RU2022115696A

RU2022115696A - SELECTIVE SULPHATION OF Benzodiazepine Derivatives

Info

Publication number: RU2022115696A
Application number: RU2022115696A
Authority: RU
Inventors: Скотт А. ХИЛДЕРБРАНД; Бенджамин М. ХАТЧИНС
Original assignee: Иммуноджен, Инк.
Priority date: 2016-11-23
Filing date: 2017-11-22
Publication date: 2022-09-09

Claims

1. A method for producing a cell-binding agent-cytotoxic agent conjugate, comprising the steps:

(a) interaction of an imine moiety in an imine-containing cytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof, with sulfur dioxide, a bisulfite salt or a metabisulfite salt in an aqueous solution at a pH of 1.9 to 5.0 to form a modified cytotoxic agent containing a modified imine moiety represented by the following formula:

or a pharmaceutically acceptable salt thereof; and

(b) reacting the modified cytotoxic agent with a cell-binding agent to form a cell-binding agent-cytotoxic agent conjugate,

where D is an imine-containing benzodiazepine compound; and L is a linker.

2. The method of claim 1 wherein D is an imine-containing indolinobenzodiazepine.

3. Process according to claim 1 or 2, wherein the reaction in step (a) is carried out at a pH of 2.9 to 4.0.

4. Process according to claim 1 or 2, wherein the reaction in step (a) is carried out at a pH of 2.9 to 3.7.

5. Process according to claim 1 or 2, wherein the reaction in step (a) is carried out at a pH of 3.1 to 3.5.

6. Process according to claim 1 or 2, wherein the reaction in step (a) is carried out at a pH of 3.2 to 3.4.

7. Process according to claim 1 or 2, wherein the reaction in step (a) is carried out at pH 3.3.

8. The method according to any one of paragraphs. 1-7, where the reaction in step (a) is carried out in the presence of a buffer solution.

9. The method of claim 8 wherein the buffer solution is citrate buffer, acetate buffer, succinate buffer and phosphate buffer.

10. The method of claim 8 wherein the buffer solution is a succinate buffer.

11. The method according to any one of paragraphs. 1-7, where the reaction in step (a) is carried out in the absence of a buffer solution.

12. A method for producing a cell-binding agent-cytotoxic agent conjugate, comprising the steps:

or a pharmaceutically acceptable salt thereof, with sulfur dioxide, a bisulfite salt or a metabisulfite salt in an aqueous solution in the absence of a buffer for the formation of a modified cytotoxic agent containing a modified imine moiety represented by the following formula:

or a pharmaceutically acceptable salt thereof; and

where D is an imine-containing benzodiazepine compound; and L is a linker.

13. The method according to any one of paragraphs. 1-12, wherein 0.5 to 5 equivalents of the bisulfite salt or 0.25 to 2.5 equivalents of the metabisulfite salt are reacted with 1 equivalent of an imine-containing cytotoxic agent.

14. The method of claim 13 wherein 0.8 to 2.0 equivalents of the bisulfite salt or 0.4 to 1.0 equivalents of the metabisulfite salt is reacted with 1 equivalent of an imine-containing cytotoxic agent.

15. The method of claim 13, wherein 1.1 to 1.6 equivalents of the bisulfite salt or 0.55 to 0.8 equivalents of the metabisulfite salt are reacted with 1 equivalent of an imine-containing cytotoxic agent.

16. The method of claim 13, wherein 1.4 equivalents of the bisulfite salt or 0.7 equivalents of the metabisulfite salt are reacted with 1 equivalent of an imine-containing cytotoxic agent.

17. The process according to claim 1 or 2, wherein the reaction of step (a) is carried out at a pH of 2.9 to 3.7 and 1.0 to 1.8 equivalents of the bisulfite salt or 0.5 to 0.9 equivalents of the salt metabisulfite is reacted with 1 equivalent of an imine-containing cytotoxic agent.

18. The method according to p. 1 or 2, where the reaction of stage (a) is carried out at a pH of from 3.1 to 3.5 and from 1.1 to 1.6 equivalents of the bisulfite salt or from 0.55 to 0.8 equivalents of salt metabisulfite is reacted with 1 equivalent of an imine-containing cytotoxic agent.

19. The method of claim 1 or 2, wherein the reaction of step (a) is carried out at pH 3.3 and 1.4 equivalents of the bisulfite salt or 0.7 equivalents of the metabisulfite salt is reacted with 1 equivalent of an imine-containing cytotoxic agent.

20. The method according to any one of paragraphs. 1-19, where the reaction of step (a) is carried out in a mixture of an organic solvent and water.

21. The method of claim 20 wherein the reaction of step (a) is carried out in a mixture of dimethylacetamide (DMA) and water.

22. The method according to claim 20, wherein the reaction of step (a) is carried out in a mixture of DMA and water, where the volume ratio of DMA and water is 1:1.

23. The method according to any one of paragraphs. 1-22, where the reaction of step (a) is carried out for 1 minute to 48 hours.

24. The method according to p. 23, where the reaction in stage (a) is carried out for from 1 hour to 10 hours.

25. The method according to p. 24, where the reaction in stage (a) is carried out for from 1 hour to 4 hours.

26. The method according to any one of paragraphs. 1-25, where in step (a), an imine-containing cytotoxic agent is reacted with sodium bisulfite.

27. The method according to any one of paragraphs. 1-25, where in step (a) an imine-containing cytotoxic agent is reacted with sodium metabisulphite.

28. The method according to any one of paragraphs. 1-27, where the modified cytotoxic agent is not purified prior to reaction with the cell-binding agent in step (b).

29. The method according to any one of paragraphs. 1-27 wherein the modified cytotoxic agent is purified prior to reaction with the cell-binding agent in step (b).

