EA040562B1 - PIPERIDINOBENZODIAZEPINE COMPOUNDS WITH ANTIPROLIFERATING ACTIVITY - Google Patents

Description

Field of invention

The invention relates to pyrridinobenzodiazepines (PDD) containing three fused 6-7-6-membered rings. In particular, the invention relates to compounds containing a PDD group linked through ring A to aromatic groups, and to pharmaceutically acceptable salts thereof, which can be used as drugs, in particular as antiproliferative agents.

Background of the invention

The pyrridinobenzodiazepines (PDDs) are structurally similar to the pyrrolobenzodiazepines (PBDs). Pyrrolobenzodiazepines (PBDs) are a group of compounds, some of which have been shown to be sequence-specific DNA minor groove binders. PBDs were first found in species of the genus Streptomyces [1–5]. They are tricyclic in nature and consist of fused 6-7-5-membered rings which include anthranilate (ring A), diazepine (ring B) and pyrrolidine (ring C) [3]. They are characterized by an electrophilic imine group N10=C11 (as shown below) or a hydrated equivalent, carbinolamine [NH-CH(OH)] or carbinolamine alkyl ester ([NH-CH(OR, where R=alkyl)], which can form a covalent bond with C2-amino group of DNA guanine to obtain a DNA adduct [6].

Carbinolamine Imine Carbinolamine alkyl ester

Natural products interact in the minor groove of the DNA helix with ideal packing (i.e., good isohelix) due to the right longitudinal twist caused by the chiral CHa position, which has the ^-configuration [6]. The DNA adduct has been reported to inhibit a number of biological processes, including the binding of transcription factors [7-9] and the function of enzymes such as endonucleases [10, 11] and RNA polymerase [12]. Using footprinting [6], NMR (13, 14), molecular modeling [15] and X-ray crystallography [16], it was shown that PBD monomers (for example, anthramycin) span three base pairs and have a thermodynamic preference for the 5'-Pu -G-Pu-3' (where Pu=purine and G is reactive guanine) [17] and a kinetic preference for Py-5-Py (where Py=pyrimidine).

It is believed that PBDs interact with DNA by first locating in a low charge energy binding sequence (i.e. 5'-Pu-G-Pu-3' triplet) via van der Waals, hydrogen and electrostatic interactions [7]. Then, after placement, a nucleophilic attack by the exocyclic C2-amino group of the central guanine occurs with the formation of a covalent adduct [7]. After binding, PBD remains anchored in the DNA minor groove, avoiding DNA repair, causing slight distortion of the DNA helix [16]. The ability of PBDs to form an adduct in the minor groove and cross-link DNA allows them to interfere with the work of DNA and, therefore, allows for their use as antiproliferative agents.

A number of PBD monomeric structures have been isolated from Streptomyces samples, including anthramycin [18], the first PBD, tomycin [19], and recently uzabamycin [20] from Streptomyces seabed samples. This has led to the development of a wide range of synthetic analogs, which have been considered [1, 21]. Recently, there have been reports (WO 2007/039752, WO 2013/164593 [22-27]) on a number of PBD monomer structures that are connected via their C8 position to pyrroles and imidazoles.

WO 2010/091150 describes a 6-7-6 dimer of the ring system linked through its A-rings. WO 2015/028850 describes dimers of PBD 6-7-5 ring systems that are connected via linkers containing phosphine oxide attached to aromatic A-rings. In addition, WO 2015/028850 describes a dimeric compound containing a 6-7-6 ring system linked via linkers containing a key phosphine oxide.

Various PBDs have been shown to act in vitro as cytotoxic agents (see e.g. WO 00/12508, WO 2004/087711) and in vivo as antitumor agents in animal tumor models (see e.g. WO 2011/117882, WO 2013/164593). In addition, for the C8/C8'-linked PBD dimer SJG-136 [28, 29], phase I clinical trials on leukemia and ovarian cancer [30] were completed and a sufficient therapeutic effect was shown to continue phase II studies.

SJG-136

However, the results of a phase I clinical evaluation of SJG-136 showed that the drug caused several

- 1 040562 side effects, including swelling and heaviness in the lower extremities (31).

Thus, there is a need for additional PBD-related compounds that are therapeutically active in the treatment of a number of proliferative diseases.

Presented herein are pyrridininobenzodiazepines (PDDs) which are PBD but contain an extended 6-membered C-ring compared to the 5-membered C-ring of PBD. The inventors have found that PDD conjugates provide properties such as cytotoxicity and DNA binding, resulting in effective compounds.

The present invention is directed to overcoming the problems associated with the prior art.

The essence of the invention

The present invention relates to a compound of formula (I)

(I) and its salts and solvates, where dotted lines indicate the optional presence of a double bond between one or more C1 and C2; C2 and C3; and C3 and C4;

R1 is selected from R ₇ , ═CH ₂ , ═CH-(CH ₂ ) _m -CH3, ═O, (CH2VOR7, (CH2VCO2R7, (CH2VNR7R8, 0-No)πNR7R8, NH-C(O)-R7, O- (CH2)n-NH-C(O)-R7, O-(CH2)nC(O)-NH-R7, (CH2)m-SO2R7, O-SO2R7, (CH2)mC(O)R7 and (CH2 )mC(O)NR7R8;

R ₂ is selected from R ₉ , =CH ₂ , =CH-(CH ₂ ) _r -CH ₃ , =O, (CH ₂ ) _r -OR ₉ , (CH ₂ ) _r -CO ₂ R ₉ , (CH ₂ ) _r -NR ₉ R ₁₀ , O-(CH ₂ ) _s NR ₉ R ₁₀ , NH-C(O)-R ₉ , O-(CH ₂ ) _s -NH-C(O)-R ₉ , O-( CH ₂ ) _s -C(O)-NH-R, ₉ (CH ₂ ) _r -SO ₂ R ₉ , O-SO ₂ R ₉ , (CH ₂ ) _r -COR ₉ and (CH2) rC(O)NR9R10 ;

R ₃ is selected from H, C ₁ . ₁₂ -alkyl and CH ₂ Ph;

R ₄ is selected from phenyl and C ₅ . ₉ -heteroα-aryl groups optionally substituted with up to three optional substituents selected from OH, C ₁ . ₆ -alkyl, OC ₁ . ₆ -alkyl, (CH ₂ )j-CO ₂ R ₁₁ , O-(CH ₂ ) _k -NR ₁₁ R ₁₂ , (CH ₂ ) _j -NR ₁₁ R ₁₂ , C (=O)-NH-(CH ₂ ) _k -NR ₁₁ R ₁₂ , C (=O)-NH-R ₂₄ and C (=O)-NH-(CH ₂ ) _k -C(=NH)NR ₁₁ R ₁₂ , provided that optionally substituted C ₅ . ₉ -heteroaryl is not an indolyl;

R ₁₉ is selected from H and (CH ₂ ) _t -NR ₂₀ R ₂₁ ;

Y 1 represents N or CH;

Y ₂ is N or CH, where at least one of Y ₁ and Y ₂ is CH;

p is 0 or 1;

j, m, r and t are independently selected from an integer from 0 to 6;

k, n and s are independently selected from an integer from 1 to 6;

X1 is selected from O, S, NR13, CR13R14, CR13R14O, C(=O), C(=O)NR _B , NR _b C(=O), OC(O) and C(O)-O;

L is selected from an amino acid, a peptide chain having 2 to 6 amino acids, an alkylene chain having 1 to 12 carbon atoms, which may have one or more carbon-carbon double or triple bonds, a paraformaldehyde chain (OCH ₂ )1. ₁₂ -, polyethylene glycol chain - (OCH ₂ CH ₂ ) ₁ . ₆ - where the chains can be interrupted by one or more groups of O, S, and/or NH, and/or C ₃ . ₉ -heteroarylene and/or phenylene;

X ₂ is selected from O, S, NR ₁₅ , CR ₁₅ R ₁₆ , CR ₁₅ R ₁₆ O, C(=O), C(=O)NR ₁₅ , NR ₁₅ C(=O), OC(O) and C (O)-O or absent;

q is selected from 0, 1, 2, 3, 4, 5 and 6;

And selected from

A1 _and A2.

in each group A1, one of Y ₃ and Y ₄ is independently selected from NR ₁₇ , S and O; and the other of Y ₃ and Y ₄ is CH; and Y ₅ is independently selected from CH, N, S and CH; and in each group A2, one of Y6 and Y7 is independently selected from N and CH; and the other of Y6 and Y7 is CH;

R7 and R ₉ are independently selected from H, C1. ₁₂ -alkyl, C ₅ . ₉ -heteroaryl, C ₆ . ₁₅ -heteroarylalkyl, phenyl and C ₇ . ₁₂ -aralkyl groups, where heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with up to three optional substituents selected from C _1-6 -alkyl, OH, OC ₁ . ₆ -alkyl;

R ₂₄ is phenyl optionally substituted with up to three optional substituents selected from OH, C _1-6 alkyl, OC _1-6 alkyl, (CH2) _j -CO2R ₁₁ , O-(CH ₂ ) _k -NR ₁₁ R ₁₂ , (CH ₂ ) _j -NR ₁₁ R ₁₂ ,

C(=O)-NH-(CH2)k-NR _n Ri2 and C(=O)-NH-(CH2)kC(=NH)NR _n R12;

R ₈ , R ₁₀ , Rh, R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R2o and R ₂₁ are independently selected from H and C1_ ₆ -alkyl;

and also (i) R5 and R6 together form a double bond;

(ii) R5 is H and R6 is OH; or (iii) R5 is H and R6 is OC ₁ _ ₆ alkyl, provided that when p is 0 and A is A1 then (a) in at least one A1 group one of Y3 and Y4 selected from S and O; or (b) in at least one group A1 Y5 is S; or (c) R4 is not pyrrolyl, imidazolyl, optionally substituted pyrrolyl, or optionally substituted imidazolyl.

The present invention relates to a compound of formula (I)

and its salts and solvates, where dotted lines indicate the optional presence of a double bond between one or more C1 and C2; C2 and C3; and C3 and C4;

R1 is selected from R7, =CH ₂ , =CH-(CH2)m-CH3, =O, (CH2VOR7, (CH2)m.-CO2R, (CH^^Rg, O-(CH2)pNR7R8, NH-C( O)-R7, O-(CH2)n-NH-C(O)-R7, O-(CH2)nC(O)-NH-R7, (CH2VSO2R7, O-SO2R7, (CH2)mC(O)R7 and (CH2)mC(O)NR7R8;

R2 is selected from R9, =CH2, =CH-(CH2)g-CH3, =O, (CH2X-OR9, (CH2X-CO2R9, (CH2X-NR9R10, O-(CH2)sNR9R ₁₀ , NH-C(O) -R9, O-(CH ₂ ) _s -NH-C(O)-R ₉ , O-(CH ₂ ) _s -C(O)-NH-R ₉ , (CH ₂ ) _r -SO ₂ R ₉ , O-SO2R9, (CH ₂ ) _r -COR ₉ and (CH2) _r -C(O)NR9R10;

R ₃ is selected from H, C ₁ _ ₁₂ -alkyl and CH ₂ Ph;

R4 is selected from phenyl and C ₅ _ ₉ heteroaryl groups optionally substituted with up to three optional substituents selected from OH, C ₁ _ ₆ -alkylα, OC ₁ _ ₆ -alkyl, (CH ₂ ) _j -CO ₂ R ₁₁ , O -(CH ₂ ) _k -NR ₁₁ R ₁₂ , (CH2)j-NR11R12, C(=O)-NH-(CH2)k-NR11R12, C(=O)-NH-C6H4-(CH2)j-R18 and C(=O)-NH-(CH2)kC(=NH)NR11R12, with the proviso that the optionally substituted C _5-9 heteroaryl is not indolyl;

R ₁₉ is selected from H and (CH ₂ ) _t -NR ₂₀ R ₂₁ ;

Y1 is N or CH;

Y2 is N or CH; and where at least one of Y ₁ and Y ₂ represents CH;

p is 0 or 1;

j, m, r and t are independently selected from an integer from 0 to 6;

k, n and s are independently selected from an integer from 1 to 6;

X1 is selected from O, S, NR13, CR13R14, CR13R14O, C(=C), C(=O)NR _n , NR _n C(=O), OC(O) and C(O)-O;

L is selected from an amino acid, a peptide chain having 2 to 6 amino acids, an alkylene chain having 1 to 12 carbon atoms, which may have one or more double or triple carbon-carbon bonds, a paraformaldehyde chain (OCH ₂ ) _{1_12} -, polyethylene glycol chain -(OSH ₂ CH ₂ )1_ ₆ -, where the chain can be interrupted by one or more groups of O, S, and/or NH, and/or C3-9-heteroarylene and/or phenylene;

X2 is selected from O, S, NR15, CR15R16, CR15R16O, C(=C), C(=O)NR _b , NR15C(=O), OC(O) and C(O)-O, or absent;

q is selected from 0, 1, 2, 3, 4, 5 and 6;

And selected from

A1 _and A2.

in each group A1, one of Y3 and Y4 is independently selected from NR ₁₇ , S and O; and the other of Y3 and Y4 is CH; and Y5 is independently selected from CH, N, S and CH; and in each group A2, one of Y ₆ and Y ₇ is independently selected from N and CH; and the other of Y ₆ and Y ₇ is CH;

R7 and R9 are independently selected from H, C ₁ . ₁₂ -alkyl, C ₅ . ₉ -heteroaryl, C ₆ . ₁₅ -heteroarylalkyl, phenyl and C ₇ _ ₁₂ -aralkyl groups, wherein the heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with up to three optional substituents selected from

C1-b-alkyl, OH, OC _1-6 -alkyl;

R ₁₈ is selected from CO ₂ R ₁₁ u NR11R12;

R ₈ , R ₁₀ , R11, R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R2 ₀ and R21 are independently selected from H and C _1-6 alkyl;

and also (i) R ₅ and R 6 together form a double bond;

(ii) R ₅ is H and R6 is OH; or (iii) R ₅ is H and R ₆ is OC _1-6 alkyl;

with the proviso that when p is 0 and A is A1, then (a) in at least one group A1, one of Y ₃ and Y ₄ is selected from S and O; or (b) in at least one group A1 Y ₅ is S; or (c) R4 is not pyrrolyl, imidazolyl, optionally substituted pyrrolyl, or optionally substituted imidazolyl.

In yet another aspect, a compound of formula (I) and salts and solvates thereof are provided for use in a method of treatment.

In yet another aspect, a compound of formula (I) and salts and solvates thereof are provided for use as a medicament.

In yet another aspect, a compound of formula (I) and salts and solvates thereof are provided for use in the treatment of a proliferative disease.

In yet another aspect, a pharmaceutical composition is provided, comprising a compound of formula (I) and its salts and solvates, and a pharmaceutically acceptable excipient, carrier or diluent.

In yet another aspect, the present invention relates to the use of a compound of formula (I) and a salt and solvate thereof in the manufacture of a medicament for the treatment of a proliferative disease.

In yet another aspect, the present invention relates to a method of treating a patient suffering from a proliferative disease, comprising administering to said patient a therapeutically effective amount of a compound of formula (I) and its salt and solvates, or a pharmaceutical composition of the present invention.

In yet another aspect, the compound of formula (I) and its salts and solvates may be administered alone or in combination with other drugs, either simultaneously or sequentially, depending on the condition being treated.

The pharmaceutical composition of the present invention may also contain one or more (eg two, three or four) other active agents.

In yet another aspect, the compound of formula (I) and its salts and solvates can be associated either directly or indirectly with a targeted agent (eg, antibody, antibody fragment, hormone, etc.) to obtain a targeted conjugate. The target conjugates of the present invention may contain one or more compounds of formula (I) (or their salts and solvates). A variety of targeted conjugates are known in the art and can be used with compounds (I) and its salts and solvates. For example, in a particular aspect, the targeted conjugate is an antibody drug conjugate wherein one or more compounds of formula (I) are linked directly or indirectly to an antibody. Thus, the compound of formula (I) and its salts and solvates can be used as a payload in a targeted conjugate.

Definitions.

The description uses the following abbreviations:

Ac: acetyl;

Alloc: allyloxycarbonyl;

BAIB: bis(acetoxy)iodobenzene/ (diacetoxyiodo)benzene;

Boc: tert-butoxycarbonyl;

BPD benzopyridodiazecins;

CBz: benzyloxycarbonyl;

DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene;

DHP: dihydropyran;

DMAP: 4-dimethylaminopyridine;

DMF: dimethylformamide;

DMSO: dimethyl sulfoxide;

EDCl: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;

Et: ethyl;

Et2O: diethyl ether;

EtOAc: ethyl acetate;

EtOH ethanol;

HATU: (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate);

HMDST: hexamethyldisilathian;

iBu: isobutyl;

KotBu: potassium tert-butoxide;

Lithium L-selectride tri-sec-butyl(hydride)borate;

Me: methyl;

MeOH: methanol;

PBD: pyrrolo[2,1-c][1,4]benzodiazepines;

PDD: pyridinobenzodiazepines;

PIFA: phenyliodine(III) bis[trifluoroacetate];

Ph: phenyl;

p-TSA/PTSA: p-toluenesulfonic acid;

Pyr: pyridine;

TBAF: tetrabutylammonium fluoride;

TBS-Cl/TBDMSCl: tert-butyldimethylsilyl chloride;

TEA: triethylamine;

TEMPO: (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl;

TFA: trifluoroacetic acid;

THF: tetrahydrofuran;

THP: tetrahydropyranyl;

Troc: 2,2,2-trichloroethyl carbonate; And

Ts (tosylate): p-toluenesulfonic acid.

The term substituted, when used in connection with a chemical substituent or moiety (e.g., an alkyl group), means that one or more hydrogen atoms of the substituent or moiety have been replaced by one or more non-hydrogen atoms or groups, provided that the rules of valence and that the result of substitution is a chemically stable compound.

The term optionally substituted refers to a parent group which may be unsubstituted or which may be substituted with one or more substituents. Accordingly, unless otherwise indicated, when there are optional substituents, an optional substituted parent group includes one to three optional substituents. If a group can be optionally substituted with up to three groups, this means that the group can be substituted with 0, 1, 2, or 3 optional substituents. If a group may be optionally substituted with one or two optional substituents, this means that the group may be substituted with 0, 1 or 2 optional substituents. Suitable groups may optionally be substituted with 0 or 1 optional substituents.

The term independently selected is used in the context of stating that, for example, R1 and R ₂ are independently selected from H, C ₁ . ₁₂ -alkyl, phenyl..., and means that in each case the functional group, for example R1, is selected from the listed options, regardless of any other example of R1 or R2 in the connection. Therefore, for example, C1_ ₁₂ -alkyl can be selected for the first example of R1 in the connection; a phenyl group may be chosen for the following example R1 in a compound; and H may be selected for the first example of R ₂ in the compound.

Ci-12-alkyl refers to straight and branched saturated hydrocarbon groups, usually containing from 1 to 12 carbon atoms; preferably to C ₁ . ₇ -alkyl; more preferably Cl. ₆ -alkyl. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pent-1-yl, pent-2-yl, pent-3-yl, 3-methylbut-1- yl, 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2,2-trimethylethyl-1-yl, n-hexyl, n-heptyl, and the like.

The term alkylene refers to a divalent alkane derivative which may be linear or branched, an example of which is -CH ₂ CH ₂ CH ₂ CH ₂ -.

The term aryl refers to fully unsaturated monocyclic, bicyclic and polycyclic aromatic hydrocarbons having at least one aromatic ring and containing a certain number of carbon atoms that include ring members (for example, C ₆ . ₁₄ -aryl refers to an aryl group containing from 6 to 14 carbon atoms as ring members). The aryl group may be attached to the parent group or to the substrate at any ring atom and may include one or more non-hydrogen substituents, so long as such attachment or substitution does not violate the valence rules.

Term C ₇ . ₁₂ -aralkyl refers to an arylalkyl group containing 7 to 12 carbon atoms and having an alkyl group substituted with an aryl group. A suitable alkyl group is C ₁ . ₆ -alkyl group, and the aryl group is phenyl. Examples of C ₇ _ ₁₂ aralkyl include benzyl and phenethyl. In some instances, the C ₇ _ ₁₂ aralkyl group may optionally be substituted, and an example of an optionally substituted C ₇ _ ₁₂ aralkyl group is 4-methoxylbenzyl.

The term C ₅ _ ₉ -heteroaryl refers to unsubstituted monocyclic or bicyclic aromas

- 5 040562 tic groups containing from 5 to 9 ring atoms, whether carbon atoms or heteroatoms, of which 1 to 5 are ring heteroatoms. Accordingly, a monocyclic heteroaryl ring has 5 to 6 ring atoms and 1 to 3 ring heteroatoms. Preferably, each ring heteroatom is independently selected from nitrogen, oxygen and sulfur. Bicyclic rings include fused ring systems and in particular include bicyclic groups in which a monocyclic heterocycle containing 5 ring atoms is fused to a benzene ring. The heteroaryl group may be attached to the parent group or to the substrate at any ring atom and may contain one or more non-hydrogen substituents, so long as such addition or substitution does not violate valence requirements or result in a chemically unstable compound.

Examples of monocyclic heteroaryl groups include, but are not limited to, derivatives of the following heteroaryls:

N1: pyrrole, pyridine;

O1: furan;

S1: thiophene;

N1O1: oxazole, isoxazole, isoxazine;

N2O1: oxadiazole (eg 1-oxa-2,3-diazolyl, 1-oxa-2,4-diazolyl, 1-oxa-2,5-diazolyl, 1-oxa3,4-diazolyl);

N ₃ O1: oxatriazole;

N1S1: thiazole, isothiazole;

N2: imidazole, pyrazole, pyridazine, pyrimidine, pyrazine;

N ₃ : triazole, triazine; And

N ₄ : tetrazole.

Examples of a heteroaryl that contains fused rings include, but are not limited to, derivatives of the following heteroaryls:

O1: benzofuran, isobenzofuran;

N1: indole, isoindole, indolizine, isoindoline;

S1: benzothiofuran;

N1O1: benzoxazole, benzisoxazole;

N1S1: benzothiazole;

N ₂ : benzimidazole, indazole;

O2: benzodioxole;

N2O1: benzofurazan;

N2S1: benzothiadiazole;

N ₃ : benzotriazole; And

N4: purine (eg adenine, guanine), pteridine.

The term heteroarylene refers to a divalent radical derived from a heteroaryl group as illustrated by pyridinylene -(C ₅ H ₃ N)-.

The term C ₆ _ ₁₅ heteroarylalkyl refers to an alkyl group substituted with a heteroaryl group. Suitable alkyl is C ₁ . _{a 6} alkyl α group, and the heteroaryl group is C ₅ _ ₉ heteroaryl as defined above. Examples of C ₆ _ ₁₅ heteroarylalkyl groups include pyrrol-2ylmethyl, pyrrol-3-ylmethyl, pyrrol-4-ylmethyl, pyrrol-3-ylethyl, pyrrol-4-ylethyl, imidazol-2-ylmethyl, imidazol-4-ylmethyl, imidazole -4-ylethyl, thiophen-3-ylmethyl, furan-3-ylmethyl, pyridin-2-ylmethyl, pyridin-2-ylethyl, thiazol-2-ylmethyl, thiazol-4-ylmethyl, thiazol-2-ylethyl, pyrimidin-2 -ylpropyl, etc.

nitrogen protecting groups.

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

A large number of possible carbamate nitrogen protecting groups are listed on pages 706-771 of the review by Wuts, P.G.M. and Greene, T.W., Protective Groups in Organic Synthesis, 4th edition, WileyInterscience, 2007; and in R. Kocienski, Protective Groups, 3rd edition (2005), which are incorporated herein by reference.

Particularly preferred protecting groups include Alloc (allyloxycarbonyl), Troc (2,2,2-trichloroethyl carbonate), Teos [2-(trimethylsilyl)ethoxycarbonyl], BOC (tert-butyloxycarbonyl), Doc (2,4-dimethylpent-3-yloxycarbonyl ), Nose (cyclohexyloxycarbonyl), TcBOC (2,2,2-trichloro-tert-butyloxycarbonyl), Fmoc (9-fluorenylmethyloxycarbonyl), 1-Adoc (1-adamantyloxycarbonyl) and 2-Adoc (2-adamantyloxycarbonyl).

hydroxyl protecting groups.

Hydroxy protecting groups are well known in the art; a large number of suitable groups are described on pages 16-366 of the review by Wuts, P.G.M. and Greene, T.W., Protective Groups in Organic Synthesis, 4th edition, Wiley-Interscience, 2007; and in R. Kocienski, Protective Groups, 3rd edition (2005), which are incorporated herein by reference.

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

Particularly preferred protecting groups include THP (tetrahydropyranyl ether).

The expression compound of formula (I) and its salts and solvates refers to compounds of formula (I), salts of compounds of formula (I), solvates of compounds of formula (I) and solvates of salts of compounds of formula (I).

The term drug, drug substance, active pharmaceutical ingredient, etc. refers to a compound (eg, compounds of formula (I) and compounds specifically named above) that can be used to treat a subject in need of treatment.

The term excipient refers to a substance that can interfere with the bioavailability of a drug but is otherwise pharmacologically inactive.

The term pharmaceutically acceptable substances refers to substances which, according to sound medical judgment, are suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic reaction, etc., are commensurate with a reasonable benefit-risk ratio, and are effective for their intended use.

The term pharmaceutical composition refers to a combination of one or more drug substances and one or more excipients.

The term subject, as used herein, refers to a human and a non-human mammal. Examples of the non-human mammal include farm animals such as sheep, horses, cows, pigs, goats, rabbits and deer; and domestic animals such as cats, dogs, rodents and horses.

The expression "therapeutically effective amount of a drug" refers to the amount of drug or composition that is effective in treating a subject and thus produces the desired therapeutic, beneficial, inhibitory or prophylactic effect. A therapeutically effective amount may depend, among other things, on the weight and age of the subject and the route of administration.

The term treatment refers to reversing, alleviating, inhibiting the progression of, or preventing the disorder, disease, or condition to which the term refers, or reversing, alleviating, inhibiting, or preventing one or more of the symptoms of such disorder, disease, or condition.

Cure refers to the effect of treatment as defined immediately above.

As defined herein, the term containing means including at least to some extent and must be inclusive or unrestricted. In interpreting each statement in this specification that includes the term containing, features, elements, and/or steps other than those preceded by the term may also be present. Related terms such as contain and contains should be interpreted in the same way.

