Novel Cytotoxic Agents And Conjugates Thereof

Abstract

Provided herein are novel maytansinoid compounds of general formula I. Also provided herein are conjugates comprising the compounds linked to a binding protein via a linker, and conjugating reagents comprising the compounds attached via a linker to at least one functional group capable of reacting with a binding protein. Also provided herein are pharmaceutical compositions comprising the compounds and conjugates, therapeutic methods and uses involving the compounds and conjugates, for example in cancer therapy, and novel synthetic processes.

Claims

1-44. (canceled)

45. A compound of the general formula I or a salt thereof: ##STR00207## in which R represents a group YOH, YOR.sup.x, YSH, YSR.sup.x, YS(O).sub.2NHR.sup.x, YNHS(O).sub.2R.sup.x, YC(O)H, YCO.sub.2H, YC(O)R.sup.x, YC(O)NHR.sup.x, YNHC(O)R.sup.x, YNHR.sup.y, YNR.sup.xR.sup.y, YNR.sup.yNHR.sup.z, YCR.sup.yNOH, YC(NH.sub.2)NOH, YC(O)NH.sub.2, YC(O)NHNH.sub.2, or YS(O).sub.2NH.sub.2, in which either Y is not present or Y represents a C.sub.1-6alkylene, C.sub.2-6alkenylene, C.sub.2-6alkynylene or C.sub.1-6alkyleneoxy group which may be interrupted by an oxygen atom and/or which may optionally be substituted by OH or OC.sub.1-4alkyl, or Y represents a phenylene or C.sub.5-10heteroarylene group; R.sup.x represents a C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynyl, phenyl, C.sub.5-10heteroaryl or benzyl group which is substituted by OH, SH, NHR.sup.y, or CO.sub.2H; each of R.sup.y and R.sup.z independently represents a hydrogen atom, a C.sub.1-4alkyl group, phenyl, C.sub.5-10heteroaryl or a benzyl group; X represents OH, OC.sub.1-4alkyl, SH, S.sub.1-4alkyl, or CN; Ra represents a hydrogen atom or a C.sub.1-4alkyl group; Rb represents hydrogen, OH, C.sub.1-4alkoxy or C.sub.1-4alkylC(O)O; Rc represents hydrogen, OH, C.sub.1-4alkoxy or C.sub.1-4alkylC(O)O; Rd represents a hydrogen atom or a C.sub.1-4alkyl group; each Re independently represents a halogen atom, an optionally substituted C.sub.1-6alkyl, C.sub.2-6alkenyl, C.sub.2-6alkynyl or C.sub.1-6alkoxy group each of which may be optionally interrupted by an oxygen atom, an optionally substituted phenyl or C.sub.5-10heteroaryl group, OH, CO.sub.2R.sup.v, C(O)NR.sup.vR.sup.w, NR.sup.vC(O)R.sup.w, NR.sup.vR.sup.w, SR.sup.v, S(O)R.sup.v, S(O).sub.2R.sup.v, S(O).sub.2NR.sup.vR.sup.w, a CN group, or a NO.sub.2 group; R.sup.v and R.sup.w are each independently selected from the group consisting of hydrogen, phenyl, benzyl, and an optionally substituted C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl group each of which may be optionally interrupted by an oxygen atom; and n is 0, 1, 2, 3 or 4; Rf represents a hydrogen atom or a C.sub.1-4alkyl group; and Rg represents a hydrogen atom or an optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, or heteroaryl group.

46. The compound as claimed in claim 45, wherein R is an OH, NH.sub.2, SH, CONH.sub.2, SO.sub.2NH.sub.2, CO.sub.2H, CH.sub.2OH, or NHC(O)C.sub.1-6alkylene-SH group.

47. The compound as claimed in claim 45, wherein R is a NHC(O)C.sub.1-6alkyl which alkyl is substituted by SH.

48. The compound as claimed in claim 45, wherein any Re group present is selected from the group consisting of a halogen atom, a methoxy group, a CN group or a NO.sub.2 group; and/or wherein Rg represents C.sub.1-4alkyl which is unsubstituted or substituted by N(R.sup.i)(R.sup.ii); R.sup.i represents a C.sub.1-4alkyl group; and R.sup.ii represents a C(O)C.sub.1-6alkyl group.

49. The compound as claimed in claim 45, wherein R represents a group YOH, YOR.sup.x, YSH, YSR.sup.x, YCO.sub.2H, YCOR.sup.x, YNHR.sup.y, YNR.sup.yNHR.sup.z, or YCR.sup.yNOH, in which either Y is not present or Y represents a C.sub.1-6alkylene or C.sub.1-6alkyleneoxy group either of which may be interrupted by an oxygen atom, R.sup.x represents a C.sub.1-4alkyl group substituted by OH, SH, NHR.sup.y, or CO.sub.2H, and each of R.sup.y and R.sup.z independently represents a hydrogen atom or a C.sub.1-4alkyl group; X represents OH, OC.sub.1-4alkyl, SH, S.sub.1-4alkyl, or CN; Ra represents a hydrogen atom or a C.sub.1-4alkyl group; Rb represents hydrogen, OH, C.sub.1-4alkoxy or C.sub.1-4alkylC(O)O; Rc represents hydrogen, OH, C.sub.1-4alkoxy or C.sub.1-4alkylC(O)O; Rd represents a hydrogen atom or a C.sub.1-4alkyl group; each Re independently represents a halogen atom, a CF.sub.3 group, or a C.sub.1-4alkyl or C.sub.1-4alkoxy group, and n is 0, 1, 2, 3 or 4; Rf represents a hydrogen atom or a C.sub.1-4alkyl group; and Rg represents a hydrogen atom or an optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, or heteroaryl group.

50. The compound as claimed in claim 45, in which: i) Y is not present, or in which Y represents a C.sub.1-4alkylene or C.sub.1-4alkyleneoxy group, which may be interrupted by an oxygen atom; and/or ii) R is an OH, NH.sub.2, CONH.sub.2 or CO.sub.2H group, or a C.sub.1-4alkylene group substituted by an OH, NH.sub.2, CONH.sub.2 or CO.sub.2H group; and/or R is in the 3- or 4-position of the phenyl ring; and/or iii) any Re group present is a halogen atom or a methyl or methoxy group; and/or iv) n is 0, 1 or 2; and/or v) X represents OH; and/or vi) Ra represents C.sub.1-4alkyl; Rb represents hydrogen; Rc represents hydrogen or methoxy; Rd represents C.sub.1-4alkyl; Re represents chlorine or hydrogen; Rf represents C.sub.1-4alkyl; and Rg represents C.sub.1-4alkyl.

51. The compound as claimed in claim 45, which is a compound of the general formula Ia or a salt thereof: ##STR00208##

52. The compound as claimed in claim 45, which is a compound of the general formula Ib or a salt thereof: ##STR00209##

53. A conjugate comprising the compound as claimed in claim 45 linked to a binding protein via a linker, said linker being connected to said compound via the group R of the general formula I; wherein, optionally: i) the linker includes the group SC.sub.1-6alkylene-; and/or ii) the conjugate comprises the compound as defined in claim 45; and/or iii) the binding protein is a full length antibody or an antibody fragment comprising an antigen-binding region of the full length antibody.

54. The conjugate as claimed in claim 53, wherein the binding protein is IgG1 or IgG4 or a fragment of IgG1 or IgG4.

55. The conjugate as claimed in claim 53, which includes a portion: ##STR00210## in which W represents an electron withdrawing group or a group obtained by reduction of an electron withdrawing group, each of A and B independently represents a C.sub.1-5alkylene or alkenylene chain, and Pr represents said binding protein bonded to A and B via nucleophiles Nu; or includes a portion:
W(CHCH).sub.p(CH.sub.2).sub.2Nu-Pr in which W represents an electron withdrawing group or a group obtained by reduction of an electron withdrawing group, p is 0 or an integer from 1 to 4, and Pr represents said binding protein bonded to the rest of the molecule via a nucleophile Nu.

56. The conjugate as claimed in claim 53, which includes a portion: ##STR00211## or which includes a portion:
NHCOArCO(CH.sub.2).sub.2Nu-Pr in which Ar represents an optionally substituted aryl group.

57. The conjugate as claimed in claim 53, in which each Nu represents a sulfur atom present in a cysteine residue in the binding protein Pr; or in which each Nu represents an imidazole group present in a polyhistidine tag attached to the binding protein.

58. The conjugate as claimed in claim 53, in which: i) the linker includes a pendant polyethylene glycol chain which has a terminal end group of formula CH.sub.2CH.sub.2OR.sup.r in which R.sup.r represents a hydrogen atom, an alkyl group, or an optionally substituted aryl group; or ii) at least two (CH.sub.2CH.sub.2O) units within a ring; and/or iii) the linker includes a peptidyl group comprising at least two naturally-occurring alpha amino acids.

59. The conjugate as claimed in claim 53, wherein the linker includes the sequence Val-Cit-PAB or Val-Ala.

60. A conjugating reagent comprising the compound as claimed in claim 45, attached via a linker to at least one functional group capable of reacting with a binding protein, said linker being connected to said compound via the group R of the general formula I; and wherein optionally: i) the conjugating reagent includes a portion ##STR00212## or a salt thereof; or ii) the conjugating reagent includes a portion: ##STR00213## or iii) the functional group of the conjugating reagent has the formula: ##STR00214## in which W represents an electron-withdrawing group; each of A and B independently represents a C.sub.1-5alkylene or alkenylene chain; and either each L independently represents a leaving group, or both Ls together represent a leaving group; or ##STR00215## in which W and A have the meanings given above, L represents a leaving group, and m is 0 to 4; or
W(CHCH).sub.p(CH.sub.2).sub.2-L or W(CHCH).sub.pCHCH.sub.2 in which W represents an electron withdrawing group, p represents 0 or an integer of from 1 to 4, and L represents a leaving group.

61. The conjugating reagent as claimed in claim 60, in which said functional group has the formula: ##STR00216## in which Ar represents an optionally substituted aryl group.

62. The conjugating reagent as claimed in claim 60, in which: i) the or each leaving group includes a portion (CH.sub.2CH.sub.2O).sub.q in which q is a number of six or more; or ii) the linker a) includes a pendant polyethylene glycol chain which has a terminal end group of formula CH.sub.2CH.sub.2OR.sup.r in which R.sup.r represents a hydrogen atom, an alkyl group, or an optionally substituted aryl group; or b) has at least two (CH.sub.2CH.sub.2O) units within a ring; and/or c) includes a peptidyl group comprising at least two naturally-occurring alpha amino acids; or iii) the conjugating reagent comprises the compound.

63. A pharmaceutical composition comprising the compound as claimed in claim 45, or a conjugate comprising the compound, together with a pharmaceutically acceptable carrier, optionally together with an additional therapeutic agent.

64. A method of treating a patient in need of treatment for a proliferative, autoimmune, or infectious disease or disorder, comprising administering a pharmaceutically-effective amount of the compound as claimed in claim 45, or a conjugate comprising the compound, or a pharmaceutical composition comprising the compound, to the patient.

65. A process for the preparation of a compound of the general formula I or a salt thereof as claimed in claim 45, which comprises reacting a compound of the general formula: ##STR00217## in which X, Ra-Rd, Rf and Rg have the meanings given for the general formula I, with an aryl-organometallic reagent in which the aryl moiety is a phenyl group substituted by (Re).sub.n and by R or a protected version of R, in which R and (Re).sub.n have the meanings given for the general formula I, the reaction being carried out in the presence of a transition metal catalyst; wherein, optionally, the aryl-organometallic reagent is an aryl-boronic acid or aryl-boronate ester, and in which the reaction is carried out in the presence of a palladium catalyst in the presence of water and in the absence or substantial absence of oxygen.

66. The process as claimed in claim 65, wherein R represents a group YOH, YOR.sup.x, YSH, YSR.sup.x, YCO.sub.2H, YCOR.sup.x, YNHR.sup.y, YNR.sup.yNHR.sup.z, or YCR.sup.yNOH, in which either Y is not present or Y represents a C.sub.1-6alkylene or C.sub.1-6alkyleneoxy group either of which may be interrupted by an oxygen atom, R.sup.x represents a C.sub.1-4alkyl group substituted by OH, SH, NHR.sup.y, or CO.sub.2H, and each of R.sup.y and R.sup.z independently represents a hydrogen atom or a C.sub.1-4alkyl group; X represents OH, OC.sub.1-4alkyl, SH, S.sub.1-4alkyl, or CN; Ra represents a hydrogen atom or a C.sub.1-4alkyl group; Rb represents hydrogen, OH, C.sub.1-4alkoxy or C.sub.1-4alkylC(O)O; Rc represents hydrogen, OH, C.sub.1-4alkoxy or C.sub.1-4alkylC(O)O; Rd represents a hydrogen atom or a C.sub.1-4alkyl group; each Re independently represents a halogen atom, a CF.sub.3 group, or a C.sub.1-4alkyl or C.sub.1-4alkoxy group, and n is 0, 1, 2, 3 or 4; Rf represents a hydrogen atom or a C.sub.1-4alkyl group; and Rg represents a hydrogen atom or an optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, or heteroaryl group.

67. The process as claimed in claim 65, in which the aryl-organometallic reagent is a boronic acid of the general formula: ##STR00218## or a protected version thereof and the reaction is carried out in the presence of a palladium catalyst.

68. An intermediate useful for preparing a compound of the general formula I or a salt thereof as claimed in claim 45, which has the general formula ##STR00219## in which X, n and Ra-Rg have the meanings given for the general formula I and R.sub.prot is the group R of the general formula I carrying a protecting group; or a salt thereof; wherein optionally, R includes an OH or SH group and the protecting group is a silyl group, an acyl group, or an arylmethyl group; R includes a CO.sub.2H group and the protecting group is methyl, ethyl, t-butyl, benzyl, p-methoxybenzyl, 9-fluorenylmethyl, trimethylsilyl, t-butyldimethylsilyl, or diphenylmethyl; or R includes an NHR, NHR or NHR group and the protecting group is t-butoxycarbonyl, trityl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, formyl, trimethylsilyl, or t-butyldimethylsilyl.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0159] FIGS. 1 and 2 show the results of Example 2, showing plots of % cell viability versus compound concentration for compound 3 and for DM1 in in vitro assays against the CD30-positive human T cell lymphoma cell line Karpas-299 and the HER2-positive tumour cell line SK-BR-3.

[0160] FIG. 3 shows the results of the mouse xenograft study described in Example 16, showing a plot of mean tumour volumestandard error over time following administration of vehicle, conjugate 29 of the invention, or of Kadcyla to mice.

[0161] FIG. 4 shows the results of the mouse serum study of Example 38, showing plots of drug-related ions over time for compounds 26 and 78, and demonstrating that significant degradation of compound 78 took place in mouse serum after 7 days at 37 C., whereas no comparable degradation products were observed for compound 26.

EXAMPLES

[0162] The following Examples illustrate the invention.

General Methods:

[0163] .sup.1H NMR spectra were recorded using an appropriate NMR instrument (e.g. a Varian Inova 500 MHz NMR instrument). Chromatographic purities were determined using LC/MS (e.g. on an Agilent 1200 Series LC/MS system).

Example 1: Preparation of 4-(N-Boc amino)phenyl-AP3, 3

[0164] ##STR00114##

[0165] Compound 1 4-(N-Boc amino)phenyl boronic acid (18.7 mg), was added to solid AP3 2 (32.6 mg), with potassium phosphate tribasic (32.7 mg) and the palladium catalyst Sphos Pd G3 (1 mg) commercially available from Sigma-Aldrich. The solids were purged with Ar 3 and dry THF (150 L) added to the mixture. Ar was then bubbled through the reaction mixture for 60 sec, the reaction vessel was sealed and heated at 40 C. for 2 h, at which point the reaction was deemed complete by LC/MS. The reaction mixture was then filtered through a short pad of SiO.sub.2, washed with Et.sub.2O and concentrated. The mixture was then purified by preparative HPLC (Gemini 15030 mm column) (5.fwdarw.95% ACN in H.sub.2O, each containing 0.05% AcOH) and desired fractions lyophilized to afford 3 as a white crystalline solid (14.3 mg, 35% yield). .sup.1H NMR (500 MHz, CHLOROFORM-d) =7.41 (d, J=7.3 Hz, 2H), 7.06 (d, J=8.8 Hz, 2H), 6.88 (dd, J=1.5, 10.7 Hz, 2H), 6.58-6.46 (m, 2H), 6.27-6.18 (m, 2H), 5.49 (dd, J=9.0, 15.4 Hz, 1H), 4.85 (dd, J=2.9, 12.2 Hz, 1H), 4.36-4.28 (m, 1H), 3.83 (s, 3H), 3.61-3.52 (m, 2H), 3.39 (s, 3H), 3.28 (d, J=13.2 Hz, 1H), 3.09-2.95 (m, 3H), 2.70-2.68 (m, 3H), 2.65 (quin, J=7.0 Hz, 1H), 2.25 (dd, J=2.9, 13.7 Hz, 1H), 1.77 (s, 3H), 1.70 (d, J=13.2 Hz, 1H), 1.55 (s, 9H), 1.33 (d, J=6.3 Hz, 3H), 1.28 (d, J=7.3 Hz, 3H), 1.22 (d, J=6.8 Hz, 3H), 0.96 (s, 3H); LC/MS: retention time 3.49 min (Merck Chromolith RP-18e analytical HPLC column (monolithic, 502 mm); analytical HPLC method: injection volume 5 L; flow rate 1 mL/min; 5.fwdarw.95% acetonitrile in water over 5 mins; Agilent diode array detector at =254, or 220 nm; room temperature), (ES.sup.+) calc for C.sub.43H.sub.58N.sub.3O.sub.11: [M+H].sup.+ 792; found 792.

##STR00115##

[0166] The BOC protecting group may be removed from compound 3 to produce the corresponding compound containing a free amine group. This compound may then, in turn, be conjugated via the amine group using known methods to produce a conjugate, specifically, and antibody-drug conjugate.

Example 2: In Vitro Potency Assay of Compound 3 in Karpas-299 and SK-BR-3 Cell Lines

[0167] Loss of tumour cell viability following treatment with cytotoxic drugs or ADCs in vitro can be measured by growing cell lines in the presence of increasing concentrations of drugs or ADCs and quantifying the loss of proliferation or metabolic activity using CellTiter-Glo Luminescence reagent (Promega). The protocol describes cell seeding, drug treatment and determination of the cell viability in reference to untreated cells based on ATP synthesis, which is directly related to the number of cells present in the well.