30. The method according to any one of paragraphs. 1-29, where the reaction in step (b) is carried out at pH 4 to 9.

31. The method of claim 30, wherein the reaction in step (b) is carried out at a pH of 5 to 8.5.

32. The method of claim 30, wherein the reaction in step (b) is carried out at a pH of 5.5 to 6.5.

33. The method according to any one of paragraphs. 1-32, where the conjugate is purified by tangential flow filtration to obtain a purified conjugate.

34. The method of claim 33, wherein the purified conjugate is prepared in formulation buffer containing a bisulfite salt.

35. The method of claim 34, wherein the formulation buffer contains 10 to 200 µM sodium bisulfite.

36. The method of claim 33 wherein the formulation buffer contains 40 to 80 µM sodium bisulfite.

37. The method of claim 33 wherein the formulation buffer contains 50 μM sodium bisulfite.

38. The method according to any one of paragraphs. 34-37, wherein the formulation buffer has a pH of 4 to 5.

39. The method of claim 38 wherein the formulation buffer has a pH of 4.2.

40. The method according to any one of paragraphs. 1-39, where D is an imine-containing compound of indolinobenzodiazepine.

41. The method according to any one of paragraphs. 1-40, where D is represented by the following formula:

or

or a pharmaceutically acceptable salt thereof, where:

one of L', L'' and L''' is represented by the following formula:

or

and the other two are each independently selected from -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, polyethylene glycol unit -(CH ₂ CH ₂ O) _n -R ^c , halogen, guanidinium [ -NH(C=NH)NH ₂ ], -OR, -NR'R'', -NO ₂ , -NR'COR'', -SR, -SOR', -SO ₂ R', -SO ₃ H, -OSO ₃ H, -SO ₂ NR'R'', cyano, azido, -COR', -OCOR' and -OCONR'R';

one of Z ₁ and Z ₂ is -C(=O)- and the other is -NR ₅ -;

P ₁ is an amino acid residue or a peptide containing 2 to 20 amino acid residues;

R _x1 is an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;

R ^e represents -H, linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, or -(CH ₂ -CH ₂ -O) _n -R ^k , where R ^k represents -H, linear branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (for example, -NHR ¹⁰¹ ) or tertiary amino (-NR ¹⁰¹ R ¹⁰² ) group or a 5- or 6-membered nitrogen-containing heterocycle, where R ¹⁰¹ and R ¹⁰² is each independently linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms;

R is at each occurrence independently selected from the group consisting of -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, polyethylene glycol unit -(CH ₂ CH ₂ O) _n -R ^c , an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5-18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, or an optionally substituted 3-18 membered heterocyclic ring containing from 1 to 6 heteroatoms independently selected from O, S, N and P;

R' and R'' are each independently selected from -H, -OH, -OR, -NHR, -NR ₂ , -COR, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, polyethylene glycol link -(CH ₂ CH ₂ O) _n -R ^c and optionally substituted 3-18-membered heterocyclic ring having from 1 to 6 heteroatoms independently selected from O, S, N and P;

R ^c represents -H or optionally substituted linear or branched alkyl having from 1 to 4 carbon atoms;

n is an integer from 1 to 24;

X ₁ ' is selected from -H, an amino protecting group, an optionally substituted linear, branched or cyclic alkyl, an alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(CH ₂ CH ₂ O) _n -R ^c , an optionally substituted aryl , having from 6 to 18 carbon atoms, an optionally substituted 5-18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, and an optionally substituted 3-18-membered heterocyclic ring containing from 1 to 6 heteroatoms independently selected from O, S, N and P;

Y ₁ ' is selected from -H, oxo, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, optionally substituted 6-18 membered aryl, optionally substituted 5-18 membered heteroaryl ring, containing one or more heteroatoms independently selected from nitrogen, oxygen and sulfur, optionally substituted 3-18-membered heterocyclic ring having from 1 to 6 heteroatoms;

R ₁ , R ₂ , R ₃ , R ₄ , R ₁ ', R ₂ ', R ₃ ' and R ₄ ' are each independently selected from the group consisting of -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl , having from 1 to 10 carbon atoms, a polyethylene glycol link - (CH ₂ CH ₂ O) _n -R ^c , halogen, guanidinium [-NH (C \u003d NH) NH ₂ ], -OR, -NR'R'', - NO ₂ , -NCO, -NR'COR'', -SR, -SOR', -SO ₂ R', -SO ₃ ^- H, -OSO ₃ H, -SO ₂ NR'R'', cyano, azido, -COR', -OCOR' and -OCONR'R'';

R ₆ is -H, -R, -OR, -SR, -NR'R'', -NO ₂ or halo;

G is -CH- or N-;

A and A' are the same or different and are independently selected from -O-, oxo(-C(=O)-), -CRR'O-, -CRR'-, -S-, -CRR'S-, -NR ₅ and -CRR'N(R ₅ )-; and

R ₅ at each occurrence is independently -H or optionally substituted linear or branched alkyl having from 1 to 10 carbon atoms.

42. The method according to claim 41, where D is represented by the following formula:

or a pharmaceutically acceptable salt thereof.

43. The method according to p. 41 or 42, where one of L', L'' and L''' is represented by formula (A') or (D'), and the others are -H, linear or branched alkyl having from 1 to 6 carbon atoms, halogen, -OH, (C ₁ -C ₆ )alkoxy or -NO ₂ .