R1.

R1 is selected from R7, =¾ = CH-(CH2) _sh -CH3, =O, (CHVOR * (CH2) _m -CO2R7, (CH ₂ )m-NR ₇ R ", O-(CH2) nNR ₇ R ₈ , NH-C(O)-R ₇ , O-(CH ₂ ) _n -NH-C(O)-R ₇ , O-(CH ₂ ) _n -C(O)-NH-R ₇ , (CH ₂ ) _m -SO ₂ R ₇ , O-SO2R7, (CH2) _m -C(O)R ₇ and (CH2) _m -C(O)NR ₇ R _8. For cases where R1 is selected from =CH2, =CH -(CH ₂ ) _m -CH ₃ and =O, the carbon atom of the C-ring to which it is attached cannot have an optional double bond to meet the valency requirements in the molecule.For example, if R1 is =CH2 and is located at position C1 C-ring adjacent to the condensed carbon C-ring and R2 is H, then the resulting compound of formula (I) can be represented as

Preferably R1 is selected from R ₇ , (CH ₂ ) _m -OR ₇ , (CH ₂ ) _m -CO ₂ R ₇ , (CH2) _m -NR ₇ R ₈ , O-(CH2) _n -NR ₇ R ₈ , NH -C(O)-R ₇ , O-(CH ₂ ) _n -NH-C(O)-R ₇ , O-(CH ₂ ) _n -C(O)-NH-R ₇ , (CH ₂ ) _m -SO ₂ R ₇ , O-SO2R7, (CH ₂ ) _m -C(O)R ₇ and (CH2)mC(O)NR7R8.

Preferably R1 is selected from R ₇ , (CH ₂ ) _m -OR ₇ , (CH ₂ ) _m -CO ₂ R ₇ , (CH2) _m -NR ₇ R ₈ , O-(CH2) _n -NR ₇ R ₈ , NH -C(O)-R ₇ , O-(CH ₂ ) _n -NH-C(O)-R ₇ , O-(CH ₂ ) _n -C(O)-NH-R ₇ , (CH ₂ ) _m -C(O)R ₇ and (CH ₂ ) _m -C(O)NR ₇ R ₈ .

Preferably R1 is selected from R ₇ , OR ₇ , CO2R7, NR ₇ R ₈ , NH-C(O)-R ₇ , O-(CH2) _n -NH-C (O)-R ₇ , O-(CH ₂ ) _n -C(O)-NH-R ₇ , C(O)R ₇ and C(O)NR ₇ R ₈ .

- 7 040562

Preferably R1 is selected from R ₇ , OR ₇ , CO ₂ R ₇ , O-(CH ₂ ) _n -NH-C(O)-R ₇ , O-(CH ₂ ) _n -C(O)-NH-R ₇ , C(O)R ₇ and C(O)NR ₇ R ₈ .

Preferably R1 is selected from R ₇ , O-(CH ₂ ) _n -NH-C(O)-R ₇ and O-(CH ₂ ) _n -C(O)-NH-R ₇ .

In some embodiments, R1 is H.

R2.

R2 is selected from R9, (CH2VOR9, (CH2VCO2R9, (CH2VNR9R10, 0-(CH2)s-NR9Rio, NH-C(O)-R9, O-(CH2)sNH-C(O)-R9, O-(CH2 )s-C(O)-NH-R9, (CH2VSO2R9, O-SO2R9, (CH2)r-COR9, and (CH2)r-C(O)NR9R10.

Preferably R ₂ is selected from R ₉ , (CH ₂ ) _r -OR ₉ , (CH ₂ ) _r -CO ₂ R ₉ , (CH ₂ ) _r -NR ₉ R ₁₀ , O-(CH ₂ ) _s -NR ₉ R ₁₀ , NH-C(O)-R9, O-(CH2)s-NH-C(O)-R9, O-(CH2)sC(O)-NH-R9, (CH2X-COR9 and (CH2)tC (0)NR ₉ Rio.

Preferably R ₂ is selected from R ₉ , OR ₉ , CO ₂ R ₉ , NR ₉ R ₁₀ , NH-C(O)-R ₉ , O-(CH ₂ ) _s -NH-C(O)-R ₉ , O -(CH2)sC(O)-NH-R9, COR9 and c(o)NR9R _w .

Preferably R ₂ is selected from R ₉ , OR ₉ , CO ₂ R ₉ , O-(CH ₂ ) _s -NH-C(O)-R ₉ , O-(CH ₂ ) _s -C(O)-NH-R ₉ , COR ₉ and C(0)NR9Rio.

Preferably R ₂ is selected from R ₉ , O-(CH ₂ ) _s -NH-C(O)-R ₉ and O-(CH ₂ ) _S -C(O)-NH-R ₉ .

In some embodiments, R ₂ is H.

R3.

Preferably R ₃ is selected from H, C _1-6 -αkyl and CH ₂ Ph. Preferably R ₃ is selected from H, methyl, ethyl and CH ₂ Ph. More preferably R ₃ is selected from methyl and ethyl. More preferably R ₃ is methyl.

R4.

R4 is selected from phenyl and C _5-9 heteroaryl groups optionally substituted with up to three optional substituent groups. Therefore, any of the phenyl group or C _5-9 heteroaryl groups selected for R4 may optionally be substituted with up to three optional substituent groups.

Preferably R4 is selected from phenyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzoxazolyl and benzothiazolyl, optionally substituted with up to three optional substituents chosen from OH, C _1-6 -αkyl, OC _1-6 -αkyl, (CH ₂ )j-CO ₂ R ₁₁ , O-(CH2>k-NR _n R12, (CH2)j-NR _π R12, C( =O)-NH-(CH2)k-NR _n R12, C(=O)-NH-R24 and C(=O)-NH-(CH2)kC(=NH)NR _n R12.

Preferably R4 is selected from phenyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzoxazolyl and benzothiazolyl, optionally substituted with one or two optional substituent groups selected from from OH, C _1-6 -αkyl, OC _1-6 -αkyl, (CH2) _j -CO2Rii, O-(CH2)k-NRnR12, (CH2)j-NR11R12, C(=O)-NH-(CH2 )k-NRnR12; C(=O)-NH-R24 and C(=O)-NH(CH2)k-C(=NH)NR _n R12.

Preferably R4 is selected from phenyl, N-methylpyrrolyl, thiophenyl, N-methylimidazolyl, oxazolyl, thiazolyl, benzothiophenyl, N-methylbenzoimidazolyl and benzothiazolyl, optionally substituted with one or two optional substituent groups selected from OH, C _1-6 -αkyl, OC1- 6-αkyl, (C^j-^RnO-^k-NRnRn, (C^j-NRnRn, C(=O)-NH-(CH2)k-NR _n R12, C(=O)-NH-R24 and C(=O)-NH-(CH2)kC(=NH)NR11R _n .

Preferably R4 is optionally substituted with up to three optional substituents selected from OH, C1-6-αkyl, O^-alkyl, (CHU^Kc, O-(CH2)k-NH2, (CH2)j-NH2, C(=O) -NH-(CH2)k-NH2, C(=O)-NH-R24 and C(=O)-NH-(CH2)k-C(=NH)NH2.

Preferably R4 is optionally substituted with C(=O)-NH-R ₂₄ , where R24 is -C ₆ H ₄ -(CH ₂ )jR ₁₈ and the phenylene group -C ₆ H4- is para-substituted.

Preferably R4 is optionally substituted with up to three optional substituents selected from OH, methyl, ethyl, OCH3, OCH2CH3, CO2H, CO2CH3, CO2CH2CH3, O-(CH2)k-NH2 and (CH2)j-NH2.

Preferably R4 is optionally substituted with one or two optional substituent groups.

More preferably R4 is optionally substituted with one optional substituent group.

More preferably R4 is selected from

where Z1 is selected from NH, N-CH ₃ , S and O;

Z ₂ is selected from CH and N;

Z ₃ is selected from S and O;

Z4 is selected from CH and N;

R22 is selected from (CH2)jCO2R11, (C^NR-nR^ and C(=O)-NH-C6H4-(CH2)j-R18;

R ₁₈ is selected from CO ₂ Rh and NRnR ₁₂ ;

- 8 040562 j is selected from an integer from 0 to 6;

And ₁₁ and R12 are independently selected from H and C _1-6 alkyl; And

R23 is selected from H and C _1-6 alkyl.

The wavy line indicates the point of attachment of the above ID group to the remainder of the compound of formula (I). More preferably AND ₄ is selected from

where Z ₁ is selected from NH, N-CH3, S and O;

Z2 is selected from CH and N;

Z3 is selected from S and O;

Z4 is selected from CH and N;

And ₁₁ is selected from H and C _1-6 alkyl; And

R23 is selected from H and C _1-6 alkyl.

And ₅ and Ib.

Preferably for (iii) And ₅ is H and And ₆ is OC _1-6 alkyl selected from O-CH3 and O-CH2CH3.

More preferably (i) And ₅ and I6 together form a double bond.

I7.

Preferably, And ₇ is selected from H, C _1-12 -alkyl, C _5-9 -heteroaryl, C _6-15 -heteroarylalkyl, phenyl, benzyl and phenethyl, where the heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with up to three groups selected from C _1-6 -alkyl, OH, OS _1-6 -alkyl.

Preferably, And ₇ is selected from H, C _1-12 -alkyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, indolyl, N-methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N- methylbenzoimidazolyl, benzoxazolyl, benzothiazolyl, pyrrol-3-ylmethyl, pyrrol-4-ylmethyl, imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen3-ylmethyl, furan-3-ylmethyl, phenyl, benzyl and phenethyl, where heteroaryl, heteroarylalkyl , phenyl and aralkyl groups are optionally substituted with up to three groups selected from C _1-6 alkyl, OH, OC _1-6 alkyl.

Preferably, And ₇ is selected from H, C _1-6 -alkyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, indolyl, N-methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N- methylbenzoimidazolyl, benzoxazolyl, benzothiazolyl, pyrrol-3-ylmethyl, pyrrol-4-ylmethyl, imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen3-ylmethyl, furan-3-ylmethyl, phenyl, benzyl and phenethyl, where heteroaryl, heteroarylalkyl , phenyl and aralkyl groups are optionally substituted with up to three groups selected from C _1-6 alkyl, OH, OC _1-6 alkyl.

Preferably And ₇ is selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl , pyridyl, indolyl, N-methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzoxazolyl, benzothiazolyl, pyrrol-3-ylmethyl, pyrrol-4ylmethyl, imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen-3-ylmethyl , furan-3-ylmethyl, phenyl, benzyl and phenethyl, optionally substituted with up to three groups selected from C _1-6 alkyl, OH, OC _1-6 alkyl.

Preferably And ₇ is selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl , pyridyl, indolyl, N-methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzoxazolyl, benzothiazolyl, phenyl, benzyl and phenethyl, optionally substituted with up to three groups selected from C _1-6 -alkyl, OH, OC _1-6 -alkyl.

In some embodiments, H7 is selected from H, methyl, ethyl, n-propyl, isopropyl, nbutyl, sec-butyl, isobutyl, t-butyl.

Preferably, And ₉ is selected from H, C _1-12 -alkyl, C _5-9 -heteroaryl, C _6-15 -heteroarylalkyl, phenyl, benzyl and phenethyl, where heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optionally substituted with up to three groups selected from C _1-6 -alkyl, OH, OS _1-6 -alkyl.

Preferably, And ₉ is selected from H, C _1-12 -alkyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, indolyl, N-methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N- methylbenzoimidazolyl, benzoxazolyl, benzothiazolyl, pyrrol-3-ylmethyl, pyrrol-4-ylmethyl, imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen3-ylmethyl, furan-3-ylmethyl, phenyl, benzyl and phenethyl, where heteroaryl, heteroarylalkyl , phenyl and aralkyl groups are optionally substituted with up to three groups selected from C _1-6 alkyl, OH, OC _1-6 alkyl.

- 9 040562

Preferably R _g is selected from H, C1-6-alkyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, indolyl, N-methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl , benzoxazolyl, benzothiazolyl, pyrrol-3-ylmethyl, pyrrol-4-ylmethyl, imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen3-ylmethyl, furan-3-ylmethyl, phenyl, benzyl and phenethyl, where heteroaryl, heteroarylalkyl, the phenyl and aralkyl groups are optionally substituted with up to three groups selected from _C1-6 alkyl, OH, OC ₁ . ₆ -alkylα.

Preferably R9 is selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, indolyl, N-methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzoxazolyl, benzothiazolyl, pyrrol-3-ylmethyl, pyrrol-4ylmethyl, imidazol-2-ylmethyl, imidazol-4-ylmethyl, thiophen-3-ylmethyl, furan-3-ylmethyl, phenyl, benzyl and phenethyl, optionally substituted with up to three groups selected from _C1-6 -alkyl, OH, OC ₁ .6-alkyl.

Preferably R _g is selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl , pyridyl, indolyl, N-methylindolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzoxazolyl, benzothiazolyl, phenyl, benzyl and phenethyl, optionally substituted with up to three groups selected from _C1-6- alkyl, OH, _OC1-6 -alkyl .

In some embodiments, R _g is selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl.

^R 8, ^R 10, ^R 11, ^R 12, ^R 13, R 14, ^R 15, ^R 16, ^R 17, ^R 20 ^{and R} 21 ^.

Preferably, each of R ₈ , R ₁₀ , Rn, Ri2, R ₁₃ , R ₁₄ , R ₁₅ , Ri ₆ , Ri7, R ₂₀ and R2i is independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl.

Preferably, R ₈ , R ₁₀ , R 11 , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R ₂₀ and R ₂₁ are each independently selected from H, methyl and ethyl.

Preferably R ₈ is H.

Preferably R ₁₀ is H.

Preferably, each R11 is independently selected from H and methyl.

Preferably, each R ₁₂ is independently selected from H and methyl; more preferably each R ₁₂ is H.

Preferably R ₁₃ is H.

Preferably R ₁₄ is H.

Preferably R ₁₅ is H.

Preferably R ₁₆ is H.

Preferably R ₁₇ is methyl.

Preferably R20 is H.

Preferably R ₂₁ is H.

^{R18 .}

Preferably R ₁₈ is selected from CO ₂ H, CO ₂ CH ₃ , CO ₂ CH ₂ CH ₃ , NH(CH ₃ ) and NH2.

R19-

Preferably R _ig is selected from H, (CH ₂ ) _t -N(CH ₂ CH ₃ ) ₂ , (CH ₂ ) _t -N(CH ₃ ) ₂ , (CH ₂ ) _t -NH(CH ₂ CH ₃ ), ( CH2)t-NH(CH3) and (CH2)t-NH2.

More preferably R _ig is selected from H and (CH ₂ ) _t -NH ₂ .

R24 ^.

Preferably R24 is phenyl optionally substituted with up to three optional substituents selected from OH, methyl, ethyl, propyl, OCH3, OCH2CH3, CO2H, CO2CH3, CO2CH2CH3, O-(CH2) _k -NH2, O-(CH2) _k -NH (CH3), (CH2)j-NH2, (CH2)j-NH(CH3), C(=O)-NH-(CH2)k-NH2, C(=O)-NH-(CH2)k-NH (CH3), C(=O)-NH-(CH2)k-C(=NH)NH(CH3) and C(=O)-NH-(CH2)kC(=NH)NH2.

Preferably R ₂₄ is phenyl optionally substituted with up to three optional substituents selected from OH, methyl, ethyl, OCH ₃ , OCH ₂ CH ₃ , CO ₂ H, CO ₂ CH ₃ , CO ₂ CH ₂ CH ₃ , O-(CH ₂ ) _k -NH ₂ and (CH ₂ ) _j -NH ₂ .

Preferably R ₂₄ is a para-substituted phenyl group.

More preferably, in some aspects R ₂₄ is -C ₆ H ₄ -(CH ₂ )jR ₁₈ where R ₁₈ is selected from CO2R11 and NR11R12.

j.

In each case, j is independently selected from an integer between 0 and 6, hence each j is independently selected from 0, 1, 2, 3, 4, 5, and 6.

Preferably, each j is independently selected from 0, 1, 2, and 3.

More preferably, each j is independently selected from 0 and 1.

More preferably, each j is 0.

- 10 040562

k.

In each case, k is independently selected from an integer from 1 to 6, hence each k is independently selected from 1, 2, 3, 4, 5, and 6.

Preferably each k is independently selected from 1, 2 and 3.

More preferably, each k is 1. m.

m is chosen from an integer between 0 and 6, hence m is chosen from 0, 1, 2, 3, 4, 5, and 6.

Preferably m is selected from 0, 1, 2 and 3.

More preferably, m is selected from 0 and 1.

More preferably m is 0. n.

n is chosen from an integer between 1 and 6, hence n is chosen from 1, 2, 3, 4, 5, and 6.

Preferably n is selected from 1, 2 and 3.

More preferably n is 1. r.

r is chosen from an integer between 0 and 6, hence r is chosen from 0, 1, 2, 3, 4, 5, and 6.

Preferably R is selected from 0, 1, 2 and 3.

More preferably R is selected from 0 and 1.

More preferably R is 0.

s.

s is chosen from an integer between 1 and 6, hence s is chosen from 1, 2, 3, 4, 5, and 6.

Preferably s is selected from 1, 2 and 3.

More preferably s is 1.

t t is chosen from an integer between 0 and 6, hence t is chosen from 0, 1, 2, 3, 4, 5, and 6.

Preferably t is selected from 0, 1, 2 and 3.

More preferably, t is selected from 0 and 1.

More preferably, t is 0.

Y1.

Y 1 represents N or CH; preferably Y 1 is CH.

Y ₂ represents N or CH; preferably Y ₂ is CH.

x1.

Preferably X ₁ is selected from O, S, NH, CH ₂ , CH ₂ O, C(=O), C(=O)NR ₁₃ , NR ₁₃ C(=O), O-C(O) and C(O )-ABOUT.

Preferably X1 is selected from O, C(=O), C(=O)NR ₁₃ and NR ₁₃ C(=O).

More preferably X1 is selected from O, C(=O)NH and NHC(=O).

More preferably X1 is O.

Preferably X ₂ is selected from O, S, NH, CH ₂ , CH ₂ O, C(=O), C(=O)NR ₁₅ , NR ₁₅ C(=O), OC(O) and C(O)- O or missing.

Preferably X ₂ is selected from O, C(=O), C(=O)NR ₁₅ and NR ₁₆ C(=O) or absent.

More preferably X ₂ is selected from O, C(=O)NH and NHC(=O).

Preferably X ₂ is the same as X ₁ .

More preferably X ₂ is O.

L.

L is a linker group. Preferably, any of the peptide chain, alkylene chain, paraformaldehyde chain or polyethylene glycol chain is interrupted by one or more heteroatoms (eg N, O and S), and/or one or more C _5-9 heteroarylene groups (eg pyrrolylene , pyrazolylene, pyrazolylene, 1,2,3-triazolylene, pyridinylene), and/or one or more phenylene groups. More preferably, the chains may be terminated with one to three heteroatoms and/or one to three C _5-9 heteroarylene groups and/or one to three phenylene groups.

Preferably L is selected from a peptide chain having 2 to 5 amino acids, 2 to 4 amino acids, 2 to 3 amino acids; alkylene chain containing from 1 to 11 carbon atoms, from 1 to 10 carbon atoms, from 1 to 9 carbon atoms, from 1 to 8 carbon atoms, from 1 to 7 carbon atoms, from 1 to 6 carbon atoms, from 1 to 5 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3 carbon atoms, which may contain one or more double or triple carbon-carbon bonds; paraformaldehyde chain -(OCH ₂ )ii ₂ -, -(OCH ₂ )i-ii-, -(OCH ₂ )1.u-, -(OCH ₂ )i_9-, -(OCH2)i-8-, -( OCH ₂ )i.7-, -(OCH2)i-6-, -(OCH ₂ ) _1-5 -, -(OCH ₂ ) _1- 4-, -(OCH ₂ ) _1-3 -; polyethylene glycol chain -(OCH ₂ CH ₂ ) _1-5 -, chain -(OCH ₂ CH ₂ ) _1-4- , chain -(OCH ₂ CH ₂ ) _1-3 -, where the chains can be interrupted by one or more heteroatoms, and/or C _5-9 heteroarylene groups, and/or one to three phenylene groups.

More preferably, L may be selected from an alkylene chain containing from 1 to 12 carbon atoms, which may have one or more carbon-carbon double or triple bonds.

More preferably, L may be selected from CH=CH, CH ₂ , CH2CH2, CH ₂ CH ₂ CH ₂ ,

- 11 040562

CH2CH2CH2CH2 and CH2CH2CH2CH2CH2.

A.

In one embodiment, A is Al

A1 where in each group A1 one of Y3 and Y4 is independently selected from NR ₁₇ , S and O; and the other of Y3 and Y4 is CH; and Y5 is independently selected from CH, N, S and CH.

In this embodiment, when q is selected from 2, 3, 4, 5 and 6, then A will contain different A1 groups connected to each other.

Therefore, a 5-membered ring containing Y ₃ , Y 4 and Y ₅ is a heteroaryl ring. This Al group can be attached to the remainder of the molecule in any direction. Therefore, when A is A1, as in the above embodiment, the compound of formula (I) is selected from the following:

(P) _and (III).

More preferably, when A is Al, the compound of formula (I) is compound (II).

Therefore, the heteroaryl ring containing Y3, Y4 and Y5 is selected from one of the following groups:

H24.

More preferably A is

Az where Y5 is selected from CH and N.

In another embodiment, A is A2

ABOUT

A2 where in each group A2 one of Y6 and Y7 is independently selected from N and CH; and the other of Y6 and Y7 is CH.

Therefore, a 6-membered ring containing Y ₆ and Y ₇ is a phenyl or pyridinyl ring. The A2 group can be attached to the remainder of the molecule in any direction. According to the above embodiment, the compound is selected when A is A2 as in formula (I) from the following:

More preferably, when A is A2, the compound of formula (I) is compound (IV). Preferably A is A4

More preferably Y6 is CH; and Y ₇ is CH.

q.

Preferably, q is selected from 0, 1, 2, and 3. More preferably, q is 0 or 1. A 6-membered aromatic ring.

Preferably the 6-membered aromatic ring of formula (I) is para-substituted

More preferably, the 6-membered aromatic ring of formula (I) is

Optional double bonds in the C-ring.

The present invention relates to a compound of formula (I)

where dotted lines indicate the optional presence of a double bond between one or more C1 and C2; C2 and C3; and C3 and C4.

In one aspect, the compound of formula (I) contains a double bond between C1 and C2 to form a compound of formula (VI)

In another aspect, the compound of formula (I) contains a double bond between C2 and C3 to form a compound of formula (VII)

- 13 040562

In another aspect, the compound of formula (I) contains a double bond between C3 and C4 to form a compound of formula (VIII) r ₄

r ₂ o

(VIII)

In another aspect, the compound of formula (I) contains a double bond between C1 and C2 and a double bond between C3 and C4 to form a compound of formula (IX)

Other restrictions.

Options for compounds of formula (I) include the condition that when p is O and A is A1 then (a) in at least one A1 group, one of Y3 and Y ₄ is selected from S and O; or (b) in at least one group A1 Y ₅ is S; or (c) R4 is not an optionally substituted pyrrolyl or imidazolyl.

When q is chosen from 2, 3, 4, 5 and 6, there will be more than one A1 group.

Therefore, when p is 0 and A is A1, the convention requires the presence of at least one aryl group, or alternatively the presence of a heteroaryl group (either as part of A1 or R4) that does not contain a 5-membered pyrrole or imidazole ring or optionally substituted derivatives such as the N-methylpyrrole or N-methylimidazole ring. As a result, this condition excludes compounds of formula (I) having a pure long chain polypyrrole or polyimidazole or polypyrrolimidazole group attached to the PDD. Compounds having such long chain groups are generally relatively weakly cytotoxic.

In some aspects, suitable candidate compounds of formula (I) include the condition that when p is O and A is Al, then (a) the 5-membered ring in Al is selected from H9, H10, H11, H12, H13, H14, H15, H16, H17, H19, H20, H21, H22, H23 and H24; or (b) the 5-membered ring in A1 is selected from H5 and H6; or (c) R ₄ is selected from phenyl, furanyl, thiophenyl, oxazolyl, thiazolyl, pyridyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzoxazolyl and benzothiazolyl, optionally substituted with up to three optional substituents selected from OH, C _1-6 - alkyl, OC1-6-alkyl, (CH2)j-CO2R11, O-(CH2)k-NR _n R12, (CH2)j-NK _n R12, C(=O)-NH-(CH2)k-NK11R12, C(=O)-NaC6H4-(CH2)j-R18 and C(=O)-NH-(CH2)kC(=NH)NR11R12.

In some aspects, suitable candidate compounds of formula (I) include the condition that when p is O and A is Al, then (a) the 5-membered ring in Al is selected from H9, H10, H11, H12, H13, H14, H15, H16, H17, H19, H20, H21, H22, H23 and H24; or (b) the 5-membered ring in A1 is selected from H5 and H6; or (c) R4 is selected from phenyl and C ₉ heteroaryl groups optionally substituted with up to three optional substituents selected from OH, C _1-6 alkyl, OC _1-6 alkyl, (CH ₂ )j-CO ₂ R ₁₁ , O-(CH ₂ ) _k -NR ₁₁ R ₁₂ , (CH2)j-NR _n R12, C(=O)-NH-(CH2)k-NR _n R12, С(=O)-NH-C6H4- (CH2)j-R18, C(=O)-NH-(CH2)kC(=NH)NR11R12, provided that C _5-9 -heteroaryl is not indolyl.

suitable structures.

Compound of formula (I)

depicted without indicating the position of R1 and R2 on the C-ring. Therefore, R1 and R2 can be present at any position on the C-ring, as long as the valency requirements are met. Since the fused carbon and nitrogen of the C-ring have all of their substituents shown, this means that R1 and R2 can be present on any of the unfused carbons of the C-ring (i.e., at positions

C1, _C2 , C3 or _C4 as above). Suitable R1 and R2 are present on two different unfused carbons of the C-ring.

In one aspect, the compound of formula (I) is selected from the following:

In another aspect, the compound of formula (I) is selected from the following:

(hsh); And

(XIV).