[0168] The CD30-positive human T cell lymphoma cell line Karpas-299 was obtained from Dr Abraham Karpas at the University of Cambridge. The cells were grown in RPMI medium supplemented with 10% foetal bovine serum, 100 U/mL Penicillin and 100 g/mL Streptomycin. Karpas 299 cells were seeded at 2,500 cells per well (50 L/well) into clear bottom white-walled 96-well plates and incubated for 24 h at 37 C. and 5% CO.sub.2.

[0169] HER2-positive tumour cell line, SK-BR-3 (ATCC-HTB-30), was grown in McCoy's 5A medium complemented with 10% foetal bovine serum, 100 u/mL Penicillin and 100 g/mL Streptomycin. SK-BR-3 cells were detached, seeded at 5,000 cells per well (100 L/well) into poly-D-lysine coated clear bottom white-walled 96-well plates and incubated for 24 h at 37 C. and 5% CO.sub.2.

[0170] Serial dilutions of compounds were prepared in triplicate using the relevant cell culture medium as diluent. The CD30-positive Karpas-299 cells were treated with 50 L compounds at 2 assay concentration. Medium from the SK-BR-3 assay plates was removed and replaced by 100 L serially diluted compounds at 1 assay concentration. The assay concentrations are specified in Table 1. The cells were then incubated with the compounds (total volume 100 L/well), at 37 C. and 5% CO.sub.2 for a further 96 h.

TABLE-US-00001 TABLE 1 Cell line Compound Concentration range Karpas-299 3 200 nM-20 fM SK-BR-3 3 50 nM-640 fM

[0171] At the end of the incubation, cell viability was measured using the CellTiter-Glo Luminescence reagent, as described by the manufacturer's instructions. The data was subsequently analysed using a four parameter non-linear regression model.

[0172] Viability was expressed as % of untreated cells and calculated using the following formula:

[00001] $% .Math. .Math. Viability = 100 \frac{{Luminescence}_{Sample} - {Luminescence}_{No .Math. .Math. cell .Math. .Math. Control}}{{Luminescence}_{Untreated} - {Luminescence}_{No .Math. .Math. cell .Math. .Math. Control}}$

[0173] The % viability (Y-axis) was plotted against the logarithm of drug concentration in nM (X-axis) to extrapolate the IC.sub.50 values for all compounds.

[0174] Results are shown in Table 2 and FIGS. 1 and 2. In both cell lines compound 3 has higher potency than the control DM1.

TABLE-US-00002 TABLE 2 IC50 (nM) SKBR3 IC50 (nM) Karpas- Compound number/name Cell line 299 Cell line 3 4 0.7 DM1 10 9

Example 3: Preparation of Maytansinoid Compound 4

[0175] ##STR00116##

[0176] The aryl boron reagent, 4-aminophenylboronic acid (215 mg), tripotassium phosphate (668 mg), the catalyst SPhos Pd G3 (15.4 mg) and AP3 (500 mg) were sequentially added to an argon purged reaction vessel. The vessel was then sealed and the solids purged with argon (4evacuation/purge cycles). THF (6 mL) and water (0.6 mL), which had been rigorously deoxygenated by purging with argon, were then added and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was then diluted with ethyl acetate (60 mL) and washed with brine (20 mL). The layers were separated and the organic layer was concentrated under reduced pressure. The residue was then purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v) and the desired fractions lyophilised to give compound 4 as a white solid (449 mg). .sup.1H NMR (500 MHz; CDCl.sub.3) 6.90 (d, J=8.1 Hz, 2H), 6.84 (d, J=10.1 Hz, 2H), 6.69 (d, J=8.6 Hz, 2H), 6.48 (dd, J=15.4, 11.0 Hz, 1H), 6.24 (s, 1H), 6.18 (d, J=11.0 Hz, 1H), 5.49-5.44 (m, 1H), 4.84 (dd, J=11.9, 2.8 Hz, 1H), 4.32-4.27 (m, 1H), 3.82 (s, 3H), 3.55 (d, J=13.0 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.37 (s, 3H), 3.25 (d, J=13.0 Hz, 1H), 3.06 (s, 1H), 3.00 (d, J=9.9 Hz, 1H), 2.97-2.94 (m, 1H), 2.68 (s, 3H), 2.64-2.61 (m, 1H), 2.23 (dd, J=13.8, 2.7 Hz, 1H), 1.74 (s, 3H), 1.68 (d, J=13.6 Hz, 1H), 1.52-1.47 (m, 1H), 1.30 (d, J=6.5 Hz, 3H), 1.26 (d, J=7.2 Hz, 3H), 1.25-1.23 (m, 1H), 1.20 (d, J=6.5 Hz, 3H), 0.93 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (692, 100%).

Example 4: Preparation of Maytansinoid Compound 5

[0177] ##STR00117##

[0178] Compound 5 was synthesised in an analogous way to compound 4 of Example 3. Briefly, 4-amino-3-chlorophenylboronic acid pinacol ester (100 mg), tripotassium phosphate (674 mg), SPhos Pd G3 (15.2 mg) and AP3 (500 mg) were sequentially added to an argon purged reaction vessel. The vessel was then sealed and the solids purged with argon (4 evacuation/purge cycles). THF (6 mL) and water (0.6 mL), which had been rigorously deoxygenated by purging with argon, were then added and the reaction mixture was stirred at room temperature for 5 h. The reaction mixture was then diluted with ethyl acetate (60 mL) and washed with brine (20 mL). The layers were separated and the organic layer was concentrated under reduced pressure. The residue was then purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v) and the desired fractions lyophilised to give compound 5 as a white solid (80 mg). NMR (500 MHz; CDCl.sub.3) 7.05 (s, 1H), 6.85 (d, J=11.4 Hz, 2H), 6.77 (s, 2H), 6.48 (dd, J=15.4, 11.2 Hz, 1H), 6.24 (s, 1H), 6.18 (d, J=11.2 Hz, 1H), 5.46 (dd, J=15.4, 9.0 Hz, 1H), 4.84 (dd, J=11.8, 2.8 Hz, 1H), 4.32-4.27 (m, 1H), 4.11 (d, J=0.6 Hz, 2H), 3.83 (s, 3H), 3.55 (d, J=13.0 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.37 (s, 3H), 3.25 (d, J=13.0 Hz, 1H), 3.02-2.99 (m, J=7.8 Hz, 2H), 2.93 (dd, J=13.7, 12.2 Hz, 1H), 2.72 (s, 3H), 2.65-2.60 (m, 1H), 2.24-2.21 (m, 1H), 1.74 (s, 3H), 1.68 (d, J=13.3 Hz, 1H), 1.53-1.47 (m, 1H), 1.30 (d, J=6.4 Hz, 3H), 1.30-1.27 (m, 1H), 1.26 (d, J=7.2 Hz, 3H), 1.20 (d, J=6.7 Hz, 3H), 0.93 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (726, 100%).

Example 5: Preparation of Maytansinoid Compound 6

[0179] ##STR00118##

Step 1: Synthesis of Compound 7.

[0180] ##STR00119##

[0181] To a solution of 2,2-dithiobis(5-nitropyridine) (930 mg) and 4-mercapto-pentanoic acid (197 mg) in THF:DMF (8 mL, 1:1 v/v) was added pyridine (150 L) and the solution was stirred at room temperature for 15 h. The reaction mixture was then diluted with ethyl acetate (50 mL) and washed with brine (20 mL). The organic layer was separated, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was then purified by normal phase chromatography eluting with hexane:ethyl acetate (100:0 v/v to 60:40 v/v). The solvent was removed in vacuo to give compound 7 as a pale yellow solid (194 mg). .sup.1H NMR (500 MHz; CDCl.sub.3) 9.26 (d, J=2.1 Hz, 1H), 8.39 (dd, J=8.8, 2.1 Hz, 1H), 7.89 (d, J=8.8 Hz, 1H), 3.13-3.06 (m, 1H), 2.64-2.53 (m, 2H), 2.04-1.89 (m, 2H), 1.37 (d, J=6.8 Hz, 3H). LC/MS: (ES+) [M+H].sup.+ (289, 100%).

Step 2: Synthesis of Compound 8.

[0182] ##STR00120##

[0183] To a stirred solution of compound 4 (237 mg), compound 7 (102 mg) and HATU (405 mg) in DMF (8 mL) at 0 C. was slowly added N,N-diisopropylethylamine (DIPEA, 240 L). The reaction mixture was allowed to warm to room temperature and was stirred for 16 h. The solution was diluted with water (20 mL) and brine (20 mL) and the resulting mixture was extracted with ethyl acetate (50 mL). The organic layer was separated and concentrated in vacuo. The residue was dissolved in DMF (8 mL) and purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v). The desired fractions were lyophilised to give compound 8 as a pale yellow solid (253 mg). .sup.1H NMR (500 MHz; CDCl.sub.3) 9.27 (d, J=2.2 Hz, 1H), 8.40 (dt, J=8.8, 2.9 Hz, 1H), 7.95-7.93 (m, 1H), 7.56-7.54 (m, 2H), 7.47-7.45 (m, 1H), 7.10 (dd, J=7.6, 0.4 Hz, 2H), 6.89 (d, J=7.7 Hz, 2H), 6.54-6.48 (m, 1H), 6.24 (d, J=0.4 Hz, 1H), 6.21 (d, J=11.1 Hz, 1H), 5.49 (dd, J=15.2, 9.1 Hz, 1H), 4.85 (dd, J=12.0, 2.8 Hz, 1H), 4.34-4.29 (m, 1H), 3.84 (s, 3H), 3.59 (d, J=13.0 Hz, 1H), 3.54 (d, J=9.0 Hz, 1H), 3.39 (s, 3H), 3.29 (d, J=12.9 Hz, 1H), 3.22-3.17 (m, 1H), 3.04-2.97 (m, 3H), 2.69 (d, J=2.8 Hz, 3H), 2.67-2.56 (m, 3H), 2.27-2.24 (m, 1H), 2.12-2.07 (m, 2H), 1.77 (s, 3H), 1.70 (d, J=13.0 Hz, 1H), 1.55-1.48 (m, 1H), 1.42-1.41 (m, 3H), 1.32 (d, J=6.4 Hz, 3H), 1.27-1.26 (m, 1H), 1.28 (d, J=7.2 Hz, 3H), 1.22 (d, J=6.7 Hz, 3H), 0.96 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (962, 100%).

Step 3: Synthesis of Compound 6.

[0184] To a solution of compound 8 (22 mg) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP.HCl, 67 mg) in acetonitrile:water (1.8 mL, 5:4 v/v) was slowly added saturated sodium hydrogen carbonate solution (1.2 mL) until a pH of 7-8 was achieved. The reaction mixture was then stirred at room temperature for 2 h before the solution was concentrated in vacuo and the residue dissolved in acetonitrile (50 mL). The acetonitrile solution was then washed with brine (20 mL), the layers were separated and the organic layer was concentrated in vacuo. The residue was then purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v) and the desired fractions lyophilised to give compound 6 as a white solid (14 mg). .sup.1H NMR (500 MHz; CDCl.sub.3) 7.58-7.57 (m, 1H), 7.51-7.47 (m, 1H), 7.09 (d, J=8.2 Hz, 2H), 6.89 (d, J=9.0 Hz, 2H), 6.51 (dd, J=15.4, 11.1 Hz, 1H), 6.24 (s, 1H), 6.22-6.20 (m, 1H), 5.49 (dd, J=15.3, 9.0 Hz, 1H), 4.85 (dd, J=12.0, 2.8 Hz, 1H), 4.34-4.29 (m, 1H), 3.83 (s, 3H), 3.58 (d, J=12.9 Hz, 1H), 3.54 (d, J=8.9 Hz, 1H), 3.39 (s, 3H), 3.28 (d, J=12.8 Hz, 1H), 3.07-2.96 (m, 4H), 2.69 (s, 3H), 2.66-2.62 (m, 1H), 2.61-2.53 (m, 2H), 2.27-2.24 (m, 1H), 2.19-2.13 (m, 1H), 1.86-1.80 (m, 1H), 1.77 (s, 3H), 1.70 (d, J=13.5 Hz, 1H), 1.54-1.48 (m, 2H), 1.43 (d, J=6.7 Hz, 3H), 1.32 (d, J=6.3 Hz, 3H), 1.28 (d, J=7.2 Hz, 3H), 1.27-1.26 (m, 1H), 1.22 (d, J=6.7 Hz, 3H), 0.96 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (808, 100%).

Example 6: Preparation of Maytansinoid Compound 9

[0185] ##STR00121##

Step 1: Synthesis of Compound 10.

[0186] ##STR00122##

[0187] Compound 10 was synthesised in an analogous way to compound 7 of Example 5 using 4-mercapto-4-methyl-pentanoic acid instead of 4-mercapto-pentanoic acid. Compound 10 was isolated as a pale yellow solid. LC/MS: (ES+) [M+H].sup.+ (303, 100%).

Step 2: Synthesis of Compound 11.

[0188] ##STR00123##

[0189] Compound 11 was synthesised in an analogous way to compound 8 of Example 5 using compound 10 instead of compound 7. Compound 11 was isolated as a pale yellow solid. .sup.1H NMR (500 MHz; CDCl.sub.3) 9.35-9.24 (m, 1H), 8.44-8.36 (m, 1H), 7.88 (dd, J=46.8, 8.7 Hz, 1H), 7.54-7.44 (m, 2H), 7.35-7.34 (m, 1H), 7.09-7.07 (m, 1H), 6.87-6.86 (m, 2H), 6.51-6.46 (m, 1H), 6.26 (s, 1H), 6.20-6.17 (m, 1H), 5.47 (dd, J=15.6, 9.1 Hz, 1H), 4.85-4.82 (m, 1H), 4.32-4.27 (m, 1H), 3.82 (s, 3H), 3.58-3.55 (m, 1H), 3.52 (d, J=8.9 Hz, 1H), 3.37 (s, 3H), 3.28-3.25 (m, 1H), 3.16-3.12 (m, 1H), 3.03-2.94 (m, 3H), 2.67 (s, 3H), 2.66-2.60 (m, 1H), 2.56-2.53 (m, 2H), 2.25-2.22 (m, 1H), 2.15-2.07 (m, 2H), 1.75 (s, 3H), 1.69-1.66 (m, 1H), 1.52-1.48 (m, 1H), 1.37 (s, 6H), 1.30 (d, J=6.3 Hz, 3H), 1.26 (d, J=7.1 Hz, 3H), 1.25-1.24 (m, 1H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (976, 100%).

Step 3: Synthesis of Compound 9.

[0190] Compound 9 was synthesised in an analogous way to compound 6 of Example 5 using compound 11 instead of compound 8. Compound 9 was isolated as a white solid. .sup.1H NMR (500 MHz; CDCl.sub.3) .sup.1H NMR (500 MHz; CDCl.sub.3) 7.56-7.54 (m, 2H), 7.34 (s, 1H), 7.08-7.07 (m, 2H), 6.86 (d, J=9.8 Hz, 2H), 6.51-6.46 (m, 1H), 6.21-6.18 (m, 2H), 5.49-5.44 (m, 1H), 4.85-4.82 (m, 1H), 4.32-4.27 (m, 1H), 3.81 (s, 3H), 3.56 (d, J=12.7 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.37 (s, 3H), 3.26 (d, J=13.6 Hz, 1H), 3.01-2.94 (m, 3H), 2.67 (s, 3H), 2.64-2.61 (m, 1H), 2.59-2.56 (m, 2H), 2.25-2.22 (m, 1H), 2.04-2.01 (m, 2H), 1.75 (s, 3H), 1.69-1.67 (m, 1H), 1.52-1.48 (m, 1H), 1.43 (s, 6H), 1.30 (d, J=6.3 Hz, 3H), 1.26 (d, J=7.1 Hz, 3H), 1.25-1.23 (m, 1H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (822, 100%).

Example 7: Preparation of Maytansinoid Compound 12

[0191] ##STR00124##

Step 1: Synthesis of Compound 13.

[0192] ##STR00125##

[0193] To a stirred solution of 2,2-dithiobis(5-nitropyridine) (682 mg) and 4-methylmorpholine (NMM, 600 L) in THF (10 mL) was slowly added a solution of 4-mercaptobutyric acid (222 mg) in ethyl acetate (2 mL) and the combined solution was stirred at room temperature for 1 h. The reaction mixture was then diluted with saturated sodium hydrogen carbonate solution (10 mL), water (10 mL) and ethyl acetate (30 mL). The organic layer was separated and washed with saturated sodium hydrogen carbonate solution (20 mL). The aqueous layers were combined and acidified with 1 M HCl (70 mL) before extracting with ethyl acetate (250 mL). The combined organic layers were then dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by normal phase chromatography eluting with hexane (0.5% acetic acid):ethyl acetate (100:0 v/v to 60:40 v/v). The solvent was removed in vacuo to give compound 13 as a pale yellow solid (82 mg). .sup.1H NMR (500 MHz, CDCl.sub.3) 9.28 (s, 1H), 8.40 (dd, J=8.8, 2.6 Hz, 1H), 7.87 (d, J=8.8 Hz, 1H), 2.91 (t, J=7.1 Hz, 2H), 2.54 (t, J=7.1 Hz, 2H), 2.05 (quintet, J=7.1 Hz, 2H). LC/MS: (ES+) [M+H].sup.+ (275, 100%).

Step 2: Synthesis of Compound 14.

[0194] ##STR00126##

[0195] Compound 14 was synthesised in an analogous way to compound 8 of Example 5 using compound 13 instead of compound 7. Compound 14 was isolated as a pale yellow solid. .sup.1H NMR (500 MHz; CDCl.sub.3) 9.30 (d, J=2.2 Hz, 1H), 8.43 (dd, J=8.8, 2.5 Hz, 1H), 7.92 (d, J=9.0 Hz, 1H), 7.57-7.54 (m, 1H), 7.44-7.41 (m, 1H), 7.11-7.08 (m, 2H), 6.90-6.88 (m, 2H), 6.53-6.48 (m, 1H), 6.23-6.19 (m, 2H), 5.52-5.47 (m, 1H), 4.86-4.83 (m, 1H), 4.31-1.29 (m, 1H), 3.84 (s, 3H), 3.59 (d, J=13.1 Hz, 1H), 3.54 (d, J=9.0 Hz, 1H), 3.39 (s, 3H), 3.29 (d, J=12.7 Hz, 1H), 3.04-2.97 (m, 5H), 2.69 (s, 3H), 2.67-2.62 (m, 1H), 2.58-2.55 (m, 2H), 2.27-2.24 (m, 1H), 2.21-2.15 (m, 2H), 1.77 (s, 3H), 1.70 (ddd, J=13.0, 1.4, 0.6 Hz, 1H), 1.54-1.50 (m, 1H), 1.32 (d, J=6.4 Hz, 3H), 1.28 (d, J=11.2 Hz, 4H), 1.27-1.26 (m, 1H), 1.22 (d, J=6.7 Hz, 3H), 0.96 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (948, 100%).