44. The method according to p. 43, where L' is represented by formula (A'); a L'' and L''' are both -H.

45. The method according to p. 43, where L' is represented by formula (D'); a L'' and L''' are both -H.

46. The method according to any one of paragraphs. 41-45, where R _x1 is a linear, branched or cyclic alkyl having from 1 to 6 carbon atoms, optionally substituted with halogen, -OH, -SO ₃ H, (C ₁ -C ₃ ) alkyl, (C ₁ -C ₃ ) alkoxy, halogen (C ₁ -C ₃ ) alkyl, or a charged substituent or an ionizable group Q.

47. The method according to any one of paragraphs. 43-46, where L' is represented by the following formula:

or

where:

each of R _a and R _b at each occurrence independently represents -H, (C ₁ -C ₃ )alkyl or a charged substituent or an ionizable group Q;

s is an integer from 1 to 6;

s1' is 0 or an integer from 1 to 6; and

Cy is cyclic alkyl with 5 or 6 ring carbon atoms, optionally substituted with halogen, -OH, (C ₁ -C ₃ )alkyl, (C ₁ -C ₃ )alkoxy, or halo(C ₁ -C ₃ )alkyl.

48. The method according to p. 47, where R _a and R _b both represent H; Cy in formulas (B2') and (C2') is cyclohexane; and R ₅ is H or Me.

49. The method according to claim 47 or 48, where s1' is 0 or 1.

50. The method according to any one of paragraphs. 41-49, where the charged substituent or ionizable group Q is i) -SO ₃ H, -Z'-SO ₃ H, -OPO ₃ H ₂ , -Z'-OPO ₃ H ₂ , -PO ₃ H ₂ , - Z'-PO ₃ H ₂ , -CO ₂ H, -Z'-CO ₂ H, -NR ₁₁ R ₁₂ or -Z'-NR ₁₁ R ₁₂ or a pharmaceutically acceptable salt thereof; or ii) -N ⁺ R ₁₄ R ₁₅ R ₁₆ X ^- or -Z'-N ⁺ R ₁₄ R ₁₅ R ₁₆ X ^- ; Z' is an optionally substituted alkylene, an optionally substituted cycloalkylene or an optionally substituted phenylene; each R ₁₄ -R ₁₆ is independently optionally substituted alkyl; and X ^- is a pharmaceutically acceptable anion.

51. The method of claim 50 wherein Q is -SO ₃ H or a pharmaceutically acceptable salt thereof.

52. The method according to any one of paragraphs. 41-51, where P ₁ is a peptide containing 2 to 10 amino acid residues.

53. The method according to p. 52, where P ₁ is a peptide containing from 2 to 5 amino acid residues.

54. The method according to p. 53, where P ₁ is Gly-Gly-Gly, Ala-Val, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit , Ile-Cit, Trp, Cit, Phe-Ala, Phe-N ⁹ -tosyl-Arg, Phe-N ⁹ -nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys , Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2) , Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D- Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D- Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe and Gln-Ala.

55. The method of claim 54 wherein P ₁ is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

56. The method according to any one of paragraphs. 41-55, where X ₁ ' is selected from the group consisting of -H, -OH, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, and phenyl; and Y ₁ ' is selected from the group consisting of -H, oxo, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms.

57. The method according to p. 56, where X ₁ ' represents -H, -OH, (C ₁ -C ₃ )alkyl, halo(C ₁ -C ₃ )alkyl or phenyl; and Y ₁ ' represents -H, oxo, (C ₁ -C ₃ )alkyl or halo(C ₁ -C ₃ )alkyl.

58. The method according to p. 56, where X ₁ ' represents -H, -OH or -Me; and Y ₁ ' represents -H or oxo.

59. The method according to p. 56, where X ₁ 'represents-H; and Y ₁ ' is -H.

60. The method according to any one of paragraphs. 41-59, where A and A' are the same or different and are selected from -O- and -S-.

61. The method of claim 60 wherein A and A' are -O-.

62. The method according to any one of paragraphs. 41-61, where R ₆ is -OMe.

63. The method according to any one of paragraphs. 41-62, where R ₁ , R ₂ , R ₃ , R ₄ , R ₁ ', R ₂ ', R ₃ ' and R ₄ ' independently represent -H, halogen, -NO ₂ , -OH, (C ₁ -C ₃ )alkyl, halo(C ₁ -C ₃ )alkyl or (C ₁ -C ₃ )alkoxy.

64. The method according to p. 63, where R ₁ , R ₂ , R ₃ , R ₄ , R ₁ ', R ₂ ', R ₃ ' and R ₄ ' are all -H.

65. The method according to any one of paragraphs. 41-64, where each of R, R', R'' and R ₅ independently represents -H or (C ₁ -C ₃ )alkyl.

66. The method according to any one of paragraphs. 41-55, where:

R ₁ , R ₂ , R ₃ , R ₄ , R ₁ ', R ₂ ', R ₃ ' and R ₄ ' are all -H;

R ₆ is -OMe;

X ₁ ' and Y ₁ ' are both -H; and

A and A' are -O-.

67. The method according to claim 41, where D is represented by the following structural formula:

or

or a pharmaceutically acceptable salt thereof.

68. The method according to any one of paragraphs. 1-67, where -L- is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, where:

s1 is a site covalently linked to D; s2 is a site covalently linked to the maleimide group;

R ₂₃ and R ₂₄ are each independently H or optionally substituted alkyl;

m' is an integer from 0 to 10; and

R ^h' is H or optionally substituted alkyl.

69. The method according to p. 68, where R ₂₃ and R ₂₄ both represent H; and m' is an integer from 1 to 6.

70. The method according to p. 68 or 69, where R ^h' represents H.

71. The method according to claim 68, where L is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof.

72. The method according to any one of paragraphs. 1-40, where the imine-containing cytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof, and the modified cytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof.

73. The method according to p. 72, where the modified cytotoxic agent is represented by the following formula:

or their sodium or potassium salt.