More preferably, the compound of formula (I) has the following structure:

For compounds of formula (XV) wherein R1 and/or R2 are substituents other than H, the C-ring carbon atoms to which any such substituents are attached will be steric centers. In formula (XV), R1 and R2 are shown without indicating the stereochemistry of the carbon atoms on the C-ring to which they are attached.

- 15 040562

More preferably, the compound of formula (I) is selected from the following:

(XIX) where q is selected from 0, 1, 2, 3, 4, 5 or 6;

p is 0 or 1;

L is an alkylene chain containing from 1 to 12 carbon atoms;

Y 1 represents N or CH;

Y ₂ is N or CH, where at least one of Y1 and Y ₂ is CH;

Y5 is selected from CH and N;

Z ₁ is selected from O, S, NH and N-CH ₃ ;

Z ₂ is selected from CH and N;

Z ₃ is selected from S and O;

Z ₄ is selected from CH and N;

R22 is selected from (CH2)jCO2H, (CH2) _J CO2C1-6-alkyl, (C^^n^ and C(^-NH-C^C^jR^;

R ₁₈ is selected from CO2R11 and NR11R12;

R19 is selected from H and (CH ₂ ) _t -NR ₂₀ R ₂₁ ;

j and t are independently selected from an integer from 0 to 6; And

R11, R ₁₂ and R ₂₃ are independently selected from H and C1. ₆ -alkyl, and also (i) R5 and R6 together form a double bond;

(ii) R5 is H and R6 is OH; or (iii) R5 is H and R6 is OC _1-6 alkyl, provided that when the compound is (XVI) and p is 0, then Z1 is selected from O and S.

More preferably, the compound of formula (I) is selected from the following:

- 16 040562

(XXIII);

where q is selected from 0, 1, 2, 3, 4, 5 or 6;

p is 0 or 1;

L is an alkylene chain containing from 1 to 12 carbon atoms;

Y 1 represents N or CH;

Y ₂ is N or CH, where at least one of Y1 and Y ₂ is CH;

Y ₅ is selected from CH and N;

Z1 is selected from O, S, NH and N-CH ₃ ;

Z ₂ is selected from CH and N;

Z ₃ is selected from S and O;

Z ₄ is selected from CH and N;

R19 is selected from H and (CH ₂ ) _t -NR ₂₀ R ₂₁ ;

t is selected from an integer from 0 to 6;

Rn, R ₂₀ , R ₂₁ and R ₂₃ are independently selected from H and C ₁ . ₆ -alkyl;

and also (i) R ₅ and R 6 together form a double bond;

(ii) R ₅ is H and R6 is OH; or (iii) R ₅ is H and R6 is OC ₁ . ₆ -alkyl, provided that when the compound is (XX) and p is 0, then Z1 is selected from O and S.

More preferably, the compound of formula (I) is selected from the following:

(a) methyl (S)-5-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a] [ 1,4]diazepin-3 yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)benzo-[b]thiophene-2-carboxylate (13) o

(13);

(b) methyl (S)-5-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a] [ 1,4]diazepin-3 yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)benzo[b]thiophene-2-carboxylate (17) o

(17)

- 17 040562 (c) methyl (S)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[ 1,2-a][1,4]diazepin3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate (20 ) (d)

(S)-4-(4-(4-(4-((2-MemoKCu-12-oxo-6а,7,8,9,10,12-hexahydrobenzo[e]pyrido[ 1,2methyl

a] [ 1,4]diazepin-3 -yl)oxy)butanamido)-1 -methyl-1 H-pyrrole-2-carboxamido)phenyl)-1 -methyl-1 H-pyrrole2-carboxylate (24)

(e) methyl ^)-4-(4-(4-(4-((2-methoxy-12-oxo-6а,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepin3-yl)oxy)butanamido)-benzamido)phenyl)-1-methyl-III-pyrrol-2-carboxylate (28)

(f) methyl (S)-5-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a] [ 1,4]diazepin3-yl)oxy)butanamido)-benzamido)benzo[b]thiophene-2-carboxylate (30)

(g) methyl (S)-4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a] [1,4]diazepin-3yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)benzoate (34)

(h) methyl (S)-4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a] [ 1,4]diazepin-3 yl)oxy)butanamido)-1-methyl-1 H-pyrrole-2-carboxamido)benzoate (38)

(i) (S)-N-(4-aminophenyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydro- benzo[e]pyrido[1,2a] [ 1,4]diazepin-3 -yl)oxy)butanamido)-1 -methyl-1 H-pyrrole-2-carboxamido)phenyl)-1 -methyl-1 H-pyrrole2 -carboxamide (41)

- 18 040562 (j) (S)-N-(2-((4-aminophenyl)carbamoyl)benzo[b]thiophen-5-yl)-4-(4-((2-methoxy-12-oxo-6a ,7,8,9,10,

12-hexahydrobenzo[e]pyrido[1,2-а][1,4]diazepin-3-yl)oxy)-butanamido)-1-methyl-1H-pyrrole-2-carboxamide (47)

(k) Methyl 5-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)benzo[b]thiophene-2carboxylate(62)

(l) (S)-N-(4-aminophenyl)-4-(6-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1, 2-a]

[1,4]diazepin-3-yl)oxy)hexanamido)-1-methyl-1H-pyrrole-2-carboxamide (66)

(m) (S)-N-(2-aminoethyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6а,7,8,9,10,12-hexahydro- benzo[e]pyrido[1,2a] [ 1,4]diazepin-3 -yl)oxy)butanamido)-1 -methyl-1 H-pyrrole-2-carboxamido)phenyl)-1 -methyl-1 H-pyrrole2 -carboxamide (68)

In yet another aspect, a compound of formula (I) is provided.

and its salts and solvates, where dotted lines indicate the optional presence of a double bond between one or more C1 and C2; C2 and C3; and C3 and C4;

R1 is selected from R7, CH, =CH-(CH2) _w -CH3, =O, (CHV-OR7. (CH^-OD^ (CH^-NRiRfo G^CH^-NRR, NH-C(O)- R ₇ , O-(CH2) _n -NH-C(O)-R7, G-(CH2) _n -C(O)-NH-R7, (CH2VSO2R7, O-SO2R7, (CH2) _m -C(O )R7 and (CH2) _m -C(O)NR7R8;

R2 is selected from R* =CH2, =CH-(CH2) _r -CH3, =O, (CH^-OR^ (CHX-CORfc (CH ₂ ) _t -NR ₉ R ₁ o, O-(CH2) _S - NR9R _W , NH-C(O)-R9, O-(CH ₂ ) _S -NH-C(G)-R ₉ , G-(CH ₂ ) _S -C(O)-NH-R ₉ , (CH ₂ ) _r -SO ₂ R ₉ , O-SO2R9, (CH ₂ ) _r -COR ₉ and (CH2)rC(O)NR9R1o;

R ₃ is selected from H, C _1-12 -alkyl and CH ₂ Ph;

R4 is selected from phenyl and C _5-9 heteroaryl groups optionally substituted with up to three optional substituents selected from OH, C _1-6 alkyl, OC _1-6 alkyl, (CH ₂ ) _J -CO ₂ R ₁₁ , O -(CH ₂ ) _k -NR ₁₁ R ₁₂ , (CH ₂ )j-NR11R1 ₂ , C(=O)-NH-(CH2)k-NR _n R12, C(=O)-NH-C6H4-(CH2 )j-R18 and C(=O)-NH-(CH ₂ )kC(=NH)NR11R1 ₂ ;

R ₁₉ is selected from H and (CH ₂ )t-NR ₂₀ R ₂₁ ;

Y 1 represents N or CH;

Y ₂ is N or CH, where at least one of Y1 and Y ₂ is CH;

p is 0 or 1;

j, m, r and t are independently selected from an integer from 0 to 6;

k, n and s are independently selected from an integer from 1 to 6;

X1 is selected from G, S, NR13, CR13R14, CR13R14O, C(=O), C(=O)NR13, NR _n C(=O), GC(G) and C(G)-G;

- 19 040562

L is selected from an amino acid, a peptide chain having 2 to 6 amino acids, an alkylene chain having 1 to 12 carbon atoms, which may have one or more double or triple carbon-carbon bonds, a paraformaldehyde chain (OCH ₂ ) _1-12 - , polyethylene glycol chain

-(OCH ₂ CH ₂ ) _1-6 - where the chains can be interrupted by one or more groups of O, S, and/or NH, and/or

With _3-9 -heteroarylene and/or phenylene;

X2 is selected from O, S, NR15, CR15R16, CR15R16O, C(=O), C(=O)NR15, NR15C(=O), O-C(O) and C(O)-O, or absent;

q is selected from 0, 1, 2, 3, 4, 5 and 6;

And selected from

Αι _and A2.

in each group A1, one of Y3 and Y ₄ is independently selected from NR ₁₇ , S and O; and the other of Y3 and Y ₄ is CH; and Y5 is independently selected from CH, N, S and CH; and in each group A2, one of Y ₆ and Y ₇ is independently selected from N and CH; and the other of Y ₆ and Y ₇ is CH;

R ₇ and R9 are independently selected from H, C _1-12 alkyl, C _5-9 heteroaryl, C _6-15 heteroarylalkyl, phenyl and C _7-12 aralkyl groups, where heteroaryl, heteroarylalkyl, phenyl and aralkyl groups are optional substituted with up to three optional substituents selected from C _1-6 -alkyl, OH, OC _1-6 -alkylα;

R ₁₈ is selected from CO2R11 and NR11R12;

R ₈ , R ₁₀ , R11, R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R ₂₀ and R ₂₁ are independently selected from H and C _1-6 alkyl; and (i) R5 and _R6 together form a double bond; or (ii) R ₅ is H and R ₆ is OH; or (iii) R5 is H and R ₆ is OC _1-6 alkyl.

Application.

The present invention finds use in the treatment of proliferative diseases.

In some aspects, a method of treating a proliferative disease is provided, wherein the method comprises administering to a subject a therapeutically effective amount of a compound of formula (I) and a salt and solvate thereof, or a composition comprising a compound of formula (I) and a salt and solvate thereof.

In some aspects, a method of treating a proliferative disease is provided, wherein the method comprises administering to a subject a therapeutically effective amount of a targeted conjugate comprising a compound of formula (I) and salts and solvates thereof.

In some aspects, a method of treating a proliferative disease is provided, wherein the method comprises administering to a subject a therapeutically effective amount of an antibody-drug conjugate comprising a compound of formula (I) and salts and solvates thereof.

The term proliferative disease refers to unwanted or uncontrolled cellular proliferation of excess or abnormal cells that is undesirable, such as neoplastic or hyperplastic growth, either in vitro or in vivo. Examples of proliferative conditions include, but are not limited to, benign, premalignant, and malignant cell proliferation including, but not limited to, neoplasms and tumors (eg, histocytoma, glioma, astrocytoma, osteoma), cancer (eg, lung cancer, small cell lung cancer). , hepatocellular cancer, gastric or gastric cancer, including cancer of the gastrointestinal tract, bowel cancer, colon cancer, hepatoma, breast cancer, glioblastoma, cervical cancer, ovarian cancer, pharynx (or esophagus) cancer, oral cancer, prostate cancer , testicular cancer, liver cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, uterine cancer, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocarcinoma, anal carcinoma, penile carcinoma , head and neck cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemia, psoriasis, bone loss, fibroproliferative disorders (eg, connective tissue), and atherosclerosis.

The predominantly proliferative disease is selected from bladder cancer, bone cancer, bowel cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, esophageal cancer, cancer oral cavity, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, retinoblastoma, sarcoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer and uterine cancer. Preferably the proliferative disease is selected from breast cancer and cervical cancer.

Any cell type may be treated, including, but not limited to, bone, eye, head and neck, lung, gastrointestinal (including, for example, mouth, esophagus, intestine, colon,

- 20 040562 intestines), breast, cervix, ovary, uterus, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain and skin.

One skilled in the art can readily determine for any particular cell type whether or not a candidate compound can treat a proliferative condition.

Suitable subjects are humans, farm animals and pets.

In yet another aspect, the compound of formula (I) and its salts and solvates can be associated either directly or indirectly with a targeted agent (eg, antibody, antibody fragment, hormone, etc.) to obtain a targeted conjugate. The target conjugates of the present invention may contain one or more compounds of formula (I) (or their salts and solvates). Various targeted conjugates are known in the art and can be used with a compound of formula (I) and its salts and solvates. For example, in a particular aspect, the targeted conjugate is an antibody drug conjugate wherein one or more compounds of formula (I) are linked, directly or indirectly, to an antibody. Thus, the compound of formula (I), and its salts and solvates, can be used as a payload on a targeted conjugate.

Accordingly, a compound of formula (I) and its salts and solvates for use as a drug in a targeted conjugate is prepared by attaching a compound of formula (I) and its salts and solvates to the target agent, either directly or through an optional linker group. Preferably, the compound of formula (I) and its salts and solvates are attached to the target agent via a linker group. Preferably, the targeted conjugate is used to treat a disease, more specifically a proliferative disease. Preferably, the drug may be attached, via the functional group it contains, to the target agent, either directly or via a linker group. Typically, the drug contains, or may be modified to contain, one or more functional groups, such as amine, hydroxyl, or carboxylic acid groups, to attach the drug to the targeted agent, either directly or via a linker group. In some aspects, one or more atoms or groups of compounds of formula (I) may be eliminated during the binding of the drug to the antibody. In some aspects, the targeting agent binds to the cell surface of a receptor or tumor-associated antigen. In some aspects, the target agent is an antibody. In some aspects, the target agent is a hormone. In some aspects, the target agent is a protein. In some aspects, the target agent is a polypeptide. In some aspects, the targeting agent is a small molecule (eg, folic acid).

Compounds of formula (I) find use as payloads of antibodies or antibody fragments. The compounds of formula (I) are readily conjugated with antibodies or antibody fragments.

Antibody-Drug Conjugates. Antibacterial therapy has been developed for the treatment of patients with oncological, immunological and angiogenic disorders (Carter, P. (2006), Nature Reviews Immunology, 6:343-357). The use of antibody-drug conjugates (ADCs), i.e. immunoconjugates, for local delivery of cytotoxic or cytostatic agents, ie. drugs to kill or inhibit tumor cells in the treatment of cancer, targets tumor drug delivery and intracellular accumulation, while systemic administration of these unconjugated drugs can lead to unacceptable levels of toxicity to normal cells (Xie et al. (2006), Expert. Opin Biol Ther., 6(3):281-291; Kovtun et al. (2006), Cancer Res., 66(6):3214-3121; Law et al. (2006), Cancer Res., 66 (4):23282337; Wu et al. (2005), Nature Biotech., 23(9):1137-1145; Lambert J. (2005), Current Opin. in Pharmacol., 5:543-549; Hamann P. (2005), Expert Opin. Ther. Patents, 15(9):1087-1103; Payne, G. (2003), Cancer Cell, 3:207-212; Trail et al. (2003), Cancer Immunol. Immunother. , 52:328-337; Syrigos and Epenetos (1999), Anticancer Research, 19:605-614).

Thus, maximum efficiency is achieved with minimum toxicity. Efforts to develop and improve ADCs have focused on the selectivity of monoclonal antibodies (mAbs), as well as the mechanism of drug action, drug binding, drug/antibody ratio (load), and drug release properties (Junutula, et al., 2008b, Nature Biotech., 26(8):925-932; Doman et al. (2009), Blood 114(13):2721-2729; US 7521541; US 7723485; WO 2009/052249; McDonagh (2006), Protein Eng. Design & Sel., 19(7):299-307; Doronina et al. (2006), Bioconj. Chem., 17:114-124; Erickson et al. (2006), CancerRes. 66(8):1- 8 et al (2005) Clin CancerRes 11:843-852 Jeffrey et al (2005) J Med Chem 48:1344-1358 Hamblett et al (2004) Clin Cancer Res., 10:7063-7070).

In some aspects, the present invention provides a compound of formula (I) and salts and solvates thereof for use as a drug in an antibody-drug conjugate. Accordingly, a compound of formula (I) and its salts and solvates for use as a drug in an antibody-drug conjugate is prepared by attaching a compound of formula (I) and its salts and solvates to an antibody, either directly or through an optional linker group. Preferably, the compound of formula (I) and its salts and solvates are attached to the antibody via a linker group. Accordingly, the antibody-drug conjugate is for the treatment of a disease, more particularly a proliferative disease. Accordingly, the antibody-drug conjugate is for the treatment of a disease, more particularly a proliferative disease. Preferably, the drug may be attached to the antibody via the appropriate functional group it contains, either directly or via a linker group. Typically, the drug contains or may be modified to contain one or more functional groups, such as amine, hydroxyl, or carboxylic acid groups, to attach the drug to the antibody, either directly or via a linker group. In some aspects, the antibody in the antibody drug conjugate is an antibody fragment, such as, but not limited to, a single chain antibody. In some aspects, one or more atoms or groups of a compound of formula (I) may be eliminated upon attachment of a drug to an antibody. In some aspects, the antibody binds to a cell surface receptor or a tumor-associated antigen.

In some aspects, the present invention relates to the use of a compound of formula (I) and salts and solvates thereof as a drug in an antibody-drug conjugate. Accordingly, the use of a compound of formula (I) and its salts and solvates as a drug in an antibody-drug conjugate is accomplished by attaching the compound of formula (I) and its salts and solvates to the antibody, either directly or through an optional linker group. Preferably, the compound of formula (I) and its salts and solvates are attached to the antibody via a linker group. Accordingly, the antibody-drug conjugate is for the treatment of a disease, more particularly a proliferative disease. Preferably, the drug can be attached to the antibody using a suitable functional group that it contains, either directly or through a linker group. Typically, the drug contains or may be modified to contain one or more functional groups, such as amine, hydroxyl, or carboxylic acid groups, to attach the drug to the antibody, either directly or via a linker group. In some aspects, the antibody in the antibody-drug conjugate is an antibody fragment, such as, but not limited to, a single chain antibody. In some aspects, one or more atoms or groups of a compound of formula (I) may be eliminated upon attachment of a drug to an antibody. In some aspects, the antibody binds to a cell surface receptor or a tumor-associated antigen.

Substituent groups of compounds of formula (I) may interact with DNA sequences and may be chosen to target specific sequences. In particular, the following groups in the compounds of formula (I)

can be tailored to specific target sequences. Therefore, when the substituent groups are adapted in this way, the compounds of formula (I) find use in targeted chemotherapy.

Antibodies and antibody fragments.

The term antibody specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), intact antibodies, and antibody fragments as long as they exhibit the desired biological activity, such as the ability to bind the desired antigen on a target cell or tissue. The antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein produced by the immune system that is able to recognize and bind to a specific antigen (Janeway, C., Travers, P., Walport, M., Shlomchik (2001), Immunobiology, 5th ed., Garland Publishing, New York) . The target antigen typically has multiple binding sites, also called epitopes, recognized by the CDRs on the antibody. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody comprises a full length immunoglobulin molecule or an immunologically active portion of a full length immunoglobulin molecule, i. a molecule that contains an antigen binding site that immunospecifically binds an antigen of the target of interest, or a portion thereof, such targets include, but are not limited to, cancer cells or cells that produce autoimmune antibodies associated with an autoimmune disease. An immunoglobulin can be of any species (eg, IgG, IgE, IgM, IgD, and IgA), class (eg, lgG1, lgG2, lgG3, lgG4, lgA1, and 1gA2), or subclass or allotype (eg, human G1m1,

G1m2, G1m3, non-G1m1 (this is any allotype other than G1m1), G1m17, G2m23, G3m21, G3m28,

G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2,

Km1, Km2 and Km3) immunoglobulin molecules. Immunoglobulins can be obtained from any species, including human, mouse or rabbit.

As used herein, the expression binds an epitope is used to denote that an antibody binds an epitope with higher affinity than a non-specific partner such as bovine serum albumin (BSA, Genbank accession number GCBA76847, version number CAA76847.1 G1:3336842, date entry updated: January 7, 2011 02:30 PM). In some embodiments, the antibody binds an epitope with an association constant (Ka) at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 104, 105, or 106 times higher than the antibody association constant for BSA, when measured under physiological conditions.

The term antibody fragment refers to a portion of a full-length antibody, such as an antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and scFv fragments; dimers; linear antibodies; fragments produced by the Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementarity determining region), single chain antibody molecules; and multispecific antibodies formed from antibody fragments and epitope-binding fragments of any of the above, which immunospecifically bind to target antigens such as, for example, cancer cell antigens, viral antigens, or microbial antigens. The term monoclonal antibody, as used herein, refers to an antibody derived from a population of substantially homogeneous antibodies, ie. the individual antibodies that make up a population are identical except for possible naturally occurring mutations that may be present in minor amounts.

Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, unlike polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant or epitope on the antigen. In addition to their specificity, monoclonal antibodies are convenient in that they can be synthesized without containing other antibodies. The modifier monoclonal indicates the nature of the antibody, which is obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the production of antibodies in any particular way. For example, monoclonal antibodies that are used in accordance with the present invention can be obtained using the hybridoma method, first described by Kohler et al. (1975) Nature 256:495; or can be obtained by recombinant DNA methods (see US 4816567). Monoclonal antibodies can also be isolated from phage antibody libraries using the methods described by Clackson et al. (1991), Nature, 352:624-628; Marks et al. (1991), J. Mol. Biol., 222:581-597; or from transgenic mice carrying the complete human immunoglobulin system (Lonberg (2008), Curr. Opinion, 20(4):450-459).

Antibodies, including monoclonal antibodies, as used herein specifically include chimeric antibodies in which a portion of the antibody structure, such as the heavy and/or light chain, is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass. antibodies, while the rest of the chain(s) is identical or homologous to the corresponding sequences in antibodies obtained from other samples or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (US 4816567 and Morrison et al. (1984), Proc. Natl. Acad. Sci., USA, 81:6851-6855). Chimeric antibodies include primatized antibodies containing antigen-binding variable domain sequences derived from a non-human primate (eg, Old World monkey or monkey) and human constant region sequences. An intact antibody as used herein is an antibody containing VL and VH domains, as well as light chain (CL) constant domains and heavy chain constant domains, CH1, CH2 and CH3. The constant domains can be native sequence constant domains (eg, human native sequence constant domains) or amino acid sequences of variants thereof. An intact antibody may have one or more effector functions that relate to those biological activities that are inherent in the constant Fc region of the antibody (native sequence Fc region or amino acid sequence of a variant Fc region). Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors such as the B cell receptor and BCR.

The antibodies described herein may be modified. For example, to make them less immunogenic in relation to humans. This can be achieved using any of a number of methods known to those skilled in the art, such as humanization.

- 23 040562

Introduction and dose.

The compounds of formula I can be administered alone or in combination with one or another (either one or more) pharmacologically active compound, which is different from the compounds of formula I.

The compounds of the invention may be combined with various components, as appropriate, to form compositions of the invention. Preferably, the compositions are combined with a pharmaceutically acceptable carrier or diluent to provide a pharmaceutical composition that can be used in humans or animals. Suitable carriers and diluents include isotonic saline solutions, such as phosphate buffered saline. Used pharmaceutical compositions and methods for their preparation can be found in the well-known pharmaceutical literature. See, for example, Handbook for Pharmaceutical Additives, 3rd edition (eds. M. Ash and I. Ash), 2007 (Synapse Information Resources, Inc., Endicott, New York, USA); and Remington: The Science and Practice of Pharmacy, 21st edition (ed. D.B. Troy) 2006 (Lippincott, Williams and Wilkins, Philadelphia, USA), which are incorporated herein by reference.

The compounds of the invention may be administered by any suitable route. Preferably, the compounds of the invention are usually administered orally or by any parenteral route as pharmaceutical preparations containing the active ingredient, optionally as addition salts of non-toxic organic or inorganic acids or bases, in a pharmaceutically acceptable dosage form.

The compounds of the invention, their pharmaceutically acceptable salts, and pharmaceutically acceptable solvates of any type may be administered alone, but are usually administered in admixture with a suitable pharmaceutical excipient, diluent or carrier, chosen with respect to the intended route of administration and standard pharmaceutical practice.

For example, the compounds of the invention, or salts or solvates thereof, may be administered orally, buccally, or sublingually as tablets, capsules (including soft gelatin capsules), vaginal suppositories, elixirs, solutions, or suspensions, which may contain flavoring or coloring agents, for immediate use. , delayed, modified, sustained, controlled or pulsatile release. The compounds of the invention may also be administered via rapidly dispersing or rapidly dissolving dosage forms.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and some complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin and gum arabic. In addition, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type can also be used as fillers in gelatin capsules.

Preferred excipients in this case include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavoring agents, coloring or coloring agents, emulsifying and/or suspending agents and diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

Modified-release and pulsatile-release dosage forms may contain excipients, such as those detailed for immediate release dosage forms, together with additional excipients that act as release rate modifiers, which are coated and/or incorporated into the body of the device. . Release rate modifiers include, but are not limited to, hydroxypropyl methylcellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, polyethylene oxide, xanthan gum, carbomer, ammonium methacrylate copolymer, hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, methacrylic copolymer acids and their mixtures. Modified release and pulsatile dosage forms may contain one or a combination of rate-modifying excipients. The release rate modifying excipients can be present both within the dosage form, i. e. within the matrix, and/or on the dosage form, i. e. on the surface or coating.

Rapidly dispersing or dissolving dosage forms (FDDF) may contain the following ingredients: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diacorbic acid, ethyl acrylate, ethyl cellulose, gelatin, hydroxypropyl methylcellulose, magnesium stearate, mannitol, methyl methacrylate, mint flavor, polyethylene glycol, highly dispersed silica, silica, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol.

The compounds of the invention may also be administered parenterally, eg intravenously, intra-arterially, or by infusion methods. For such parenteral administration, they are best used as a sterile aqueous solution which may contain other substances, such as sufficient salts or glucose, to render the solution isotonic to blood. Aqueous solutions should be suitably buffered (preferably to pH 3 to 9) if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical methods well known to those skilled in the art.

For the mode of administration, eg oral, intravenous, etc., a suitable composition according to the invention is optimized.

Administration during treatment may be in a single dose, continuously or intermittently (eg, in divided doses at regular intervals). Methods for determining the most effective agents and doses are well known to those skilled in the art and will vary with the formulation used for treatment, the purpose of treatment, the target cell being treated, and the subject being treated. Single or multiple administration may be at the dosage level and dosage regimen selected by the attending physician, veterinarian or clinician.