Step 3: Synthesis of Compound 12.

[0196] Compound 12 was synthesised in an analogous way to compound 6 of Example 5 using compound 14 instead of compound 8. Compound 12 was isolated as a white solid. .sup.1H NMR (500 MHz; CDCl.sub.3) 7.56-7.54 (m, 1H), 7.49 (d, J=3.9 Hz, 1H), 7.08-7.07 (m, 2H), 6.86 (d, J=8.8 Hz, 2H), 6.49 (dd, J=15.3, 11.0 Hz, 1H), 6.21-6.18 (m, 2H), 5.47 (dd, J=15.3, 9.0 Hz, 1H), 4.81-1.81 (m, 1H), 4.32-4.27 (m, 1H), 3.81 (s, 3H), 3.56 (d, J=13.0 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.37 (s, 3H), 3.28-3.25 (m, 1H), 3.04-2.95 (m, 3H), 2.67 (s, 3H), 2.65-2.61 (m, 2H), 2.53 (t, J=7.2 Hz, 2H), 2.26-2.22 (m, 1H), 2.07-2.02 (m, 2H), 1.75 (s, 3H), 1.69 (d, J=0.4 Hz, 1H), 1.54-1.48 (m, 1H), 1.38 (t, J=8.0 Hz, 1H), 1.30 (d, J=6.3 Hz, 3H), 1.26 (d, J=7.2 Hz, 3H), 1.25-1.24 (m, 1H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (794, 100%).

Example 8: Preparation of Maytansinoid Compound 15

[0197] ##STR00127##

Step 1: Synthesis of Compound 16.

[0198] ##STR00128##

[0199] To a stirred solution of 3-mercapto-propionic acid (0.41 mL) in water (15 mL) at 0 C. was added a solution of S-methyl methanethiolsulfonate (0.49 mL) in ethanol (7.5 mL). The reaction mixture was allowed to warm to room temperature and was stirred for 18 h before concentrating in vacuo. Saturated sodium hydrogen carbonate solution (8 mL) was added to the residue until a pH of 7-8 was achieved and the resulting mixture was then extracted with dichloromethane (210 mL). The layers were separated and the aqueous layer was acidified with 0.1 M HCl solution (12 mL) and then 1 M HCl solution (6 mL) to achieve a solution pH of 2-3. The aqueous layer was then extracted with ethyl acetate (240 mL). The organic layers were separated and combined, dried over sodium sulfate, filtered and concentrated in vacuo to give compound 16 as a white solid (0.71 g). .sup.1H NMR (500 MHz; CDCl.sub.3) 2.94 (t, J=6.8 Hz, 2H), 2.83 (d, J=6.8 Hz, 2H), 2.42 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (152, 100%).

Step 2: Synthesis of Compound 17.

[0200] ##STR00129##

[0201] Compound 17 was synthesised in an analogous way to compound 8 of Example 5 using compound 16 instead of compound 7. Compound 17 was isolated as a white solid. LC/MS: (ES+) [M+H].sup.+ (826, 100%).

Step 3: Synthesis of Compound 15.

[0202] Compound 15 was synthesised in an analogous way to compound 6 of Example 5 using compound 17 instead of compound 8. Compound 15 was isolated as a white solid. .sup.1H NMR (500 MHz; CDCl.sub.3) 7.63 (s, 1H), 7.57-7.55 (m, 1H), 7.09-7.07 (m, 2H), 6.87 (d, J=8.2 Hz, 2H), 6.49 (dd, J=15.3, 11.1 Hz, 1H), 6.22-6.18 (m, 2H), 5.47 (dd, J=15.3, 9.1 Hz, 1H), 4.85-4.82 (m, 1H), 4.32-4.27 (m, 1H), 3.82 (s, 3H), 3.57 (d, J=13.0 Hz, 1H), 3.52 (d, J=8.9 Hz, 1H), 3.37 (s, 3H), 3.26 (d, J=12.7 Hz, 1H), 3.04-2.95 (m, 3H), 2.90 (q, J=7.4 Hz, 2H), 2.70 (dd, J=8.7, 4.5 Hz, 2H), 2.67 (s, 3H), 2.64-2.60 (m, 1H), 2.24 (dd, J=13.9, 2.6 Hz, 1H), 1.75 (s, 3H), 1.72 (d, J=8.5 Hz, 1H), 1.70-1.66 (m, 1H), 1.54-1.48 (m, 1H), 1.30 (d, J=6.4 Hz, 3H), 1.26 (d, J=7.2 Hz, 3H), 1.24-1.23 (m, 1H), 1.20 (d, J=6.8 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (780, 100%).

Example 9: Preparation of Maytansinoid Compound 18

[0203] ##STR00130##

Step 1: Synthesis of Compound 19.

[0204] ##STR00131##

[0205] To a solution of 4-mercapto-pentanoic acid (0.97 g) in ethanol:water (10 mL, 1:1 v/v) was added S-methyl methanethiolsulfonate (0.72 mL) and the reaction mixture was stirred under an argon atmosphere at room temperature for 22 h. The ethanol was removed under reduced pressure before the reaction solution was diluted with brine (10 mL) and extracted with ethyl acetate (315 mL). The combined organic layers were then washed with brine (15 mL), the organic layer was separated, dried over sodium sulfate, filtered and concentrated in vacuo to give compound 19 as a yellow oil (0.9 g). .sup.1H NMR (300 MHz; CDCl.sub.3): 9.09 (s, 1H), 2.89 (m, 1H), 2.52 (t, J=7.6 Hz, 2H), 2.41 (s, 3H), 2.03-1.85 (m, 2H), 1.35 (d, J=6.8 Hz, 3H).

Step 2: Synthesis of Compound 20.

[0206] ##STR00132##

[0207] To a solution of compound 19 (33 mg) in anhydrous THF (2 mL) at 0 C. under an argon atmosphere was added isobutyl chloroformate (25 L) and NMM (50 L). After stirring for 20 min at 0 C., a solution of compound 5 (42 mg) in anhydrous THF (4 mL) was slowly added to the reaction mixture which was allowed to warm to room temperature and was stirred for a further 16 h. A solution of compound 19 (33 mg) and isobutyl chloroformate (25 L) in anhydrous THF (1 mL), which had been stirred under an argon atmosphere at room temperature for 1 h, was then added to the reaction mixture. The reaction solution was then stirred for 24 h before the solvent was removed in vacuo and the residue purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.05% acetic acid and buffer B (v/v): acetonitrile:0.05% acetic acid (80:20 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give compound 20 as a white solid (20 mg). .sup.1H NMR (500 MHz; CDCl.sub.3): 8.42 (s, 1H), 7.70 (s, 1H), 7.12 (s, 1H), 7.04 (d, J=7.0 Hz, 1H), 6.88 (s, 1H), 6.85 (s, 1H), 6.48 (dd, J=15.4, 11.1 Hz, 1H), 6.21-6.17 (m, 2H), 5.47 (dd, J=15.5, 8.9 Hz, 1H), 4.83 (dd, J=11.9, 2.3 Hz, 1H), 4.30 (t, J=10.8 Hz, 1H), 3.82 (s, 3H), 3.56 (d, J=13.0 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.37 (s, 3H), 3.27 (d, J=12.4 Hz, 2H), 3.00 (d, J=9.5 Hz, 2H), 2.94-2.89 (m, 3H), 2.73 (s, 3H), 2.64 (dq, J=21.7, 7.3 Hz, 3H), 2.43 (d, J=3.6 Hz, 1H), 2.23-2.21 (m, 1H), 2.17-2.15 (m, 1H), 2.06 (dd, J=13.1, 7.4 Hz, 1H), 1.75 (s, 3H), 1.67 (d, J=13.7 Hz, 1H), 1.50 (d, J=6.6 Hz, 3H), 1.40 (d, J=6.7 Hz, 1H), 1.31 (d, J=6.2 Hz, 3H), 1.26 (q, J=8.2 Hz, 5H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (888, 100%).

Step 3: Synthesis of Compound 18.

[0208] Compound 18 was synthesised in an analogous way to compound 6 of Example 5 using compound 20 instead of compound 8. .sup.1H NMR (500 MHz; CDCl.sub.3): 8.41 (s, 1H), 7.70 (s, 1H), 7.12 (s, 1H), 7.04 (d, J=6.8 Hz, 1H), 6.88 (s, 1H), 6.85 (s, 1H), 6.48 (dd, J=15.2, 11.2 Hz, 1H), 6.21-6.17 (m, 2H), 5.47 (dd, J=15.4, 9.0 Hz, 1H), 4.84-4.80 (m, 1H), 4.30 (t, J=11.0 Hz, 1H), 3.82 (s, 3H), 3.56 (d, J=12.8 Hz, 1H), 3.52 (d, J=8.9 Hz, 1H), 3.37 (s, 3H), 3.27 (d, J=12.7 Hz, 1H), 2.99 (t, J=8.4 Hz, 1H), 2.92 (t, J=12.8 Hz, 1H), 2.73 (s, 3H), 2.67-2.59 (m, 2H), 2.22 (d, J=13.9 Hz, 1H), 2.08-2.04 (m, 1H), 1.75 (s, 3H), 1.67 (d, J=14.3 Hz, 2H), 1.50 (d, J=6.6 Hz, 3H), 1.36 (d, J=6.8 Hz, 2H), 1.30 (t, J=6.6 Hz, 6H), 1.20 (d, J=6.6 Hz, 3H), 0.94 (s, 3H), 0.88 (dd, J=11.6, 5.5 Hz, 3H). LC/MS: (ES+) [M+H].sup.+ (842, 100%).

Example 10: Preparation of Maytansinoid Compound 21

[0209] ##STR00133##

Step 1: Synthesis of Compound 22.

[0210] ##STR00134##

[0211] Compound 22 was synthesised in an analogous way to compound 8 of Example 5 using compound 5 instead of compound 4 and using compound 10 instead of compound 7. Compound 22 was isolated as a white solid. LC/MS: (ES+) [M+H].sup.+ (1010, 100%).

Step 2: Synthesis of Compound 21.

[0212] Compound 21 was synthesised in an analogous way to compound 6 of Example 5 using compound 22 instead of compound 8. Compound 21 was isolated as a white solid. .sup.1H NMR (500 MHz; CDCl.sub.3) 8.44-8.42 (m, 1H), 7.69 (d, J=0.3 Hz, 1H), 7.12 (d, J=0.6 Hz, 1H), 7.06-7.04 (m, 1H), 6.86 (d, J=11.8 Hz, 2H), 6.51-6.45 (m, 1H), 6.22-6.17 (m, 2H), 5.47 (dd, J=15.4, 9.0 Hz, 1H), 4.84-4.82 (m, 1H), 4.84-4.82 (m, 1H), 4.32-4.27 (m, 1H), 3.82 (s, 3H), 3.56 (d, J=13.0 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.37 (s, 3H), 3.27 (d, J=12.7 Hz, 1H), 3.00 (d, J=9.5 Hz, 2H), 2.92 (t, J=12.9 Hz, 1H), 2.72 (s, 3H), 2.66-2.60 (m, 3H), 2.24-2.21 (m, 1H), 2.04 (dd, J=9.2, 6.8 Hz, 2H), 1.75 (s, 3H), 1.67 (d, J=11.3 Hz, 1H), 1.54-1.48 (m, 1H), 1.44 (s, 6H), 1.31 (d, J=6.3 Hz, 3H), 1.25 (s, 3H), 1.25-1.23 (m, 1H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (856, 100%).

Example 11: Preparation of Maytansinoid Compound 23

[0213] ##STR00135##

Step 1: Synthesis of Compound 24.

[0214] ##STR00136##

[0215] To a stirred solution of compound 5 (50 mg), compound 16 (29 mg) and HATU (79 mg) in DMF (1.5 mL) at 0 C. was slowly added DIPEA (50 L). The reaction mixture was allowed to warm to room temperature and was stirred for 14 h. Additional quantities of compound 16 (47 mg) in DMF (500 L), HATU (129 mg) and DIPEA (100 L) were added and the reaction mixture was stirred at room temperature for a further 6 h. The reaction mixture was then directly purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give compound 24 as a white solid (15 mg). LC/MS: (ES+) [M+H].sup.+ (860, 100%).

Step 2: Synthesis of Compound 23.

[0216] Compound 23 was synthesised in an analogous way to compound 6 of Example 5 using compound 24 instead of compound 8. Compound 23 was isolated as a white solid. NMR (500 MHz; CDCl.sub.3) .sup.1H NMR (500 MHz; CDCl.sub.3) 8.45-8.43 (m, 1H), 7.75 (s, 1H), 7.13 (s, 1H), 7.06 (d, J=8.2 Hz, 1H), 6.88-6.85 (m, 2H), 6.48 (dd, J=15.4, 11.0 Hz, 1H), 6.21-6.17 (m, 2H), 5.47 (dd, J=15.5, 9.0 Hz, 1H), 4.83 (dd, J=11.8, 2.9 Hz, 1H), 4.32-4.27 (m, 1H), 3.82 (s, 3H), 3.56 (d, J=12.9 Hz, 1H), 3.52 (d, J=8.9 Hz, 1H), 3.37 (s, 3H), 3.27 (d, J=12.8 Hz, 1H), 3.02-2.99 (m, 2H), 2.94-2.90 (m, 3H), 2.78 (t, J=6.5 Hz, 2H), 2.73 (s, 3H), 2.65-2.60 (m, 1H), 2.23 (dd, J=13.7, 2.8 Hz, 1H), 1.74 (s, 3H), 1.67 (d, J=13.1 Hz, 1H), 1.53-1.47 (m, 1H), 1.31 (d, J=6.3 Hz, 3H), 1.26 (d, J=7.2 Hz, 3H), 1.25-1.23 (m, 1H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (814, 100%).

Example 12: Preparation of Conjugation Reagent 25 Comprising a Maytansinoid Cytotoxic Payload

[0217] ##STR00137##

Step 1: Synthesis of Compound 26.

[0218] ##STR00138##

[0219] A mixture of compound 8 (17 mg) and 4-mercapto-pentanoic acid (10 mg) in DMF (2 mL) was stirred at room temperature for 16 h. The reaction mixture was then diluted with ethyl acetate (20 mL) and washed with brine (10 mL). The organic layer was separated and concentrated in vacuo before the residue was dissolved in DMF (6 mL) and purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give compound 26 as an off-white solid (23 mg). .sup.1H NMR (500 MHz; CDCl.sub.3) 8.17 (s, 1H), 7.59-7.58 (m, 2H), 7.06-7.05 (m, 2H), 6.86 (s, 2H), 6.48 (dd, J=15.3, 11.1 Hz, 1H), 6.41 (s, 1H), 6.18 (d, J=10.9 Hz, 1H), 5.47 (dd, J=15.4, 8.9 Hz, 1H), 4.79 (d, J=12.0 Hz, 1H), 4.29 (t, J=11.3 Hz, 1H), 3.81 (s, 3H), 3.56 (d, J=13.0 Hz, 1H), 3.52 (d, J=8.9 Hz, 1H), 3.37 (s, 3H), 3.26 (d, J=12.9 Hz, 1H), 2.96 (d, J=9.2 Hz, 2H), 2.90-2.84 (m, 2H), 2.66 (d, J=3.7 Hz, 3H), 2.62 (dd, J=14.0, 7.0 Hz, 2H), 2.55-2.40 (m, 4H), 2.32-2.25 (m, 1H), 2.07-1.95 (m, 3H), 1.79 (t, J=6.9 Hz, 1H), 1.74 (s, 3H), 1.68-1.65 (m, 1H), 1.53-1.46 (m, 1H), 1.34 (d, J=6.4 Hz, 3H), 1.27 (d, J=6.6 Hz, 6H), 1.24 (d, J=7.2 Hz, 3H), 1.24-1.23 (m, 1H), 1.19 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (940, 100%).

Step 2: Synthesis of Reagent 25.

[0220] A mixture of compound 26 (23 mg), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC.HCl, 12 mg) and N-hydroxysuccinimide (5 mg) in anhydrous dichloromethane (2 mL) was stirred at room temperature for 4 h. The reaction mixture was then concentrated in vacuo and the residue dissolved in DMSO (5 mL) before purification by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give reagent 25 as a white solid (25 mg). .sup.1H NMR (500 MHz; CDCl.sub.3) 7.75-7.69 (m, 1H), 7.58-7.54 (m, 2H), 7.08-7.06 (m, 2H), 6.87-6.85 (m, 2H), 6.48 (dd, J=15.4, 11.0 Hz, 1H), 5.47 (dd, J=15.4, 9.0 Hz, 1H), 4.84-1.81 (m, 1H), 4.32-4.27 (m, 1H), 3.81 (s, 3H), 3.56 (d, J=13.0 Hz, 1H), 3.52 (d, J=8.9 Hz, 1H), 3.37 (s, 3H), 3.26 (d, J=13.0 Hz, 1H), 3.01-2.84 (m, 6H), 2.82 (d, J=1.0 Hz, 3H), 2.80-2.73 (m, 2H), 2.67 (s, 3H), 2.64-2.60 (m, 1H), 2.53-2.50 (m, 2H), 2.25-2.21 (m, 1H), 2.15-2.11 (m, 1H), 2.06-1.96 (m, 3H), 1.75 (s, 3H), 1.68 (d, J=13.5 Hz, 1H), 1.53-1.47 (m, 1H), 1.37 (d, J=6.8 Hz, 3H), 1.34-1.30 (m, 6H), 1.26 (d, J=7.2 Hz, 3H), 1.24-1.23 (m, 1H), 1.20 (d, J=6.8 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (1037, 100%).

Example 13: Preparation of Conjugation Reagent 27 Comprising a Maytansinoid Cytotoxic Payload

[0221] ##STR00139##

Step 1: Synthesis of Compound 28.

[0222] ##STR00140##

[0223] Compound 28 was synthesised in an analogous way to compound 26 of Example 12 using 4-mercapto-4-methyl-pentanoic acid instead of 4-mercapto-pentanoic acid. Compound 28 was isolated as a white solid. .sup.1H NMR (500 MHz; CD.sub.3OD) 7.63-7.62 (m, 2H), 6.96 (s, 1H), 6.71-6.66 (m, 1H), 6.29-6.27 (m, 1H), 5.58-5.53 (m, 1H), 4.76-1.73 (m, 1H), 4.26-4.22 (m, 1H), 3.83 (s, 3H), 3.63-3.60 (m, 2H), 3.39 (s, 3H), 3.36 (m, 1H), 3.07-3.02 (m, 1H), 2.99-2.94 (m, 1H), 2.89-2.87 (m, 1H), 2.79-2.74 (m, 1H), 2.70 (s, 3H), 2.63-2.54 (m, 2H), 2.40-2.37 (m, 2H), 2.27-2.24 (m, 1H), 2.06-1.90 (m, 5H), 1.80 (s, 3H), 1.65-1.54 (m, 3H), 1.36 (d, J=6.8 Hz, 3H), 1.29-1.27 (d, 13H), 1.22 (d, J=6.7 Hz, 3H), 1.02 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (954, 100%).

