Hydrazinyl-pyrrolo compounds and methods for producing a conjugate

1. A method of producing a polypeptide conjugate, the method comprising:

(a) combining in a reaction mixture:

(i) a compound of formula (V):

wherein

one of Q 3 and Q 4 is —(CH 2 ) m NR 3 NHR 2 and the other is Y 4 ;

m is 0 or 1;

R 2 and R 3 are each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R 2 and R 3 are optionally cyclically linked to form a 5 or 6-membered heterocyclyl;

X 1 , X 2 , X 3 and X 4 are each C;

Y 1 , Y 2 , Y 3 and Y 4 are each independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

one of R 16 , Y 1 , Y 2 , Y 3 or Q 4 is -L-W 1 , wherein if Q 4 is -L-W 1 , then Q 3 is —(CH 2 ) m NR 3 NHR 2 and Y 4 is absent; and wherein if one of Y 1 , Y 2 , Y 3 or Q 4 is -L-W 1 , then R 16 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

L is a linker of the formula -(T 1 -V 1 ) a -(T 2 -V 2 ) b -(T 3 -V 3 ) c -(T 4 -V 4 ) d -(T 5 -V 5 ) e -, wherein a, b and c are each 1, and d and e are each independently 0 or 1, where the sum of a, b, c, d and e is 3 to 5;

T 1 , T 2 , and T 3 are each independently selected from (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, (EDA) w , (PEG) n , (AA) p , —(CR 13 OH) h —, piperidin-4-amino (P4A), para-amino-benzyloxycarbonyl (PABC), a meta-amino-benzyloxycarbonyl (MABC), a para-amino-benzyloxy (PABO), a meta-amino-benzyloxy (MABO), para-aminobenzyl, an acetal group, a disulfide, a hydrazine, a carbohydrate, a beta-lactam, an ester, (AA) p -MABC-(AA) p , (AA) p ,-MABO-(AA) p , (AA) p -PABO-(AA) p and (AA) p -PABC-(AA) p ;

T 4 is selected from (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, (EDA) w , (PEG) n , (AA) p , —(CR 13 OH) h —, piperidin-4-amino (P4A), para-amino-benzyloxycarbonyl (PABC), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), meta-amino-benzyloxy (MABO), para-aminobenzyl, an acetal group, a disulfide, a hydrazine, a carbohydrate, a beta-lactam, an ester, (AA) p -MABC-(AA) p , (AA) p ,-MABO-(AA) p , (AA) p ,-PABO-(AA) p , (AA) p ,-PABC-(AA) p , PABC-(AA) p , (AA) p -PABO, and MABC-(AA) p ;

T 5 is selected from (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, (EDA) w , (PEG) n , (AA) p , —(CR 13 OH) h —, piperidin-4-amino (P4A), para-amino-benzyloxycarbonyl (PABC), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), meta-amino-benzyloxy (MABO), para-aminobenzyl, an acetal group, a disulfide, a hydrazine, a carbohydrate, a beta-lactam, an ester, (AA) p -MABC-(AA) p , (AA) p ,-MABO-(AA) p , (AA) p ,-PABO-(AA) p , (AA) p ,-PABC-(AA) p , and PABC-(AA) p ;

wherein:

EDA is an ethylene diamine moiety, PEG is a polyethylene glycol or a modified polyethylene glycol, and AA is an amino acid residue;

w is an integer from 1 to 20;

n is an integer from 1 to 30;

p is an integer from 1 to 20;

h is an integer from 1 to 12;

V 1 and V 2 are each independently selected from the group consisting of —CO—, —NR 11 —, —CONR 11 —, —NR 11 CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO 2 —, —SO 2 NR 11 —, —NR 11 SO 2 - and —P(O)OH—;

V 3 , V 4 and V 5 are each independently selected from the group consisting of a covalent bond, —CO—, —NR 11 —, —CONR 11 —, —NR 11 CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO 2 —, —SO 2 NR 11 —, —NR 11 SO 2 — and —P(O)OH—;

each R 11 and R 13 are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

W 1 is selected from a polypeptide and a drug, and

(ii) a second compound comprising a reactive aldehyde group or a reactive ketone group,

wherein the combining is under reaction conditions suitable to promote reaction between the compound and the reactive aldehyde group or reactive ketone group of the second compound to form the conjugate; and

(b) isolating the conjugate from the reaction mixture.