Depending on the disorder and the patient to be treated, as well as the route of administration, the compositions may be administered at various dosages. For example, a typical adult dose may be 100 ng to 25 mg (preferably about 1 μg to about 10 mg) per kg of patient body weight per day.

When evaluating the starting dose for humans, suitable recommendations can be obtained from studies in test animals. For example, when a specific dose is identified for mice, a suitable starting dose for humans may be from about 0.5x to 2x the mg/kg values given to mice.

Other forms.

Unless otherwise indicated, the above includes the well-known ionic, salt, solvate and protected forms of these substituents. For example, reference to a carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-COO ^- ), its salt or solvate, as well as conventional protected forms. Similarly, reference to an amino group includes the protonated form (-N+HR1R ² ), a salt or solvate of the amino group, such as the hydrochloride salt, as well as conventional protected forms of the amino group. Similarly, reference to a hydroxyl group also includes the anionic form (-O ^- ), its salt or solvate, as well as conventional protected forms.

Isomers, salts and solvates.

Some compounds may exist in one or more particular geometric, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomeric, 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 mesoforms; D- and L-forms; d- and l-forms; (+) and (-) shapes; keto, enol and enolate forms; syn- and anti-forms; synclinal and anticlinal forms; alpha and beta forms; axial and equatorial forms; boat-, chair-, twist-, semi-cross-shaped envelopes; and combinations thereof, hereinafter collectively referred to as isomers (or isomeric forms).

It should be noted that, except as described below for tautomeric forms, specifically excluded from the term isomers as used herein are structural (or constitutional) isomers (i.e., isomers that differ in the bonding of atoms, and not just the position of atoms in space). For example, reference to the methoxy group -OCH ₃ should not be construed as referring to its structural isomer, the hydroxymethyl group, -CH2OH.

Reference to a class of structures may also include structural isomeric forms within that class (e.g., _Cl.7 -αkyl includes n-propyl and isopropyl, butyl includes n-, iso-, sec-, and tert-butyl, methoxyphenyl includes ortho-, meta- and para-methoxyphenyl).

The above exclusion does not apply to tautomeric forms, e.g. keto, enol and enolate forms, such as in the following tautomeric pairs: keto/enol, imine/enamine, amide/iminoalcohol, amidine/amidine, nitroso/oxime, thioketone/enthiol , N-nitroso/hydroxyazo and nitro/acinitro.

It should be noted that specifically included in the term isomer are compounds with one or more isotopic substituents. For example, H can be in any isotopic form, including 1H, ^2H (D), and ^3H (T), C can be in any isotopic form, including ^12C , ^13C , and ^14C , O can be in any isotopic form. , including ¹⁶ O and ¹⁸ O, etc.

Unless otherwise indicated, reference to a specific compound includes all such isomeric forms, including (in whole or in part) racemic and other mixtures thereof.

Methods for the preparation (eg, asymmetric synthesis) and separation (eg, fractional crystallization and chromatographic means) of such isomeric forms are either known in the art or can be easily accomplished by adapting in a known manner the methods described herein or known ways.

Unless otherwise indicated, reference to a specific compound also includes ionic, salt, solvate, and protected forms, for example, as described below.

In some embodiments, the implementation of the compound of formula (I) and its salts and solvates contain pharmaceutically acceptable salts of the compounds of formula (I).

The compounds of formula (I), which include the compounds specifically named above, may form pharmaceutically acceptable complexes, salts, solvates and hydrates. These salts include non-toxic acid salts (including diacids) and base salts.

If the compound is cationic or has a functional group that may be cationic (eg, -NH2 may be -NH ₃ ⁺ ), then an acid addition salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, anions derived from the following inorganic acids: hydrochloric acid, nitric acid, nitrous acid, phosphoric acid, sulfuric acid, sulphurous acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, phosphoric acid, and phosphorous acid . Examples of suitable organic anions include, but are not limited to, anions derived from the following organic acids: 2-acetylbenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, ethylenediaminetetraacetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucinic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, tartaric, toluenesulfonic and valeric. Examples of suitable polymeric organic anions include, but are not limited to, anions derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. Such salts include acetate, adipate, aspartate, benzoate, besylate, bicarbonate, carbonate, bisulfate, sulfate, borate, camsylate, citrate, cyclamate, edisylate, azilate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzat, hydrochloride/chloride. , hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfonate, naphthylate, 2-napsilate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate, hydrogen phosphate, dihydrophosphate, pyroglutamate, saccharate , stearate, succinate, tanate, tartrate, tosylate, trifluoroacetate and sinofoate.

For example, if the compound is anionic or has a functional group that may be anionic (eg, -COOH may be -COO ^- ), then the base salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, metal cations such as an alkali or alkaline earth metal cation, ammonium and substituted ammonium cations, and amines. Examples of suitable metal cations include sodium (Na ⁺ ), potassium (K ⁺ ), magnesium (Mg ²⁺ ), calcium (Ca ²⁺ ), zinc (Zn ²⁺ ) and aluminum (Al ³⁺ ) cations. Examples of suitable organic cations include, but are not limited to, ammonium ion (ie NH4 ⁺ ) and substituted ammonium ions (eg NH3R ⁺ , NH2R2 ⁺ , NHR3 ⁺ , NR4 ⁺ ). Examples of some suitable substituted ammonium ions are amine derivatives: 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 ⁺ . Examples of suitable amines include arginine, N,N'-dibenzylethylenediamine, chlorprocaine, choline, diethylamine, diethanolamine, dicyclohexylamine, ethylenediamine, glycine, lysine, N-methylglucamine, olamine, 2-amino-2-hydroxymethyl-propane-1,3-diol and procaine. For a discussion of acid and base addition salts used (see SM Berge et al., J. Pharm. Sci. (1977), 66:1-19; see also Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (2011)).

Pharmaceutically acceptable salts can be obtained in various ways. For example, a compound of formula 1 may be reacted with an appropriate acid or base to form the desired salt. It is also possible to react the precursor of the compound of formula 1 with an acid or base to remove the mobile protecting group for the acid or base group, or to open the lactone or lactam group of the precursor. It is also possible to convert a salt of a compound of formula 1 to another salt by treatment with an appropriate acid or base, or by contact with an ion exchange resin. After the reaction, the salt can then be isolated by filtration if it precipitates out of solution, or by evaporation to recover the salt. The degree of ionization of a salt can vary from fully ionized to almost non-ionized.

It may be convenient or desirable to obtain, purify and/or process an appropriate solvate of the active compound. The term solvate describes a molecular complex comprising a compound and one or more pharmaceutically acceptable solvent molecules (eg, EtOH). Ter- 26 040562 min hydrate is a solvate in which the solvent is water. Pharmaceutically acceptable solvates include compounds in which the solvent may be isotopically substituted (eg D2O, acetone-d ₆ , DMSO-d ₆ ).

The currently accepted classification system for solvates and hydrates of organic compounds is one that distinguishes between solvates and hydrates with a dedicated site, channel hydrates, or hydrates coordinated by a metal ion. See, for example, K.R. Morris (H. G. Brittain ed.), Polymorphism in Pharmaceutical Solids (1995). Solvates and hydrates with a dedicated site are those in which the solvent molecules (eg, water) are isolated from direct contact with each other through the interaction of organic compound molecules. In channel solvates, the solvent molecules are in the channels of the crystal lattice, where they are adjacent to other solvent molecules. In solvates coordinated by a metal ion, the solvent molecules are bound to the metal ion.

When the solvent or water is firmly bound, the complex will have a well-defined stoichiometry, regardless of humidity. However, when the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water or solvent content will depend on the humidity and drying conditions. In such cases, there is usually a lack of stoichiometry.

Compounds of formula I include the imine, carbinolamine and carbinolamine ester forms of PDD. Carbinolamine or carbinolamine ester is formed when a nucleophilic solvent (H ₂ O, ROH) is added via the imine bond of the PDD moiety. The balance of these equilibria between these forms depends on the conditions under which the compounds are found, as well as on the nature of the fragment itself.

These compounds can be isolated in solid form, for example by lyophilization.

Additional specific and preferred aspects are set forth in the appended independent and dependent claims. Features of dependent claims may be combined with features of independent claims, if necessary, and in combinations other than those expressly indicated in the claims.

Synthesis strategies.

The compounds of formula (I) can be obtained using the methods described below. Some schemes and examples may omit details of common reactions, including oxidation, reduction, etc., separation methods (extraction, evaporation, precipitation, chromatography, filtration, trituration, crystallization, etc.) and analytical procedures that are known to those skilled in the art. in the field of organic chemistry. Details of such reactions and methods can be found in a number of reviews, including Richard Larock, Comprehensive Organic Transformations, A Guide to Functional Group Preparations, 2nd ed (2010), in the multi-volume Compendium of Organic Synthetic Methods, edited by Michael B. Smith et al. 1974 onwards). Starting materials and reagents may be obtained from commercial sources or may be obtained using literature methods. In some reaction schemes, indications of unimportant products resulting from chemical transformations (for example, alcohol during the hydrolysis of an ester, CO2 during the decarboxylation of a diacid, etc.) may be omitted. In addition, in some cases, reaction intermediates can be used in subsequent steps without isolation or purification (ie, in situ).

In some reaction schemes and examples below, some compounds can be prepared using protecting groups that prevent unwanted chemical reaction at other reactive sites. Protecting groups can also be used to increase the solubility or otherwise change the physical properties of the compound. Discussion of protecting group strategies, description of materials and methods for maintaining and removing protecting groups, and selection of useful protecting groups for common functional groups including amines, carboxylic acids, alcohols, ketones, aldehydes, etc. (Reviewed by T.W. Greene, P.G. Wuts, Protecting Groups in Organic Chemistry, 4th edition, (2006); and P. Kocienski, Protective Groups, 3rd edition (2005)).

Typically, the chemical transformations disclosed in the description can be carried out using essentially stoichiometric amounts of reagents, although in some reactions it may be effective to use an excess of one or more reagents. In addition, many of the reactions disclosed in the description can be carried out at room temperature (RT) and ambient pressure, but depending on the reaction kinetics, yield, etc. some reactions may be carried out at elevated pressures, or higher temperatures (eg, reflux conditions) or lower temperatures (eg, -78 to 0° C.) may be used. Any reference in the description to stoichiometric range, temperature range, pH range, etc. whether directly or indirectly uses the word range also includes the specified boundary values.

Many chemical transformations may also use one or more compatible solvents, which may affect the reaction rate and yield. Depending on the nature of the reactants, one or more solvents may be polar protic solvents (including water), polar aprotic solvents, non-polar solvents, or some combination. Typical solvents include saturated aliphatic hydrocarbons (e.g. n-pentane,

- 27 040562 n-hexane, n-heptane, n-octane); aromatic hydrocarbons (eg benzene, toluene, xylenes); halogenated hydrocarbons (eg methylene chloride, chloroform, carbon tetrachloride); aliphatic alcohols (e.g. methanol, ethanol, propan-1-ol, propan-2-ol, butan-1-ol, 2-methylpropan-1-ol, butan2-ol, 2-methylpropan-2-ol, pentan-1 -ol, 3-methylbutan-1-ol, hexan-1-ol, 2-methoxyethanol, 2-ethoxyethanol, 2-butoxyethanol, 2-(2-methoxy-ethoxy)ethanol, 2-(2-ethoxyethoxy)ethanol, 2 -(2-butoxyethoxy)ethanol); ethers (e.g. diethyl ether, diisopropyl ether, dibutyl ether, 1,2-dimethoxyethane, 1,2-diethoxyethane, 1-methoxy-2-(2-methoxyethoxy)ethane, 1-ethoxy-2-(2-ethoxyethoxy) ethane, tetrahydrofuran, 1,4-dioxane); ketones (eg acetone, methyl ethyl ketone); esters (methyl acetate, ethyl acetate); nitrogen-containing solvents (eg formamide, N,N-dimethylformamide, acetonitrile, N-methylpyrrolidone, pyridine, quinoline, nitrobenzene); sulfur-containing solvents (eg carbon disulfide, dimethyl sulfoxide, tetrahydrothiophene-1,1-dioxide); and phosphorus-containing solvents (eg, hexamethylphosphoric triamide).

Additional specific and preferred aspects are set forth in the appended independent and dependent claims. Features of dependent claims may be combined with features of independent claims, if necessary, and in combinations other than those expressly indicated in the claims.

Brief description of the drawings

Embodiments of the present invention are described below with reference to the accompanying drawings, in which FIG. 1 shows an HPLC chromatogram that confirms the formation of a DNA adduct with a sequence linking the transcription factor NFkB and the C8-linked PDD monomer 13;

in fig. 2 shows an HPLC chromatogram that confirms the formation of a DNA adduct with a sequence linking transcription factor NFkB and C8-linked PDD monomer 17;

in fig. 3 shows an HPLC chromatogram that confirms the formation of a DNA adduct with a sequence linking the transcription factor NFkB and the C8-linked PDD 20 monomer;

in fig. 4 shows an HPLC chromatogram that confirms the formation of a DNA adduct with a sequence linking the transcription factor NFkB and the C8-linked PDD 24 monomer.

Examples

Basic remarks.

Synthetic building blocks and reagents were purchased from Maybridge Chemicals (UK), Fluorochem (USA), ChemShuttle Inc (USA), and Sigma-Aldrich (UK). Solvents were purchased from Sigma-Aldrich (UK) and Fisher Scientific (UK). Anhydrous reactions were carried out in pre-dried glassware under an inert nitrogen atmosphere. Anhydrous solvents were used purchased without additional drying. Thin layer chromatography (TLC) was performed on silica gel aluminum plates (Merck 60, F254) and column chromatography was performed either manually using silica gel (Merck 9385, 230-400 mesh ASTM, 40-63 μM) (while monitoring with TLC: UV (254 nm) and aqueous alkaline potassium permanganate as spots) or using the Grace Reveleris® X2 Automated Flash Chromatography System. All NMR spectra were obtained at room temperature using a Bruker DPX400 spectrometer, where chemical shifts are expressed in ppm relative to solvent and binding constants are expressed in Hz.

All liquid chromatography-mass spectroscopy (LCMS) analysis was performed on a Waters Alliance 2695 instrument with a mobile phase containing water (A) and acetonitrile (B). Formic acid (0.1%) was added to acetonitrile to maintain an acidic environment throughout the assay. Type of function: diode array (535 scans). Column type: Monolithic C18 50x4.60 mm. Mass spectrometry data was recorded using a Waters Micromass ZQ instrument connected to a Waters 2695 HPLC using a Waters 2996 PDA. Used parameters Waters Micromass ZQ: capillary (kV), 3.38; cone (B), 35; extractor (B), 3.0; Source temperature (°C), 100; desolvation temperature (°C), 200; cone flow rate (l/h), 50; gas flow rate (l/h), 250. Microwave reactions were carried out in an Anton Paar Monowave 300 microwave synthesis reactor. Output data refers to isolated material (homogeneous by TLC or NMR) unless otherwise indicated and names are given according to IUPAC nomenclature . The LCMS gradient conditions are described as follows.

Method A (10 min): 95% A/5% B to 50% B in 3 min. Then from 50% B to 80% B for 2 min. Then from 80% B to 95% B over 1.5 min and kept constant for 1.5 min. It was then reduced to 5% B over 0.2 min and kept at 5% B for 1.8 min. The flow rate was 0.5 ml/min, 200 μl was separated through the zero dead volume of the T-piece, then transferred to the mass spectrometer. The wavelength range of the UV detector was 220–400 nm.

Method B (5 min): 95% A/5% B to 90% B in 3 min. Then from 90% B to 95% B over 0.5 min and kept constant for 1 min. It was then reduced to 5% B over 0.5 min. The flow rate was 1.0 ml/min, 100 μl was separated through the zero dead volume of the T-piece, then transferred to the mass spectrometer. The wavelength range of the UV detector was 220–500 nm.

- 28 040562

General synthesis scheme.

i) K ₂ CO ₃ , DMF, methyl 4-bromobutyrate, room. pace.;

ii) KNO3, TFA, 0-5°C;

iii) KMnO ₄ , acetone, H ₂ O, reflux;

iv) oxalyl chloride, (S)-piperidin-2-ylmethanol, cat. DMF, Et ₃ N, CH ₂ Cl ₂ , 0°C room pace.;

v) H ₂ , Pd/C, EtOH;

vi) allyl chloroformate, pyridine, CH ₂ Cl ₂ , -10°C-room. pace.;

vii) TEMPO, BAIB, CH ₂ Cl ₂ , room pace.;

viii) pTSA, DHP, EtOAc, room. pace.;

ix) NaOH, dioxane, H ₂ O, room. pace.;

x) RNH2, EDCl, DMAP, DMF, room. pace.;

xi) PPh ₃ , Pd(PPh ₃ ) ₄ , pyrrolidine, CH ₂ Cl ₂ , room pace.

Example 1 Methyl 4-(4-formyl-2-methoxyphenoxy)butanoate (1).

A mixture of vanillin (20.0 g, 131 mmol), methyl 4-bromobutanoate (17.5 ml, 139 mmol) and potassium carbonate (27.2 g, 197 mmol) in N,N-dimethylformamide (100 ml) was stirred at room temperature for 18 h. The reaction mixture was diluted with water (500 mL) to give the title compound (30.2 g, 91%) by filtration as a white solid.

The product was used in the next step without any further purification.

1H NMR (400 MHz, CDCl ₃ ) δ 9.84 (s, 1H), 7.46-7.37 (m, 2H), 6.98 (d, J=8.2 Hz, 1H), 4, 16 (t, J=6.3 Hz, 2H), 3.91 (s, 3H), 3.69 (s, 3H), 2.56 (t, J=7.2 Hz, 2H), 2, 20 (quin, J=6.7 Hz, 2H);

¹³ C NMR (100 MHz, CDCl3) δ 190.9, 173.4, 153.8, 149.9, 130.1, 126.8, 111.6, 109.2, 67.8, 56.0, 51.7, 30.3, 24.2;

MS m/z (EIMS)=271.9 (M+Na)+;

LCMS (method A): tR=6.48 min.

Example 2 Methyl 4-(4-formyl-2-methoxy-5-nitrophenoxy)butanoate (2).

A solution of methyl 4-(4-formyl-2-methoxyphenoxy)butanoate (1) (20, 0 g, 79.2 mmol) in TFA (50 ml). The reaction mixture was stirred at room temperature for 1 h. It was then concentrated in vacuo and diluted with ethyl acetate (400 ml). The organic layer was washed with brine (3x100 ml) and saturated aqueous sodium hydrogen carbonate (2x80 ml), dried over sodium sulfate, filtered and concentrated to give the title

- 29 040562 compounds (23.5 g, 100%) as a yellow solid. The product was used in the next step without any further purification.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.42 (s, 1H), 7.60 (s, 1H), 7.39 (s, 1H), 4.21 (t, J=6.3 Hz , 2H), 3.98 (s,

3H), 3.70 (s, 3H), 2.61-2.53 (m, 2H), 2.22 (quin, J=6, 6Hz, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 187.8, 173.2, 153.5, 151.7, 143.8, 125.5, 109.9, 108.1, 68.6, 56.6 , 51.8, 30.2, 24.1;

MS m/z (EIMS)=296.1 (MH) ^- ;

LCMS (method A): t _R =6.97 min.

Example 3 5-Methoxy-4-(4-methoxy-4-oxobugoxy)-2-nitrobenzoic acid (3).

To a solution of methyl 4-(4-formyl-2-methoxy-5-nitrophenoxy)butanoate (2) (23.0 g, 77.4 mmol) in acetone (600 ml) was quickly added a hot (70°C) solution of potassium permanganate (46.0 g, 291 mmol) in water (400 ml). The reaction mixture was stirred at 70° C. for 3 hours. The reaction mixture was cooled to room temperature and passed through celite. The Celite layer was washed with hot water (200 ml). A solution of sodium bisulfite in HCl (200 ml) was added to the filtrate, which was extracted with dichloromethane (2x400 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica gel) eluting with methanol/dichloromethane (0 to 50%) to give the title compound (17.0 g, 70%) as a pale yellow solid.

¹ H NMR (400 MHz, MeOD) δ 7.47 (s, 1H), 7.25 (s, 1H), 4.13 (t, J=6.2 Hz, 2H), 3.94 (s, 3H), 3.68 (s, 3H), 2.54 (t, J=7.2 Hz, 2H), 2.17-2.06 (m, 2H);

¹³ C NMR (100 MHz, MeOD) δ 175.3, 168.6, 153.8, 151.3, 143.1, 122.8, 112.4, 109.2, 69.6, 57.0, 52.2, 31.2, 25.5;

MS m/z (EIMS)=311.9 (MH) ^- ;

LCMS (method A): t _R = 6.22 min.

Example 4 Methyl (S)-4-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5nitrophenoxy)butanoate (4).

4

A mixture of 5-methoxy-4-(4-methoxy-4-oxobutoxy)-2-nitrobenzoic acid (3) (8.0 g, 25.5 mmol), oxalyl chloride (6.6 ml, 77.0 mmol) and anhydrous N,N-dimethylformamideα (2 drops) in anhydrous dichloromethane (100 ml) was stirred at room temperature for 1 h. Anhydrous toluene (20 ml) was added to the reaction mixture, which was then concentrated in vacuo. To a solution of (S)-piperidin-2-ylmethanol (3.8 g, 33.4 mmol) and triethylamine (10.7 ml, 77.0 mmol) in anhydrous dichloromethane (90 ml) at -10°C was added dropwise a solution of the resulting residue in anhydrous dichloromethane (10 ml). The reaction mixture was stirred at room temperature for 2 h and then washed with hydrochloric acid (1 M, 50 ml) and brine (50 ml), dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica gel), eluting with methanol/dichloromethane (0 to 5%) to give the title compound (9.2 g, 73%) as a yellow oil.

¹ H NMR (400 MHz, CDCl3) δ 7.68-7.64 (m, 1H), 6.77-6.70 (m, 1H), 4.16-4.07 (m, 3H), 3 .93-3.89 (m, 3H), 3.83 (s, 1H), 3.67 (s, 3H), 3.15 (d, J=1.4 Hz, 1H), 3.11 ( s, 1H), 2.78 (s, 1H), 2.56-2.50 (m, 3H), 2.21-2.12 (m, 4H), 1.74-1.55 (m, 4H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 173.3, 168.1, 154.6, 148.2, 137.4, 127.6, 111.4, 108.3, 68.3, 60.6 , 56.7, 53.5, 51.7, 43.3, 38.0, 34.9, 30.3, 24.1, 19.7;

MS m/z (EIMS)=411.0 (M+H)+;

LCMS (method A): tR=6.28 min.

- 30 040562

Example 5 Methyl (S)-4-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2methoxyphenoxy)butanoate (5).

To a solution of methyl ^)-4-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5nitrophenoxy)butanoate (4) (9.2 g, 22.4 mmol) in ethanol (40 ml ) and ethyl acetate (10 ml) was added palladium on activated carbon (10 wt.% basis) (920 mg). The reaction mixture was hydrogenated at 35 psi for 3 hours on a Parr apparatus. The reaction mixture was filtered through celite and the resulting filter layer was washed with ethyl acetate. The filtrate was concentrated in vacuo to give the title compound (9.0 g, 90%) as a pink solid. The product was used in the next step without any further purification.

1H NMR (400 MHz, CDCl ₃ ) δ 6.69 (s, 1H), 6.27-6.18 (m, 1H), 4.03-3.94 (m, 3H), 3.94-3 .82 (m, 3H), 3.81-3.76 (m, 1H), 3.74 (s, 3H), 3.73-3.68 (m, 1H), 3.67-3.65 (m, 3H), 3.56 (d, J=4.8 Hz, 1H), 3.03 (s, 1H), 2.51 (t, J=7.2 Hz, 2H), 2.11 (quin, J=6.7 Hz, 2H), 1.68-1.59 (m, 4H), 1.55-1.40 (m, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 173.6, 171.2, 150.3, 141.8, 141.1, 113.2, 112.3, 102.4, 67.5, 60.8 , 60.4, 56.8, 51.6, 30.4, 25.8, 24.3, 21.0, 19.9, 14.2;

MS m/z (EIMS)=381.0 (M+H)+;

LCMS (method A): tR=5.52 min.

Example 6 Methyl (S)-4-(5-(((allyloxy)karbonyl)amino)-4-(2-(hydroxyl-methyl)n-piperidine-1carbonyl)-2-methoxyphenoxy)butanoate (6).

6

To a solution of methyl (S)-4-(5-amino-4-(2-(hydrokCimethyl)piperidine-1-karbonyl)-2methoxyphenoxy)butanoate (5) (9.0 g, 23.7 mmol) and pyridine (4 .4 ml, 54.4 mmol) in anhydrous dichloromethane (100 ml) at -10° C. was added dropwise a solution of allyl chloroformate (2.6 ml, 24.8 mmol) in anhydrous dichloromethane (20 ml). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was washed successively with saturated aqueous copper(II) sulfate (80 ml), water (80 ml) and saturated aqueous sodium hydrogen carbonate (80 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The resulting residue (2.0 g from 11.0 g crude) was purified by column chromatography (silica gel) eluting with methanol/dichloromethane (0 to 1%) to give the title compound (930 mg, 47% calculated per amount purified) in the form of yellow oil.

1H NMR (400 MHz, CDCl ₃ ) δ 8.30 (br s, 1H), 7.63 (br s, 1H), 6.76 (br s, 1H), 5.92 (ddt, J =17.2, 10.6, 5.4, 5.4 Hz, 1H), 5.37-5.28 (m, 1H), 5.20 (dKV, J=10.4, 1.3 Hz , 1H), 4.65-4.56 (m, 2H), 4.06 (t, J=6.2 Hz, 2H), 3.94-3.82 (m, 1H), 3.79 ( s, 3H), 3.66 (s, 3H), 3.62-3.54 (m, 1H), 3.40 (br s, 1H), 3.10-2.88 (m, 1H) , 2.52 (t, J=7.4 Hz, 2H), 2.22-2.04 (m, 3H), 1.64 (br s, 4H), 1.56-1.31 (m , 2H);

¹³ C NMR (100 MHz, CDCl3) δ 173.5, 170.6, 153.9, 149.7, 144.8, 132.6, 130.1, 117.6, 116.9, 110.8, 107.1, 106.0, 67.7, 65.6, 60.7, 56.3, 53.5, 51.6, 43.1, 30.5, 25.7, 24.4, 19, 7;

MS m/z (EIMS)=465.1 (M+H) ⁺ ;

LCMS (method A): t _R =6.47 min.