Step 2: Synthesis of Reagent 27.

[0224] Reagent 27 was synthesised in an analogous way to reagent 25 of Example 12 using compound 28 instead of compound 26. Reagent 27 was isolated as a white solid. .sup.1H NMR (500 MHz; CDCl.sub.3) 7.89 (d, J=4.9 Hz, 1H), 7.58-7.56 (m, 2H), 7.07 (d, J=8.1 Hz, 2H), 6.86 (d, J=6.4 Hz, 2H), 6.48 (dd, J=15.3, 11.1 Hz, 1H), 6.23 (s, 1H), 6.18 (d, J=10.9 Hz, 1H), 5.47 (dd, J=15.4, 9.0 Hz, 1H), 4.84-4.81 (m, 1H), 4.32-4.27 (m, 1H), 3.81 (s, 3H), 3.56 (d, J=12.9 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.37 (s, 3H), 3.26 (d, J=12.8 Hz, 1H), 3.06 (s, 1H), 3.00-2.94 (m, 2H), 2.87-2.83 (m, 6H), 2.77-2.71 (m, 2H), 2.66 (s, 3H), 2.64-2.60 (m, 1H), 2.55-2.48 (m, 2H), 2.24 (d, J=13.4 Hz, 1H), 2.09-1.96 (m, 4H), 1.75 (s, 3H), 1.68 (d, J=13.5 Hz, 1H), 1.54-1.46 (m, 1H), 1.34 (d, J=6.7 Hz, 3H), 1.30-1.28 (m, 9H), 1.25-1.24 (m, 1H), 1.26 (d, J=7.2 Hz, 3H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (1051, 100%).

Example 14: Conjugation of Reagents 25 and 27 to Trastuzumab to Produce Antibody Drug Conjugates (ADCs) 29 and 30, Respectively

[0225] Conjugation reagents 25 and 27 were conjugated to Trastuzumab, giving rise to ADCs 29 and 30, respectively. Briefly, the reagents were dissolved in DMSO to give 10 mM stock solutions. To a solution of Trastuzumab in 100 mM sodium phosphate buffer, 100 mM sodium chloride, pH 8.0 was added propylene glycol (40% v/v) and the solution mixed gently to give a final antibody concentration of 4.8 mg/mL. Conjugation reagents (13 eq. per mAb) were then added to the antibody solutions and the reaction mixtures were mixed gently and incubated at 24 C. for 3 h. Activated charcoal powder (70% w/w of mAb) was then added to the reaction solutions which were gently agitated for 30 min at room temperature to remove unreacted drug related species. The reaction mixtures were then filtered (0.22 m PES membrane) and the purified sample was buffer exchanged into 10 mM succinic acid, 6% w/v trehalose, 0.01% v/v Tween 20, pH 5.5 using PD-10 desalting columns.

[0226] The DARs of the conjugates were determined by mass spectrometry following deglycosylation of the samples using PNGase F. The average DARs of the conjugates were calculated from the relative peak intensities of the individual DAR species. DAR assignments of 3.3 and 2.8 were determined for antibody drug conjugates 29 and 30, respectively.

Example 15: In Vitro Potency Assay of Compounds in SK-BR-3 Cell Lines

[0227] Loss of tumour cell viability following treatment with compounds of the invention was tested by growing SK-BR-3 cell lines in the presence of increasing concentrations of compounds of the invention and quantifying the loss of proliferation or metabolic activity as described in Example 2. The average IC.sub.50 values for compounds of the invention are shown in Table 3 and the assay concentrations are specified in Table 4. The IC.sub.50 value for a comparator compound 31:

##STR00141##

from Chem. Eur. J. 2012, 18, 880-886 is also provided.

TABLE-US-00003 TABLE 3 Compound number Average IC.sub.50 (nM) SK-BR-3 Cell line 4 1.2 (n = 4) 5 1.2 (n = 2) 6 2.7 (n = 3) 18 0.9 (n = 2) 31 (comparator) 33.4 (n = 3)

TABLE-US-00004 TABLE 4 Cell line Compound Concentration range SK-BR-3 4 200 nM-2.6 pM SK-BR-3 5 200 nM-2.6 pM SK-BR-3 6 200 nM-0.2 pM SK-BR-3 18 200 nM-2.6 pM SK-BR-3 31 (comparator) 1000 nM-457 pM

[0228] Compounds of the invention, which contain a biphenyl moiety, unexpectedly have lower IC.sub.50 values with respect to inhibiting proliferation of SK-BR-3 cells, than an allylamine-containing comparator compound.

Example 16: JIMT-1 Mouse Xenograft Study Comparing Trastuzumab-Drug Conjugate 29 to Kadcyla (Comparative)

[0229] Conjugate 29 was prepared as described in Example 14. Healthy female NMRI nude mice (RjOrl:NMRI-Foxn1.sup.nu/Foxn1.sup.nu) aged 6 weeks at arrival were used for cell inoculation.

[0230] Tumours were induced by subcutaneous injection of 510.sup.6 JIMT-1 cells (breast carcinoma) in 200 L of cell suspension in PBS into the right flank. Matrigel (40 L Matrigel per 200 L cell suspension) was added shortly before inoculation of tumour cells. Tumours were measured twice a week with calipers, and the volume was estimated using the formula:

[00002] $Tumor .Math. .Math. Volume .Math. .Math. ({mm}^{3}) = \frac{{width}^{2} length}{2}$

[0231] When the tumour volumes reached a mean tumour volume of approximately 134 mm.sup.3, the animals were randomised into groups of eight mice and treatment was initiated (Day 0). All test substances were injected via the tail vein (i.v. bolus). A single dose of 30 mg/kg of ADC was given in 10 mL/kg and PBS was used for the vehicle group.

[0232] Mice viability and behaviour were recorded every day. Body weights were measured twice a week. The animals were euthanized when a humane endpoint was reached (e.g. calculated tumour weight of >10% body weight and/or tumour volume >2000 mm.sup.3 animal body weight loss of >20% compared to the body weight at group distribution, ulceration of tumours, lack of mobility, general signs of pain), or at a pre-determined study end date.

[0233] The mean tumour volumesstandard error at Day 0 and Day 14 post treatment are represented in FIG. 3. Results show that for vehicle treated animals, the tumour volume approximately doubled between Day 0 and Day 14 (135 Vs 268 mm.sup.3). For animals treated with conjugate 29, (30 mg/Kg dose), by Day 14 the tumour volume was reduced to almost half the value recorded at Day 0 (134 Vs 73 mm.sup.3), whereas the clinical product Kadcyla displayed an increase in tumour volume over the same time period (134 Vs 165 mm.sup.3). All compounds were well tolerated.

Example 17: Preparation of Maytansinoid Compound 32

[0234] ##STR00142##

[0235] Compound 32 was synthesised in an analogous way to compound 4 of Example 3. Briefly, 4-aminophenylboronic acid pinacol ester (16 mg), tripotassium phosphate (33 mg), SPhos Pd G3 (6 mg) and maytansine (available from Toronto Research Chemicals, 25 mg) were sequentially added to an argon purged reaction vessel. The vessel was then sealed and the solids purged with argon (4evacuation/purge cycles). THF (600 L) and water (60 L), which had been rigorously deoxygenated by purging with argon, were then added and the reaction mixture was stirred at room temperature for 17 h. The reaction mixture was then diluted with ethyl acetate (20 mL) and washed with brine (10 mL). The layers were separated and the organic layer was concentrated under reduced pressure. The residue was then purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v) and the desired fractions lyophilised to give compound 32 as a white solid (17.3 mg). .sup.1H NMR (500 MHz; CDCl.sub.3) 6.90 (d, J=7.9 Hz, 2H), 6.82 (s, 1H), 6.72-6.66 (m, 3H), 6.66 (s, 1H), 6.46 (dd, J=15.4, 11.2 Hz, 1H), 6.23 (s, 1H), 5.70 (dd, J=15.3, 9.0 Hz, 1H), 5.35-5.31 (m, 1H), 4.82 (dd, J=12.0, 2.7 Hz, 1H), 4.32-4.28 (m, 1H), 3.81 (s, 3H), 3.68 (d, J=12.8 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.42-3.41 (m, 1H), 3.37 (s, 3H), 3.15 (d, J=12.7 Hz, 1H), 3.10-3.03 (m, 2H), 2.81 (s, 3H), 2.68 (s, 3H), 2.26 (dd, J=14.3, 2.6 Hz, 1H), 2.07 (s, 3H), 1.68 (s, 3H), 1.65 (d, J=13.7 Hz, 1H), 1.54-1.48 (m, 1H), 1.32-1.30 (m, 6H), 1.26 (d, J=13.0 Hz, 1H), 0.92 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (749, 100%).

Example 18: Preparation of Maytansinoid Compound 33

[0236] ##STR00143##

Step 1: Synthesis of Compound 34.

[0237] ##STR00144##

[0238] Compound 34 was synthesised in an analogous way to compound 8 of Example 5 using compound 32 instead of compound 4. Compound 34 was isolated as a pale yellow solid. LC/MS: (ES+) [M+H].sup.+ (1019, 100%).

Step 2: Synthesis of Compound 33.

[0239] Compound 33 was synthesised in an analogous way to compound 6 of Example 5 using compound 34 instead of compound 8. Compound 33 was isolated as a white solid. .sup.1H NMR (500 MHz; CDCl.sub.3) 7.56-7.53 (m, 2H), 7.28 (dq, J=3.1, 1.0 Hz, 1H), 7.07-7.05 (m, 2H), 6.83 (s, 1H), 6.72-6.69 (m, 1H), 6.67 (d, J=0.3 Hz, 1H), 6.48-6.42 (m, 1H), 6.21 (s, 1H), 5.71-5.66 (m, 1H), 5.35-5.31 (m, 1H), 4.82-4.79 (m, 1H), 4.31-4.27 (m, 1H), 3.80 (s, 3H), 3.69 (d, J=12.7 Hz, 1H), 3.51 (d, J=9.0 Hz, 1H), 3.40-3.39 (m, 1H), 3.36 (s, 3H), 3.17-3.14 (m, 1H), 3.07 (d, J=9.8 Hz, 1H), 3.03-2.98 (m, 2H), 2.80 (s, 3H), 2.65 (s, 3H), 2.57-2.52 (m, 2H), 2.26-2.23 (m, 1H), 2.17-2.11 (m, 1H), 2.06 (s, 3H), 1.82-1.75 (m, 1H), 1.68 (s, 3H), 1.64-1.61 (m, 1H), 1.46-1.44 (m, 1H), 1.41-1.40 (m, 3H), 1.31-1.29 (m, 6H), 1.26-1.23 (m, 1H), 0.91 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (865, 100%).

Example 19: Preparation of Conjugation Reagent 35 Comprising a Maytansinoid Cytotoxic Payload

[0240] ##STR00145##

Step 1: Synthesis of Compound 36.

[0241] ##STR00146##

[0242] A solution of 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (1.0 g, Nature Protocols, 2006, 1(54), 2241-2252) was added to N-hydroxybenzotriazole hydrate (306 mg) in anhydrous THF (10 mL) under a nitrogen atmosphere. The resulting solution was cooled to 0 C. and diisopropylcarbodiimide (310 L) was added dropwise. The reaction mixture was stirred for 20 min at 0 C. before being warmed to room temperature. Additional THF (10 mL) was added to the reaction mixture after 1 h. After 18 h, the formed precipitate was filtered and washed with cold THF (25 mL) before being dried in vacuo. The solid was stirred with methanol (10 mL) for 1 h at room temperature, collected by filtration and washed sequentially with methanol (25 mL) and diethyl ether (5 mL). The solid was then dried in vacuo to give compound 36 as a white solid (1.1 g). LC/MS: (ES+) [M+H].sup.+ (618, 100%).

Step 2: Synthesis of Compound 37.

[0243] ##STR00147##

[0244] To a stirred suspension of L-Glutamic acid 5-tert-butyl ester (198 mg) in anhydrous DMF (20 mL) under a nitrogen atmosphere was added NMM (107 L). The reaction mixture was cooled to 0 C. before compound 36 (603 mg) was added. The resulting suspension was stirred at 0 C. for 1 h, after which the reaction mixture was allowed to warm to room temperature. After 19 h, the resulting solution was concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:5% acetonitrile:0.1% formic acid and buffer B (v/v): acetonitrile:0.1% formic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give compound 37 as a white solid (198 mg). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.98 (1H, d), 7.86 (2H), 7.71-7.65 (6H, m), 7.36 (4H, d), 4.68 (1H, ddd), 4.34 (1H, q), 3.62 (2H, ddd), 3.50 (2H, ddd), 2.69 (1H ddd), 2.55-2.45 (1H, m), 2.48 (6H, s), 2.34-2.16 (2H, m), 1.46 (9H, s). LC/MS: (ES+) [2M+H].sup.+ (1371, 70%), [2M+H-tBu].sup.+ (1315, 70%), [M+H-tBu].sup.+ (630, 100%).

Step 3: Synthesis of Compound 38.

[0245] ##STR00148##

[0246] Compound 37 (50 mg) and (benzotriazol-1-yloxy)tris-(dimethylamino)phosphonium hexafluorophosphate (BOP) (40 mg) were dissolved in anhydrous DMF (3 mL), cooled to 0 C. and added to a solution of NH.sub.2PEG(24u)-OMe (99 mg) and NMM (10 L) in anhydrous DMF (2 mL). The reaction mixture was stirred at 0 C. and after 4 h, additional amounts of BOP (10 mg) and NMM (2.5 L) were added to the reaction mixture which was stirred for a further 15 min before being stored at 20 C. for 18 h. The reaction mixture was then concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:5% acetonitrile:0.1% formic acid and buffer B (v/v): acetonitrile:0.1% formic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give bis-tolylsulfonyl-propanoyl-benzamide-L-Glu-[OtBu]-[PEG(24u)-OMe] as a colourless oil (128 mg). LC/MS: (ES+) [M+H].sup.+ (1757 Da, 100%), [M+2H].sup.2+ (879, 100%). Bis-tolylsulfonyl-propanoyl-benzamide-L-Glu-[OtBu]-[PEG(24u)-OMe] (126.5 mg) was dissolved in formic acid (2.5 mL) and stirred under a nitrogen atmosphere at room temperature. After 20 h, the reaction mixture was concentrated in vacuo and dried under high vacuum for 18 h to give compound 38 as a colourless oil (122 mg, assumed quantitative yield). LC/MS: (ES+) [M+H].sup.+ (1700, 100%).

Step 4: Synthesis of Compound 39.

[0247] ##STR00149##

[0248] To a solution of compound 4 (50 mg), Fmoc-Val-Cit-PAB-PNP (Levena Biopharma, 85 mg), and 1-hydroxy-7-azabenzotriazole (HOAt) (11.5 mg) in DMF (3 mL), cooled to 0 C., was added DIPEA (40 L). The reaction mixture was allowed to warm to room temperature and was stirred for 19 h. Additional HOAt (14 mg) was added and the mixture was stirred at room temperature for 9 h before storing at 20 C. for 72 h. Further HOAt (18 mg) was then added, and the mixture was stirred at room temperature for 6 h. The reaction mixture was then directly purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The product fractions were combined and concentrated under reduced pressure and the aqueous solution was extracted with ethyl acetate (100 mL). The layers were separated and the organic layer was washed with brine. The ethyl acetate layer was then separated, dried over sodium sulfate, filtered and concentrated in vacuo. The product was further purified by normal phase chromatography eluting with dichloromethane:methanol (100:0 v/v to 85:15 v/v). The solvent was removed in vacuo to give compound 39 as a white solid (44 mg). .sup.1H NMR (500 MHz, CD.sub.3OD) ppm 0.83-1.03 (m, 14H), 1.12-1.34 (m, 17H), 1.46-1.67 (m, 7H), 1.79 (s, 6H), 1.92 (br. s., 2H), 2.03-2.14 (m, 2H), 2.24 (d, J=14.17 Hz, 2H), 2.69 (s, 4H), 2.76 (dt, J=13.92, 6.72 Hz, 2H), 2.87 (d, J=9.77 Hz, 2H), 2.99-3.16 (m, 3H), 3.16-3.24 (m, 2H), 3.37 (d, J=6.35 Hz, 8H), 3.57-3.63 (m, 3H), 3.82 (s, 4H), 3.98 (d, J=7.33 Hz, 1H), 4.17-4.27 (m, 3H), 4.34-4.46 (m, 3H), 4.50-4.61 (m, 1H), 4.74 (d, J=9.77 Hz, 2H), 5.15 (s, 3H), 5.55 (dd, J=15.14, 8.79 Hz, 1H), 6.27 (d, J=10.75 Hz, 1H), 6.67 (dd, J=15.63, 11.23 Hz, 1H), 6.95 (s, 1H), 7.04-7.15 (m, 4H), 7.27-7.43 (m, 3H), 7.49 (d, J=7.82 Hz, 5H), 7.61 (d, J=8.3, 3H), 7.67 (t, J=8.3, 3H), 7.80 (d, J=7.33 Hz, 3H). LC/MS: (ES+) [M+H].sup.+ (1320, 100%).

Step 5: Synthesis of Compound 40.

[0249] ##STR00150##

[0250] To a solution of compound 39 (52 mg) in anhydrous methanol (2 mL) under an argon atmosphere was added diethylamine (250 L) and the mixture was stirred at room temperature for 1 h. Additional diethylamine (250 L) was then added and the mixture was stirred at room temperature for a further 2 h. The reaction mixture was then concentrated under reduced pressure and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give compound 40 as a white solid (25 mg). .sup.1H NMR (500 MHz; DMSO-d6) 10.23 (s, 1H), 9.80 (s, 1H), 8.69 (d, J=7.5 Hz, 1H), 8.10 (s, 3H), 7.63 (d, J=8.5 Hz, 2H), 7.48 (d, J=8.2 Hz, 2H), 7.39 (d, J=8.5 Hz, 2H), 7.13 (s, 1H), 7.08 (d, J=8.0 Hz, 1H), 6.87 (s, 1H), 6.81 (s, 1H), 6.62 (dd, J=15.2, 11.3 Hz, 1H), 6.22 (d, J=11.0 Hz, 1H), 6.08 (s, 1H), 5.89-5.86 (m, 1H), 5.43 (dd, J=15.2, 8.9 Hz, 1H), 5.11 (s, 2H), 4.59-4.52 (m, 2H), 4.11 (t, J=11.2 Hz, 1H), 3.76 (s, 3H), 3.68-3.66 (m, J=5.3 Hz, 1H), 3.54-3.51 (m, J=7.7 Hz, 2H), 3.25 (s, 3H), 3.06-3.05 (m, 1H), 2.98-2.97 (m, 1H), 2.85 (t, J=13.1 Hz, 1H), 2.70 (d, J=9.7 Hz, 1H), 2.66-2.63 (m, 1H), 2.22 (d, J=12.4 Hz, 1H), 2.09 (q, J=6.7 Hz, 1H), 1.76-1.73 (m, 1H), 1.69 (s, 3H), 1.66-1.62 (m, 1H), 1.51-1.38 (m, 4H), 1.15-1.13 (m, 5H), 1.10 (d, J=6.7 Hz, 3H), 0.97-0.94 (m, 9H). LC/MS: (ES+) [M+H].sup.+ (1098, 100%).