74. The method according to p. 73, where the modified cytotoxic agent is represented by the following formula:

75. The method according to any one of paragraphs. 1-40, where D is represented by the following formula:

or

or a pharmaceutically acceptable salt thereof, where:

X' is selected from the group consisting of -H, -OH, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, phenyl and an amino protecting group;

Y' is selected from the group consisting of -H, oxo, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;

A and A' are selected from -O- and -S-;

W' is absent or selected from -O-, -N(R ^e )-, -N(R ^e )-C(=O)-, -N(C(=O)R ^e )-, -S- or - CH ₂ -S-, -CH ₂ NR ^e -;

R ^x is absent or selected from linear, branched or cyclic alkyl having from 1 to 10 carbon atoms;

n is an integer from 1 to 24;

G is selected from -CH- or -N-;

R ₆ is -H, -R, -OR, -SR, -NR'R'', -NO ₂ or halo; and

R represents -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, or a PEG group -(CH ₂ CH ₂ O) _n -R ^c , where n is an integer from 1 to 24, and R ^c represents a linear or branched alkyl with 1-4 carbon atoms;

R' and R' are each independently selected from -H, -OH, -OR, -NRR ^g' , -COR, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, optionally substituted aryl , having from 6 to 18 carbon atoms, optionally substituted 3-18-membered heterocyclic ring, having from 1 to 6 heteroatoms selected from O, S, N and P, PEG group -(CH ₂ CH ₂ O) _n -R ^c , a R ^g' represents -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, or a PEG group -(CH ₂ CH ₂ O) _n -R ^c .

76. The method according to claim 75, where D is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof.

77. The method according to paragraph 75 or 76, where:

X' and Y' are both -H;

A and A' are both -O-;

R ₆ is -OMe;

W' represents -N(R ^e )- or -N(R ^e )-C(=O)-;

R ^e represents -H, linear or branched alkyl with 1-4 carbon atoms or -(CH ₂ -CH ₂ -O) _n -R ^k , where R ^k represents -H, linear or branched alkyl with 1-4 atoms carbon;

n is an integer from 2 to 6; and

R ^x is straight or branched alkyl having 1 to 6 carbon atoms.

78. The method according to p. 75, where D is represented by the following structural formula:

or

or a pharmaceutically acceptable salt thereof.

79. The method according to any one of paragraphs. 75-78, where L is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, where:

s1 is a site covalently linked to D and s2 is a site covalently linked to a maleimide group;

E represents -(CR ₁₀ R ₁₁ ) _q -, cycloalkyl or cycloalkylalkyl;

Z is absent, represents -SO ₂ NR ₉ -, -NR ₉ SO ₂ -, -C (=O) -NR ₉ -, -NR ₉ -C (=O) -, -C (=O) -O- , -O-C (= O) -, -C (= O) -NR ₉ - (CH ₂ CH ₂ O) _p -, -NR ₉ -C (= O) - (CH ₂ CH ₂ O) _p - , -(OCH ₂ CH ₂ ) _p -C(=O)NR ₉ - or -(OCH ₂ CH ₂ ) _p -NR ₉ -C(=O)-;

p is an integer from 1 to 24;

Q is H, a charged substituent, or an ionizable group;

R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ are each independently H or optionally substituted alkyl; and

q and r are each independently an integer from 0 to 10.

80. The method according to p. 79, where E represents -(CR ₁₀ R ₁₁ ) _q -.

81. The method according to p. 79, where E represents

82. The method according to any one of paragraphs. 79-81, where Z is -C(=O)-NR ₉ - or NR ₉ -C(=O)-.

83. The method according to p. 82, where R ₉ represents -H.

84. The method according to any one of paragraphs. 79-83, where Q is:

i) H;

ii) -SO ₃ H, -Z'-SO ₃ H, -OPO ₃ H ₂ , -Z'-OPO ₃ H ₂ , -PO ₃ H ₂ , -Z'-PO ₃ H ₂ , -CO ₂ H, -Z'-CO ₂ H, -NR ₁₁ R ₁₂ or -Z'-NR ₁₄ R ₁₅ , or a pharmaceutically acceptable salt thereof; or

iii) -N ⁺ R ₁₄ R ₁₅ R ₁₆ X ^- or -Z'-N ⁺ R ₁₄ R ₁₅ R ₁₆ X ^- ;

Z' is an optionally substituted alkylene, an optionally substituted cycloalkylene or an optionally substituted phenylene;

R ₁₄ , R ₁₅ and R ₁₆ are each independently optionally substituted alkyl; and

X ^- is a pharmaceutically acceptable anion.

85. The method of claim 84 wherein Q is H or -SO ₃ H or a pharmaceutically acceptable salt thereof.

86. The method according to any one of paragraphs. 78-85, where R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ are all H; and q and r are each independently an integer from 1 to 6.

87. The method according to item 79, where:

each R ₁₂ and R ₁₃ at each occurrence independently represents H or (C ₁ -C ₃ )alkyl;

Q is H or -SO ₃ H or a pharmaceutically acceptable salt thereof;

Z is -C(=O)-NR ₉ - or -NR ₉ -C(=O)-;

R ₉ is H or (C ₁ -C ₃ )alkyl;

E represents -(CR ₁₀ R ₁₁ ) _q -.

R ₁₀ and R ₁₁ are each independently H or (C ₁ -C ₃ )alkyl; and

q and r are integers from 1 to 5.

88. The method according to p. 79, where L is any of the following structural formulas:

and

or a pharmaceutically acceptable salt thereof.