Example 7 Allyl (6aS)-6-hydroxy-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10hexahydrobenzo[e]pyrido[1, 2-a] [ 1,4]diazepine-5(12Н)-carboxylate (7).

7

To a solution of methyl (S)-4-(5-(((allyloxy)carbonyl)amino)-4-(2-(hydroxymethyl)-n-piperidine-1carbonyl)-2-methoxyphenoxy)butanoate (6) (930 mg, 2, 0 mmol) in dichloromethane (45 ml) was added TEMPO (32 mg, 0.20 mmol) and (diacetoxyiodine)benzene (773 mg, 2.4 mmol). The reaction mixture was stirred at room temperature for 16 h and then washed successively with saturated aqueous sodium metabisulphite (20 ml), saturated aqueous sodium hydrogen carbonate (20 ml), water (20 ml) and brine (20 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica gel) eluting with methanol/dichloromethane (0 to 5%) to give the title compound (825 mg, 89%) as an off-white solid.

MS m/z (EIMS)=462.9 (M+H)+;

LCMS (method A): t _R =6.30 min.

Example 8 Allyl (6aS)-2-methoxy-3-(4-methoxu-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-6.6a.7.8 ,9,10-hexahydrobenzo[e]pyrido[1,2-а][1,4]diazepine-5(12H)-carboxylate (8).

8

A mixture of allyl (6aS)-6-hydroxyu-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6,6a,7,8,9,10hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepine-5(12H)-carboxylateα (7) (825 mg, 1.8 mmol), 3,4-dihydro2H-pyran (1.7 ml, 18.2 mmol) and pTSA (8 .5 mg, 1% w/w) in ethyl acetate (12 ml) was stirred at room temperature for 16 h. The reaction mixture was then diluted with ethyl acetate (50 ml) and washed with saturated aqueous sodium hydrogen carbonate (20 ml) and brine solution (30 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica gel), eluting with methanol/dichloromethane (0 to 2%) to give the title compound (820 mg, 84%) as an off-white solid.

MS m/z (EIMS)=546.7 (M+H) ⁺ ;

LCMS (method A): tR=7.70 min.

Example 9 7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (9).

9

To a solution of allyl (6aS)-2-methoxy-3-(4-methoxy-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-puran-2yl)oxy)-6, 6a, 7, 8, 9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (8) (770 mg, 1.4 mmol) in 1,4-dioxane (10 ml ) was added 0.5 M aqueous sodium hydroxide solution (10 ml, 5.0 mmol). The reaction mixture was stirred at room temperature for 2 h, then concentrated in vacuo, after which water (20 ml) was added and the aqueous layer was acidified to pH with 11 M citric acid solution (5 ml). The aqueous layer was then extracted with ethyl acetate (2x50 ml). The combined organic extracts were then washed with brine (50 ml), dried over sodium sulfate, filtered and concentrated to give the title compound (700 mg, 93%) as a yellow oil. The product was used in the next step without any further purification.

MS m/z (EIMS)=532.9 (M+H) ⁺ ;

LCMS (method A): tR=6.98 min.

Example 10 Methyl 5-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2kαrboxamido)benzo[b]thiophene-2-carboxylαt (10).

NHBoc

1O

Hydrochloride 1-(3 -dimethylaminopropyl)-3ethylcarbodiimide (725 mg, 3.8 mmol) and 4-(dimethylamino)pyridine (577 mg, 4.7 mmol). The reaction mixture was stirred at room temperature for 2 hours. Methyl 5-amunobenzo[b]thiophene-2-carboxylate (392 mg, 1.9 mmol) was then added and the resulting mixture was stirred at room temperature for 16 hours. It was then poured into ice water (20 ml) and extracted with ethyl acetate (3x50 ml). The combined organic extracts were washed successively with 1 M citric acid (30 ml), saturated aqueous sodium hydrogen carbonate (35 ml), water (35 ml) and brine (35 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica gel) eluting with ethyl acetate/hexane (0 to 50%) to give the title compound (610 mg, 75%) as a beige solid.

MS m/z (EIMS)=430.2 (M+H)+;

LCMS (method A): 1r=7.90 min.

Example 11 Methyl 5-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)benzo[b]thiophene-2carboxylate hydrochloride (11).

1O 11

Methyl 5-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-npyrrol-2-carboxamido)benzo-[b]thiophene-2carboxylate (10) (610 mg, 1.4 mmol) was dissolved in hydrochloric acid (4 M in dioxane) (3.6 ml) and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo to give the title compound (600 mg, 99%) as a brown solid. The product was used in the next step without any further purification.

MS m/z (EIMS)=329.9 (M+H) ⁺ ;

LCMS (method A): t _R = 5.52 min.

Example 12 Allyl (6aS)-2-methoxy-3-(4-((5-((2-(methoxycarbonyl)benzo-[b]thiophen-5yl)carbamoyl)-1-methyl-1H-pyrrole-3- yl)amino)-4-oxo-butoxy)-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1 ,2-a][1,4]diazepine-5(12Н)-carboxylate (12).

12

In a solution of 4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)5,6,6a,7 ,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (9) (150 mg, 0.28 mmol) in N ,N-dimethylformamide (4 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (100 mg, 0.52 mmol) and 4-(dimethylamino)pyridine (80 mg, 0.65 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then methyl 5-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)benzo[b]thiophene-2-carboxylate hydrochloride (11) (95 mg, 0.26 mmol) was added and the resulting mixture was stirred at room temperature. temperature for 16 hours Then it was poured into ice water (20 ml) and was extracted with ethyl acetate (3x50 ml). The combined organic extracts were washed successively with 1 M citric acid (30 ml), saturated aqueous sodium hydrogen carbonate solution (35 ml), water (35 ml) and brine (35 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound (190 mg, 87%) as a yellow oil. The product was used in the next step without any further purification.

MS m/z (EIMS)=844.0 (M+H) ⁺ ;

LCMS (method A): t _R = 8.10 min.

Example 13 Methyl (S)-5-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2a] [1 ,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1 H-pyrrole-2-carboxamido)benzo[b]thiophene-2-carboxylate (13).

13

To a solution of allyl (6aS)-2-methoxy-3-(4-((5-((2-(methoxycarbonyl)benzo[b]thiophen-5-yl)carbamoyl)1-methyl-1H-pyrrol-3-yl )amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)- 33 040562

6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (12) (190 mg, 0.22 mmol) in dichloromethane (10 ml) was added tetrakis(triphenylphosphine)palladium(0) (13 mg, 5 mol.%), triphenylphosphine (15 mg, 25 mol.%) and pyrrolidine (22 μl, 0.27 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was placed under high vacuum for 30 min until excess pyrrolidine was completely removed. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 70%) to give the title compound (60 mg, 40%) as a yellow solid.

1H NMR (CDCl3, 400 MHz) δ 8.35 (s, 1H), 8.28 (s, 1H), 8.02 (s, 1H), 7.94 (s, 1H), 7.90 (d , J=5.7 Hz, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.58 (dd, J=8.7, 2.1 Hz, 1H), 7.42 -7.41(m, 1H), 7.13(d, J=1.6Hz, 1H), 6.78(s, 1H), 6.56(d, J=1.6Hz, 1H) , 4.25-4.18 (m, 1H), 4.08 (t, J=6.0 Hz, 2H), 3.93 (s, 3H), 3.88 (s, 3H), 3. 83 (s, 3H), 3.79-3.75 (m, 1H), 3.23-3.16 (m, 1H), 2.52-2.47 (m, 2H), 2.21 ( d, J=6.4 Hz, 1H), 2.18 (d, J=2.1 Hz, 1H), 1.96 (br s, 2H), 1.86-1.81 (m, 2H ), 1.77-1.66 (m, 2H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 170.0, 167.6, 163.4, 163.2, 160.0, 150.7, 148.0, 140.0, 139.2, 137.6 , 135.8, 134.2, 130.6, 123.0, 122.9, 121.5, 121.0, 120.1, 116.2, 111.7, 110.3, 104.3, 68 .1, 56.1, 53.5, 52.5, 49.7, 40.0, 36.8, 33.0, 24.9, 24.5, 22.9, 18.3;

MS m/z (EIMS)=658.0 (M+H)+;

LCMS (method A): t _R =6.92 min.

Example 14 Allyl (6aS)-3-(4-((2-(ethoxycarbonyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)2-methoxy-12-oxo-6- ((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2a][1,4]diazepine-5(12H)-carboxylate (14).

\\

14

In a solution of 4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)5,6,6a,7 ,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (9) (340 mg, 0.64 mmol) in N ,N-dimethylformamide (10 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (222 mg, 1.2 mmol) and 4-(dimethylamino)pyridine (177 mg, 1.4 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Ethyl 4-amino-1-methyl-1H-imidazole-2-carboxylate hydrochloride (120 mg, 0.58 mmol) was then added and the resulting mixture was stirred at room temperature for 16 hours. It was then poured into ice water (40 ml) and extracted with ethyl acetate (3x100 ml). The combined organic extracts were washed successively with 1 M citric acid (60 ml), saturated aqueous sodium hydrogen carbonate solution (70 ml), water (70 ml) and brine (70 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound (350 mg, 80%) as a yellow oil. The product was used in the next step without any further purification.

MS m/z (EIMS)=683.7 (M+H)+;

LCMS (method A): t _R = 7.35 min.

Example 15 4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-5.6, 6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl1H-imidazole-2-carboxylic acid (15).

15

To a solution of allyl (6aS)-3-(4-((2-(ethoxycarbonyl)-1-methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)2-methoxy-12-oxo-6-( (tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2a][1,4]diazepine-5(12H)-carboxylate ( 14) (350 mg, 0.46 mmol) in 1,4-dioxane (10 ml) was added 0.5 M aqueous sodium hydroxide solution (10 ml, 5.0 mmol). The reaction mixture was stirred at room temperature for 2 h and then concentrated in vacuo, after which water (20 ml) was added and the aqueous layer was acidified with 1 M citric acid solution (10 ml) to pH 1. The aqueous layer was then extracted with ethyl acetate (2x50 ml ). The combined organic extracts were then washed with brine (50 ml), dried over sodium sulfate, filtered and concentrated. The resulting residue was triturated in hexane, filtered and dried to give the title compound (220 mg, 74%) as a beige solid. The product was used on the next

- 34 040562 stage without any additional purification.

MS m/z (EIMS)=656.2 (M+H)+;

LCMS (method A): tR=6.53 min.

Example 16 Allyl (6aS)-2-methoxy-3-(4-((2-((2-(methoxycarbonyl)benzo[b]thiophen-5-yl)carbamoyl)1-methyl-III-imidazole-4- yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1, 2-a] [ 1,4]diazepine-5(12Н)-carboxylate (16).

16

Into a solution of 4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-5.6.6a ,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl1H-imidazole-2-carboxylic acid ( 15) (110 mg, 0.17 mmol) in N,N-dimethylformamide (4 ml) were placed 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (59 mg, 0.31 mmol) and 4-(dimethylamino)pyridine (47 mg, 0.38 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then methyl 5-aminobenzo[b]thiophene-2-carboxylate (32 mg, 0.15 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours. It was then poured into ice water (40 ml) and extracted with ethyl acetate (3x100 ml). The combined organic extracts were washed successively with 1 M citric acid (60 ml), saturated aqueous sodium hydrogen carbonate solution (70 ml), water (70 ml) and brine (70 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated. The resulting residue was then purified by column chromatography (silica gel) eluting with ethyl acetate/dichloromethane (0 to 100%) followed by methanol/dichloromethane (0 to 10%) to give the title compound (50 mg, 39% ) as a yellow oil.

MS m/z (EIMS)=844.9 (M+H) ⁺ ;

LCMS (method A): tR=8.22 min.

Example 17 Methyl (S)-5-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2a][1 ,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)benzo[b]thiophene-2carboxylate (17).

17

To a solution of allyl (6aS)-2-methoxy-3-(4-((2-((2-(methoxycarbonyl)benzo[b]thiophen-5-yl)carbamoyl)1 -methyl-1 H-imidazol-4- yl)amino)-4-oxobutoxy)-12-oxo-6-((teΊrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10heksahydrobenzo[e]pyrido[1,2 -a][1,4]diazepine-5(12H)-carboxylate (16) (50 mg, 0.06 mmol) in dichloromethane (3 ml) was added tetrakis(triphenylphosphine)palladium(0) (3.5 mg, 5 mol.%), triphenylphosphine (3.9 mg, 25 mol.%) and pyrrolidine (5.8 μl, 0.07 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was placed under high vacuum for 30 min until excess pyrrolidine was completely removed. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 50%) to give the title compound (10 mg, 26%) as a yellow solid.

1H NMR (CDCl3, 400 MHz) δ 9.07 (s, 1H), 8.36 (d, J=2.0 Hz, 1H), 8.13 (s, 1H), 8.03 (s, 1H ), 7.90 (d, J=5.7 Hz, 1H), 7.82 (d, J=8.7 Hz, 1H), 7.56 (dd, J=8.7, 2.1 Hz , 1H), 7.49-7.43 (m, 2H), 6.81 (s, 1H), 4.26-4.17 (m, 2H), 4.10-4.06 (m, 3H ), 3.98-3.93 (m, 6H), 3.93-3.85 (m, 1H), 3.74 (td, J=5.8, 4.0 Hz, 1H), 3, 27-3.16 (m, 1H), 2.68-2.60 (m, 2H), 2.29 (quin, J=6.4 Hz, 2H), 2.10-2.02 (m, 1H), 1.97-1.89 (m, 1H), 1.83-1.77 (m, 2H), 1.76 (s, 2H);

¹³ C NMR (100 MHz, CDCl3) δ 169.7, 167.5, 163.3, 163.2, 160.3, 156.7, 150.4, 148.0, 140.0, 139.3, 135.8, 135.0, 130.6, 123.2, 120.1, 115.4, 114.9, 110.3, 98.0, 67.8, 65.2, 56.1, 52, 6, 49.6, 39.8, 35.9, 32.9, 31.0, 29.3, 24.7, 24.6, 22.9, 18.4;

MS m/z (EIMS)=659.1 (M+H) ⁺ ;

LCMS (method A): tR=7.00 min.

- 35 040562

Example 18 Methyl 4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxylate (18).

Methyl 4-bromo-1-methyl-1H-pyrrole-2-carboxylate mixture (750 mg, 3.44 mmol), 4-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl) aniline (905 mg, 4.13 mmol) and potassium carbonate (1.43 g, 10.3 mmol) in toluene/ethanol/water (9:3:1) (total 13 mL) were degassed with nitrogen for 5 minutes. Tetrakis(triphenylphosphine)palladium(0) (230 mg, 6 mol%) was then charged and the reaction mixture was subjected to microwave radiation at 100° C. for 15 min. Then water (10 ml) was added to the reaction mixture, extracted with ethyl acetate (3x40 ml). The combined organic extracts were then dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica gel) eluting with ethyl acetate/hexane (0 to 50%) to give the title compound (145 mg, 18%) as a yellow solid.

MS m/z (EIMS)=230.9 (M+H)+;

LCMS (method A): t _R =5.17 min.

Example 19 Allyl (6S,6aS)-2-methoxy-3-(4-((2-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3yl)phenyl)carbamoyl)-1 -methyl-1H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2Hpyran-2-yl)oxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a] [1,4]diazepine-5(12Н)-carboxylate (19).

In a solution of 4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran2-yl)oxy)-5.6.6a ,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)1-methyl-1H-imidazole-2-carboxylic acid (15) (110 mg, 0.17 mmol) in N,N-dimethylformamide (4 ml) were placed 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (59 mg, 0.31 mmol) and 4-(dimethylamino) pyridine (47 mg, 0.38 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then methyl 4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxylate (18) (35 mg, 0.15 mmol) was added and the resulting mixture was stirred at room temperature for 16 h.

Then it was poured into ice water (40 ml) and extracted with ethyl acetate (3x100 ml). The combined organic extracts were washed successively with 1 M citric acid (60 ml), saturated aqueous sodium hydrogen carbonate solution (70 ml), water (70 ml) and brine (70 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated. The resulting residue was then purified by column chromatography (silica gel) eluting with cetone/dichloromethane (0 to 50%) to give the title compound (54 mg, 37%) as a yellow oil.

MS m/z (EIMS)=868.1 (M+H) ⁺ ;

LCMS (method A): tR=8.22 min.

Example 20 Methyl (S)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2a ] [ 1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)phenyl)-1-methyl-1Hpyrrole-2-carboxylate (20).

MeO ₂ C

20

To a solution of allyl (6S,6aS)-2-methoxy-3-(4-((2-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3yl)phenyl)carbamoyl)-1 - methyl-1 H-imidazol-4-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H

- 36 040562 pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-5(12H)-carboxylate ( 19) (54 mg, 0.06 mmol) in dichloromethane (3 ml) was added tetrakis(triphenylphosphine)palladium(0) (3.6 mg, 5 mol.%), triphenylphosphine (4.1 mg, 25 mol.%) and pyrrolidine (6.2 µl, 0.07 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was placed under high vacuum for 30 min until excess pyrrolidine was completely removed. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 50%) to give the title compound (22 mg, 52%) as a yellow solid.

¹ H NMR (CDCl ₃ , 400 MHz) δ 8.95 (s, 1H), 8.27 (s, 1H), 7.89 (d, J=5.7 Hz, 1H), 7.59 (d , J=8.6 Hz, 2H), 7.47-7.41 (m, 4H), 7.19 (d, J=2.0 Hz, 1H), 7.05 (d, J=1, 9 Hz, 1H), 6.79 (s, 1H), 4.25-4.18 (m, 1H), 4.17-4.12 (m, 1H), 4.12-4.06 (m , 1H), 4.04 (s, 3H), 3.95 (s, 3H), 3.93 (s, 3H), 3.84 (s, 3H), 3.76-3.71 (m, 1H), 3.26-3.16 (m, 1H), 2.65-2.57 (m, 2H), 2.26 (t, J=6.4 Hz, 2H), 2.09-2 .01 (m, 2H), 1.96-1.89 (m, 1H), 1.85-1.77 (m, 2H), 1.67 (dd, J=10.9, 5.5 Hz , 1Н);

¹³ C NMR (100 MHz, CDCl3) δ 169.7, 167.5, 163.3, 161.7, 156.5, 150.4, 148.0, 140.0, 135.8, 135.6, 133.7, 130.6, 126.1, 125.5, 123.1, 122.8, 120.0, 114.6, 111.6, 110.2, 67.8, 56.1, 51, 2, 49.6, 39.8, 37.0, 35.8, 32.8, 31.0, 29.7, 24.7, 24.5, 22.9, 18.4;

MS m/z (EIMS)=682.1 (M+H)+;

LCMS (method A): tR=7.03 min.

Example 21 Allyl (6aS)-2-methoxy-3-(4-((5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)amino)-4oxobutoxy)-12-oxo-6-( (tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2a] [1,4]diazepin-5(12H)-carboxylate ( 21).

Chemical formula: C34H44N4O10

Molecular weight: 668.74

21

In a solution of 4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)5,6,6a,7 ,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (9) (150 mg, 0.64 mmol) in N ,N-dimethylformamide (4 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (98 mg, 0.51 mmol) and 4-(dimethylamino)pyridine (79 mg, 0.64 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then 4-amino-1-methyl-1H-pyrrole-2-carboxylate methyl hydrochloride (49 mg, 0.26 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours. It was then poured into ice water (40 ml ) and extracted with ethyl acetate (3x100 ml). The combined organic extracts were washed successively with 1 M citric acid (60 ml), saturated aqueous sodium hydrogen carbonate solution (70 ml), water (70 ml) and brine (70 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound (150 mg, 88%) as a yellow oil. The product was used in the next step without any further purification.

MS m/z (EIMS)=668.8 (M+H)+;

LCMS (method A): tR=7.42 min.

Example 22 4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-5.6, 6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a] [1,4]diazepin-3-yl)oxy)butanamido)-1-methyl1H-pyrrole-2-carboxylic acid (22).

Chemical formula: Ο ₃₃ Η ₄₂ Ν ₄ Ο _ια

Molecular weight: 654.72

22

To a solution of allyl (6aS)-2-methoxy-3-(4-((5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)amino)-4oxobutoxy)-12-oxo-6-(( tetrahydro-2H-pyran-2-yl)oxy)-6,6а,7,8,9,10-hexahydrobenzo[e]pyrido[1,2- 37 040562

a][1,4]diazepine-5(12H)-carboxylate (21) (150 mg, 0.22 mmol) in 1,4-dioxane (5 ml) was added 0.5 M aqueous sodium hydroxide solution (5 ml, 5.0 mmol). The reaction mixture was stirred at room temperature for 2 h, then concentrated in vacuo, after which water (20 ml) was added and the aqueous layer was acidified with 1 M citric acid solution (10 ml) to pH 1. The aqueous layer was then extracted with ethyl acetate (2x50 ml) . The combined organic extracts were then washed with brine (50 ml), dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound (140 mg, 90%) as a beige solid. The product was used in the next step without any further purification.

MS m/z (EIMS)=677.0 (M+Na)+;

LCMS (method A): t _R =6.92 min.

Example 23 Allyl (6S,6aS)-2-methoxy-3-(4-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3yl)phenyl)carbamoyl)-1 -methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran2-yl)oxy)-6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-а][1,4]diazepine-5(12Н)-carboxylate (23).

Into a solution of 4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-5.6.6a ,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl1H-pyrrole-2-carboxylic acid ( 22) (140 mg, 0.21 mmol) in N,N-dimethylformamide (4 ml) were placed 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (74 mg, 0.39 mmol) and 4-(dimethylamino)pyridine (59 mg, 0.48 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Methyl 4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxylate (18) (45 mg, 0.19 mmol) was then added and the resulting mixture was stirred at room temperature for 16 hours. It was then poured into ice water (40 ml) and extracted with ethyl acetate (3x100 ml). The combined organic extracts were washed successively with 1 M citric acid (60 ml), saturated aqueous sodium hydrogen carbonate solution (70 ml), water (70 ml) and brine (70 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 50%) to give the title compound (160 mg, 95%) as a yellow solid.

MS m/z (EIMS)=867.0 (M+H)+;

LCMS (method A): t _R = 8.10 min.

Example 24 Methyl (S)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2a ][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole2-carboxylate (24).

24

To a solution of allyl (6S,6aS)-2-methoxy-3-(4-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3yl)phenyl)carbamoyl)-1 - methyl-1 H-pyrrol-3 -yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran2-yl)oxy)-6,6a,7,8,9,10- tetrakis(triphenylphosphine)palladium( 0) (5.3 mg, 5 mol%), triphenylphosphine (6.1 mg, 25 mol%) and pyrrolidine (9.1 µl, 0.11 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was placed under high vacuum for 30 min until excess pyrrolidine was completely removed. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 50%) to give the title compound (23 mg, 37%) as a yellow solid.

- 38 040562

1H NMR (CDCl ₃ , 400 MHz) δ 8.09 (s, 1H), 8.04-8.01 (m, 1H), 7.90 (d, J=5.8 Hz, 1H), 7, 58 (s, 1H), 7.56 (s,

1H), 7.44-7.40 (m, Zn), 7.18 (d, J=2.0 Hz, 1H), 7.12 (d, J=1.8 Hz, 1n), 7, 04 (d, J=2.0 Hz, 1n), 6.78 (s, 1n), 6.50 (d, J=1.9 Hz, 1H), 4.26-4.18 (m, 1H ), 4.07 (t, J=6.0 Hz, 2H), 3.94 (s, 3H), 3.87 (s, 3H), 3.84 (d, J=2.9 Hz, 6H ),

3.76 (td, J=5.7, 3.9 Hz, 1H), 3.25-3.15 (m, 1H), 2.49 (t, J=7.0 Hz, 2H), 2 .24-2.18 (m, 2H), 2.10-2.03 (m, 1H),

2.01-1.93 (m, 2H), 1.86-1.80 (m, 2H), 1.73-1.66 (m, 1H);

¹³ C NMR (100 MHz, CDCl ₃ ) δ 169.9, 167.6, 163.5, 161.7, 159.7, 150.7, 147.9, 139.9, 136.4, 130.2 , 126.1, 125.4, 123.3, 123.0, 120.6, 119.8, 114.6, 111.7 110.2, 103.9, 68.1, 56.1, 53, 8, 51.2, 49.7, 39.9, 37.0, 36.7, 33.0,

31.0, 29.3, 24.9, 24.5, 22.9, 18.4;

MS m/z (EIMS)=681.0 (M+H)+;

LCMS (method A): t _R =6.98 min.

Example 25 Allyl (6aS)-2-methoxy-3-(4-((4-(methoxycarbonyl)phenyl)amino)-4-oxobutoxy)-12oxo-6-((tetrahydro-2H-pyran-2-yl )hydroxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine 5(12H)-carboxylate (25).

25

Into a solution of 4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-5,6,6a,7, 8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (9) (200 mg, 0.376 mmol) in anhydrous dichloromethane (5 ml) was placed N-[(dimethylamino)1H-1,2,3-triazolo-[4,5-b]-pyridin-1-ylmethylene]-N-methylmethanaminium N-oxide hexafluorophosphate (150 mg, 0.394 mmol) and anhydrous triethylamine (220 µl, 1.58 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then methyl 4-aminobenzoate (57.0 mg, 0.376 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate (20 ml) and extracted with dichloromethane (2x50 ml). The combined organic extracts were washed with water containing a few drops of acetic acid (30 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by column chromatography (silica gel) eluting with methanol/dichloromethane (0 to 10%) to give the title compound (110 mg, 44%) as a yellow solid.

MS (ES+): m/z=666 (M+H)+;

LCMS (method A): t _R = 7.88 min.

Example 26 4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-5.6, 6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)benzoic acid (26).

26

To a solution of allyl (6aS)-2-methoxy-3-(4-((4-(methoxycarbonyl)phenyl)amino)-4-oxobutoxy)-12oxo-6-((tetrahydro-2H-pyran-2-yl)oxy )-6,6a,7,8,9,10-hexahydrobenzo-[e]pyrido[1,2-a][1,4]diazepine 5(12H)-carboxylate (25) (90 mg, 0.14 mmol) in 1,4-dioxane (2.5 ml) was added an aqueous solution of sodium hydroxide (0.5 M, 2.5 ml, 1.3 mmol). The reaction mixture was stirred at room temperature for 16 h and then concentrated in vacuo, after which water (20 ml) was added and the aqueous layer was acidified to pH 1 with aqueous citric acid (1 M, 10 ml). The aqueous layer was then extracted with ethyl acetate (2x50 ml). The combined organic extracts were then washed with brine (50 ml), dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound (86 mg, 98%) as an off-white solid. The product was used in the next step without any further purification.