Step 6: Synthesis of Reagent 35.

[0251] To a solution of compound 38 (25.3 mg) in anhydrous DMF (400 L) cooled to 0 C. was added HATU (5.7 mg). After stirring for 25 min, NMM (1.5 L) was added and the solution was stirred for 15 min before allowing to warm to room temperature and stirring for a further 10 min. To a separate solution of compound 40 (15 mg) in anhydrous DMF (300 L) was added NMM (1.5 L) and the solution was stirred for 10 min at room temperature. The solutions were then combined and additional HATU (5.7 mg) and NMM (0.8 L) were added to the reaction mixture. After stirring for 1 h at room temperature, the reaction mixture was concentrated in vacuo and purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water: 0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (95:5 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give reagent 35 as a colourless solid (27.2 mg). LC/MS: (ES+) [M+2Na].sup.2+ (1412 Da, 40%), [M+H+Na].sup.2+ (1401, 50%), [M+2H+Na].sup.3+ (935, 80%), [MH.sub.2O+3H].sup.3+ (921, 100%).

Example 20: Preparation of Conjugation Reagent 41 Comprising a Maytansinoid Cytotoxic Payload

[0252] ##STR00151##

Step 1: Synthesis of Compound 42.

[0253] ##STR00152##

[0254] To a stirred solution of diethanolamine (2.5 g) and triethylamine (6.05 g) in dichloromethane (15 mL) was slowly added a solution of tosyl chloride (3.8 g) in dichloromethane (15 mL) at room temperature. After 2 h, water (25 mL) was added to the reaction mixture and the product was extracted with dichloromethane (530 mL). The combined organic extracts were dried over magnesium sulfate, the solution was then filtered and the volatiles removed in vacuo to yield compound 42 as a white solid (4.7 g). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.68 (d, J=8.3 Hz, 2H), 7.30 (d, J=8.3 Hz, 2H), 3.84 (t, J=5.0 Hz, 4H), 3.56 (s, 2H), 3.24 (t, J=5.0 Hz, 4H), 2.41 (s, 3H). LC/MS: (ES+) [M+Na].sup.+ (282, 95%), [M+H].sup.+ (260, 100%).

Step 2: Synthesis of Compound 43.

[0255] ##STR00153##

[0256] A solution of compound 42 (176 mg) in anhydrous THF (2 mL) was added dropwise over a period of 1 h to a solution of sodium hydride (80 mg, 60% dispersion in mineral oil) in anhydrous THF (8 mL) at room temperature. After stirring for 1 h, a solution of hexaethyleneglycol di-p-toluenesulfonate (400 mg) in anhydrous THF (2 mL) was added over a period of 2 h and the reaction mixture was stirred at room temperature for 72 h. Water (30 mL) was added and the THF was removed in vacuo. The aqueous solution was extracted with chloroform (425 mL), the organic phases were combined and dried over magnesium sulfate before the solution was filtered and concentrated in vacuo. The residue was then purified by reverse phase C18-column chromatography eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v):acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent was removed by lyophilisation to give compound 43 as a colourless oil (78 mg). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.68 (d, J=8.3 Hz, 2H), 7.26 (d, J=8.3 Hz, 4H), 3.67-3.58 (m, 24H), 3.58-3.53 (m, 4H), 3.38 (t, J=6.0 Hz, 4H), 2.40 (s, 3H). LC/MS: (ES+) [M+Na].sup.+ (528, 80%), [M+H].sup.+ (506, 50%).

Step 3: Synthesis of Compound 44.

[0257] ##STR00154##

[0258] To a solution of compound 43 (78 mg) in anhydrous THF (6 mL) was added lithium aluminium hydride (1.13 mL, 1 M solution in THF) and the solution was heated at reflux for 16 h before the reaction mixture was cooled to 0 C. and quenched by the dropwise addition of water. The suspension was filtered and the precipitate washed with chloroform:ethanol (9:1 v/v, 56 mL). The filtrate and washings were combined and concentrated in vacuo to give compound 44 as a colourless oil (50 mg). LC/MS: (ES+) [M+Na].sup.+ (374, 70%), [M+H].sup.+ (352, 100%).

Step 4: Synthesis of Compound 45.

[0259] ##STR00155##

[0260] To a solution of Fmoc-Glu(OtBu)-OH (78 mg) in anhydrous DMF (500 L) at 0 C. was added HATU (108 mg) and NMM (34 L) and the mixture was stirred at 0 C. for 10 min. To this was added a solution of compound 44 (44 mg) in anhydrous DMF (500 L) and the mixture was stirred at 0 C. under an argon atmosphere for 15 min. The reaction mixture was then concentrated in vacuo and the residue dissolved in anhydrous DMF (500 L). Piperidine (70 L) was added and the solution stirred for 90 min at room temperature. The reaction solution was concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:5% acetonitrile:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (100:0 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give compound 45 as an orange oil (44 mg). LC/MS: (ES+) [M+H].sup.+ (537, 45%).

Step 5: Synthesis of Compound 46.

[0261] ##STR00156##

[0262] To a solution of 4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (37.5 mg) in anhydrous DMF (500 L) at 0 C. was added HATU (65 mg) and NMM (20 L) and the mixture was stirred at 0 C. for 10 min. To this was added a solution of compound 45 (44.3 mg) in anhydrous DMF (500 L) and the mixture was stirred at 0 C. under an argon atmosphere for 1 h. The reaction mixture was then concentrated in vacuo, the residue dissolved in DMF (1 mL) and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (60:40 v/v to 0:100 v/v). The solvent was removed by lyophilisation to give bis-tolylsulfonyl-propanoyl-benzamide-L-Glu-(OtBu)-aza-24-crown-8 as a white solid (28.5 mg). LC/MS: (ES+) [M+Na].sup.+ (1041, 20%), [M+H].sup.+ (1019, 5%). To a solution of bis-tolylsulfonyl-propanoyl-benzamide-L-Glu-(OtBu)-aza-24-crown-8 (26.5 mg) in anhydrous dichloromethane (1 mL) was added trifluoroacetic acid (500 L) and the solution stirred at room temperature under an argon atmosphere for 1 h. The volatiles were removed in vacuo to give compound 46 as a white solid (assumed quantitative yield). LC/MS: (ES+) [M+Na].sup.+ (985, 35%), [M+H].sup.+ (963, 30%).

Step 6: Synthesis of Reagent 41.

[0263] To a solution of compound 46 (10.5 mg) in DMF (300 L) cooled to 0 C. was added HATU (4 mg). After stirring for 20 min, NMM (1 L) was added and the reaction solution was stirred for a further 30 min at 0 C. To a separate solution of compound 40 (10 mg) in DMF (200 L) cooled to 0 C. was added NMM (1 L) and the solution was stirred for 40 min. The solutions were then combined before additional quantities of HATU (4 mg) and NMM (1 L) were added and the reaction mixture was allowed to warm to room temperature and was stirred for 3.25 h. The reaction solution was then concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (70:30 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give reagent 41 as a white solid (8.2 mg). LC/MS: (ES+) [M+2Na].sup.2+ (1043, 30%), [M+Na+H].sup.2+ (1033, 60%), [M+2H].sup.2+ (1021, 100%), [M+3H].sup.3+ (682, 30%).

Example 21: Preparation of Conjugation Reagent 47 Comprising a Maytansinoid Cytotoxic Payload

[0264] ##STR00157##

Step 1: Synthesis of Compound 48.

[0265] ##STR00158##

[0266] To a stirred solution of compound 4 (50 mg), Fmoc-Val-Ala-OH (Creagen, 67 mg) and HATU (85 mg) in anhydrous DMF (2 mL) at 0 C. was added DIPEA (50 L). The solution was allowed to warm to room temperature and was stirred for 16 h. The reaction mixture was then directly purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v) and the desired fractions lyophilised to give compound 48 as a white solid (57 mg). LC/MS: (ES+) [M+H].sup.+ (1084, 100%).

Step 2: Synthesis of Compound 49.

[0267] ##STR00159##

[0268] To a stirred solution of compound 48 (30 mg) in dichloromethane (2 mL) was added diethylamine (140 L) and the reaction mixture was stirred at room temperature for 16 h before the solution was concentrated in vacuo. The residue was dissolved in DMF (5 mL) and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give compound 49 as a white solid (20 mg). LC/MS: (ES+) [M+H].sup.+ (862, 100%).

Step 3: Synthesis of Reagent 47.

[0269] To a solution of compound 38 (15.6 mg), compound 49 (8 mg) and HATU (6.6 mg) in DMF (2 mL) at 0 C. was added NMM (30 L) and the mixture was stirred at 0 C. for 90 min. The reaction mixture was then directly purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give reagent 47 as a white solid (15 mg). .sup.1H NMR (500 MHz; DMSO-d.sub.6) 9.99 (s, 0.5H), 9.92 (s, 0.5H), 8.73 (dd, J=7.2, 5.1 Hz, 1H), 8.40-8.39 (m, 0.5H), 8.25 (d, J=6.5 Hz, 0.5H), 8.05-8.01 (m, 1H), 7.97-7.87 (m, 2H), 7.67-7.51 (m, 6H), 7.46-7.44 (m, 3H), 7.13-7.09 (m, 2H), 6.86 (s, 1H), 6.81 (s, 1H), 6.65-6.60 (m, 1H), 6.52 (s, 1H), 6.23-6.21 (m, 1H), 5.87 (s, 1H), 5.42 (dd, J=15.2, 8.9 Hz, 1H), 4.59-4.57 (m, 1H), 4.46-1.39 (m, 2H), 4.27-4.17 (m, 1H), 4.13-4.09 (m, 1H), 4.01-3.98 (m, 1H), 3.84-3.80 (m, 2H), 3.75-3.71 (m, 6H), 3.51 (s, 96H), 3.44-3.42 (m, 5H), 3.38-3.36 (m, 1H), 3.25 (d, J=4.0 Hz, 6H), 2.89-2.83 (m, 1H), 2.70 (d, J=9.4 Hz, 1H), 2.46 (s, 6H), 2.40-2.22 (m, 4H), 2.08-1.90 (m, 3H), 1.69 (s, 3H), 1.51-1.45 (m, 2H), 1.40-1.38 (m, 1H), 1.33-1.31 (m, 4H), 1.14 (d, J=7.1 Hz, 6H), 1.10 (d, J=6.6 Hz, 4H), 0.93 (s, 3H), 0.90-0.84 (m, 6H). LC/MS: (ES+) [M+2H].sup.2+ (1272, 30%), [M+3H].sup.3+ (848, 100%).

Example 22: Preparation of Conjugation Reagent 50 Comprising a Maytansinoid Cytotoxic Payload

[0270] ##STR00160##

Step 1: Synthesis of Compound 51.

[0271] ##STR00161##

[0272] To a stirred suspension of amino-PEG(2u)-acid (50 mg) and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (60 mg) in anhydrous DMF (2 mL) was added DIPEA (80 L) and the reaction mixture was stirred at room temperature for 2 h. The mixture was then directly purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give compound 51 as a white solid (43 mg). .sup.1H NMR (500 MHz; DMSO-d.sub.6) 7.83-7.80 (m, 1H), 7.02 (s, 2H), 3.61 (t, J=6.4 Hz, 3H), 3.41-3.36 (m, 6H), 3.34-3.33 (m, 1H), 3.18 (q, J=5.8 Hz, 3H), 2.45 (t, J=6.3 Hz, 2H), 2.05 (t, J=7.4 Hz, 2H), 1.48 (dt, J=14.9, 7.4 Hz, 4H), 1.23-1.13 (m, 2H). LC/MS: (ES+) [M+H].sup.+ (371, 100%).

Step 2: Synthesis of Reagent 50.

[0273] To a solution of compound 40 (12 mg), compound 51 (5.6 mg) and HATU (8.2 mg) in DMF (1.5 mL) cooled to 0 C. was added NMM (20 L) and the mixture was stirred at 0 C. for 2.5 h. The reaction mixture was then purified directly by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give reagent 50 as a white solid (12 mg). .sup.1H NMR (500 MHz; DMSO-d.sub.6) 10.06 (s, 1H), 9.79-9.78 (m, 1H), 8.18 (d, J=7.3 Hz, 1H), 7.91 (d, J=8.8 Hz, 1H), 7.82-7.80 (m, 1H), 7.63 (d, J=8.5 Hz, 2H), 7.47 (d, J=8.5 Hz, 2H), 7.37 (d, J=8.5 Hz, 2H), 7.13 (s, 1H), 7.08 (d, J=8.0 Hz, 2H), 7.00 (s, 2H), 6.86 (s, 1H), 6.80 (s, 1H), 6.62 (dd, J=15.3, 11.2 Hz, 1H), 6.21 (d, J=11.1 Hz, 1H), 6.04-6.01 (m, 1H), 5.87 (s, 1H), 5.45-5.40 (m, 3H), 5.10 (s, 2H), 4.59-4.56 (m, 1H), 4.41-4.37 (m, 1H), 4.25-4.22 (m, 1H), 4.13-4.09 (m, 1H), 3.76 (s, 3H), 3.63-3.59 (m, 2H), 3.54-3.50 (m, 2H), 3.48-3.46 (m, 4H), 3.38-3.36 (m, 4H), 3.25 (s, 3H), 3.17 (q, J=5.8 Hz, 2H), 3.04-2.92 (m, 2H), 2.88-2.83 (m, 1H), 2.70 (d, J=9.7 Hz, 1H), 2.67-2.61 (m, 1H), 2.47-2.45 (m, 1H), 2.41-2.36 (m, 1H), 2.23 (dd, J=13.0, 0.8 Hz, 1H), 2.04 (t, J=7.4 Hz, 2H), 2.01-1.94 (m, 1H), 1.73-1.65 (m, 5H), 1.63-1.58 (m, 1H), 1.51-1.42 (m, 7H), 1.40-1.35 (m, 2H), 1.20-1.12 (m, 8H), 1.10 (d, J=6.6 Hz, 3H), 0.94 (s, 3H), 0.87 (d, J=6.7 Hz, 3H), 0.83 (d, J=6.8 Hz, 3H). LC/MS: (ES+) [M+H].sup.+ (1449, 100%).

Example 23: Preparation of a Series of Maytansinoid Compounds

[0274] A series of maytansinoid compounds of the general formula (XV) were prepared using an analogous procedure to that described in Example 3, by replacing 4-aminophenylboronic acid with a range of aryl boron reagents to produce compounds 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 and 64. The compound structures and modifications to the synthetic protocol described in Example 3, are shown in Table 5.

##STR00162##

TABLE-US-00005 TABLE 5 Catalyst Compound Aryl boron reagent (molar Reaction No. Sub group (molar equivalents*) equivalents*) time (h) 52 [00163] embedded image [00164] SPhos Pd G3 (0.025) 18 53*** [00165] [00166] SPhos Pd G3 (0.025) 8 54 [00167] [00168] XPhos Pd G3 (0.025) 18 55 [00169] [00170] XPhos Pd G3 (0.025) 18 56 [00171] [00172] SPhos Pd G3 (0.125) 18 57 [00173] [00174] SPhos Pd G3 (0.125) + XPhos Pd G3 (0.025) 18 58 [00175] [00176] SPhos Pd G3 (0.025) 18 59 [00177] embedded image [00178] SPhos Pd G3 (0.125) 18 60 [00179] [00180] SPhos Pd G3 (0.125) 18 61 [00181] [00182] XPhos Pd G3 (0.025) 18 62 [00183] [00184] SPhos Pd G3 (0.125) 18 63*** [00185] [00186] XPhos Pd G3 (0.125) 5 64*** [00187] [00188] SPhos Pd G3 (0.14) + XPhos Pd G3 (0.12) 23 *molar equivalents relative to 1 molar equivalent of AP3. **3 molar equivalents of tripotassium phosphate were used to prepare compound 52. 4 molar equivalents of tripotassium phosphate were used to prepare compounds 53-64. ***product isolated as a mixture of atropisomers.

[0275] Characterisation data for compound 52: .sup.1H NMR (500 MHz; CDCl.sub.3) 7.21-7.15 (m, 1H), 6.85 (d, J=2.2 Hz, 2H), 6.68-6.65 (m, 1H), 6.51-6.45 (m, 3H), 6.22-6.17 (m, 2H), 5.49-5.44 (m, 1H), 4.84-4.81 (m, 1H), 4.31-4.27 (m, 1H), 3.82 (s, 3H), 3.57-3.50 (m, 2H), 3.37 (s, 3H), 3.29-3.24 (m, 1H), 3.02-2.90 (m, 3H), 2.73 (s, 3H), 2.69-2.61 (m, 1H), 2.27-2.22 (m, 1H), 1.75 (s, 3H), 1.69-1.66 (m, 1H), 1.50-1.48 (m, 1H), 1.30 (d, J=6.3 Hz, 3H), 1.26 (d, J=7.1 Hz, 3H), 1.24-1.23 (m, 1H), 1.20 (d, J=6.7 Hz, 3H), 0.93 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (692, 100%).

[0276] Characterisation data for compound 53: LC/MS: (ES+) [M+H].sup.+ (722, 100%).