89. The method according to any one of paragraphs. 75-79, where L is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, where:

R ₁₉ , R ₂₀ , R ₂₁ and R ₂₂ are each independently H or optionally substituted alkyl;

m and n are each independently an integer from 0 to 10;

R ^h is H or optionally substituted alkyl;

P _L is an optionally substituted alkylene, -(CH ₂ -CH ₂ -O) _j - (wherein the oxygen atom is bonded to a -(C=O)-group associated with P), an amino acid residue or a peptide containing from 2 to 20 amino acid residues; and

j is an integer from 1 to 24.

90. The method according to p. 89, where R ₁₉ , R ₂₀ , R ₂₁ and R ₂₂ each represent H; am and n are each independently an integer from 1 to 6.

91. The method according to p. 89 or 90, where P _L is an amino acid residue or a peptide containing from 2 to 10 amino acid residues.

92. The method according to p. 91, where P _L is a peptide containing from 2 to 5 amino acid residues.

93. The method according to p. 92, where P _L is selected from the group consisting of: Ala-Val, Val-Ala, Val-Cit, Cit-Val, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Phe-N ⁹ -tosyl-Arg, Phe-N ⁹ -nitro-Arg, Phe-Phe-Lys, D-Phe-Phe- Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D -Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala -Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala., Ala-Met, Met-Ala, Gln-Val, Asn-Ala, Gln-Phe, Gln-Ala, Gly- Gly-Gly, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-D-Ala, Ala-Val-Cit, Ala-Val-Ala and β-Ala- Gly-Gly-Gly.

94. The method according to p. 93, where P _L represents Gly-Gly-Gly, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Val-Ala or β-Ala -Gly-Gly-Gly.

95. The method according to p. 89, where L is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof.

96. The method according to any one of paragraphs. 1-40, where the imine-containing cytotoxic agent is represented by the following formula:

or

or

or a pharmaceutically acceptable salt thereof.

97. The method according to p. 96, where the modified cytotoxic agent is represented by the following formula:

or

or its sodium or potassium salt.

98. The method according to p. 97, where the modified cytotoxic agent is represented by the following formula:

or

99. The method according to any one of paragraphs. 1-39, where D is an imine compound of pyrrolobenzodiazepine (PBB).

100. The method according to claim 99, where D is represented by the following formula:

or

or a pharmaceutically acceptable salt thereof, where:

W is selected from C=O, C=S, CH ₂ , BH, SO and SO ₂ ;

R ₆ is -H, -R, -OR, -SR, -NR'R'', -NO ₂ or halo;

A and A' are the same or different and are independently selected from -O-, oxo (-C(=O)-), -CRR'O-, -CRR'-, -S-, -CRR'S-, -NR ₅ and -CRR'N(R ₅ )-,

R ^c represents -H or a substituted or unsubstituted linear or branched alkyl having from 1 to 4 carbon atoms, or a linking group with a reactive group associated with it;

n is an integer from 1 to 24;

R ₅ at each occurrence is independently —H or optionally substituted linear or branched alkyl having from 1 to 10 carbon atoms;

D ₀ and D ₀ ' are the same or different and are independently absent or selected from the group consisting of an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, an amino acid, a peptide containing from 2 to 6 amino acids, and a polyethylene glycol link (-OCH ₂ CH ₂ ) _n -;

L is absent, is a linker, a polyethylene glycol unit (-OCH ₂ CH ₂ ) _n -, optionally substituted linear, branched or cyclic alkyl or alkenyl having from 1 to 10 carbon atoms, an optionally substituted phenyl group, an optionally substituted 3-18-membered a heterocyclic ring or a 5-18 membered heteroaryl ring having 1 to 6 heteroatoms independently selected from O, S, N and P;

R _a , R _a ', R _b and R _b ' are the same or different and independently selected from the group consisting of -H, halide or optionally substituted branched, linear or cyclic alkyl having from 1 to 10 carbon atoms; or R _a and R _a' and/or R _b and R _b' together form a double bond containing the group ═B and ═B', respectively;

═B and ═B' are the same or different and are independently selected from an optionally substituted branched or linear alkenyl or carbonyl group;

Q is Q ₁ -Ar-Q ₂ ;

Q' is Q ₁ '-Ar'-Q ₂ ;

Q ₁ and Q ₁ ' each independently absent, represents a linear, branched or cyclic alkyl with 1-6 carbon atoms or-CH=CH link;

Ar and Ar' are each independently absent or represent an aryl group;

Q ₂ and Q ₂ ' are each independently selected from -H, linker, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, polyethylene glycol unit -R ^c' -(OCH ₂ CH ₂ ) _n -R ^c or a substituent selected from halogen, guanidinium [-NH(C=NH)NH ₂ ], -R, -OR, -NR'R'', -NO ₂ , -NCO, -NR'COR'' , NR'(C=O)OR'' -SR, sulfoxide represented by -SOR', sulfone represented by -SO ₂ R', sulfonate represented by -SO ₃ M, sulfate -OSO ₃ M, sulfonamide represented by SO ₂ NR'R ''cyano, azido, -COR', -OCOR' or -OCONR'R''; and

R ^c' is absent or selected from linear, branched alkyl, alkenyl or alkynyl having 1 to 5 carbon atoms.

101. The method of claim 100, wherein D is selected from one of the following:

and

or their pharmaceutically acceptable salt, where:

one of L', L'' and L''' in the formula (PBD5), (PBD6), (PBD7) or (PBD8) is represented by the following formula:

or

one of Q ₂ and Q ₂ ' in the formula (PBD9) or (PBD10) is represented by the following formula:

or

and the other is selected from -H, -R, -OR, -NR'R'', -NO ₂ , -NR'(C=O)OR'', -SR or -NO ₂ ;

one of Z ₁ and Z ₂ is -C(=O)- and the other is -NR ₅ -;

R _a '' and R _b '' are the same or different and are selected from -H and -Me; and

n' is chosen from 0, 1, 2 and 3.