MS (ES+): m/z=652 (M+H)+;

LCMS (method A): tR=7.13 min.

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Example 27 Allyl (6aS)-2-methoxyu-3-(4-((4-((4-(5-(methoxycarbonyl)-1-methyl-III-pyrrol-3yl)phenyl)carbamoyl)phenyl)amino) -4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a] [1,4]diazepine-5(12Н)-carboxylate (27).

27

Into a solution of 4-(4-(((6aS)-5-((αlyloxu)carbonyl)-2-methoxu-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxu)5.6, 6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)benzoic acid (26) (40 mg, 0.061 mmol ) in anhydrous dichloromethane (1 ml) was placed N-[(dimethylamino)1H-1,2,3-triazolo-[4,5-b]-pyridin-1-ylmethylene]-N-methylmethanaminium N-oxide hexafluorophosphate (25 mg , 0.064 mmol) and anhydrous triethylamine (36 µl, 0.26 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then methyl 4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxylate (18) (14 mg, 0.061 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated aqueous solution sodium bicarbonate (20 ml) and extracted with dichloromethane (2x50 ml). The combined organic extracts were washed with water containing a few drops of acetic acid (30 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by column chromatography (silica gel) eluting with methanol/dichloromethane (0 to 10%) to give the title compound (43 mg, 63%) as a yellow oil.

MS (ES+): m/z=864 (M+H) ⁺ ;

LCMS (method A): tR=8.10 min.

Example 28 Methyl (S)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2a ][1,4]diazepin-3-yl)oxy)butanamido)-benzamido)phenyl)-1-methyl-1H-pyrrol-2-carboxylate (28).

28

To a solution of allyl (6aS)-2-methoxy-3-(4-((4-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3yl)phenyl)carbamoyl)phenyl)amino)- 4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10hexahydrobenzo[e]pyrido[1,2-a][1 ,4]diazepine-5(12H)-carboxylate (27) (33 mg, 0.038 mmol) in dichloromethane (3 ml) was added tetrakis-(triphenylphosphine)palladium(0) (2.2 mg, 5 mol.%), triphenylphosphine (2.5 mg, 25 mol%) and pyrrolidine (4 µl, 0.11 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was placed under high vacuum for 30 min until excess pyrrolidine was completely removed. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 100%) to give the title compound (5.8 mg, 21%) as a yellow solid.

1H NMR (CDCI3, 400 MHz) δ 8.14 (br s, 1H), 8.06 (br s, 1H), 7.91 (d, J=5.7 Hz, 1H), 7.81 (d, J=8.7 Hz, 2H), 7.65 (d, J=8.6 Hz, 2H), 7.60 (d, J=8.4 Hz, 2H), 7.46-7 .50 (m, 2H), 7.41 (s, 1H), 7.21 (d, J=2.1 Hz, 1H), 7.08 (d, J=1.9 Hz, 1H), 6 .78-6.82 (m, 1H), 4.24 (d, J=14.0 Hz, 1H), 4.11-4.18 (m, 2H), 3.95-3.98 (m , 3H), 3.83-3.86 (m, 6H), 3.74-3.79 (m, 2H), 3.18-3.30 (m, 2H), 2.60-2.66 (m, 2H), 2.28 (t, J=6.3 Hz, 2H), 1.97 (d, J=6.3 Hz, 2H), 1.82-1.88 (m, 2H) ;

¹³ C NMR (100 MHz, CDCI3) δ 215.5, 171.1, 167.5, 165.0, 163.4, 161.7, 150.3, 147.8, 141.3, 140.0, 136.2, 130.8, 128.1, 125.6, 123.5, 123.1, 121.5, 120.6, 119.3, 114.7, 111.7, 110.2, 67, 9, 56.1, 51.2, 49.7, 39.8, 37.0, 34.3, 30.9, 25.6, 24.5, 23.0, 18.4;

MS (ES+): m/z=678 (m+H) ⁺ ;

LCMS (method A): tR=7.05 min.

- 40 040562

Example 29 Allyl (6aS)-2-methoxy-3-(4-((4-((2-(methoxycarbonyl)benzo-[b]thiophen-5yl)carbamoyl)phenyl)amino)-4-oxobutoxy)-12 -oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10hexahydrobenzo[e]pyrido [1,2-a]-[1,4]diazepin- 5( 12Н)-carboxylate (29).

29

Into a solution of 4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-5.6.6a ,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)benzoic acid (26) (40mg, 0.0 61 mmol) in anhydrous dichloromethane (1 ml) was placed N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]-pyridin-1-ylmethylene]-N-methylmethanaminium N-oxide (25 mg, 0.064 mmol) and anhydrous triethylamine (36 µl, 0.26 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Methyl 5-aminobenzo[b]thiophene-2carboxylate (13 mg, 0.063 mmol) was then added and the resulting mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate (20 ml) and extracted with dichloromethane (2x50 ml) . The combined organic extracts were washed with water containing a few drops of acetic acid (30 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by column chromatography (silica gel), eluting with methanol/dichloromethane (0 to 10%) to give the title compound (34 mg, 45%) as a brown oil.

MS (ES+): m/z=841 (M+H)+;

LCMS (method A): tR=8.15 min.

Example 30 Methyl (S)-5-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2a][1 ,4]diazepin-3-yl)oxy)butanamido)-benzamido)benzo[b]thiophene-2-carboxylate (30).

thirty

To a solution of allyl (6aS)-2-methoxy-3-(4-((4-((2-(methoxycarbonyl)benzo[b]thiophen-5yl)carbamoyl)phenyl)amino)-4-oxobutoxy)-12-oxo -6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-5(12H )-carboxylate (29) (23 mg, 0.028 mmol) in dichloromethane (1.5 ml) was added tetrakis(triphenylphosphine)palladium(0) (1.6 mg, 5 mol%), triphenylphosphine (1.8 mg, 25 mol %) and pyrrolidine (3.0 µl, 0.11 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was placed under high vacuum for 30 min until excess pyrrolidine was completely removed. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 100%) followed by methanol/dichloromethane (0 to 100%) to give the title compound (5.4 mg, 30%) as a pink solid.

¹ H NMR (CDCl3, 400 MHz) δ 8.48 (br s, 1H), 8.39 (d, J=1.9 Hz, 1H), 8.31 (s, 1H), 7.97 ( s, 1H), 7.91 (d, J=5.8 Hz, 1H), 7.77-7.84 (m, 3H), 7.65 (dd, J=8.8, 2.0 Hz , 1H), 7.57 (d, J=8.6 Hz, 2H), 7.38 (s, 1H), 6.79 (s, 1H), 4.24 (dt, J=13.7, 4.1 Hz, 1H), 4.09-4.17 (m, 2H), 3.95 (s, 3H), 3.79-3.82 (m, 3H), 3.74-3.79 (m, 1H), 3.49 (d, J=3.9 Hz, 1H), 3.29-3.41 (m, 1H), 3.17-3.28 (m, 1H), 2, 58-2.64 (m, 2H), 2.26 (quin, J=6.2 Hz, 2H), 2.05-2.13 (m, 1H), 1.92-2.01 (m, 1H), 1.83-1.87 (m, 1H), 1.07-1.19 (m, 1H);

¹³ C NMR (CDCls, 100 MHz) δ 171.2, 167.5, 165.5, 163.4, 163.2, 150.4, 147.8, 141.5, 140.0, 139.3, 138.0, 135.1, 134.4, 130.6, 128.2, 123.0, 121.4, 120.9, 119.2, 116.4, 111.7, 110.1, 67, 9, 56.0, 52.6, 49.7, 39.8, 34.2, 30.9, 24.7, 24.5, 22.9, 18.3;

MS (ES+): m/z=655 (M+H)+;

LCMS (method A): tR=7.00 min.

Example 31 Methyl 4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-imidazole-2carboxamido)benzoate (31).

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N-[(dimethylamino)-1H- 1,2,3triazolo-[4,5-b]n-pyridin-1-ylmethylene]-N-methyl-methanaminium N-oxide (165 mg, 0.435 mmol) and anhydrous triethylamine (242 μl, 1.74 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then methyl 4-aminobenzoate (63 mg, 0.42 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate (20 ml) and extracted with dichloromethane (2x50 ml). The combined organic extracts were washed with water containing a few drops of acetic acid (30 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by column chromatography (silica gel) eluting with methanol/dichloromethane (0 to 10%) to give the title compound (40 mg, 26%) as an off-white solid.

¹ H NMR (CDCl3, 400 MHz) δ 9.16 (s, 1H), 8.01-8.07 (m, 2H), 7.69-7.75 (m, 2H), 7.21 (br s, 1H), 6.84 (s, 1H), 4.07 (s, 3H), 3.92 (s, 3H), 1.53 (s, 9H);

MS (ES-): m/z=373 (M-H) ^- ;

LCMS (method A): tR=7.68 min.

Example 32 Methyl 4-(4-amino-1-methyl-1H-imidazole-2-carboxamido)benzoate hydrochloride (32).

Methyl 4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-imidazole-2-carboxamido)benzoate (31) (40 mg, 0.11 mmol) was dissolved in hydrochloric acid (4 M in 1, 4-dioxane) (2 ml) and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo to give the title compound (33 mg, 99%) as a brown solid. The product was used in the next step without any further purification.

1H NMR (MeOD, 400 MHz) δ 7.89-7.95 (m, 2H), 7.72-7.78 (m, 2H), 7.31 (s, 1H), 4.01 (s, 3H), 3.80 (s, 3H);

¹³ C NMR (MeOD, 100 MHz) δ 168.0, 143.6, 132.5, 131.6, 126.9, 123.3, 120.6, 92.6, 68.1, 52.3, 36.7;

MS (ES+): m/z=275 (M+H) ⁺ ;

LCMS (method A): t _R =5.43 min.

Example 33 Allyl (6aS)-2-methoxy-3-(4-((2-((4-(methoxycarbonyl)phenyl)carbamoyl)-1-methyl-1nimidazol-4-yl)amino)-4-oxobutoxy) -12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine -5(12H)-carboxylate (33).

In a solution of 4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)5,6,6a,7 ,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (9) (50 mg, 0.094 mmol) in anhydrous dichloromethane ( 0.5 ml) was placed N-[(dimethylamino)-1H1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium N-oxide hexafluorophosphate (38 mg, 0.099 mmol) and anhydrous triethylamine (55 μl, 0.40 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then methyl 4-(4-amino-1-methyl-1H-imidazole-2carboxamido)benzoate hydrochloride (32) (30 mg, 0.094 mmol) was added and the resulting mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with saturated aqueous solution sodium bicarbonate (20 ml) and extracted with dichloromethane (2x50 ml). The combined organic extracts were washed with water containing a few drops of acetic acid (30 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by column chromatography (silica gel) eluting with methanol/dichloromethane (0 to 10%) to give the title compound (72 mg, 97%) as a brown oil.

MS (ES+): m/z=789 (M+H) ⁺ ;

LCMS (method A): tR=7.87 min.

- 42 040562

Example 34 4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-imidazole-2-carboxamido)benzoate (34).

To a solution of allyl (6aS)-2-methoxy-3-(4-((2-((4-(methoxycarbonyl)phenyl)carbamoyl)-1-methyl-1Himidazol-4-yl)amino)-4-oxobutoxy)- 12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin- 5(12H)-carboxylate (33) (72 mg, 0.091 mmol) in dichloromethane (2 ml) was added tetrakis(triphenylphosphine)palladium(0) (5.3 mg, 5 mol.%), triphenylphosphine (6.0 mg, 25 mol%) and pyrrolidine (9.0 μl, 0.11 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was placed under high vacuum for 30 min until excess pyrrolidine was completely removed. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 100%) to give the title compound (15 mg, 27%) as a yellow solid.

¹ H NMR (CDCl3, 400 MHz) δ 9.13 (s, 1H), 8.24 (s, 1H), 8.03 (d, J=8.7 Hz, 2H), 7.90 (d, J=5.7 Hz, 1H), 7.65-7.75 (m, 2H), 7.43-7.50 (m, 2H), 6.77-6.83 (m, 1H), 4 .12-4.23(m, 2H), 4.07(s, 3H), 3.93(s, 3H), 3.91(s, 3H), 3.18-3.27(m, 1H ), 2.80 (s, 3H), 2.56-2.68 (m, 3H), 2.23-2.31 (m, 2H), 1.85 (d, J=10.l Hz, 4H);

¹³ C NMR (CDCl3, 100 MHz) δ 169.3, 167.1, 166.2, 162.9, 156.2, 150.1, 147.6, 147.4, 141.4, 139.6, 135.6, 132.8, 130.5, 130.3, 125.2, 121.1, 118.2, 114.8, 111.2, 109.9, 94.1, 67.4, 63, 5, 55.7, 53.4, 51.6, 49.2, 39.4, 38.2, 35.5, 32.5, 31.6, 30.9, 28.9, 24.9, 24.3, 24.1, 22.5, 19.9, 18.0;

MS (ES+): m/z=603 (M+H)+;

LCMS (method A): tR=6.57 min.

Example 35 Methyl 4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2carboxamido)benzoate (35).

In a solution of 4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxylic acid (100 mg, 0.416 mmol) in N,N-dimethylformamide (3 ml) was placed )-3ethylcarbodiimide (145 mg, 0.756 mmol) and 4-(dimethylamino)pyridine (115 mg, 0.941 mmol). The reaction mixture was stirred at room temperature for 3 hours. Then methyl 4-aminobenzoate (57 mg, 0.38 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours. Then poured into ice water (40 ml) and extracted ethyl acetate (3x100 ml). The combined organic extracts were washed successively with aqueous citric acid (1 M, 60 ml), saturated aqueous sodium hydrogen carbonate (70 ml), water (70 ml) and brine (70 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated. The resulting residue was then purified by column chromatography (silica gel) eluting with methanol/dichloromethane (0 to 10%) to give the title compound (90 mg, 58%) as a white solid.

1H NMR (CDCl3, 400 MHz) δ 7.99-8.07 (m, 2H), 7.69 (s, 1H), 7.61-7.67 (m, 2H), 6.88 (s, 1H), 6.69 (br s, 1H), 6.25 (br s, 1H), 3.93 (s, 3H), 3.91 (s, 3H), 1.52 (s, 9H );

¹³ C NMR (CDCl ₃ , 100 MHz) δ 166.6, 159.4, 153.4, 142.3, 130.9, 125.5, 123.1, 122.5, 119.2, 118.7 , 140.1, 80.5, 52.0, 36.8, 28.4;

MS (ES+): m/z=374 (M+H)+;

LCMS (method A): tR=7.52 min.

Example 36 Methyl 4-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)benzoate hydrochloride (36).

Methyl 4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)benzoate (35) (90 mg, 0.24 mmol) was dissolved in hydrochloric acid (4 M in 1, 4-dioxane) (3 ml) and the reaction mixture

- 43 040562 was stirred at room temperature for 16 hours. The reaction mixture was concentrated in vacuo to give the title compound (79 mg, 99%) as a cream-colored solid. The product was used in the next step without any further purification.

¹ H NMR (MeOD, 400 MHz) δ 7.99 (d, J=8.7 Hz, 2H), 7.80 (d, J=8.7 Hz, 2H), 7.13 (d, J= 1.9 Hz, 1H), 7.09 (d, J=1.9 Hz, 1H), 3.96 (s, 3H), 3.89 (s, 3H);

¹³ C NMR (MeOD, 100 MHz) δ 168.2, 161.2, 144.5, 131.5, 126.9, 126.4, 123.7, 120.8, 114.2, 109.0, 52.5, 37.5;

MS (ES+): m/z=274 (M+H)+;

LCMS (method A): tR=4.98 min.

Example 37 Allyl (6aS)-2-methoxy-3-(4-((5-((4-(methoxycarbonyl)phenyl)carbamoyl)-1-methyl-1Hpyrrol-3-yl)amino)-4-oxobutoxy) -12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine -5(12H)-carboxylate (37).

In a solution of 4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)5,6,6a,7 ,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (9) (50 mg, 0.094 mmol) in anhydrous dichloromethane ( 0.5 ml) was placed N-[(dimethylamino)-1H1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium N-oxide hexafluorophosphate (38 mg, 0.099 mmol) and anhydrous triethylamine (55 μl, 0.40 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then methyl 4-(4-amino-1-methyl-1H-pyrrole-2carboxamido)benzoate hydrochloride (36) (30 mg, 0.094 mmol) was added and the resulting mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with saturated aqueous solution sodium bicarbonate (20 ml) and extracted with dichloromethane (2x50 ml). The combined organic extracts were washed with water containing a few drops of acetic acid (30 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by column chromatography (silica gel) eluting with methanol/dichloromethane (0 to 10%) to give the title compound (72 mg, 97%) as a brown oil.

MS (ES+): m/z=788 (M+H)+;

LCMS (method A): tR=7.77 min.

Example 38 Methyl (S)-4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2a] [ 1 ,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrol-2-carboxamido)benzoate (38).

To a solution of allyl (6aS)-2-methoxy-3-(4-((5-((4-(methoxycarbonyl)phenyl)carbamoyl)-1-methyl-1Hpyrrol-3-yl)amino)-4-oxobutoxy)- 12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin- 5(12H)-carboxylate (37) (72 mg, 0.091 mmol) in dichloromethane (2 ml) was added tetrakis(triphenylphosphine)palladium(0) (5.3 mg, 5 mol.%), triphenylphosphine (6.0 mg, 25 mol%) and pyrrolidine (9.0 μl, 0.11 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was placed under high vacuum for 30 min until excess pyrrolidine was completely removed. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 100%) to give the title compound (15.0 mg, 27%) as a yellow solid.

¹ H NMR (CDCls, 400 MHz) δ 8.41 (s, 1H), 8.00 (s, 2H), 7.98 (s, 1H), 7.90 (d, J=5.8 Hz, 1H), 7.72-7.74 (m, 1H), 7.70-7.72 (m, 1H), 7.41 (s, 1H), 7.14 (d, J=1.8 Hz , 1H), 6.79 (s, 1H), 6.57 (d, J=1.8 Hz, 1H), 4.22 (d, J=14.l Hz, 1H), 4.09 (t , J=6, 0 Hz, 2H), 3.89 (s, 3H), 3.88 (s, 3H), 3.83 (s, 3H), 3.74-3.79 (m, 2H) , 3.21 (d, J=3.3 Hz, 1H), 2.47-2.52 (m, 2H), 2.17-2.23 (m, 2H), 1.93 (br. s , 3H), 1.79-1.85 (m, 2H);

- 44 040562 ¹³ C NMR (CDC13, 100 MHz) δ 170.0, 167.6, 166.8, 163.6, 159.8, 150.7, 147.9, 142.9, 139.9, 130 .7, 124.9,

122.8, 121.6, 121.5, 120.8, 119.1, 111.8, 110.4, 104.6, 68.1, 56.1, 52.0, 49.7, 39, 9, 36.9, 33.0, 31.0, 25.0, 24.5,

22.9, 18.3;

MS (ES+): m/z=602 (M+H)+;

LCMS (method A): t _R =6.52 min.

Example 39 4-(4-(4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran2-yl)oxy )-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a] [1,4]diazepin-3-yl)oxy)butanamido)-1 methyl-1 N-pyrrole-2-carboxamido)phenyl)-1-methyl-1 N-pyrrole-2-carboxylic acid (3 9).

_MeO MeO ₂ C

To a solution of allyl (6aS)-2-methoxy-3-(4-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3yl)phenyl)carbamoyl)-1 -methyl- 1 H-pyrrol-3 -yl)amino)-4-oxobutoxy)-12-oxo-6-((tetrahydro-2H-pyran2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[ e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (23) (195 mg, 0.225 mmol) in 1,4-dioxane (5 ml) was added an aqueous solution of sodium hydroxide (0 .5 M, 5 ml, 2.5 mmol). The reaction mixture was stirred at room temperature for 16 h and then concentrated in vacuo, after which water (20 ml) was added and the aqueous layer was acidified to pH 1 with aqueous citric acid (1 M, 5 ml). The aqueous layer was then extracted with ethyl acetate (2x50 ml). The combined organic extracts were then washed with brine (50 ml), dried over sodium sulfate, filtered and concentrated to give the title compound (190 mg, 99%) as an off-white solid. The product was used in the next step without any further purification.

MS (ES+): m/z=853 (M+H)+;

LCMS (method B): tR=3.83 min.

Example 40 Allyl (6aS)-3-(4-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3yl)phenyl)carbamoyl)-1- methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6((tetrahydro-2H-pyran-2-yl)oxy)-6.6a,7.8, 9,10-hexahydrobenzo[e]pyrido[1,2-а][1,4]diazepine-5(12Н)carboxylate (40).

—N but ₂ s

To a solution of 4-(4-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy) -5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl1H-pyrrole -2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylic acid (39) (320 mg, 0.375 mmol) in anhydrous dichloromethane (1.5 ml) was placed N-[(dimethylamino)-1H- 1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium N-oxide (150 mg, 0.395 mmol) and anhydrous triethylamine (220 µl, 1.58 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then benzene-1,4-diamine (41 mg, 0.38 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate (20 ml) and extracted with dichloromethane (2x50 ml). The combined organic extracts were washed with water containing a few drops of acetic acid (30 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by column chromatography (silica gel) eluting with methanol/dichloromethane (0 to 10%) to give the title compound (250 mg, 71%) as an off-white solid.

MS (ES+): m/z=944 (M+H)+;

LCMS (method B): tR=3.45 min.

Example 41 (S)-N-(4-Aminophenyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12hexahydrobenzo[e] pyrido [1,2-a][1,4]diazepin-3 -yl)oxy)butanamido)-1-methyl-1 H-pyrrole-2carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide ( 41).

41

To a solution of allyl (6aS)-3-(4-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3yl)phenyl)carbamoyl)-1-methyl -1 H-pyrrol-3 -yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro2H-pyran-2-yl)oxy)-6,6а,7,8,9 ,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (40) (250 mg, 0.265 mmol) in dichloromethane (3 ml) was added tetrakis(triphenylphosphine)palladium (0) (15 mg, 5 mol%), triphenylphosphine (17 mg, 25 mol%) and pyrrolidine (26 µl, 0.32 mmol). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was placed under high vacuum for 30 minutes until excess pyrrolidine was completely removed. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 100%) followed by methanol/acetone (0 to 100%) to give the title compound (118 mg, 59% ) as a yellow solid.

¹ H NMR (DMSO-d6, 400 MHz) δ 9.88-9.96 (m, 1H), 9.81 (s, 2H), 9.50 (s, 1H), 8.32 (br s , 2H), 8.00 (d, J=5.7 Hz, 1H), 7.67-7.73 (m, 2H), 7.48 (d, J=8.6 Hz, 2H), 7 .39 (d, J=1.8 Hz, 1H), 7.31-7.35 (m, 2H), 7.30 (d, J=1.6 Hz, 1H), 7.27 (s, 1H), 7.22 (d, J=1.5 Hz, 1H), 6.96 (d, J=1.6 Hz, 1H), 6.80 (s, 1H), 6.51-6, 55 (m, 2H), 4.09-4.17 (m, 1H), 3.99-4.05 (m, 1H), 3.90-3.97 (m, 1H), 3.88 ( s, 3H), 3.83 (s, 3H), 3.82 (s, 3H), 3.68-3.72 (m, 1H), 3.05-3.16 (m, 2H), 2 .44 (t, J=7.3 Hz, 2H), 2.02-2.07 (m, 2H), 1.81-1.91 (m, 1H), 1.68-1.78 (m , 2H), 1.56 (d, J=4.9 Hz, 2H);

¹³ C NMR (DMSO-a ₆ , 100 MHz) δ 168.8, 166.3, 164.7, 159.5, 159.2, 150.2, 147.1, 144.7, 139.8, 137 .0, 129.6, 128.2, 126.1, 124.6, 124.3, 122.0, 121.8, 120.4, 120.2, 118.8, 113.7, 111.3 , 109.6, 104.7, 67.7, 67.2, 55.6, 51.1, 49.2, 38.5, 36.2, 36.1, 35.4, 31.8, 30 .2, 24.7, 23.7, 22.5, 17.7;

MS (ES+): m/z=757 (M+H)+;

LCMS (method A): tR=5.80 min.

Example 42 Methyl 5-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2carboxamido)benzo[b]thiophene-2-carboxylate (42).

In a solution of 4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxylic acid (127 mg, 0.530 mmol) in N,N-dimethylformamide (1 ml) was placed )-3ethylcarbodiimide (185 mg, 0.960 mmol) and 4-(dimethylamino)pyridine (147 mg, 1.20 mmol). The reaction mixture was stirred at room temperature for 4 hours. Methyl 5-aminobenzo[b]thiophene-2-carboxylate (100 mg, 0.480 mmol) was then added and the resulting mixture was stirred at room temperature for 16 hours. Then poured into ice water (40 ml) and extracted with ethyl acetate (3x100 ml). The combined organic extracts were washed successively with aqueous citric acid (1 M, 60 ml), saturated aqueous sodium hydrogen carbonate (70 ml), water (70 ml) and brine (70 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated to give the title compound (185 mg, 90%) as an off-white solid. The product was used in the next step without any further purification.

MS (ES+): m/z=430 (M+H)+;

- 46 040562

LCMS (method B): tR=4.07 min.

Example 43 Methyl 5-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)benzo[b]thiophene-2carboxylate hydrochloride (43).

Methyl 5-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)benzo-[b]thiophene-2carboxylate (42) (150 mg, 0.340 mmol) was dissolved in hydrochloric acid ( 4 M in 1,4-dioxane) (1 ml) and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated in vacuo to give the title compound (118 mg, 95%) as a pale brown solid colors. The product was used in the next step without any further purification.

MS (ES+): m/z=364 (M+H) ⁺ ;

LCMS (method B): t _R = 2.78 min.

Example 44 Allyl (6aS)-2-methoxy-3-(4-((5-((2-(methoxycarbonyl)benzo-[b]thiophen-5yl)carbamoyl)-1-methyl-1H-pyrrole-3- yl)amino)-4-oxo-butoxy)-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1 ,2-a] [ 1,4]diazepine-5(12Н)-carboxylate (44).