[0277] Characterisation data for compound 54: .sup.1H NMR (500 MHz; CDCl.sub.3) 7.98 (s, 1H), 7.08 (dd, J=8.5, 1.5 Hz, 1H), 6.89 (s, 1H), 6.86 (s, 1H), 6.82 (d, J=8.6 Hz, 1H), 6.48 (dd, J=15.4, 11.0 Hz, 1H), 6.21-6.16 (m, 4H), 5.47 (dd, J=15.4, 9.0 Hz, 1H), 4.84 (dd, J=11.9, 2.8 Hz, 1H), 4.30 (t, J=10.6 Hz, 1H), 3.84 (s, 3H), 3.57 (d, J=12.9 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.37 (s, 3H), 3.27 (d, J=13.0 Hz, 1H), 3.00 (d, J=9.7 Hz, 1H), 2.94 (t, J=12.9 Hz, 1H), 2.74 (s, 3H), 2.63 (dt, J=13.9, 6.9 Hz, 1H), 2.25 (dd, J=13.8, 2.7 Hz, 1H), 1.75 (s, 3H), 1.67 (d, J=13.5 Hz, 1H), 1.53-1.47 (m, 1H), 1.30 (d, J=6.3 Hz, 3H), 1.26 (d, J=7.1 Hz, 3H), 1.25-1.24 (m, 1H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (737, 100%).

[0278] Characterisation data for compound 55: .sup.1H NMR (300 MHz; CDCl.sub.3) 6.99 (dd, J=10.9, 8.4 Hz, 1H), 6.85 (d, J=2.7 Hz, 2H), 6.54-6.47 (m, 2H), 6.44-6.39 (m, 1H), 6.21-6.16 (m, 2H), 5.47 (dd, J=15.4, 9.1 Hz, 1H), 4.83 (dd, J=11.9, 2.9 Hz, 1H), 4.33-4.25 (m, 1H), 3.82 (s, 3H), 3.73 (s, 2H), 3.58-3.50 (m, 2H), 3.37 (s, 3H), 3.25 (d, J=13.1 Hz, 1H), 3.02-2.86 (m, 2H), 2.73 (s, 3H), 2.67-2.58 (m, 1H), 2.22 (dd, J=13.8, 2.7 Hz, 1H), 1.74 (s, 3H), 1.67 (d, J=13.7 Hz, 1H), 1.53-1.46 (m, 1H), 1.30 (d, J=6.2 Hz, 3H), 1.26 (d, J=7.0 Hz, 3H), 1.25-1.23 (m, 1H), 1.20 (d, J=6.7 Hz, 3H), 0.93 (s, 3H). LC/MS: (ES+) [2M+H].sup.+ (1420, 100%), [M+H].sup.+ (710, 70%).

[0279] Characterisation data for compound 56: .sup.1H NMR (500 MHz; CDCl.sub.3) 7.83 (d, J=8.3 Hz, 2H), 7.23-7.21 (m, 2H), 6.89 (d, J=10.0 Hz, 2H), 6.49 (dd, J=15.5, 11.0 Hz, 1H), 6.21-6.19 (m, 2H), 5.48 (dd, J=15.5, 9.1 Hz, 1H), 4.84 (dd, J=11.9, 2.9 Hz, 1H), 4.32-4.27 (m, 1H), 3.82 (s, 3H), 3.58 (d, J=12.7 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.37 (s, 3H), 3.30-3.27 (m, 1H), 3.02-2.94 (m, 2H), 2.66 (s, 3H), 2.64-2.60 (m, 1H), 2.25 (dd, J=13.6, 2.8 Hz, 1H), 1.76 (s, 3H), 1.69-1.67 (m, 1H), 1.53-1.48 (m, 1H), 1.31 (d, J=6.4 Hz, 3H), 1.26 (d, J=7.2 Hz, 4H), 1.25-1.24 (m, 1H), 1.20 (d, J=6.8 Hz, 3H), 0.95 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (720, 100%).

[0280] Characterisation data for compound 57: .sup.1H NMR (500 MHz; CDCl.sub.3) 7.94 (d, J=8.7 Hz, 2H), 6.89 (d, J=9.0 Hz, 2H), 6.48 (dd, J=15.3, 11.0 Hz, 1H), 6.21-6.18 (m, 2H), 5.47 (dd, J=15.3, 9.0 Hz, 1H), 4.85 (s, 2H), 4.82 (dd, J=11.9, 2.9 Hz, 1H), 4.31-4.27 (m, 1H), 3.81 (s, 3H), 3.57 (d, J=12.8 Hz, 1H), 3.52 (d, J=8.9 Hz, 1H), 3.37 (s, 3H), 3.28 (d, J=12.7 Hz, 1H), 3.01-2.91 (m, 2H), 2.67 (s, 3H), 2.65-2.59 (m, 1H), 2.23 (dd, J=13.8, 2.8 Hz, 1H), 1.75 (s, 3H), 1.66 (dd, J=13.7, 1.5 Hz, 1H), 1.52-1.47 (m, 1H), 1.30 (d, J=6.3 Hz, 3H), 1.25 (d, J=7.2 Hz, 3H), 1.25-1.23 (m, 1H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (756, 100%).

[0281] Characterisation data for compound 58: .sup.1H NMR (500 MHz; CDCl.sub.3) 6.96 (d, J=7.9 Hz, 2H), 6.86 (d, J=10.4 Hz, 2H), 6.81 (d, J=8.6 Hz, 2H), 6.49 (dd, J=15.4, 11.1 Hz, 1H), 6.24 (s, 1H), 6.19 (d, J=11.1 Hz, 1H), 5.47 (dd, J=15.5, 9.0 Hz, 1H), 4.83 (dd, J=11.9, 2.8 Hz, 1H), 4.32-4.28 (m, 1H), 3.82 (s, 3H), 3.56 (d, J=13.0 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.37 (s, 3H), 3.26 (d, J=12.8 Hz, 1H), 3.02-2.96 (m, 2H), 2.67 (s, 3H), 2.64-2.60 (m, 1H), 2.27 (dd, J=13.6, 2.3 Hz, 1H), 1.75 (s, 3H), 1.68 (d, J=13.5 Hz, 1H), 1.53-1.48 (m, 1H), 1.30 (d, J=6.3 Hz, 3H), 1.26 (d, J=7.2 Hz, 3H), 1.25-1.24 (m, 1H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (693, 100%).

[0282] Characterisation data for compound 59: .sup.1H NMR (300 MHz; CDCl.sub.3) 7.22 (d, J=7.9 Hz, 1H), 6.86 (s, 2H), 6.83-6.80 (m, 1H), 6.70 (d, J=7.6 Hz, 1H), 6.62 (s, 1H), 6.48 (dd, J=15.3, 11.0 Hz, 1H), 6.39 (s, 1H), 6.19 (d, J=10.7 Hz, 1H), 5.51 (dd, J=15.3, 9.0 Hz, 1H), 4.86 (dd, J=11.9, 2.5 Hz, 1H), 4.44-4.36 (m, 1H), 3.82 (s, 3H), 3.56 (d, J=13.0 Hz, 1H), 3.49 (d, J=9.0 Hz, 1H), 3.36 (s, 3H), 3.24 (d, J=13.1 Hz, 1H), 3.00 (d, J=9.6 Hz, 1H), 2.91 (t, J=13.0 Hz, 1H), 2.65 (s, 2H), 2.63-2.58 (m, 1H), 2.41-2.35 (m, 1H), 1.74 (s, 3H), 1.71-1.65 (m, 1H), 1.57-1.45 (m, 1H), 1.31 (d, J=6.3 Hz, 3H), 1.28-1.27 (m, 1H), 1.23 (d, J=7.1 Hz, 3H), 1.19 (d, J=6.7 Hz, 3H), 0.95 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (693, 100%).

[0283] Characterisation data for compound 60: .sup.1H NMR (300 MHz; CDCl.sub.3) 7.39 (d, J=8.0 Hz, 2H), 7.11 (d, J=7.8 Hz, 2H), 6.88 (d, J=4.2 Hz, 2H), 6.49 (dd, J=15.4, 10.9 Hz, 1H), 6.22-6.17 (m, 2H), 5.47 (dd, J=15.3, 9.2 Hz, 1H), 4.82 (dd, J=12.0, 3.0 Hz, 1H), 4.73 (s, 2H), 4.33-4.26 (m, 1H), 3.82 (s, 3H), 3.57 (d, J=12.9 Hz, 1H), 3.53-3.50 (m, 1H), 3.37 (s, 3H), 3.27 (d, J=13.3 Hz, 1H), 3.03-2.91 (m, 3H), 2.67 (s, 3H), 2.67-2.58 (m, 1H), 2.23 (dd, J=13.8, 3.0 Hz, 1H), 1.75 (d, J=5.5 Hz, 3H), 1.70-1.65 (m, 1H), 1.55-1.46 (m, 1H), 1.31 (d, J=6.3 Hz, 3H), 1.26-1.24 (m, 1H), 1.26 (d, J=7.2 Hz, 3H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (707, 100%).

[0284] Characterisation data for compound 61: .sup.1H NMR (300 MHz; CDCl.sub.3) 7.40-7.30 (m, 2H), 7.13 (s, 1H), 7.07-7.04 (m, 1H), 6.88 (d, J=6.2 Hz, 2H), 6.49 (dd, J=15.4, 11.0 Hz, 1H), 6.21-6.17 (m, 2H), 5.47 (dd, J=15.2, 9.0 Hz, 1H), 4.84-4.80 (m, 1H), 4.70 (s, 2H), 4.33-4.25 (m, 1H), 3.82 (s, 3H), 3.57 (d, J=12.7 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.37 (s, 3H), 3.27 (d, J=12.6 Hz, 1H), 3.04-2.91 (m, 3H), 2.66 (s, 3H), 2.63-2.58 (m, 1H), 2.28-2.22 (m, 1H), 1.86-1.82 (m, 1H), 1.75 (s, 3H), 1.70-1.65 (m, 1H), 1.52-1.47 (m, 1H), 1.30 (d, J=6.2 Hz, 3H), 1.27-1.24 (m, 1H), 1.26 (d, J=7.0 Hz, 3H), 1.20 (d, J=6.7 Hz, 3H), 0.95 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (707, 100%).

[0285] Characterisation data for compound 62: .sup.1H NMR (500 MHz; CDCl.sub.3) 7.11 (s, 1H), 7.00 (d, J=8.4 Hz, 1H), 6.89-6.85 (m, J=10.7 Hz, 3H), 6.48 (dd, J=15.4, 11.0 Hz, 1H), 6.23 (s, 1H), 6.18 (d, J=11.0 Hz, 1H), 6.09 (s, 1H), 5.47 (dd, J=15.4, 9.0 Hz, 1H), 4.83 (dd, J=11.9, 2.9 Hz, 1H), 4.32-4.27 (m, 1H), 3.83 (s, 3H), 3.56 (d, J=12.9 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.37 (s, 3H), 3.26 (d, J=12.9 Hz, 1H), 3.11 (d, J=2.0 Hz, 1H), 3.00 (d, J=9.7 Hz, 1H), 2.94 (dd, J=13.7, 12.1 Hz, 1H), 2.71 (s, 3H), 2.65-2.59 (m, 1H), 2.24 (dd, J=13.8, 2.7 Hz, 1H), 1.74 (s, 3H), 1.67 (d, J=13.5 Hz, 1H), 1.52-1.46 (m, 1H), 1.30 (d, J=6.4 Hz, 3H), 1.26 (d, J=7.2 Hz, 3H), 1.27-1.25 (m, 1H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (727, 100%).

[0286] Characterisation data for compound 63: .sup.1H NMR (500 MHz; CDCl.sub.3) 7.05 (d, J=8.3 Hz, 0.33H), 6.98 (d, 0.66H, J=2.4 Hz, 0.66H), 6.94-6.91 (m, 1H), 6.89-6.85 (m, 2.33H), 6.79 (d, J=8.3 Hz, 0.66H), 6.51-6.44 (m, 1H), 6.23-6.15 (m, 2H), 5.50-5.41 (m, 1H), 4.84 (dd, J=11.9, 3.0 Hz, 0.66H), 4.74 (dd, J=12.1, 3.1 Hz, 0.33H), 4.32-4.23 (m, 1H), 3.94-3.92 (m, 2H), 3.88 (s, 2H), 3.84 (s, 1H), 3.59-3.50 (m, 2H), 3.37 (s, 2H), 3.36 (s, 1H), 3.31-3.25 (m, 1H), 3.02-2.78 (m, 4H), 2.69 (s, 2H), 2.65-2.60 (m, 1H), 2.27-2.23 (m, 0.66H), 2.14-2.11 (m, 0.33H), 1.77 (s, 1H), 1.74 (s, 2H), 1.70-1.62 (m, 1H), 1.52-1.46 (m, 1H), 1.32-1.25 (m, 7H), 1.21-1.19 (m, 3H), 0.95 (s, 2H), 0.90 (s, 1H). LC/MS: (ES+) [M+H].sup.+ (717, 100%).

[0287] Characterisation data for compound 64: .sup.1H NMR (500 MHz; CDCl.sub.3) 7.30 (d, J=2.4 Hz, 0.2H), 7.24 (d, J=2.4 Hz, 0.8H), 6.94 (d, J=8.1 Hz, 0.2H), 6.90 (s, 0.8H), 6.83-6.79 (m, 2H), 6.74 (s, 1H), 6.71 (d, J=8.2 Hz, 1H), 6.51-6.46 (m, 1H), 6.22-6.15 (m, 2H), 5.49-5.41 (m, 1H), 4.86 (dd, J=11.8, 2.7 Hz, 0.8H), 4.68-4.65 (m, 0.2H), 4.33-4.24 (m, 1H), 3.85-3.81 (m, 2H), 3.76-3.73 (m, 3H), 3.68-3.66 (m, 3H), 3.59-3.49 (m, 2H), 3.37-3.36 (m, 3H), 3.28-3.22 (m, 1H), 3.11-3.02 (m, 2.4H), 2.93 (s, 0.6H), 2.91-2.85 (m, 0.6H), 2.70 (s, 2.4H), 2.67-2.60 (m, 1H), 2.29-2.26 (m, 0.8H), 1.80 (s, 0.6H), 1.78-1.74 (m, 0.2H), 1.73 (s, 2.4H), 1.72-1.63 (m, 1H), 1.54-1.46 (m, 1H), 1.32-1.30 (m, 3H), 1.28-1.24 (m, 4H), 1.21-1.18 (m, 3H), 0.97 (s, 2.4H), 0.91 (s, 0.6H). LC/MS: (ES+) [M+H].sup.+ (750, 100%).

Example 24: Preparation of Maytansinoid Compound 65

[0288] ##STR00189##

[0289] Compound 61 (36 mg), iron(III) nitrate nonahydrate (13 mg), TEMPO (4.8 mg), potassium chloride (6.2 mg) and 1,2-dichloroethane (2 mL) were sequentially added to a reaction vessel and the mixture was stirred under an oxygen atmosphere for 5 days. The reaction mixture was then concentrated in vacuo, the residue was dissolved in DMF (10 mL) and then purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give compound 65 as a white solid (4.5 mg). .sup.1H NMR (500 MHz; CD.sub.3OD) 8.01 (d, J=7.4 Hz, 1H), 7.82-7.80 (m, 1H), 7.53-7.50 (m, 1H), 7.42-7.39 (m, 1H), 7.17 (s, 1H), 6.99 (s, 1H), 6.72-6.67 (m, 1H), 6.30 (d, J=11.0 Hz, 1H), 5.59-5.51 (m, 1H), 4.75 (d, J=11.7 Hz, 1H), 4.27-4.22 (m, 1H), 3.85 (s, 3H), 3.65-3.61 (m, 3H), 3.39 (s, 3H), 3.11-3.06 (m, 1H), 2.88 (d, J=9.4 Hz, 1H), 2.80-2.74 (m, 1H), 2.67 (s, 3H), 2.32-2.29 (m, 1H), 1.81 (s, 3H), 1.66-1.57 (m, 3H), 1.31-1.27 (m, 7H), 1.23 (d, J=6.5 Hz, 3H), 1.04 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (721, 100%).

Example 25: Preparation of Conjugation Reagent 66 Comprising a Maytansinoid Cytotoxic Payload

[0290] ##STR00190##

Step 1: Synthesis of Compound 67.

[0291] ##STR00191##

[0292] To a stirred solution of compound 58 (328 mg), Boc-Val-Cit-PAB (BroadPharm, 688 mg) and tri-n-butylphosphine (700 L) in DMF (15 mL) at 0 C. was slowly added diisopropyl azodicarboxylate (560 L). The mixture was allowed to gradually warm to room temperature and was stirred for 2.5 h. The reaction mixture was then directly purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give compound 67 as a pale yellow solid (92 mg). LC/MS: (ES+) [M+H].sup.+ (1154, 100%).

Step 2: Synthesis of Compound 68.

[0293] ##STR00192##

[0294] A solution of compound 67 (92 mg) in formic acid (2 mL) was stirred at room temperature for 80 min. The reaction mixture was then concentrated in vacuo, the residue was dissolved in DMF (5 mL) and then purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give compound 68 as a white solid (47 mg). LC/MS: (ES+) [M+H].sup.+ (1054, 100%).

Step 3: Synthesis of Reagent 66.

[0295] Reagent 66 was synthesised in an analogous way to reagent 47 of Example 21 (Step 3) using compound 68 instead of compound 49. Reagent 66 was isolated as a white solid. .sup.1H NMR (500 MHz; DMSO-d.sub.6) 10.02 (s, 1H), 8.76 (d, J=7.8 Hz, 0.5H), 8.16-8.14 (m, 0.5H), 8.05-8.02 (m, 1H), 7.92-7.79 (m, 2H), 7.69-7.61 (m, 2H), 7.57-7.51 (m, 5H), 7.47-7.43 (m, 4H), 7.41-7.31 (m, 2H), 7.12 (d, J=0.4 Hz, 1H), 7.09-7.08 (m, 2H), 7.00 (d, J=8.8 Hz, 2H), 6.86 (s, 1H), 6.81 (s, 1H), 6.65-6.60 (m, 0.5H), 6.53 (s, 0.5H), 6.23-6.20 (m, 0.5H), 5.99-5.97 (m, 0.5H), 5.89-5.87 (m, 1H), 5.45-5.40 (m, 3H), 5.06-5.00 (m, 1H), 4.59-4.53 (m, 0.5H), 4.44-4.39 (m, 1H), 4.27-4.24 (m, 0.5H), 4.14-4.07 (m, 0.5H), 4.02-3.97 (m, 0.5H), 3.86-3.80 (m, 1H), 3.76 (s, 3H), 3.74-3.71 (m, 1.5H), 3.68-3.63 (m, 0.5H), 3.51 (s, 96H), 3.46-3.42 (m, 5H), 3.25 (d, J=4.4 Hz, 7H), 3.05-2.94 (m, 1.5H), 2.90-2.85 (m, 0.5H), 2.72-2.70 (m, 1H), 2.66-2.63 (m, 2H), 2.46 (s, 6H), 2.40-2.37 (m, 2H), 2.34-2.27 (m, 2H), 2.20-2.17 (m, 1H), 2.05-1.91 (m, 2H), 1.72-1.68 (m, 4H), 1.62-1.59 (m, 0.5H), 1.52-1.35 (m, 4.5H), 1.14 (d, J=6.9 Hz, 6H), 1.10 (d, J=6.6 Hz, 3H), 0.94 (s, 3H), 0.88 (d, J=6.5 Hz, 3H), 0.84 (d, J=6.7 Hz, 4H). LC/MS: (ES+) [M+2H].sup.2+ (1368, 20%), [M+3H].sup.3+ (912, 70%), [M+4H].sup.4+ (684, 100%).

