nitro, Me₃Sn and halo; where R and R' are independently selected from optionally substituted Ci_-7 alkyl, C_3-2O heterocyclyl and C_5-20 aryl groups;

Y is selected from O, S and NH and R^γ is H or C_1-7 alkyl or H and SO_xM, where x is 2 or

3, and M is a monovalent pharmaceutically acceptable cation; each X is independently a C_5-6 heteroarylene group; n is from 1 to 6.

2. A Compound according to claim 1 , wherein each X is independently an optionally substituted C₅ heteroarylene group.

3. A compound according to claim 1 , wherein each fragment

x L. is independently selected from:

4. A compound according to any one of claims 1 to 3, wherein n is 1 , 2 or 3.

5. A compound according to any one of claims 1 to 4, wherein R-O and R¹ ^ together form a double bond between N10 and C11.

6. The compound according to any one of claims 1 to 5, wherein R⁷ is selected from H, OH, OR, SH, SR, NH₂, NHR, NRR', and halo.

7. A compound according to claim 6, wherein R⁷ is selected from OMe and OCH₂Ph.

8. A compound according to any one of claims 1 to 7, wherein R² is H.

9. A compound according to any one of claims 1 to 7, wherein R² is an optionally substituted C_5-20 aryl group.

10. A compound according to any one of claims 1 to 7, wherein R² is =CH₂.

11. A compound according to any one of claims 1 to 7, wherein R² is =CH-R, and R is selected from C_1-7 alkyl and C_5-20 aryl.

12. A compound of formula II:

wherein X, n, R² and R⁷ are as defined in any one of claims 1 to 11 , R^10A is a nitrogen protecting group and R^11A is OH or OProt⁰, where Prot⁰ is a hydroxy protecting group.

13. A compound according to claim 12, wherein R^10A is selected from Alloc, Troc, Teoc, BOC, Doc, Hoc, TcBOC, Fmoc, 1-Adoc and 2-Adoc.

14. A compound according to claim 13, wherein R^10A is selected from boc, Troc or alloc.

15. A compound according to any one of claims 12 to 14, wherein R^11A is selected from THP or TBS.

16. A pharmaceutical composition comprising a compound according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier or diluent.

17. A compound according to any one of claims 1 to 11 for use in a method of medical treatment.

18. The use of a compound according to any one of claims 1 to 11 in the manufacture of a medicament for treating a disease or condition ameliorated by the down-regulation of the Androgen Receptor.

19. The use according to claim 18, wherein the disease or condition ameliorated by the down-regulation of the Androgen Receptor is prostate cancer

20. A compound according to any one of claims 1 to 11 for use in a method of treatment of a disease or condition ameliorated by the down-regulation of the Androgen Receptor.

21. A compound according to claim 20, wherein the disease or condition ameliorated by the down-regulation of the Androgen Receptor is prostate cancer.