4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetra-hydro-2H-pyran-2-yl)oxy) In solution

5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanoic acid (9) (300 mg, 0.560 mmol) in N,N-dimethylformamide (3 ml) was placed 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (238 mg, 1.23 mmol) and 4-(dimethylamino)pyridine (189 mg, 1.55 mmol). The reaction mixture was stirred at room temperature for 4 hours. Methyl 5-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)benzo[b]thiophene-2-carboxylate hydrochloride (43) (225 mg, 0.620 mmol) and the resulting mixture was stirred at room temperature for 16 h. Then poured into ice water (40 ml) and extracted with ethyl acetate (3x100 ml). The combined organic extracts were washed successively with aqueous citric acid (1 M, 60 ml), saturated aqueous sodium hydrogen carbonate (70 ml), water (70 ml) and brine (70 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 30%) to give the title compound (348 mg, 66%) as a brown solid.

MS (ES+): m/z=844 (M+H) ⁺ ;

LCMS (method B): tR 4.23 min.

Example 45 5-(4-(4-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-5 ,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl1H-pyrrol-2 -carboxamido)benzo[b]thiophene-2-carboxylic acid (45).

45

To a solution of 4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-5,6,6a,7, 8,9,10,12-octahydrobenzo[e]pyrido[1,2-a] [1,4]diazepin-3-yl)oxy)butanoic acid (44) (327 mg, 0.387 mmol) in 1,4- dioxane (5 ml) was added an aqueous solution of sodium hydroxide (0.5 M, 5 ml, 2.5 mmol). The reaction mixture was stirred at room temperature for 3 hours and then concentrated in vacuo, after which water (20 ml) was added and the aqueous layer was acidified to pH 1 with aqueous citric acid (1 M, 5 ml). The aqueous layer was then extracted with ethyl acetate (2x50 ml). The combined organic extracts were then washed with brine (50 ml), dried over sodium sulfate, filtered and concentrated to give the title compound (315 mg, 99%) as a brown solid. The product was used in the next step without any further purification.

MS (ES+): m/z=831 (M+H)+;

LCMS (method B): tR=3.82 min.

Example 46 Allyl (6aS)-3-(4-((5-((2-((4-aminophenyl)carbamoyl)benzo-[b]thiophen-5-yl)carbamoyl)1-methyl-1H-pyrrole- 3-yl)amino)-4-oxo-butoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)6,6a,7,8,9,10- hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12Н)-carboxylate (46).

46

In a solution of 5-(4-(4-(((6aS)-5-((αlyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-5, 6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl1H-pyrrol-2- carboxamido)benzo[b]thiophene-2-carboxylic acid (45) (50 mg, 0.060 mmol) in anhydrous dichloromethane (1 ml) was placed ,5-b]pyridin1-ylmethylene]-M-methylmethanaminium N-oxide (28 mg, 0.072 mmol) and anhydrous triethylamine (35 μl, 0.25 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then benzene-1,4-diamine (7.0 mg, 0.066 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate (20 ml) and extracted with dichloromethane (2x50 ml). The combined organic extracts were washed with water containing a few drops of acetic acid (30 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 50%) to give the title compound (6.8 mg, 12%) as a yellow solid.

MS (ES+): m/z=921 (M+H)+;

LCMS (method B): tR=3.48 min.

Example 47 (S)-N-(2-((4-Aminophenyl)carbamoyl)benzo[b]thiophen-5-yl)-4-(4-((2-methoxy-12-oxo6a,7,8, 9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole2-carboxamide (47).

47

To a solution of allyl (6aS)-3-(4-((5-((2-((4-aminophenyl)carbamoyl)benzo[b]thiophen-5-yl)carbamoyl)-1methyl-1H-pyrrol-3-yl )amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)6,6a,7,8,9,10-hexahydrobenzo[e]pyrido [1,2-a][1,4]diazepine-5(12H)-carboxylate (46) (6.8 mg, 0.0074 mmol) in dichloromethane (1 ml) was added tetrakis(triphenylphosphine)palladium(0) ( 0.4 mg, 5 mol.%), triphenylphosphine (0.5 mg, 25 mol.%) and pyrrolidine (1 μl, 0.012 mmol). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was placed under high vacuum for 30 minutes until excess pyrrolidine was completely removed. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 100%) followed by methanol/dichloromethane (0 to 5%) to give the title compound (1.7 mg, 31%) as a pale yellow solid.

1H NMR (DMSOph, 400 MHz) δ 10.13 (s, 1H), 9.98-10.03 (m, 1H), 9.95 (s, 1H), 8.35 8.42 (m, 1H ), 8.19 (s, 1H), 8.01 (d, J=5.7 Hz, 1H), 7.95 (d, J=8.9 Hz, 1H), 7.33-7.40 (m, 2H), 7.23-7.28 (m, 2H), 7.02 (s, 1H), 6.81 (s, 1H), 6.57 (d, J=8.7 Hz, 2H), 5.00 (br s, 2H), 4.10-4.14 (m, 1H), 3.86 (s, 3H), 3.83 (s, 3H), 3.65-3 .74 (m, 2H), 3.15-3.19 (m, 1H), 3.06-3.14 (m, 1H), 2.45 (t, J=7.5 Hz, 3H), 2.11-2.13 (m, 1H), 2.00-2.08 (m, 4H) 1.74 (dd, J=9.0, 5.3 Hz, Zn);

MS (ES+): m/z=734 (M+H)+;

- 48 040562

LCMS (method A): t _R =5.63 min. General synthesis scheme.

i) K2CO3, DMF, ethyl 6-bromohexanoate, rt. pace.;

ii) KNO3, TFA, 0-5°C;

iii) KMnO ₄ , acetone, H2O, reflux;

iv) HATU, (S)-piperidin-2-ylmethanol, Et ₃ N, CH ₂ Cl ₂ , 0°C-room. pace.;

v) H2, Ni/Ra, MeOH;

vi) allyl chloroformate, pyridine, CH ₂ Cl ₂ , -10°C-room. pace.;

vii) TEMPO, BAIB, CH2Q2, room pace.;

viii) pTSA, DHP, EtOAc, room. pace.;

ix) NaOH, dioxane, H2O, room pace.;

x) RNH ₂ , EDCl, DMAP, DMF, room. pace.;

xi) PPh ₃ , Pd(PPh ₃ ) ₄ , pyrrolidine, CH ₂ Cl ₂ , room pace.

Example 48 Ethyl 6-(4-formyl-2-methoxyphenoxy)hexanoate (48).

A mixture of vanillin (6.5 g, 42.7 mmol), ethyl 6-bromohexanoate (8.0 ml, 45.0 mmol) and potassium carbonate (8.70 g, 63.0 mmol) in N,N-dimethylformaldehyde ( 50 ml) was stirred at room temperature for 18 hours. The reaction mixture was diluted with water (100 ml), separated and extracted with ethyl acetate (120 ml). The combined organic extracts were washed successively with water (100 ml), brine (100 ml), dried over magnesium sulfate, filtered and concentrated to give the title compound as a pale yellow oil (12.5 g, 99%). The product was used in the next step without any further purification.

¹ H NMR (400 MHz, CDCl3) δ 9.84 (s, 1H), 7.42-7.44 (dd, J=8.2, 1.9 Hz, 1H), 7.40 (d, J =1.9 Hz, 1H), 6.96 (d, J=8.1 Hz, 1H), 4.08-4.15 (m, 4H), 3.92 (s, 3H), 2.34 (t, J=7.5 Hz, 2H), 1.87-1.94 (m, 2H), 1.68-1.75 (m, 2H), 1.49-1.56 (m, 2H ), 1.25 (t, J=7.2 Hz, 3H);

MS (ES+): m/z=317 (M+Na) ⁺ ;

LCMS (method B): t _R =3.82 min.

Example 49 Ethyl 6-(4-formyl-2-methoxy-5-nitrophenoxy)hexanoate (49).

A solution of ethyl 6-(4-formyl-2-methoxyphenoxy)hexanoate (48) (12.5 ml, 42 mmol) in trifluoroacetic acid (25 ml). The reaction mixture was stirred for 1 h. Then concentrated in vacuo and the residue was dissolved in ethyl acetate (200 ml). It was washed with brine (3x50 ml) followed by saturated aqueous sodium bicarbonate

- 49 040562 (2x40 ml), dried over magnesium sulfate, filtered and concentrated in vacuo to give the title compound as a yellow solid (14.4 g, 100%). The product was used in the next step without any further purification.

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.43 (s, 1H) 7.58 (s, 1H), 7.40 (s, 1H), 4.10-4.16 (m, 4H), 4.00 (s, 3H), 2.35 (t, J=7.4 Hz, 2H), 1.84-1.96 (m, 2H), 1.69-1.76 (m, 2H) , 1.50-1.58 (m, 2H), 1.25 (t, J=7.2 Hz, 3H);

MS (ES+): m/z=340 (M+H)+;

LCMS (method B): tR=4.02 min.

Example 50 4-((6-Ethoxy-6-oxohexyl)oxy)-5-methoxy-2-nitrobenzoic acid (50).

oh oh

50

A hot (70°C) solution of potassium permanganate ( 13.6 g, 86.0 mmol) in water (100 ml). The mixture was then stirred at 70° C. for 4 hours. The reaction mixture was cooled to room temperature and passed through celite. The filter bed was then washed with hot water (100 ml). A solution of sodium bisulfite in hydrochloric acid (100 ml) was added to the filtrate and extracted with dichloromethane (2x200 ml). The combined organic extracts were dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound as a yellow solid (5.0 g, 61%) which was used in the next step without further purification.

¹ H NMR (400 MHz, CDCl3) δ 7.34 (s, 1H), 7.14 (s, 1H), 3.96-4.03 (m, 4H), 3.84 (s, 3H), 2.24 (t, J=7.4 Hz, 2H), 1.70-1.77 (m, 2H), 1.55-1.62 (m, 2H), 1.39-1.45 ( m, 2H), 1.13 (t, J=7.1 Hz, 3H);

MS (ES+): m/z=354 (MH) ⁺ ;

LCMS (method B): t _R =3.63 min.

Example 51 Ethyl (S)-6-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5nitrophenoxy)hexanoate (51).

ABOUT

51

To a stirred solution of 4-((6-ethoxy-6-oxohexyl)oxy)-5-methoxy-2-nitrobenzoic acid (50) (2.0 g, 5.6 mmol) and trimethylamine (4.70 ml, 33. 8 mmol) in dichloromethane (40 ml) O-(7-azabenzotriazol-1-yl)-N,N,N,N'-tetramethyluronium hexafluorophosphate (2.2 g, 5.9 mmol) was added in one portion and the resulting mixture was stirred for 2 h at room temperature. Then, a solution of (S)-piperidin-2-ylmethanol (647 mg, 5.63 mmol) in dichloromethane (10 ml) was added dropwise, and the resulting mixture was stirred for 16 hours at room temperature. The reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate (40 ml), the phases were separated and the aqueous layer was then extracted with dichloromethane (20 ml). The combined organic extracts were washed with brine (40 ml), dried over magnesium sulfate, filtered and concentrated to give an amber oil. Purification was carried out using column chromatography (silica gel) eluting with ethyl acetate/hexane (0 to 100%) to give the title compound (1.2 g, 48%) as a colorless oil.

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.60-7.63 (m, 1H), 6.75-6.77 (m, 1H), 4.02-4.13 (m, 4H), 3.93 (s, 3H), 3.70-3.78 (m, 1H), 3.39-3.68 (m, 1H), 3.11-3.18 (m, 3H), 2. 32 (t, J=7.6 Hz, 2H), 1.83-1.91 (m, 2H), 1.39-1.72 (m, 11H), 1.26 (t, J=7, 1 Hz, 3H);

MS (ES+): m/z=453 (M+H)+;

LCMS (method B): t _R =3.63 min.

Example 52 Ethyl (S)-6-(5-amino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2methoxyphenoxy)hexanoate (52).

Oh Oh

52

To a solution of ethyl (S)-6-(4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2-methoxy-5nitrophenoxy)hexanoate (51) (1.2 g, 2.7 mmol) in methanol (20 ml) was added Raney Nickel® (suspension in

- 50 040562

H2O) (120 mg). The resulting mixture was hydrogenated at a pressure of 50 psi. inch for 1.5 hours on a Parr apparatus, then filtered through a pad of celite and concentrated in vacuo to give the title compound (991 mg, 87%) as a gray oil which solidified on standing. The resulting material was used in the next step without further purification.

1H NMR (400 MHz, CDCl ₃ ) δ 6.69 (s, 1H), 6.32 (s, 1H), 4.13 (m, 4H), 3.98 (t, J=6.5 Hz, 2H), 3.79 (s, 3H), 3.67-3.57 (m, 1H), 3.19-3.22 (m, 4H), 2.87 (s, 2H), 2.32 -2.36 (m, 2H), 1.82-1.89 (m, 2H), 1.65-1.73 (m, 6H), 1.47-1.55 (m, 3H), 1 .27 (t, J=7.1 Hz, 3H);

MS (ES+): m/z=423 (M+H) ⁺ ;

LCMS (method B): tR=3.23 min.

Example 53 Ethyl (S)-6-(5-(((αlyloxy)karbonyl)amino)-4-(2-(hydroxy-methyl)piperidine-1carbonyl)-2-methoxyphenoxy)hexanoate (53).

53

To a solution of ethyl (S)-6-(5-αmino-4-(2-(hydroxymethyl)piperidine-1-carbonyl)-2methoxyphenoxy)hexanoate (52) (1.23 g, 2.91 mmol) and pyridine (542 µl, 6.69 mmol) in anhydrous dichloromethane (20 ml) at -10° C. was added dropwise a solution of allyl chloroformate (278 µl, 2.62 mmol) in dichloromethane (12 ml). The resulting reaction mixture was stirred at room temperature for 0.5 h, quenched with saturated aqueous copper (II) sulfate (25 ml), diluted with dichloromethane (10 ml), separated and washed successively with water (20 ml), saturated aqueous sodium hydrogen carbonate (20 ml) and brine (20 ml). The organic layer was then dried over magnesium sulfate, filtered and concentrated in vacuo to give the title compound (588 mg, 40%) as an orange oil. The resulting material was used in the next step without further purification.

¹ H NMR (400 MHz, CDCl3) δ 8.23 (br s, 1H), 7.70 (br s, 1H), 6.78 (s, 1H), 5.90-6.00 (m , 1H), 5.33-5.38 (m, 1H), 5.24 (dd, J=10.4, 1.3 Hz, 1H), 4.63 (m, 2H), 4.12 ( q, J=1.1 Hz, 2H) 4.05 (t, J=6.6 Hz, 2H), 3.83 (s, 3H), 3.64-3.72 (m, 1H), 3 .02-3.12 (m, 1H), 2.33 (t, J=7.6 Hz, 2H), 1.84-1.91 (m, 2H), 1.67-1.74 (m , 10H), 1.66-1.54 (m, 4H), 1.26 (t, J=7.1 Hz, 3H);

MS (ES+): m/z=507 (M+H) ⁺ ;

LCMS (method B): tR=3.70 min.

Example 54 Allyl (6aS)-3-((6-ethoxy-6-oxohexyl)oxy)-6-hydroxy-2-methoxy-12-oxo6,6a,7,8,9,10-hexahydrobenzo[e]pyrido [1,2-а][1,4]diazepine-5(12Н)-carboxylate (54).

54

To a solution of ethyl (S)-6-(5-(((allyloxy)karbonyl)amino)-4-(2-(hydroxymethyl)-piperidine-1carbonyl)-2-methoxyphenoxy)hexanoate (53) (1.7 g, 3.4 mmol) in dichloromethane (80 ml) was added 2,2,6,6 tetramethyl-1-piperidinyloxy (53 mg, 0.30 mmol) and (diacetoxyiodine)benzene (1.3 g, 4.0 mmol). The reaction mixture was stirred at room temperature for 16 h, then placed in an ice bath and quenched with saturated aqueous sodium metabisulphite (35 ml). The mixture was diluted with dichloromethane (30 ml), separated and washed successively with saturated aqueous sodium hydrogen carbonate (30 ml), water (30 ml) and brine (30 ml). The organic layer was then dried over magnesium sulfate, filtered and concentrated in vacuo. Purification was carried out using column chromatography (silica gel) eluting with ethyl acetate/hexane (0 to 80%) to give the desired compound (1.1 g, 66%) as a colorless oil.

1H NMR (400 MHz, CDCl3) δ 7.70-7.72 (m, 1H), 7.09-7.13 (m, 1H), 5.80-5.98 (m, 1H), 5, 25-5.38 (m, 1H), 5.14-5.19 (m, 2H), 4.63-4.72 (m, 2H), 4.35-4.50 (m, 1H), 4.13 (q, J=7.1 Hz, 2H), 4.03-4.08 (m, 1H), 3.96-4.01 (m, 2H), 3.91 (s, 3H) , 3.81-3.83 (m, 1H), 3.45-3.53 (m, 1H), 3.03-3.10 (m, 1H), 2.33 (t, J=7, 6 Hz, 2H), 1.83-1.90 (m, 2H), 1.62-1.74 (m, 10H), 1.48-1.53 (m, 2H);

MS (ES+): m/z=505 (M+H) ⁺ ;

LCMS (method B): tR=3.57 min.

- 51 040562

Example 55 Allyl (6aS)-3-((6-ethoxy-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2Hpyran-2-yl)oxy)-6.6a, 7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12Н)-carboxylate (55).

To a solution containing allyl (6aS)-3-((6-ethoxy-6-oxohexyl)oxy)-6-hydroxy-2-methoxy-12oxo-6,6a,7,8,9,10-hexahydrobenzo[e] pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (54) (1.1 g, 2.2 mmol) in dichloromethane (50 ml) was added 3,4-dihydro-2H- pyran (2.00 ml, 22.4 mmol) and p-toluenesulfonic acid monohydrate (113 mg, 1% w/w) and the resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was then diluted with dichloromethane (50 ml) and washed with saturated aqueous sodium hydrogen carbonate solution (50 ml) and brine (50 ml). The organic layer was dried over magnesium sulfate, filtered and concentrated to give the title compound as a yellow oil (863 mg, 66%) after purification by column chromatography (silica gel) eluting with ethyl acetate/hexane (0 to 70%) .

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.16 (m, 1H), 6.50 (s, 1H), 6.10 (m, 1H), 5.76-5.81 (m, 1H) , 5.03-5.14 (m, 2H), 4.57-4.69 (m, 2H), 4.37-4.49 (m, 1H), 4.26-4.34 (m, 1H), 4.12 (q, J=7.1 Hz, 2H), 3.94-4.01 (m, 3H), 3.90 (s, 3H), 3.62-3.68 (m , 1H), 3.46-3.68 (m, 2H), 3.03-3.12 (m, 1H), 2.33 (t, J=7.4 Hz, 2H), 1.66- 1.89 (m, 11H), 1.47-1.57 (m, 6H), 1.25 (t, J=7.1 Hz, 3H);

MS (ES+): m/z=589 (M+H)+;

LCMS (method B): tR=4.32 min.

Example 56 6-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyrαn-2yl)oxy)-5,6,6a,7 ,8,9,10,12-Octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanoic acid (56).

To a solution of allyl (6aS)-3-((6-ethoxy-6-oxohexyl)oxy)-2-methoxy-12-oxo-6-((tetrahydro-2Hpyran-2-yl)oxy)-6,6a,7 ,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (55) (200 mg, 0.34 mmol) in 1,4-dioxane (3 ml) was added an aqueous solution of sodium hydroxide (0.5 M, 1.2 ml). The reaction mixture was stirred at room temperature for 2 h and then concentrated in vacuo, after which water (6 ml) was added and the aqueous layer was then acidified to pH 1 with acetic acid. The aqueous layer was then extracted with ethyl acetate (2x40 ml). The combined organic extracts were then washed with brine (40 ml), dried over sodium sulfate, filtered and concentrated to give the title compound as a yellow oil (181 mg, 95%) which was used in the next step without further purification.

1H NMR (400 MHz, CDCl ₃ ) δ 7.18 (s, 1H), 6.19 (s, 1H), 5.99-6.19 (m, 1H), 5.71-5.81 (m , 1H), 5.02-5.12 (m, 2H), 4.51-4.67 (m, 1H), 4.36-4.48 (m, 1H), 4.23-4.31 (m, 1H), 3.88-4.00 (m, 7H), 3.46-3.66 (m, 2H), 3.02-3.12 (m, 1H), 2.36 (t , J=7.4 Hz, 2H), 1.79-1.81 (m, 2H), 1.65-1.75 (m, 10H), 1.49-1.55 (m, 7H);

MS (ES+): m/z=561 (M+H)+;

LCMS (method B): tR=3.78 min.

Example 57 Methyl 4-(4-(4-((tert-Butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate (57).

Hydrochloride 1 -(3-dimethylaminopropyl)-3-ethylcarbodiimide (67 mg, 0.36 mmol) and 4-(dimethylamino)pyridine (65 mg, 0.53 mmol). The reaction mixture was stirred at room temperature for 2 hours. Methyl 4-(4-aminophenyl)-1-methyl-1H-pyrrole-2-carboxylate (41 mg, 0.18 mmol) was added to the reaction mixture, followed by stirring at room temperature within 16 hours the Reaction mixture was poured into ice water (40 ml) and was extracted with ethyl acetate (3x100 ml). The combined organic layer was washed successively with 1 M citric acid (60 ml), saturated aqueous sodium hydrogen carbonate solution (70 ml), water (70 ml) and brine (70 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by column chromatography (silica gel) eluting with ethyl acetate/dichloromethane (0 to 50%) to give the title compound (36 mg, 45%) as an off-white solid.

MS (ES+): m/z=453 (M+H) ⁺ ;

LCMS (method B): tR=4.07 min.

Example 58 Methyl 4-(4-(4-amino-1-methyl-1H-pyrrole-2-carboxamido)-phenyl)-1-methyl-1H-pyrrole2-carboxylate (58).

4-(4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1 H-pyrrole-2-carboxamido)-phenyl)-1 Methyl methyl-1H-n-pyrrol-2-carboxylate (57) (150 mg, 0.330 mmol) was dissolved in hydrochloric acid (4 M in 1,4-dioxane) (1 ml) and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated in vacuo to give the title compound (114 mg, 99%) as a brown solid. The product was used in the next step without further purification.

MS (ES+): m/z=353 (M+H) ⁺ ;

LCMS (method B): tR=2.88 min.

Example 59 Allyl (6aS)-2-methoxy-3-((6-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3yl)phenyl)carbamoyl)-1- methyl-1H-pyrrol-3-yl)amino)-6-oxohexyl)oxy)-12-oxo-6-((tetrahydro-2Hpyran-2-yl)oxy)-6,6a,7,8,9,10 -hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12Н)-carboxylate (59).

In a solution of 6-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)5,6,6a,7 ,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanoic acid (56) (194 mg, 0.360 mmol) in N,N -dimethylformamide (5 ml) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (126 mg, 0.660 mmol) and 4-(dimethylamino)pyridine (121 mg, 0.990 mmol). The reaction mixture was stirred at room temperature for 3 h. Then methyl 4-(4-(4amino-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylate (58 ) (150 mg, 0.330 mmol) and the resulting mixture was stirred at room temperature for 16 h. Then poured into ice water (20 ml) and extracted with ethyl acetate (3x75 ml). The combined organic extracts were washed successively with aqueous citric acid (1 M, 50 ml), saturated aqueous sodium hydrogen carbonate (50 ml), water (50 ml) and brine (50 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound (133 mg, 45%) as a yellow oil. The product was used in the next step without further purification.

MS (ES+): m/z=896 (M+H) ⁺ ;

LCMS (method B): tR=4.25 min.

Example 60 4-(4-(4-(6-(((6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran2-yl)oxy )-5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanamido)-1methyl-1H- pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylic acid (60).

(6aS)-2-methoxy-3-((6-((5-((4-(5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3allyl)phenyl)carbamoyl)-1-methyl-1H -pyrrol-3-yl)amino)-6-oxohexyl)oxy)-12-oxo-6-((tetrahydro-2HK solution

- 53 040562 pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-5(12H)-carboxylate ( 59) (200 mg, 0.340 mmol) in 1,4-dioxane (3 ml) was added an aqueous solution of sodium hydroxide (1 M, 1.2 ml). The reaction mixture was stirred at room temperature for 2 h and then concentrated in vacuo, after which water (6 ml) was added and the aqueous layer was acidified to pH 1 with acetic acid. The aqueous layer was then extracted with ethyl acetate (2x40 ml). The combined organic extracts were then washed with brine (40 ml), dried over sodium sulfate, filtered and concentrated to give the title compound as a yellow oil (181 mg, 95%) which was used in the next step without further purification.

MS (ES+): m/z=882 (M+H)+;

LCMS (method B): tR=3.92 min.

Example 61 Allyl (6aS)-3-((6-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3yl)phenyl)carbamoyl)-1 -methyl-1H-pyrrol-3-yl)amino)-6-oxohexyl)oxy)-2-methoxy-12-oxo-6((tetrahydro-2H-pyran-2-yl)oxy)-6.6a.7 ,8,9,10-hexahydrobenzo[e]pyrido[1,2-а][1,4]diazepine-5(12Н)carboxylate (61).

6θ 61

To a solution of 4-(4-(4-(6-(((6aS)-5-((αlyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy) -5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanamido)-1-methyl1H-pyrrole -2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylic acid (60) (123 mg, 0.14 mmol) in anhydrous dichloromethane (2 ml) was placed N-[(dimethylamino)-1H- 1,2,3-triazolo-[4,5b]pyridin-1-ylmethylene]-N-methylmethanaminium N-oxide (54 mg, 0.14 mmol) and anhydrous triethylamine (117 μl, 0.84 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then benzene-1,4-diamine (15.1 mg, 0.14 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate (20 ml) and extracted with dichloromethane (2x50 ml ). The combined organic extracts were washed with water containing a few drops of acetic acid (30 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 50%) to give the title compound (63 mg, 46%) as a yellow solid.

MS (ES+): m/z=972 (M+H)+;

LCMS (method B): tR=3.55 min.

Example 62 Methyl 5-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2carboxamido)benzo[b]thiophene-2-carboxylate (62).