Example 26: Preparation of Conjugation Reagent 69 Comprising a Maytansinoid Cytotoxic Payload

[0296] ##STR00193##

[0297] Reagent 69 was synthesised in an analogous way to reagent 66 of Example 25 using compound 62 instead of compound 58 (step 1). Reagent 69 was isolated as a white solid. .sup.1H NMR (500 MHz; CDCl.sub.3) 9.15 (s, 0.5H), 7.96-7.88 (m, 2.5H), 7.80-7.72 (m, 1H), 7.75-7.66 (m, 8H), 7.47-7.44 (m, 1H), 7.37-7.32 (m, 7H), 7.17 (s, 1H), 6.97 (d, J=8.4 Hz, 1H), 6.89 (d, J=8.1 Hz, 1H), 6.85 (d, J=4.7 Hz, 2H), 6.48 (dd, J=15.3, 11.0 Hz, 0.5H), 6.25-6.17 (m, 1.5H), 5.97-5.86 (m, 0.5H), 5.50-5.45 (m, 0.5H), 5.11-5.00 (m, 4H), 4.81 (dd, J=11.6, 1.9 Hz, 1H), 4.67-4.65 (m, 0.5H), 4.56-1.55 (m, 0.5H), 4.38-4.20 (m, 2H), 3.81 (s, 3H), 3.63 (s, 96H), 3.57-3.52 (m, 12H), 3.37 (d, J=5.5 Hz, 6H), 3.27-3.20 (m, 3H), 2.94 (dd, J=27.1, 11.3 Hz, 2H), 2.69 (s, 3H), 2.65-2.61 (m, 1H), 2.47 (s, 6H), 2.41-2.34 (m, 1.5H), 2.22-2.13 (m, 2.5H), 1.98-1.95 (m, 1H), 1.83-1.79 (m, 3H), 1.74 (s, 3H), 1.66 (d, J=13.5 Hz, 1H), 1.58-1.48 (m, 2H), 1.29 (d, J=6.1 Hz, 3H), 1.25 (d, J=7.1 Hz, 4H), 1.19 (d, J=6.7 Hz, 3H), 1.00 (t, J=6.4 Hz, 5H), 0.93 (s, 3H). LC/MS: (ES+) [M+2H].sup.2+ (1385, 15%), [M+3H].sup.3+ (923, 75%), [M+4H].sup.4+ (693, 100%).

Example 27: Preparation of Conjugation Reagent 70 Comprising a Maytansinoid Cytotoxic Payload

[0298] ##STR00194##

Step 1: Synthesis of Compound 71.

[0299] ##STR00195##

[0300] A solution of compound 18 (18 mg) and compound 7 (19 mg) in anhydrous DMF (1 mL) was stirred under an argon atmosphere at room temperature for 18 h. The reaction solution was then diluted with water (1 mL) and purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.05% acetic acid and buffer B (v/v): acetonitrile:0.05% acetic acid (70:30 v/v to 10:90 v/v). The desired fractions were combined, concentrated in vacuo and lyophilised to give compound 71 as a white solid (12 mg). NMR (500 MHz; CDCl.sub.3): 8.41 (s, 1H), 7.70 (s, 1H), 7.12 (s, 1H), 7.04 (d, J=7.3 Hz, 1H), 6.87 (dd, J=10.8, 5.9 Hz, 2H), 6.48 (dd, J=15.2, 10.9 Hz, 1H), 6.31 (s, 1H), 6.19 (dd, J=11.0, 0.5 Hz, 1H), 5.47 (dd, J=15.4, 8.8 Hz, 1H), 4.84-4.81 (m, 1H), 4.32-4.28 (m, 1H), 3.82 (d, J=5.0 Hz, 311), 3.56 (d, J=12.7 Hz, 1H), 3.52 (d, J=9.0 Hz, 1H), 3.37 (s, 3H), 3.28-3.21 (m, 2H), 3.00 (dd, J=10.0, 4.8 Hz, 1H), 2.91 (t, J=13.0 Hz, 1H), 2.73 (d, J=8.7 Hz, 3H), 2.67-2.56 (m, 4H), 2.44 (d, J=8.5 Hz, 1H), 2.23 (dd, J=13.8, 3.1 Hz, 1H), 2.16 (dt, J=12.7, 6.6 Hz, 1H), 2.03 (ddt, J=25.3, 12.2, 6.5 Hz, 4H), 1.50 (d, J=6.7 Hz, 3H), 1.45 (d, J=6.7 Hz, 3H), 1.36 (dt, J=6.9, 3.6 Hz, 1H), 1.31 (q, J=6.1 Hz, 5H), 1.25 (dt, J=7.5, 4.1 Hz, 4H), 1.20 (d, J=6.7 Hz, 3H), 0.93 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (974, 100%), [M+Na].sup.+ (996, 45%)

Step 2: Synthesis of Reagent 70.

[0301] A mixture of compound 71 (11 mg), EDC.HCl (15 mg) and N-hydroxysuccinimide (10 mg) in anhydrous dichloromethane (3 mL) was stirred under an argon atmosphere at room temperature for 72 h. The reaction mixture was then concentrated in vacuo and the residue dissolved in dichloromethane:acetonitrile (2 mL, 1:1 v/v) before purification by normal phase chromatography eluting with dichloromethane:acetonitrile (100:0 v/v to 0:100 v/v). The desired fractions were combined and the solvent was removed in vacuo to give reagent 70 as a white solid (12 mg). .sup.1H NMR (500 MHz; CDCl.sub.3): 8.41 (d, J=7.5 Hz, 1H), 7.79-7.70 (m, 1H), 7.12 (s, 1H), 7.06-7.04 (m, 1H), 6.86 (d, J=11.8 Hz, 2H), 6.48 (dd, J=15.4, 11.0 Hz, 1H), 6.19 (d, J=14.5 Hz, 2H), 5.49-5.45 (m, 1H), 4.83 (dd, J=11.7, 2.6 Hz, 1H), 4.30 (t, J=11.2 Hz, 1H), 3.81 (s, 3H), 3.56 (d, J=12.8 Hz, 1H), 3.52 (d, J=8.8 Hz, 1H), 3.37 (s, 3H), 3.27 (d, J=12.5 Hz, 2H), 3.00 (d, J=9.8 Hz, 2H), 2.93 (dd, J=13.0, 4.8 Hz, 2H), 2.87-2.79 (m, 7H), 2.72 (d, J=3.3 Hz, 3H), 2.62 (tt, J=13.4, 6.5 Hz, 3H), 2.23 (d, J=14.2 Hz, 1H), 2.07 (dddd, J=31.6, 23.4, 16.2, 7.7 Hz, 4H), 1.74 (s, 3H), 1.67 (d, J=13.6 Hz, 1H), 1.50 (d, J=6.7 Hz, 1H), 1.47 (d, J=6.7 Hz, 1H), 1.37 (dd, J=6.8, 1.7 Hz, 2H), 1.34 (d, J=6.9 Hz, 2H), 1.31 (d, J=6.3 Hz, 3H), 1.26 (q, J=8.3 Hz, 5H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (1071, 100%), [M+Na].sup.+ (1093, 80%).

Example 28: Preparation of Conjugation Reagent 72 Comprising a Maytansinoid Cytotoxic Payload

[0302] ##STR00196##

Step 1: Synthesis of Compound 73.

[0303] ##STR00197##

[0304] To a solution of Fmoc-Lys(Boc)-OH (470 mg) in anhydrous DMF (5 mL) at 0 C. was added HATU (1.4 g). To this was added a solution of NH.sub.2PEG(24u)-OMe (1 g) and NMM (340 L) in anhydrous DMF (5 mL) at 0 C. before the solution was allowed to warm to room temperature and was stirred for 1 h. The reaction mixture was then concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (95:5 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give Fmoc-Lys(Boc)-[PEG(24u)-OMe]. LC/MS: (ES+) [M+Na].sup.+ (1560, 100%), [M+H].sup.+ (1539, 80%). To a solution of the Fmoc-Lys(Boc)-[PEG(24u)-OMe] in DMF (10 mL) was added piperidine (0.9 mL) and the solution was stirred for 10 min at room temperature. The reaction solution was concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (95:5 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give compound 73 as a white solid (1 g). LC/MS: (ES+) [M+H].sup.+ (1316, 10%), [M+2H-Boc].sup.2+ (609, 100%).

Step 2: Synthesis of Compound 74.

[0305] ##STR00198##

[0306] To a solution of compound 36 (420 mg) in anhydrous DMF (2 mL) was added a solution of compound 73 (1 g) and NMM (92 L) in anhydrous DMF (3 mL). After stirring for 2.5 at room temperature, the reaction mixture was concentrated in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (95:5 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give bis-tolylsulfonyl-propanoyl-benzamide-L-Lys-[Boc]-[PEG(24u)-OMe] as a white solid (0.84 g). LC/MS: (ES+) [M+2H-Boc].sup.2+ (850, 95%). Bis-tolylsulfonyl-propanoyl-benzamide-L-Lys-[Boc]-[PEG(24u)-OMe] (0.84 g) was then dissolved in formic acid (3 mL) and the solution stirred at room temperature for 3 h. The volatiles were removed in vacuo and the residue washed with toluene (36 mL) before the residue was dried in vacuo and purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (95:5 v/v to 0:100 v/v). The organic solvent was removed in vacuo and the aqueous solvent removed by lyophilisation to give compound 74 as a white solid (0.59 g). LC/MS: (ES+) [M+H].sup.+ (1699, 5%), [M+2H].sup.2+ (850, 100%).

Step 3: Synthesis of Reagent 72.

[0307] To a solution of compound 74 (52 mg) in anhydrous DMF (0.9 mL) was added reagent 25 (21 mg) followed by NMM (9 L) and the mixture was stirred at room temperature. Additional quantities of NMM (29 L) were added after 2 and 4 h and then after 6 h, the reaction solution was stored at 20 C. for a further 16 h. The reaction solution was then concentrated in vacuo, the residue dissolved in acetonitrile (400 L) and then purified by reverse phase C18-column chromatography, eluting with buffer A (v/v): water:0.05% trifluoroacetic acid and buffer B (v/v): acetonitrile:0.05% trifluoroacetic acid (70:30 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give reagent 72 as a white solid (17 mg). MS: (ES+) [M+2Na].sup.2+ (1333, 10%), [M+3H].sup.3+ (874, 100%), [M+4H].sup.4+ (656, 30%).

Example 29: Preparation of Conjugation Reagent 75 Comprising a Maytansinoid Cytotoxic Payload

[0308] ##STR00199##

Step 1: Synthesis of Compound 76.

[0309] ##STR00200##

[0310] A mixture of compound 34 (40 mg) and 4-mercapto-pentanoic acid (27 mg) in DMF (5 mL) was stirred at room temperature for 16 h. The reaction mixture was then directly purified by reverse phase C-18 column chromatography eluting with buffer A (v/v): water:0.1% acetic acid and buffer B (v/v): acetonitrile:0.1% acetic acid (100:0 v/v to 0:100 v/v). The desired fractions were combined and lyophilised to give compound 76 as a white solid (36 mg). LC/MS: (ES+) [M+H].sup.+ (998, 100%), [M+2H].sup.2+ (499, 20%).

Step 2: Synthesis of Reagent 75.

[0311] Reagent 75 was synthesised in an analogous way to reagent 25 of Example 12 using compound 76 instead of compound 26. Reagent 75 was isolated as a white solid. .sup.1H NMR (500 MHz; CDCl.sub.3) 7.76-7.70 (m, 1H), 7.58-7.56 (m, 2H), 7.08-7.06 (m, 2H), 6.84 (d, J=0.4 Hz, 1H), 6.72-6.68 (m, 2H), 6.46 (dd, J=15.3, 11.3 Hz, 1H), 6.25 (s, 1H), 5.72-5.67 (m, 1H), 5.36-5.32 (m, 1H), 4.82-4.79 (m, 1H), 4.30 (td, J=11.2, 1.4 Hz, 1H), 3.81 (s, 3H), 3.70 (d, J=12.9 Hz, 1H), 3.52 (d, J=9.1 Hz, 1H), 3.37 (s, 3H), 3.18-3.15 (m, 1H), 3.09-3.02 (m, 2H), 2.96-2.73 (m, 6H), 2.66 (s, 3H), 2.54-2.51 (m, 2H), 2.28-2.24 (m, 1H), 2.15-1.95 (m, 6H), 1.69 (s, 3H), 1.66 (d, J=0.4 Hz, 1H), 1.53-1.47 (m, 1H), 1.37 (d, J=6.7 Hz, 3H), 1.34-1.25 (m, 13H), 0.92 (s, 3H). LC/MS: (ES+) [M+H].sup.+ (1094, 100%).

Example 30: Preparation of Conjugation Reagent 77 (Comparator) Comprising a Maytansinoid Cytotoxic Payload

[0312] ##STR00201##

Step 1: Synthesis of Compound 78.

[0313] ##STR00202##

[0314] Compound 78 was synthesised in an analogous way to compound 71 of Example 27 using N2-deacetyl-N2-(4-mercapto-1-oxopentyl)-maytansine (available from BOC Sciences) instead of compound 18. Compound 78 was isolated as a white solid. .sup.1H NMR (500 MHz; CDCl.sub.3): 6.83 (s, 1H), 6.69-6.67 (m, 1H), 6.42 (ddd, J=14.9, 11.3, 3.4 Hz, 1H), 6.32 (d, J=6.4 Hz, 1H), 5.69-5.62 (m, 1H), 4.92-4.86 (m, 1H), 4.32 (quintet, J=9.8 Hz, 1H), 3.99 (s, 3H), 3.62 (ddd, J=20.7, 13.1, 7.4 Hz, 1H), 3.48 (td, J=10.4, 4.2 Hz, 1H), 3.35 (d, J=2.8 Hz, 3H), 3.21 (s, 3H), 3.17-3.12 (m, 1H), 3.01-2.95 (m, 2H), 2.91 (dd, J=13.8, 8.8 Hz, 2H), 2.84 (td, J=14.1, 6.7 Hz, 2H), 2.67-2.60 (m, 2H), 2.55-2.36 (m, 4H), 2.20 (dd, J=14.4, 2.6 Hz, 1H), 1.95 (td, J=13.2, 6.9 Hz, 2H), 1.81 (dt, J=20.0, 5.7 Hz, 2H), 1.73 (dd, J=19.2, 14.2 Hz, 1H), 1.65 (s, 3H), 1.48-1.45 (m, 2H), 1.39 (dd, J=12.6, 6.9 Hz, 2H), 1.34 (dd, J=9.0, 7.0 Hz, 2H), 1.32-1.25 (m, 12H), 0.82 (dd, J=11.9, 5.7 Hz, 3H). LC/MS: (ES+) [MH.sub.2O+H].sup.+ (880, 100%), [M+H].sup.+ (898, 15%), [M+Na].sup.+ (920, 60%).

Step 2: Synthesis of Reagent 77.

[0315] Reagent 77 was synthesised in an analogous way to reagent 25 of Example 12 using compound 78 instead of compound 26. Reagent 77 was isolated as a white solid. .sup.1H NMR (500 MHz; CDCl.sub.3): 6.86-6.83 (m, 1H), 6.75 (t, J=8.8 Hz, 1H), 6.64 (d, J=4.8 Hz, 1H), 6.43 (dd, J=15.3, 11.2 Hz, 1H), 6.20 (s, 1H), 5.67 (ddd, J=15.0, 9.4, 5.2 Hz, 1H), 5.42 (q, J=6.7 Hz, 1H), 4.78 (dd, J=12.0, 2.7 Hz, 1H), 4.27 (t, J=11.3 Hz, 1H), 3.98 (s, 3H), 3.65 (d, J=12.7 Hz, 1H), 3.50 (d, J=9.0 Hz, 1H), 3.35 (s, 3H), 3.22 (s, 3H), 3.13 (t, J=10.2 Hz, 1H), 3.04 (d, J=9.7 Hz, 1H), 2.85 (d, J=3.4 Hz, 9H), 2.72 (t, J=6.9 Hz, 2H), 2.61 (t, J=13.2 Hz, 1H), 2.55-2.49 (m, 1H), 2.46-2.35 (m, 1H), 2.17 (dd, J=14.3, 2.4 Hz, 1H), 2.04-1.83 (m, 4H), 1.64 (s, 3H), 1.57 (d, J=13.6 Hz, 1H), 1.51 (s, 2H), 1.48-1.44 (m, 1H), 1.30-1.23 (m, 14H), 0.80 (s, 3H). LC/MS: (ES+) [MH.sub.2O+H].sup.+ (977, 100%), [M+H].sup.+ (995, 15%), [M+Na].sup.+ (1017, 55%).

Example 31: Preparation of Maytansinoid Compound 79

[0316] ##STR00203##

[0317] Compound 79, which was synthesised as a mixture of atropisomers, was prepared in a similar way to compound 4 of Example 3. Briefly, 4-amino-2-chlorophenylboronic acid pinacol ester (66 mg), tripotassium phosphate (148 mg), SPhos Pd G3 (13.6 mg) and AP3 (100 mg) were sequentially added to an argon purged reaction vessel. The vessel was then sealed and the solids purged with argon (4evacuation/purge cycles). THF (1.2 mL) and water (120 L), which had been rigorously deoxygenated by purging with argon, were then added and the reaction mixture was stirred at room temperature for 12 h. Analysis of the crude reaction mixture by LC/MS identified two peaks with the expected molecular mass for compound 79. The two peaks resolved at 2.81 and 3.00 min correspond to two atropisomer species. Henceforth, the atropisomers eluting at 2.81 and 3.00 min will be referred to as compounds 80 and 81, respectively.

[0318] Separation of the atropisomers was achieved by reverse phase preparative HPLC. Firstly, the crude reaction mixture was diluted with ethyl acetate (40 mL) and then washed with brine (20 mL). The organic layer was separated and concentrated in vacuo and the residue dissolved in DMF (4 mL). Atropisomers 80 and 81 were separated by reverse phase preparative HPLC using a Luna C18(2) column (250 mm L50 mm ID, 5 m) eluting with buffer A (v/v): water:0.05% acetic acid and buffer B (v/v): acetonitrile:0.05% acetic acid (90:10 v/v to 10:90 v/v, 35 min, room temperature). Fractions corresponding to the two atropisomers (as confirmed by LC/MS) were separated and lyophilised to give compounds 80 and 81 as white solids. Each compound was then further purified by normal phase chromatography eluting with dichloromethane:acetone (100:0 v/v to 50:50 v/v) to give compound 80 (15 mg) and compound 81 (10 mg) as white solids. The HPLC chromatograms of compounds 80 and 81 following normal phase purification indicate that both compounds achieved a purity >96% by peak area.