102. The method according to p. 101, where one of L', L'' and L''' in the formula (PBD5), (PBD6), (PBD7) or (PBD8) is represented by formula (A') or (D') , and the other two are -H, linear or branched alkyl having from 1 to 6 carbon atoms, halogen, -OH, (C ₁ -C ₆ )alkoxy or -NO ₂ ; or one of Q ₂ and Q ₂ ' in the formula (PBD9) or (PBD10) is represented by formula (A'), (D') or (E'); and the other is -H, linear or branched alkyl having 1 to 6 carbon atoms, halogen, -OH, (C ₁ -C ₆ )alkoxy or -NO ₂ .

103. The method according to item 102, where:

L'' and L''' are both -H; a L' in the formula (PBD5), (PBD6), (PBD7) or (PBD8) is represented by the following formula:

or

where:

s is an integer from 1 to 6;

s1' is 0 or an integer from 1 to 6; and

104. The method according to p. 103, where R _a and R _b both represent H; Cy in formulas (B2') and (C2') is cyclohexane; R ₅ is H or Me; and s1' is 0 or 1.

105. The method according to any one of paragraphs. 101-104, where Q is -SO ₃ H or a pharmaceutically acceptable salt thereof.

106. The method according to any one of paragraphs. 101-105, where P ₁ is a peptide containing 2 to 5 amino acid residues.

107. The method according to p. 106, where P ₁ is Gly-Gly-Gly, Ala-Val, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit , Ile-Cit, Trp, Cit, Phe-Ala, Phe-N ⁹ -tosyl-Arg, Phe-N ⁹ -nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys , Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2) , Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D- Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D- Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, Gln-Val, Asn-Ala, Gln-Phe and Gln-Ala.

108. The method according to p. 107, where P ₁ denotes Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala or D-Ala-D-Ala.

109. The method according to any one of paragraphs. 100-108, where:

R ₆ is -OMe;

X ₁ ' and Y ₁ ' are both -H; and

A and A' are -O-.

110. The method according to p. 100, where D is represented by the following structural formula:

or

or a pharmaceutically acceptable salt thereof.

111. The method according to any one of paragraphs. 100-110, where -L- is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, where:

R ₂₃ and R ₂₄ are each independently H or optionally substituted alkyl;

m' is an integer from 0 to 10; and

R ^h' is H or optionally substituted alkyl.

112. The method according to p. 111, where R ₂₃ and R ₂₄ both represent H; and m' is an integer from 1 to 6.

113. The method according to p. 111 or 112, where R ^h' represents H.

114. The method according to p. 113, where L is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof.

115. The method according to any one of paragraphs. 100-114, where the imine-containing cytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof.

116. The method according to item 100, where D is represented by the following formula:

and

or a pharmaceutically acceptable salt thereof, where:

X ₁ ' is selected from the group consisting of -H, -OH, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, phenyl and an amine protecting group;

Y ₁ ' is selected from the group consisting of -H, oxo, substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;

one of Q ₂ and Q ₂ ' in the formula (PBD15) or (PBD16) is -W'-R ^x -S-; and the other is selected from -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(CH ₂ CH ₂ O) _n -R ^c , halogen, guanidinium [-NH( C=NH)NH ₂ ], -OR, -NR'R'', -NO ₂ , -NR'COR'', -SR, -SOR', -SO ₂ R', -SO ₃ H, -OSO ₃ H, -SO ₂ NR'R'', cyano, azido, -COR', -OCOR' and -OCONR'R'';

n is an integer from 1 to 24;

R ₆ is -H, -R, -OR, -SR, -NR'R'', -NO ₂ or halo; and

117. The method according to p. 116, where

X ₁ ' and Y ₁ ' are both -H;

R ₆ is -OMe;

W' represents -N(R ^e )- or -N(R ^e )-C(=O)-;

n is an integer from 2 to 6; and

R ^x is straight or branched alkyl having 1 to 6 carbon atoms.

118. The method according to claim 116 or 117, where L is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, where:

E represents -(CR ₁₀ R ₁₁ ) _q -, cycloalkyl or cycloalkylalkyl;

p is an integer from 1 to 24;

Q is H, a charged substituent, or an ionizable group;

q and r are each independently an integer from 0 to 10;

119. The method according to p. 118, where E represents -(CR ₁₀ R ₁₁ ) _q -.

120. The method of claim 118 or 119, wherein Z is -C(=O)-NR ₉ - or -NR ₉ -C(=O)-.

121. The method according to p. 120, where R ₉ represents -H.

122. The method according to any one of paragraphs. 118-121, where Q is H or -SO ₃ H or a pharmaceutically acceptable salt thereof.

123. The method according to any one of paragraphs. 118-122, where R ₉ , R ₁₀ , R ₁₁ , R ₁₂ and R ₁₃ are all H; and q and r are each independently an integer from 1 to 6.

124. The method according to clause 118, where:

Q is H or -SO ₃ H or a pharmaceutically acceptable salt thereof;

Z is -C(=O)-NR ₉ - or -NR ₉ -C(=O)-;

R ₉ is H or (C ₁ -C ₃ )alkyl;

E represents -(CR ₁₀ R ₁₁ ) _q -.

R ₁₀ and R ₁₁ are each independently H or (C ₁ -C ₃ )alkyl; and

q and r are integers from 1 to 5.

125. The method according to p. 118, where L is represented by any of the following structural formulas:

and

or a pharmaceutically acceptable salt thereof.

126. The method according to p. 116 or 117, where L is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, where:

m and n are each independently an integer from 0 to 10;

R ^h is H or optionally substituted alkyl;

j is an integer from 1 to 24.