62

To a solution of allyl (6aS)-3-((6-((5-((4-(5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3yl)phenyl)carbamoyl)-1- methyl-1H-pyrrol-3-yl)amino)-6-oxohexyl)oxy)-2-methoxy-12-oxo-6((tetrahydro-2H-pyran-2-yl)oxy)-6.6a.7, 8,9,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)carboxylate (61) (25 mg, 0.026 mmol) in dichloromethane (1 ml) )palladium(0) (2.5 mg, 5 mol%), triphenylphosphine (1.7 mg, 25 mol%) and pyrrolidine (21 µl, 0.260 mmol). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was placed under high vacuum for 30 minutes until excess pyrrolidine was completely removed. The resulting residue was then purified by column chromatography (silica gel) eluting with ethyl acetate/hexane (0 to 100%) to give the title compound (6.8 mg, 33%) as a pale yellow solid.

1H NMR (DMSO-d ₆ , 400 MHz) δ 9.81-9.85 (m, 1H), 9.58 (s, 1H), 9.51 (s, 2H), 8.00 (d, J = 5.7 Hz, 1H), 7.69-7.72 (m, 2H), 7.47-7.49 (m, 2H), 7.38-7.43 (m, 1H), 7, 30-7.35 (m, 2H), 7.18-7.24 (m, 1H), 7.11-7.13 (m, 1H), 7.07 (s, 1H), 6.94- 6.98 (m, 1H), 6.80 (br s, 1H), 6.63-6.72 (m, 2H), 6.52-6.54 (m, 1H), 3.95- 4.14 (m, 3H), 3.89 (s, 3H), 3.83 (s, 3H), 3.70 (s, 3H), 3.65-3.69 (m, 1H), 3 .17 (d, J=5.2 Hz, 2H), 2.28 (t, J=6.5 Hz, 2H), 1.72-1.78 (m, 4H), 1.62-1, 68 (m, 4H), 1.42-1.48 (m, 3H);

¹³ C NMR (DMSO-d ₆ , 100 MHz) δ 169.5, 166.3, 164.6, 159.5, 159.2, 150.3, 147.1, 144.8, 139.8, 137 .0, 129.6, 128.2, 126.6, 124.6, 124.3, 122.7, 122.1, 121.8, 121.7, 120.5, 120.4, 118.7 , 113.7, 111.3, 109.6, 109.3,

- 54 040562

104.7, 68.1, 55.6, 36.3, 36.1, 35.5, 28.3, 25.2, 25.1, 23.7, 22.5, 17.7;

MS (ES+): m/z=785 (M+H) ⁺ ;

LCMS (method A): tR=3.0 8 min.

Example 63 Allyl (6S,6aS)-2-methoxy-3-((6-((5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)amino)6-oxohexyl)oxy)-12 -oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-5 (12H)-carboxylate (63).

In a solution of 6-(((6S,6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-5.6.6a, 7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanoic acid (56) (109 mg, 0.190 mmol) in anhydrous dichloromethane (3 ml) N-[(dimethylamino)1H-1,2,3-triazolo-[4,5-b]-pyridin-1-ylmethylene]-N-methylmethanaminium N-oxide hexafluorophosphate (76 mg, 0.20 mmol) and anhydrous triethylamine (115 μl, 1.14 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then methyl 4-amino-1-methyl-1H-pyrrole-2-carboxylate (37 mg, 0.24 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate (20 ml ) and extracted with dichloromethane (2x50 ml). The combined organic extracts were washed with water containing a few drops of acetic acid (30 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (0 to 30%) to give the title compound (82 mg, 62%) as a white solid.

MS (ES+): m/z=691 (M+H) ⁺ ;

LCMS (method B): t _R =3.98 min.

Example 64 4-(6-(((6S,6aS)-5-((Allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-5, 6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanamido)-1-methyl1H-pyrrol-2- carboxylic acid (64).

To a solution of allyl (6S,6aS)-2-methoxy-3-((6-((5-(methoxycarbonyl)-1-methyl-1H-pyrrol-3-yl)amino)-6oxohexyl)oxy)-12-oxo -6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10-hexahydrobenzo[e]pyrido[1,2a][1,4]diazepin-5(12H )-carboxylate (63) (76 mg, 0.11 mmol) in 1,4-dioxane (1 ml) was added an aqueous solution of sodium hydroxide (0.5 M, 1.0 ml, 0.50 mmol). The reaction mixture was stirred at room temperature for 16 h and then concentrated in vacuo, after which water (20 ml) was added and the aqueous layer was acidified to pH 1 with aqueous citric acid (1 M, 10 ml). The aqueous layer was then extracted with ethyl acetate (2x50 ml). The combined organic extracts were then washed with brine (50 ml), dried over sodium sulfate, filtered and concentrated in vacuo to give the title compound (74 mg, 98%) as an off-white solid. The product was used in the next step without any further purification.

MS (ES+): m/z=683 (M+H) ⁺ ;

LCMS (method B): t _R =3.68 min.

Example 65 Allyl (6S,6aS)-3-((6-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)6-oxohexyl)oxy)- 2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10hexahydrobenzo[e]pyrido[1,2-a][1, 4]diazepine-5(12Н)-carboxylate (65).

65

- 55 040562

To a solution of 4-(6-(((6S,6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy)-5.6 ,6а,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)hexanamido)-1-methylIII-pyrrole-2-carboxylic acid (64) (60 mg, 0.090 mmol) in anhydrous dichloromethane (1 ml) was placed N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene] hexafluorophosphate -N-methylmethanaminium

N-oxide (67.0 mg, 0.175 mmol) and anhydrous triethylamine (73 µl, 0.52 mmol).

The reaction mixture was stirred at room temperature for 30 minutes. Then benzene-1,4-diamine (10 mg, 0.10 mmol) was added and the resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate (20 ml) and extracted with dichloromethane (2x50 ml). The combined organic extracts were then washed with water containing a few drops of acetic acid (30 ml). The organic layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by column chromatography (silica gel) eluting with acetone/dichloromethane (30 to 50+5% MeOH) to give the title compound (18 mg, 26%) as a brown solid.

MS (ES+): m/z=113 (M+H) ⁺ ;

LCMS (method B): tR=3.27 min.

Example 66 (S)-N-(4-Aminophenyl)-4-(6-((2-methoxy-12-oxo-6a,7,8,9,10,12hexahydrobenzo[e]pyrido[1,2- a][1,4]diazepin-3-yl)oxy)hexan-amido)-1-methyl-1H-pyrrole-2-carboxamide (66).

Alloc OTNR

66

To a solution of allyl (6S,6aS)-3-((6-((5-((4-aminophenyl)carbamoyl)-1-methyl-1H-pyrrol-3-yl)amino)-6oxohexyl)oxy)-2- methoxy-12-oxo-6-((tetrahydro-2H-pyran-2-yl)oxy)-6,6a,7,8,9,10hexahydrobenzo[e]pyrido[1,2-a][1, 4] diazepine-5(12H)-carboxylate (65) (18 mg, 0.020 mmol) in dichloromethane (1 ml) was added tetrakis(triphenylphosphine)palladium(0) (1.3 mg, 5 mol%) and pyrrolidine (2.3 μl , 0.030 mmol). The reaction mixture was stirred at room temperature for 30 min and then placed under high vacuum for 30 min until excess pyrrolidine was completely removed. The resulting residue was then purified by column chromatography (silica gel) eluting with methanol/dichloromethane (0 to 100%) to give the title compound (11.6 mg, 86%) as an off-white solid.

1H NMR (DMSO-d ₆ , 400 MHz) δ 9.78 (s, 1H), 9.48 (s, 1H), 8.00 (d, J=5.7 Hz, 1H), 7.32 ( d, J=8.8 Hz, 2H), 7.25 (s, 1H), 7.17 (d, J=1.8 Hz, 1H), 6.82 (d, J=1.9 Hz, 1H), 6.79 (s, 1H), 6.56 (d, J=8.6 Hz, 2H), 4.13 (dd, J=5.7, 3.4 Hz, 5H), 3, 80 (s, 3H), 3.79 (s, 3H), 3.17 (s, 1H), 3.07-3.11 (m, 1H), 2.26 (t, J=7.2 Hz , 3H), 1.75 (dd, J=13.8, 7.0 Hz, 6H), 1.60-1.65 (m, 5H);

MS (ES+): m/z=587 (M+H) ⁺ ;

LCMS (method B): tR=2.72 min;

MS (ES+): m/z=587 (M+H) ⁺ ;

LCMS (method A): tR=5.23 min.

Example 67 Allyl (6aS)-3-(4-((5-((4-(5-((2-aminoethyl)carbamoyl)-1-methyl-1H-pyrrol-3yl)phenyl)carbamoyl)-1- methyl-1H-pyrrol-3-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-(tetrahydro2H-pyran-2-yl)oxy)-6,6a,7,8,9, 10-hexahydrobenzo[e]pyrido[1,2-а][1,4]diazepine-5(12Н)-carboxylate (67).

67

To a solution of 4-(4-(4-(4-(((6aS)-5-((allyloxy)carbonyl)-2-methoxy-12-oxo-6-((tetrahydro-2H-pyran-2yl)oxy) -5,6,6a,7,8,9,10,12-octahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl1H-pyrrole -2-carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxylic acid (39) (270 mg, 0.317 mmol) in anhydrous dichloromethane (6 ml) was placed N-[(dimethylamino)-1H-1 hexafluorophosphate, 2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium N-oxide (126 mg, 0.333 mmol) and anhydrous triethylamine (185 µl, 1.33 mmol). The reaction mixture was stirred at room temperature for 30 minutes. Then ethane-1,2-diamine (379 mg, 6.33 mmol) was added and the resulting mixture was stirred at room temperature for 16 h. The reaction mixture was quenched with saturated aqueous sodium hydrogen carbonate (20 ml) and extracted with dichloromethane (2x50 ml) . The combined organic extracts were washed with water containing a few drops of acetic acid (30 ml). Organic

- 56 040562 layer was then dried over sodium sulfate, filtered and concentrated in vacuo. The resulting residue was then purified by column chromatography (silica gel) eluting with ammonia in methanol (2 M)/dichloromethane (0 to 10%) to give the title compound (180 mg, 63%) as a white solid. colors.

MS (ES+): m/z=896 (M+H)+;

LCMS (method B): t _R =3.12 min.

Example 68 (S)-N-(2-Aminoethyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12hexahydrobenzo[e] pyrido[1,2-a] [1,4]diazepin-3 -yl)oxy)butanamido)-1-methyl-1 H-pyrrole-2carboxamido)phenyl)-1-methyl-1H-pyrrole-2-carboxamide ( 68).

68

To a solution of allyl (6aS)-3-(4-((5-((4-(5-((2-aminoethyl)carbamoyl)-1-methyl-1H-pyrrol-3yl)phenyl)carbamoyl)-1-methyl -1 H-pyrrol-3-yl)amino)-4-oxobutoxy)-2-methoxy-12-oxo-6-((tetrahydro2H-pyran-2-yl)oxy)-6.6a,7.8.9 ,10-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepine-5(12H)-carboxylate (67) (22 mg, 0.025 mmol) in dichloromethane (4 ml) was added tetrakis(triphenylphosphine)palladium (0) (1.4 mg, 5 mol%) and pyrrolidine (3.0 µl, 0.037 mmol). The reaction mixture was stirred at room temperature for 2 h and then placed under high vacuum for 30 min until excess pyrrolidine was completely removed. The resulting residue was then purified by column chromatography (silica gel) eluting with methanol/dichloromethane (0 to 20%) to give the title compound (11 mg, 62%) as a white solid.

1H NMR (DMSO-d ₆ , 400 MHz) δ 10.01 (s, 1H), 9.84 (s, 1H), 9.21 (br s, 2H), 8.41 (s, 1H), 8.01 (d, J=5.7 Hz, 1H), 7.70 (d, J=8.8 Hz, 2H), 7.44 (d, J=8.7 Hz, 2H), 7. 38 (d, J=1.8 Hz, 1H), 7.31 (d, J=1.9 Hz, 1H), 7.27 (s, 1H), 7.22 (d, J=1.8 Hz, 1H), 6.98 (d, J=1.8 Hz, 1H), 6.80 (s, 1H), 4.09-4.19 (m, 2H), 3.99-4.05 (m, 2H), 3.87 (s, 3H), 3.82 (m, 6H), 3.65-3.72 (m, 2H), 3.45-3.50 (m, 2H), 3.16 (d, J=5.3 Hz, 3H), 2.96 (t, J=5.8 Hz, 2H), 2.45 (t, J=7.4 Hz, 2H), 2. 00-2.09 (m, 4H); (DMSO-d ₆ , 100 MHz) δ 203.1, 168.8, 166.3, 164.7, 161.6, 159.6, 150.2, 147.1, 139.8, 137.0, 129.5, 125.9, 124.2, 122.0, 120.6, 120.4, 111.2, 109.8, 109.3, 98.8, 95.4, 85.9, 78, 8, 71.0, 67.7, 55.6, 49.2, 48.5, 36.3, 31.8, 30.2, 24.7, 22.5, 17.7;

MS (ES+): m/z=709 (M+H)+;

LCMS (method B): tR=2.80 min;

MS (ES+): m/z=709 (M+H)+;

LCMS (method A): tR=5.38 min.

Example 69 Proof of DNA adduct formation by HPLC.

The interaction of C8-linked PDD monomers with the consensus sequence transcription factor duplex was studied by HPLC analysis using an X-bridge MS C18 column 2.5 μM OST (2.3x50 mm) and a gradient mixture of 40% acetonitrile/water and 100 mM TEAB (tetraethylammonium bromide)/water as mobile phase with a flow rate of 0.5 ml/min and with UV detection at 254 nm. A 4:1 molar ratio of ligand:oligonucleotide was used, with each single-stranded oligonucleotide being dissolved in 1 M ammonium acetate to form 1 mM stock solutions. Oligonucleotides were initially annealed by heating their 1 mM solutions at 70°C for 10 minutes, then gradually cooled over 8 hours and stored overnight at -20°C. Then, working solutions of 25 μM oligonucleotides were prepared by diluting the stock solutions after annealing with 100 mM ammonium acetate. The ligands were dissolved in DMSO to obtain a stock solution of 10 mm, which was stored at -20°C for no more than four months. 100 μM drug working solutions were prepared by diluting the stock solution with 100 mM ammonium acetate. Ligand working solutions were added to the oligonucleotide working solution at room temperature, and the mixture was incubated for various time intervals at room temperature.

Example 70 Fluorescent Resonance Energy Transfer (FRET) Assay.

Oligonucleotide sequences used for FRET assays were purchased from Eurogentec, Southampton, UK: TAMRA (6-carboxytetramethyldorhodium) and FAM (6-carboxyfluorescein) are acceptor and donor fluorophores, respectively. 400 nm solutions in FRET buffer (optimized as 50 mM potassium, 50 mM cacodylate, pH 7.4) were prepared from 20 μM stock solutions before use. Oligonucleotides were annealed by heating the samples to 90°C for 10 min, followed by cooling to room temperature and stored at this temperature for 5 h. Dilutions from a stock solution of 5 mM DMSO were performed using FRET buffer. Annealed DNA solution (50 μl) and sample solution (50 μl) were added to each well of a 96-well plate (MJ Research, Waltham, MA) and processed in a DNA Engine Opticon (MJ Research). Indications

- 57 040562 fluorescence were read at intervals of 0.5°C in the range of 30-100°C, with a constant temperature maintained for 30 seconds before each reading. Incident radiation was used at 450-495 nm with detection at 515-545 nm. Raw data was imported into the Origin software (version 7.0, OringinLab Corp.) and the plots were smoothed using a 10-point mean and then normalized. The determination of melting temperatures was based on the values of the maximum of the first derivative of the smoothed melting curves using a script. The difference between the melting point of each sample and the sample (ATt) was used for comparison.

Table 1

ATT determined after 24-hour incubation with a transcription factor with _________________duplex of DNA sequences__________________

ATT at a ligand concentration of 1 μM Compound NFKB ( ^1st broadcast) NFKB ( ^2nd broadcast) AR-1 ( ^1st broadcast) AR-1 ( ^2nd broadcast) 13 12 23 eleven 19 17 eleven 26 13 18 20 9 12 8 13 24 10 14 9 15

Example 71 Cytotoxicity analysis of C8-linked PDD monomers by the MTT method.

MDA cell culture MB231 (triple negative human breast cancer) was obtained from the American Type Culture Collection. The cell line was maintained in monolayer culture in 75 cm ² flasks (TPP, Switzerland) in a humid atmosphere with 5% CO 2 at 37°C. The MDA MB231 cell line was maintained in high glucose DMEM (4.5 g/l; Invitrogen), fetal bovine serum (10%, Biosera UK), non-essential amino acids (lx, Invitrogen), L-glutamine (2 mM, Invitrogen) and penicillin-streptomycin (1% v/v, Invitrogen). The HeLa cell line was maintained in Dulbecco's Modified Eagle's Medium (IMEM, Invitrogen) supplemented with fetal bovine serum (10% v/v, Invitrogen), L-glutamine (2 mM, Invitrogen), non-essential amino acids (lx, Invitrogen), and penicillin- streptomycin (1% v/v, Invitrogen). For passage, the cells were washed with PBS (GIBCO 14040, Invitrogen, UK), incubated with trypsin (GIBCO 25300, Invitrogen, UK), and replated in fresh medium. For seeding, cells were counted using a Neubauer hemocytometer (Assistant, Germany) using a microscope (Nikon, USA) on a non-adherent cell suspension, which were washed in PBS, trypsinized, centrifuged at 8°C at a speed of 8000 rpm for 5 min and repeated suspended in fresh medium. MTT analysis. Cells were grown under normal cell culture conditions in a humid atmosphere with 5% CO2 at 37°C using the appropriate medium. The number of cells was adjusted to 10 ⁵ cells/ml and 5000-20000 cells per well, depending on the cell line. Cells were incubated for 24 h and 1 μl of appropriate inhibitor concentrations were added to the wells in triplicate. After 72 hours of continuous exposure to each compound, cytotoxicity was determined using a colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Lancaster Synthesis Ltd, UK). Absorbance was quantified by spectrophotometry at λ=570 nm (Envision Plate Reader, PerkinElmer, USA). IC50 values were calculated by dose-response analysis using GraphPad Prism® software.

table 2

IC ₅₀ values (nM) determined after 72 hours of exposure to C8-linked PDD monomers

1С ₅₀ (nanomolar) Compound MDA MB 231 (cell line triple negative breast cancer) HeLa (cervical cancer cell line) 13 64+9.6 0.6+0.4 17 21+1.8 1.2+0.8 20 0.3+0.22 0.14+0.09 24 0.8+0.66 1+0.12

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All publications mentioned in the description above are incorporated herein by reference. Although illustrative embodiments of the invention have been described in detail with reference to the accompanying drawings, it is understood that the invention is not limited to a specific embodiment and that specific

Claims (16)

various changes and modifications may be made by those skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents. CLAIM 1. Compound of formula (XV) and its salts, where R ₁ represents H; R2 is H; R3 is selected from H, C _1-12 alkyl and CH ₂ Ph; R4 is selected from phenyl, pyrrolyl, N-methylpyrrolyl, furanyl, thiophenyl, imidazolyl, N-methylimidazolyl, oxazolyl, thiazolyl, pyridyl, benzofuranyl, benzothiophenyl, benzimidazolyl, N-methylbenzoimidazolyl, benzoxazolyl and benzothiazolyl, optionally substituted with up to three optional substituents selected from OH, O _1-6 -alkyl, OC _1-6 -alkyl, (CH2)j-CO2R ₁₁ , O-(CH ₂ ) _k NR11R12, (CH ₂ ) _j -NR ₁₁ R ₁₂ , С(=O)- NH-(CH ₂ ) _k -NR ₁₁ R ₁₂ , C(=O)-NH-R ₂₄ and C(=O)-NH-(CH ₂ ) _k -C(=NH)NR ₁₁ R ₁₂ ; R ₁₉ is selected from H and (CH ₂ ) _t -NR ₂₀ R ₂₁ ; Y 1 represents N or CH; Y2 is N or CH, where at least one of Y1 and Y2 is CH; p is 0 or 1; j and t are independently selected from an integer from 0 to 6; k is independently an integer from 1 to 6; X1 is selected from O, C(=O)NH and NHC(=O); L is selected from an alkylene chain containing from 1 to 12 carbon atoms, which may have one or more carbon-carbon double or triple bonds; X ₂ is selected from O, C(=O)NH and NHC(=O); q is 0, 1, 2, 3, 4, 5 and 6; in each group A1 AI. d one of Y3 and Y4 is independently selected from NR ₁₇ , S and O; and the other of Y3 and Y4 is CH; and Y ₅ is independently selected from CH, N, S and CH; R ₂₄ is phenyl optionally substituted with up to three optional substituents selected from OH, methyl, ethyl, propyl, OCH ₃ , OCH2CH3, CO ₂ H, CO2CH3, CO2CH2CH3, O-(CH ₂ ) _k NH2, O-(CH ₂ ) _k -NH (CH ₃ ), (CH ₂ )j-NH ₂ , (CH ₂ ) _j -NH (CH ₃ ), C (= O) -NH-(CH ₂ ) _k -NH ₂ , C (= O)-NH-(CH ₂ ) _k -NH(CH ₃ ), C(=O)-NH-(CH2)kC(=NH)NH(CH3) and C(=O)-NH-(CH2)kC (=NH)NH2; R11, R ₁₂ , R ₁₇ , R ₂₀ and R ₂₁ are independently selected from H and C _1-6 alkyl; and also (i) R5 and R6 together form a double bond; (ii) R ₅ is H and R ₆ is OH; or (iii) R5 is H and R6 is OC _1-6 alkyl, provided that when p is O then (a) only one of Y3 and Y4 is selected from S and O, where Y ₅ is a CH; or (b) Y ₅ is S, with Y3 and Y4 being CH; or (c) R4 is not pyrrolyl, imidazolyl, substituted pyrrolyl, or substituted imidazolyl. 2. The compound and its salts according to claim 1, wherein R ₂₄ is -C ₆ H ₄ -(CH ₂ )jR ₁₈ , where R 18 is selected from CO2H, CO2CH3, CO2CH2CH3, NH(CH3) and NH2. 3. A compound and salts thereof according to any one of the preceding claims, wherein R ₃ is selected from methyl and ethyl. 4. The compound and its salts according to any one of the preceding claims, wherein R ₄ is selected from - 61 040562 where Z1 is selected from NH, N-CH ₃ , S and O; Z2 is selected from CH and N; Z ₃ is selected from S and O; Z ₄ is selected from CH and N; R ₂₂ is selected from (CH ₂ ) _j NR ₁₁ R ₁₂ , C(=O)-NH-C ₆ H ₄ -(CH ₂ ) _j -R ₁₈ ; R ₁₈ is selected from CO ₂ H, CO ₂ CH ₃ , CO ₂ CH ₂ CH ₃ , NH(CH ₃ ) and NH2; j is selected from an integer from 0 to 6; And R11 and R ₁₂ are independently selected from H and C _1-6 alkyl; And R ₂₃ is selected from H and C _1-6 alkyl. 5. A compound and salts thereof according to any one of the preceding claims, wherein R5 and R6 together form a double bond. 6. A compound and its salts according to any one of the preceding claims, wherein the compound is selected from where q is 0, 1, 2, 3, 4, 5 and 6; p is 0 or 1; L is an alkylene chain containing from 1 to 12 carbon atoms; Y 1 represents N or CH; Y ₂ is N or CH, where at least one of Y1 and Y ₂ is CH; Y5 is selected from CH and N; Z1 is selected from O, S, NH and N-CH3; Z2 is selected from CH and N; Z ₃ is selected from S and O; And Z ₄ is selected from CH and N; R22 is selected from (CH2)jNRiiRi2, C(=O)-NH-C6H4-(CH2)j-Ri8; R18 is selected from CO2H, CO2CH3, CO2CH2CH3, NH(CH3) and NH2; R ₁₉ is selected from H and (CH ₂ ) _t -NR ₂₀ R ₂₁ ; j and t are independently selected from an integer from 0 to 6; and Rn, R ₁₂ u R ₂₃ are independently selected from H and C _1-6 alkyl, and also (i) R5 and R6 together form a double bond; (ii) R5 is H and R6 is OH; or (iii) R5 is H and R6 is CC _1-6 alkyl; - 62 040562 provided that when the compound is (XVI) and p is 0, then Z1 is selected from Oh and S. 7. A compound and its salts according to any one of the preceding claims, wherein the compound is selected from the following: (a) (S)-N-(4-aminophenyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[ e]pyrido[1,2а][1,4]diazepin-3 -yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamido)phenyl)-1-methyl-1H-pyrrole2-carboxamide (41) (b) (S)-N-(2-((4-aminophenyl)carbamoyl)benzo[b]thiophen-5-yl)-4-(4-((2-methoxy-12-oxo-6a,7, 8,9,10,12hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)-butanamido)-1-methyl-III-pyrrole-2carboxamide (47) (c) ((8)-N-(4-Aminophenyl)-4-(4-(4-(4-((2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo [e]pyrido[1,2a] [1,4]diazepin-3-yl)oxy)-hexanamido)-1-methyl-1 H-pyrrole-2-carboxamido)phenyl)-1-methyl-1Hpyrrole-2- carboxamide (62) 8. The use of a compound and a salt thereof according to any one of claims 1 to 7 for the treatment of a proliferative disease. 9. Use according to claim 8, wherein the proliferative disease is selected from bladder cancer, bone cancer, colon cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, esophageal cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, retinoblastoma, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, and uterine cancer. 10. A pharmaceutical composition containing a compound and its salts according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient, carrier or diluent. 11. The use of a compound and its salts according to any one of paragraphs. 1-7 to obtain a medicament for the treatment of a proliferative disease. 12. A method of treating a patient suffering from a proliferative disease, comprising administering to said patient a therapeutically effective amount of a compound according to any one of paragraphs. 1-7 or a pharmaceutical composition according to claim 10. 13. The method of claim 12, wherein the proliferative disease is selected from bladder cancer, bone cancer, colon cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, esophageal cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, retinoblastoma, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, and uterine cancer. 14. The compound and its salts according to any one of claims 1 to 7, associated either directly or indirectly with a target agent to provide a targeted conjugate. 15. The compound of claim 14, wherein the target agent is an antibody. 16. The compound and its salts according to any one of claims 1 to 7 and a linker group for attaching a target agent. -