[0319] The stability of the atropisomers was investigated by heating solutions of compounds 80 and 81 in DMSO at 50 C. for 1 h. After heating for 1 h, the compounds were analysed by HPLC and no changes were observed from the HPLC chromatograms of the pre-heated samples, indicating both atropisomers are stable and do not interconvert at the indicated elevated temperature.

[0320] Characterisation data for compound 80: .sup.1H NMR (500 MHz; CDCl.sub.3) 6.92 (d, J=8.2 Hz, 1H), 6.84 (s, 2H), 6.73 (d, J=2.1 Hz, 1H), 6.57 (dd, J=8.2, 2.2 Hz, 1H), 6.51-6.45 (m, 1H), 6.21 (s, 1H), 6.18-6.16 (m, 1H), 5.47-5.42 (m, 1H), 4.76-4.74 (m, 1H), 4.30-4.25 (m, 1H), 3.81 (s, 3H), 3.55 (d, J=12.6 Hz, 1H), 3.51 (d, J=8.8 Hz, 1H), 3.36 (s, 3H), 3.28 (d, J=12.6 Hz, 1H), 3.01 (s, 1H), 2.94 (s, 3H), 2.87 (t, J=13.3 Hz, 1H), 2.64-2.59 (m, 1H), 2.19-2.15 (m, 1H), 1.77 (s, 3H), 1.66-1.63 (m, 1H), 1.50-1.45 (m, 1H), 1.29-1.24 (m, 6H), 1.19 (d, J=6.7 Hz, 3H), 0.87 (s, 3H). LC/MS: retention time 2.81 min (Acquity UPLC BEH C18 1.7 m, 2.150 mm column, eluting with water (0.05% acetic acid):acetonitrile (0.05% acetic acid) (95:5 v/v to 5:95 v/v), 5 min gradient, 0.6 mL/min, room temperature), (ES+) [M+H].sup.+ (726, 100%).

[0321] Characterisation data for compound 81: .sup.1H NMR (500 MHz; CDCl.sub.3) 6.89 (s, 1H), 6.84 (s, 1H), 6.77 (d, J=2.1 Hz, 1H), 6.67 (d, J=8.2 Hz, 1H), 6.57 (dd, J=8.2, 2.1 Hz, 1H), 6.51-6.46 (m, 1H), 6.27 (s, 1H), 6.19 (d, J=11.1 Hz, 1H), 5.47 (dd, J=15.3, 9.0 Hz, 1H), 4.84-4.82 (m, 1H), 4.32-4.27 (m, 1H), 3.84 (s, 3H), 3.58 (d, J=13.1 Hz, 1H), 3.52 (d, J=8.9 Hz, 1H), 3.37 (s, 3H), 3.26 (d, J=13.1 Hz, 1H), 3.02-2.91 (m, 2H), 2.72 (s, 3H), 2.67-2.58 (m, 1H), 2.23-2.21 (m, 1H), 1.74 (s, 3H), 1.68 (d, J=13.2 Hz, 1H), 1.53-1.47 (m, 2H), 1.30 (d, J=6.3 Hz, 3H), 1.28-1.24 (m, 4H), 1.20 (d, J=6.7 Hz, 3H), 0.94 (s, 3H). LC/MS: retention time 3.00 min, (ES+) [M+H].sup.+ (726, 100%).

[0322] The invention includes the individual atropisomers of the compound of formula 79. For example, it includes the atropisomer having a retention time of 2.81 minutes when analysed by LC/MS under the conditions indicated above in Example 31, and also includes the atropisomer having a retention time of 3.00 minutes when analysed by LC/MS under the conditions indicated above in Example 31.

[0323] Using a combination of 2D NMR (ROESY) spectroscopy and molecular modelling studies, it is proposed that compounds 80 and 81 have the following structures:

##STR00204##

Example 32: In Vitro Potency Assay of Compounds in SK-BR-3 Cell Lines

[0324] Loss of tumour cell viability following treatment with compounds of the invention was tested by growing SK-BR-3 cell lines in the presence of increasing concentrations of compounds of the invention and quantifying the loss of proliferation or metabolic activity as described in Example 2. The average IC.sub.50 values for compounds of the invention are shown in Table 6 and the assay concentrations are specified in Table 7.

TABLE-US-00006 TABLE 6 Compound Average IC.sub.50 (nM) SK-BR-3 Cell line 9 7.4 (n = 2) 12 4.2 (n = 2) 15 5.4 (n = 2) 21 1.7 (n = 2) 23 4.2 (n = 2) 32 5.5 (n = 2) 53 11.2 (n = 2) 54 2.9 (n = 3) 55 0.9 (n = 3) 56 17.0 (n = 2) 57 3.6 (n = 2) 58 0.5 (n = 3) 59 3.9 (n = 2) 60 1.3 (n = 3) 61 6.0 (n = 2) 62 1.4 (n = 3) 63 2.8 (n = 2) 64 4.9 (n = 3) 80 3.6 (n = 3) 81 0.9 (n = 3) 31 (comparator) 33.4 (n = 3)

TABLE-US-00007 TABLE 7 Cell line Compound Concentration range SK-BR-3 9 200 nM-91 pM SK-BR-3 12 200 nM-91 pM SK-BR-3 15 200 nM-91 pM SK-BR-3 21 200 nM-2.6 pM SK-BR-3 23 200 nM-2.6 pM SK-BR-3 32 500 nM-6.4 pM SK-BR-3 53 300 nM-137 pM SK-BR-3 54 800 nM-0.4 pM SK-BR-3 55 800 nM-0.4 pM SK-BR-3 56 1000 nM-61 pM.sup. SK-BR-3 57 500 nM-6.4 pM SK-BR-3 58 100 nM-6.1 pM SK-BR-3 59 1000 nM-3.6 pM SK-BR-3 60 700 nM-0.9 pM SK-BR-3 61 1000 nM-3.6 pM SK-BR-3 62 100 nM-1.3 pM SK-BR-3 63 800 nM-2.9 pM SK-BR-3 64 200 nM-12 pM SK-BR-3 80 200 nM-12 pM SK-BR-3 81 100 nM-1.3 pM SK-BR-3 31 (comparator) 1000 nM-457 pM
These data show that compounds of the invention, which contain a biphenyl moiety, unexpectedly have lower IC.sub.50 values than the allylamine-containing comparator.

Example 33: Conjugation of Reagents 35, 47, 69 and 72 to Trastuzumab to Produce Antibody Drug Conjugates (ADCs) 82, 83, 84 and 85 Respectively, with DAR 4

[0325] Conjugation reagents 35, 47, 69 and 72 were conjugated to Trastuzumab giving rise to ADCs 82, 83, 84 and 85 using methods analogous to those described in WO2014064423 and WO2014064424.

[0326] Briefly, Trastuzumab (5-7.4 mg/mL in 20 mM sodium phosphate, 150 mM NaCl, 20 mM EDTA pH 7.5) was heated to 40 C. in a heating block for 15 min. 5 mM TCEP solution (6 eq. per mAb) was added to the mAb solution, mixed gently and incubated at 40 C. for 1 h before being allowed to cool to 22 C.

[0327] For conjugations using reagents 35, 47 and 69, the reduced mAb solution was then diluted to 4.4 mg/mL with 20 mM sodium phosphate, 150 mM NaCl, 20 mM EDTA, pH 7.5. Conjugation reagents 35, 47 and 69 were dissolved in DMF to give 1.5 mM solutions. Conjugation reagent (5.6 eq. per mAb) was added to the mAb solutions to give final antibody concentrations of 4.0 mg/mL. For the conjugation using reagent 72, the reagent was dissolved in propylene glycol:DMF (3:1 v/v) to give a 0.75 mM solution. Reagent 72 (5.6 eq. per mAb) was then added to the reduced mAb solution to give a final antibody concentration of 4.0 mg/mL.

[0328] Each conjugation reaction solution was then mixed gently and incubated at 22 C. for 18-21 h. The crude reaction solutions were then mixed with equal volumes of 50 mM sodium phosphate, 4 M NaCl, pH 7 and the resulting solutions were loaded onto a ToyoPearl Phenyl-650S HIC column equilibrated with 50 mM sodium phosphate, 2 M NaCl, pH 7. Each ADC was eluted from the column with a gradient of 50 mM sodium phosphate, pH 7 (20% isopropanol). Fractions containing DAR 4 ADC were pooled and concentrated. The concentrated sample was buffer exchanged into PBS, pH 7.1-7.5, and sterile filtered (0.22 m PVDF membranes). DAR assignments were based on A248/A280 absorption ratios. The average DAR of ADCs 82, 83, 84 and 85 were calculated from the relative peak areas of individual DAR species following HIC analysis at 280 nm.

Example 34: Conjugation of Reagent 41 to Brentuximab to Produce Antibody Drug Conjugate (ADC) 86 with DAR 4

[0329] Conjugation reagent 41 was conjugated to Brentuximab giving rise to ADC 86 using a similar method to that described in Example 33. Briefly, Brentuximab (8.5 mg/mL in 20 mM sodium phosphate, 150 mM NaCl, 20 mM EDTA, pH 7.5) was heated to 40 C. in a heating block for 15 min. TCEP (6 eq. per mAb) was added to the mAb solution, mixed gently and incubated at 40 C. for 1 h before being allowed to cool to 22 C. Conjugation reagent 41 was dissolved in DMF to give a 1.6 mM solution. The reduced mAb solution was diluted to 6.7 mg/mL with 20 mM sodium phosphate, 150 mM NaCl, 20 mM EDTA, pH 7.5) followed by the addition of propylene glycol, resulting in a final reduced mAb solution concentration of 4.4 mg/mL. Conjugation reagent (6 eq. per mAb) was added to the mAb solution to give a final antibody concentration of 4 mg/mL. The reaction solution was mixed gently and incubated at 22 C. for 24 h. After this time, the reaction solution was treated with 50 mM N-acetyl-L-cysteine (20 eq. over reagent) at 22 C. for 30 min. The crude reaction solution was then mixed with an equal volume of 50 mM sodium phosphate, 4 M NaCl, pH 7 and the resulting solution was loaded onto a ToyoPearl Phenyl-650S HIC column equilibrated with 50 mM sodium phosphate, 2 M NaCl, pH 7. The ADC was eluted from the column with a gradient of 50 mM sodium phosphate, pH 7 (20% isopropanol). Fractions containing DAR 4 ADC were pooled and concentrated (Vivaspin 20, 10 kDa PES membrane). The concentrated sample was buffer exchanged into DPBS, pH 7.1-7.5, and sterile filtered (0.22 m PVDF membrane). The ADC was further purified using a Hydroxyapatite Foresight CHT column equilibrated with 10 mM sodium phosphate, pH 6.7. The ADC was eluted from the column with a gradient of 10 mM sodium phosphate, 2 M NaCl, pH 6.7. Fractions containing ADC were pooled and concentrated (Vivaspin 20, 30 kDa PES membrane) and the concentrated sample was buffer exchanged into DPBS, pH 7.1-7.5 and sterile filtered (0.22 m PVDF membrane). The DAR of the conjugate was determined using the method described in Example 33.

Example 35: Conjugation of Reagent 50 to Trastuzumab to Produce Antibody Drug Conjugate (ADC) 87

[0330] Conjugation reagent 50 was conjugated to Trastuzumab, giving rise to ADC 87. Briefly, reagent 50 was dissolved in DMSO to give a 10 mM stock solution. Trastuzumab in 100 mM HEPES buffer, 1 mM EDTA, pH 7.0 (5 mg/mL mAb concentration), was reduced with TCEP (2.2 eq. per mAb) at 37 C. for 2 h. The reduced mAb solution was allowed to cool to 25 C. and was then diluted with DMSO (10% v/v). Conjugation reagent 50 (10 eq. per mAb) was then added to the reduced mAb solution and the reaction mixture was mixed gently and incubated at 25 C. for 30 min. Excess reagent 50 was quenched by incubating the reaction solution with N-acetyl cysteine (10 eq. per mAb) at 25 C. for 30 min. Activated charcoal powder (70% w/w of mAb) was then added to the reaction solution which was gently agitated for 30 min at room temperature to remove unreacted drug related species. The reaction mixture was then filtered (0.22 m PES membrane) and the purified sample was buffer exchanged into 10 mM succinic acid, 6% w/v trehalose, 0.01% v/v Tween 20, pH 5.5 using PD-10 desalting columns. An average DAR of 4 was assigned to conjugate 87 using the method described in Example 33.

Example 36: Conjugation of Reagents 70, 75 and 77 (Comparator) to Trastuzumab to Produce Antibody Drug Conjugates (ADCs) 88, 89 and 90 (Comparator), Respectively

[0331] Conjugation reagents 70, 75 and 77 (comparator) were conjugated to Trastuzumab giving rise to ADCs 88, 89 and 90 (comparator), respectively, using the following general conjugation protocol. Briefly, the reagents were dissolved in DMF to give 1.8-4.0 mM stock solutions. To a solution of Trastuzumab in 20 mM sodium phosphate, 150 mM NaCl, 20 mM EDTA, pH 7.5 was added reagent stock solution (5-20 eq. per mAb, either as a single addition or as multiple aliquots throughout the incubation period) to give a final antibody concentration of 3.0-4.0 mg/mL (containing DMF 10% v/v). The reaction solutions were incubated at 22 C. for 1-4 h.

[0332] ADCs 88 and 89 were purified by preparative SEC chromatography using a HiLoad 16/600 Superdex 200 pg column and isocratic elution using PBS (15% isopropanol) as eluent. ADC 90 (comparator) was purified using a Foresight CHT hydroxyapatite column equilibrated with 10 mM sodium phosphate, pH 6.7 and the ADC was eluted from the column with a gradient of 10 mM sodium phosphate, 2 M sodium chloride, pH 6.7.

[0333] After column chromatography, the desired ADC containing fractions were pooled and concentrated. The concentrated samples were buffer exchanged into PBS, and sterile filtered. DAR assignments were calculated from the relative peak intensities of the individual DAR species following mass spectrometry. Average DARs of 2.0, 3.2 and 3.1 were calculated for ADCs 88, 89 and 90 (comparator), respectively.

Example 37: Analysis of ADCs by In Vitro Cell Viability Assay

[0334] Loss of tumour cell viability following treatment with ADCs incorporating maytansinoids of the invention was tested by growing cell lines in the presence of increasing concentrations of ADCs and quantifying the loss of proliferation or metabolic activity as described in Example 2. The average IC.sub.50 values for ADCs incorporating the maytansinoids of the invention are shown in Table 8 and the assay concentrations are specified in Table 9.

TABLE-US-00008 TABLE 8 Compound Average IC.sub.50 (nM) 29 0.05 (n = 4) 30 0.08 (n = 2) 82 0.18 (n = 2) 83 0.18 (n = 3) 84 0.14 (n = 2) 85 0.26 (n = 2) 86 0.11 (n = 2) 87 0.17 (n = 2) 88 0.08 (n = 2) 89 0.10 (n = 2)

TABLE-US-00009 TABLE 9 Cell line Compound Concentration range SK-BR-3 29 2 nM-3.2 pM SK-BR-3 30 10 nM-0.6 pM SK-BR-3 82 10 nM-4.6 pM SK-BR-3 83 20 nM-1.2 pM SK-BR-3 84 10 nM-0.6 pM SK-BR-3 85 50 nM-22.9 pM Karpas-299 86 50 nM-0.18 pM SK-BR-3 87 1000 nM-3.6 pM SK-BR-3 88 10 nM-0.6 pM SK-BR-3 89 10 nM-0.6 pM

[0335] The IC.sub.50 values obtained show that ADCs incorporating the novel maytansinoids of the invention have potent cell killing properties in vitro.

Example 38: Stability of Compounds 26 and 78 (Comparator) in Mouse Serum

[0336] Solutions of compounds 26 and 78 (comparator) in a 50/50 v/v mixture of DMF:buffer solution (20 mM sodium phosphate, 150 mM sodium chloride, 20 mM EDTA, pH 7.5) (0.5 mg/mL) were diluted to 0.05 mg/mL in mouse serum (90% (v/v) serum content). An aliquot of each sample corresponding to the 0 time point was immediately frozen at 80 C., while the remaining sample was incubated at 37 C. for 7 d. Additional aliquots were taken after 4 and 7 d and frozen at 80 C. Prior to analysis, the samples were removed from the freezer and maytansinoid-related species were extracted from the serum by protein precipitation. Protein precipitation was performed by adding acetonitrile (75% v/v) to each time-point aliquot, and after gentle mixing, allowing the mixtures to stand at 4 C. for 2 h. The precipitated protein was then separated by centrifugation (1400g, 30 min, 4 C.) and the supernatant containing the extracted maytansinoid-related species were analysed by LC-Orbitrap-MS.

[0337] Reverse Phase Orbitrap-MS Analysis.

[0338] The samples were further diluted with water resulting in a 15% v/v acetonitrile solution. An aliquot (2.5 L) of each solution was then injected onto a nano-liquid chromatography MS system, consisting of a Dionex ULTIMATE 3000 UPLC fitted with a PepMap C18 column (0.075150 mm), coupled online to an Orbitrap-MS instrument operated in ES positive mode at 75K resolution using lock mass. Buffer A consisted of 100% water and buffer B of 100% acetonitrile, both containing 0.1% formic acid, and a gradient from 15-80% B was performed at a flow rate of 0.3 L/min over 60 min. Data analysis was performed manually by analysing for potential degradation products using a Thermo Xcalibur Qual Browser software tool.

[0339] FIG. 4a shows that after both 4 and 7 days at 37 C. in mouse serum, significant degradation of compound 78 (comparator) has occurred, with peaks corresponding to degradation products A and B detectable by reverse phase Orbitrap-MS analysis. The amount of degradation product B in particular, is increased from day 4 to day 7. In contrast, in FIG. 4b, no degradation products equivalent to A and B (designated C and D below) were observed for compound 26 following 7 days incubation. These data are consistent with compound 26 having improved stability versus compound 78 (comparator). The structures of compound 78 and fragments A and B are shown below.

##STR00205##

[0340] The structures of compound 26 and fragments C and D are shown below.

##STR00206##

Novel Cytotoxic Agents And Conjugates Thereof

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A61K47/68033

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A61K47/6803

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C07D498/18

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Abstract

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