127. The method according to p. 126, where R ₁₉ , R ₂₀ , R ₂₁ and R ₂₂ each represent H; am and n are each independently an integer from 1 to 6.

128. The method according to p. 126 or 127, where P _L is a peptide containing from 2 to 5 amino acid residues.

129. The method according to p. 128, where P _L is selected from the group consisting of: Ala-Val, Val-Ala, Val-Cit, Cit-Val, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Phe-N ⁹ -tosyl-Arg, Phe-N ⁹ -nitro-Arg, Phe-Phe-Lys, D-Phe-Phe- Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2), Gly-Phe-Leu-Gly (SEQ ID NO: 3), Val-Arg, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D -Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala -Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala., Ala-Met, Met-Ala, Gln-Val, Asn-Ala, Gln-Phe, Gln-Ala, Gly- Gly-Gly, Ala-Ala-Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-D-Ala, Ala-Val-Cit, Ala-Val-Ala and β-Ala- Gly-Gly-Gly.

130. The method according to p. 126, where L is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof.

131. The method according to any one of paragraphs. 1-39, where the imine-containing cytotoxic agent is represented by the following formula:

or

or a pharmaceutically acceptable salt thereof, where:

dotted lines indicate the optional presence of a double bond;

R ^3'' represents a C _3-12 alkylene group,

each X' is at each occurrence independently -O-, -S- or -N(H)-;

each R ² is independently selected from -H, -OH, -CN, -R ^1' , -OR ^1' , -O-SO ₂ -R ¹ ', -CO ₂ R ^1' , -COR ^1' or halogen, or both R ² taken together are ═O, ═CH ₂ , ═CH-R ^a or ═C(R ^a ) ₂ ;

each R ^2' is independently selected from -H, -OH, -CN, -R ^1' , -OR ^1' , -O-SO ₂ -R ^1' , -CO ₂ R ^1' , -COR ^1' or halogen;

R ^4b is a leaving group selected from -OR ^6' , -OCOR ^4' , -OCOOR ^4' , -OCONR ^4' R ^5' , -NR ^4' R ^5' , -NR ^4' COR ^5' , -NR ^4' NR ^4' R ⁵ , an optionally substituted 5- or 6-membered nitrogen-containing heterocycle (e.g. piperidine, tetrahydropyrrole, pyrazole, morpholine), guanidinium represented by -NR ^4' (C=NH)NR ^4' R ^5' , amino acids or a peptide represented by -NR ^6' COP', where P' is an amino acid or a polypeptide containing from 2 to 20 amino acid units, -SR ^6' , -SOR ^4' , -SO ₂ M, -SO ₃ M, -OSO ₃ M, halo, cyano and azido;

^RL is a linker carrying a maleimide moiety that can form a covalent bond with a cell-binding agent (CCA);

R ⁶ and R ⁹ are independently selected from -H, -R ^1' , -OH, -OR ^1' , -SH, -SR ^1' , -NH ₂ , -NHR ^1' , -NR ^1' R ^3' , - NO ₂ , Me ₃ Sn and halogen; and

R ^1' and R ^3' are each independently selected from optionally substituted C _1-12 alkyl, C _3-20 heterocyclyl, or C _5-20 aryl groups, and optionally in connection with the group -NR ^1' R ^3' , R ^1' and R ^3' together with nitrogen, the atom to which they are attached forms an optionally substituted 4-, 5-, 6-, or 7-membered heterocyclic ring; or any pair of adjacent groups from R ⁶ to R ⁹ together form a group -O-(CH ₂ ) _p -O-, where p is 1 or 2;

R ^4' and R ^5' are each independently selected from -H, -OH, -OR ^6' , -NHR ^6' , -NR ^6' ₂ , -COR ⁶ , optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl, having 1 to 10 carbon atoms, a polyethylene glycol unit -(CH ₂ CH ₂ O) _n -R ^b or an optionally substituted 3-18 membered heterocyclic ring having 1 to 6 heteroatoms independently selected from O, S, N or P ;

R ^6' at each occurrence is independently selected from the group consisting of -H, optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, -(CH ₂ CH ₂ O) _n -R ^b , an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5-18 membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen or sulfur, and an optionally substituted 3-18 membered heterocyclic ring containing from 1 to 6 heteroatoms independently selected from O, S, N, or P;

R ^a is independently selected from -R ^1' , -CO ₂ R ^1' , -COR ^1' , -CHO, -CO ₂ H or halogen;

R ^b is -H or substituted or unsubstituted linear or branched alkyl having from 1 to 4 carbon atoms;

M is H or a pharmaceutically acceptable cation; and

n is an integer from 1 to 24.

132. The method according to p. 131, where the imine-containing cytotoxic agent is represented by the following formula:

or

or a pharmaceutically acceptable salt thereof, wherein R ^3'' is C _3-5 alkylene.

133. The method according to p. 131 or 132, where R ^L represents

where L ¹ is a cleavable linker, A is a connecting group bearing a maleimide capable of binding L ¹ to a cell-binding agent, L ² is a covalent bond or together with -OC(=O)- forms a self-cleaving linker.

134. The method according to p. 133, where L ¹ is a peptide containing from 2 to 5 amino acid residues.

135. The method according to p. 134, where L ¹ is selected from the group consisting of Phe-Lys, Val-Ala, Val-Lys, Ala-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Phe -Arg, Trp-Cit, Lys-Lys, Phe-Ala, Phe-N9-tosyl-Arg, Phe-N9-nitro-Arg, Val-Arg, Arg-Val, Arg-Arg, Val-D-Cit, Val -D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D -Arg, D-Arg-D-Arg, Gly-Gly-Gly, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val -Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 1), β-Ala-Leu-Ala-Leu (SEQ ID NO: 2) and Gly-Phe-Leu-Gly (SEQ ID NO: 3 ).

136. The method according to any one of paragraphs. 131-135, where -C(=O)O- and L ² together form a group:

where the asterisk indicates the point of attachment to position N10, the wavy line indicates the point of attachment to the L ¹ linker, Y' denotes -NH-, -O-, -C (=O)NH- or -C (=O)O-, and n is between 0 and 3.

137. The method according to p. 136, where -C (= O) O - and L ² together form a group:

138. The method according to p. 136, where L ¹ and L ² together with -OC (=O) constitute a group selected from:

or

where the asterisk indicates the point of attachment to position N10, and the wavy line indicates the point of attachment to the remainder of the linker L ¹ or the point of attachment to A.

139. The method according to any one of paragraphs. 131-138, where A is represented by the following structural formula:

(i)

, where the asterisk denotes the point of attachment to L ¹ and p is from 1 to 6;

(ii)

, where the asterisk indicates the attachment point to L ¹ , r is 0 or 1, as is 0 to 30.

140. The method according to p. 139, where p is from 4 to 6; r is 1; and s is between 1 and 10.

141. The method according to p. 131, where the imine-containing cytotoxic agent is represented by the following formula:

or a pharmaceutically acceptable salt thereof, where s is from 2 to 8.

142. The method of claim 141, where s is 7.

143. The method according to any one of paragraphs. 1-142, where the cell-binding agent is an antibody or antibody fragment.

144. The method according to any one of paragraphs. 1-142, where the cell-binding agent is an antibody having an engineered cysteine residue.

145. The method of claim 144, wherein the engineered cysteine residue is at position 442 according to the EU/OU numbering of one or both of the antibody heavy chains.

146. The method of claim 145, wherein the engineered cysteine residue is in both heavy chains.

147. The method of claim 145, wherein the engineered cysteine residue is in one of the heavy chains.

148. The method according to any one of paragraphs. 143-147, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a surface altered antibody, or a human antibody.

149. The method of claim 148, wherein the antibody is an anti-CD123 antibody.

150. The method of claim 149, wherein the anti-CD123 antibody comprises an immunoglobulin heavy chain region having the amino acid sequence of SEQ ID NO: 25 and an immunoglobulin light chain region having the amino acid sequence of SEQ ID NO: 26.

151. The method according to any one of paragraphs. 1-39, characterized in that it is intended to obtain a cell-binding agent-cytotoxic agent conjugate represented by the following formula:

or a pharmaceutically acceptable salt thereof, the method comprising the steps of:

(a) the interaction of the imine fragment in the imine-containing cytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof, with sulfur dioxide, a bisulfite salt or a metabisulfite salt in an aqueous solution at a pH of 3.1 to 3.5 to form a modified cytotoxic agent represented by the following formula:

or a pharmaceutically acceptable salt thereof; and

(b) reacting the modified cytotoxic agent, or a pharmaceutically acceptable salt thereof, with a cell-binding agent Ab to form a cell-binding agent-cytotoxic agent conjugate, wherein:

Ab is an anti-CD123 antibody comprising an immunoglobulin heavy chain region having the amino acid sequence of SEQ ID NO: 25 and an immunoglobulin light chain region having the amino acid sequence of SEQ ID NO: 26; and

w is 1 or 2.

152. The method according to p. 151, where the reaction in stage (a) is carried out at a pH of from 3.2 to 3.4.

153. The method according to p. 151, where the reaction in stage (a) is carried out at pH 3.3.

154. The method according to any one of paragraphs. 151-153, where the reaction in step (a) is carried out in the presence of a buffer solution.

155. The method of claim 154, wherein the buffer solution is a citrate buffer, an acetate buffer, a succinate buffer, or a phosphate buffer.

156. The method of claim 155, wherein the buffer is a succinate buffer.

157. The method according to any one of paragraphs. 151-156, wherein 1.1 to 1.6 equivalents of the bisulfite salt or 0.55 to 0.8 equivalents of the metabisulfite salt are reacted with 1 equivalent of an imine containing cytotoxic agent.

158. The method according to p. 157, where from 1.3 to 1.5 equivalents of a bisulfite salt or from 0.65 to 0.75 equivalents of a metabisulfite salt are reacted with 1 equivalent of an imine-containing cytotoxic agent.

159. The method of claim 157, wherein 1.4 equivalents of the bisulfite salt or 0.7 equivalents of the metabisulfite salt are reacted with 1 equivalent of an imine-containing cytotoxic agent.

160. The method according to any one of paragraphs. 151-159, wherein the reaction of step (a) is carried out at a pH of 3.2 to 3.4 and 1.3 to 1.5 equivalents of sodium bisulfite salt is reacted with 1 equivalent of an imine-containing cytotoxic agent.

161. The method according to any one of paragraphs. 151-160, where the reaction of step (a) is carried out in a mixture of dimethylacetamide (DMA) and water.

162. The method according to any one of paragraphs. 151-161, where the reaction in step (a) is carried out at room temperature.

163. The method according to any one of paragraphs. 151-162, where the modified cytotoxic agent is not purified prior to reaction with the cell-binding agent in step (b).

164. The method according to any one of paragraphs. 151-163, where the reaction in step (b) is carried out at a pH of 5.5 to 6.5.

165. The method according to any one of paragraphs. 151-164, where the conjugate is purified by tangential flow filtration to obtain a purified conjugate.

166. The method of claim 165 wherein the purified conjugate is prepared in formulation buffer containing 40 to 80 µM sodium bisulfite having a pH of 4 to 5.

167. The method according to p. 166, where the buffer for the composition contains 50 μm sodium bisulfite having a pH of 4.2.