2. The method of claim 1 , wherein W 1 is a polypeptide.

3. The method of claim 1 , wherein the compound is a compound of formula (VI):

wherein

Q 3 , Q 4 , X 1 , X 2 , X 3 , X 4 , L, W 1 , Y 1 , Y 2 , and Y 3 are as defined in formula (V).

4. The method of claim 1 , wherein:

T 1 is selected from a (C 1 -C 12 )alkyl and a substituted (C 1 -C 12 )alkyl;

T 2 , T 3 , T 4 and T 5 are each independently selected from (EDA) w , (PEG) n , (C 1 -C 12 )alkyl, substituted (C 1 -C 12 )alkyl, (AA) p , —(CR 13 OH) h —, piperidin-4-amino, MABC, MABO, PABO, PABC, an acetal group, a disulfide, a hydrazine, a carbohydrate, a beta-lactam, an ester, (AA) p -MABC-(AA) p , (AA) p ,-MABO-(AA) p , (AA) p ,-PABO-(AA), and (AA) p ,-PABC-(AA) p ; and

V 1 and V 2 are each independently selected from the group consisting of —CO—, —NR 11 —, —CONR 11 —, —NR 11 CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO 2 —, —SO 2 NR 11 —, —NR 11 SO 2 — and —P(O)OH—;

V 3 , V 4 and V 5 are each independently selected from the group consisting of: a covalent bond, —CO—, —NR 1 —, —CONR 11 —, —NR 11 CO—, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO 2 —, —SO 2 NR 11 —, —NR 11 SO 2 —, and —P(O)OH—;

wherein:

(PEG) n is

where n is an integer from 1 to 30;

EDA is an ethylene diamine moiety having the following structure:

where q is an integer from 1 to 6 and r is 0 or 1;

piperidin-4-amino is

each R 11 and R 12 is independently selected from hydrogen, an alkyl, a substituted alkyl, a polyethylene glycol moiety, an aryl and a substituted aryl, wherein any two adjacent R 12 groups may be cyclically linked to form a piperazinyl ring; and

R 13 is selected from hydrogen, an alkyl, a substituted alkyl, an aryl, and a substituted aryl.

5. The method of claim 1 , wherein:

a, b, c, d and e are each 1; or

a, b, c and d are each 1, and e is 0; or

a, b and c are each 1, and d and e are each 0.

6. The method of claim 1 , wherein T 1 , T 2 , T 3 and T 4 and V 1 , V 2 , V 3 and V 4 are selected from the following table:

T 1

V 1

T 2

V 2

T 3

V 3

T 4

V 4

T 5

V 5

(C 1 -C 12 )alkyl

—CONR 11 —

(PEG) n

—CO—

—

—

—

—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(PEG) n

—CO—

—

—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—

—

—

—

—

—

—

(C 1 -C 12 )alkyl

—CONR 11 —

(PEG) n

—NR 11 —

—

—

—

—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(PEG) n

—NR 11 —

—

—

—

—

(C 1 -C 12 )alkyl

—CO—

(EDA) w

—CO—

—

—

—

—

—

—

(C 1 -C 12 )alkyl

—CONR 11 —

(C 1 -C 12 )alkyl

—NR 11 —

—

—

—

—

—

—

(C 1 -C 12 )alkyl

—CONR 11 —

(PEG) n

—CO—

(EDA) w

—

—

—

—

—

(C 1 -C 12 )alkyl

—CO—

(EDA) w

—

—

—

—

—

—

—

(C 1 -C 12 )alkyl

—CO—

(EDA) w

—CO—

(CR 13 OH) h

—CONR 11 —

(C 1 -C 12 )alkyl

—CO—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(C 1 -C 12 )alkyl

—CO—

—

—

—

—

(C 1 -C 12 )alkyl

—CONR 11 —

(PEG) n

—CO—

(AA) p

—

—

—

—

—

(C 1 -C 12 )alkyl

—CONR 11 —

(PEG) n

—CO—

(AA) p

—

MABO

—

—

—

(C 1 -C 12 )alkyl

—CONR 11 —

(PEG) n

—CO—

(AA) p

—

PABO

—

—

—

(C 1 -C 12 )alkyl

—CONR 11 —

(PEG) n

—CO—

(AA) p

—

PABC

—

—

—

(C 1 -C 12 )alkyl

—CO—

(EDA) w

—CO—

(CR 13 OH) h

—CO—

(AA) p

—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(C 1 -C 12 )alkyl

—CO—

(AA) p

—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(PEG) n

—CO—

(AA) p

—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(C 1 -C 12 )alkyl

—CO—

(AA) p

—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(PEG) n

—CO—

(AA) p -

—

—

—

PABC-

(AA) p

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(PEG) n

—CO—

(AA) p -

—

PABC-

—

(AA) p

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(PEG) n

—CO—

(AA) p -

—

—

—

PABO

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(PEG) n

—CO—

(AA) p

—

PABO

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(PEG) n

—SO 2 —

(AA) p

—

—

—

(C 1 -C 12 )alkyl

—CONR 11 —

(PEG) n

—CO—

(AA) p

PABC-

—

—

—

(AA) p

(C 1 -C 12 )alkyl

—CONR 11 —

(PEG) n

—CO—

(AA) p

—

PABC

—

(AA) p

—

(C 1 -C 12 )alkyl

—CO—

(EDA) w

—CO—

(CR 13 OH) h

—CONR 11 —

(PEG) n

—CO—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(PEG) n

—CO—

MABC-

—

—

—

(AA) p -

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(PEG) n

—CO—

MABC

—

(AA) p

—

(C 1 -C 12 )alkyl

—CONR 11 —

(PEG) n

—CO—

MABO

—

—

—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(PEG) n

—CO—

MABO

—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(PEG) n

—CO—

PABO

—

—

—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—NR 11 —

(PEG) n

—CO—

PABC

—

—

—

(C 1 -C 12 )alkyl

—CONR 11 —

(PEG) n

—CO—

MABC

—

(AA) p

—

—

—

(C 1 -C 12 )alkyl

—CO—

(CR 13 OH) h

—CO—

—

—

—

—

—

—

(C 1 -C 12 )alkyl

—CONR 11 —

substituted

—NR 11 —

(PEG) n

—CO—

—

—

—

—

(C 1 -C 12 )alkyl

(C 1 -C 12 )alkyl

—SO 2 —

(C 1 -C 12 )alkyl

—CO—

—

—

—

—

—

—

(C 1 -C 12 )alkyl

—CONR 11 —

(PEG) n

—CO—

(AA) p

—

PABC

—NR 11 —

—

—

(C 1 -C 12 )alkyl

—CONR 11 —

(C 1 -C 12 )alkyl

—

(CR 13 OH) h

—CONR 11 —

—

—

—

—

(C 1 -C 12 )alkyl

—CO—

P4A

—CO—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—

PABO

—CO—

(C 1 -C 12 )alkyl

—CO—

P4A

—CO—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—

PABO

—

(C 1 -C 12 )alkyl

—CO—

P4A

—CO—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—

PABC-

—

(AA) p

(C 1 -C 12 )alkyl

—CO—

P4A

—CO—

(C 1 -C 12 )alkyl

—CO—

(AA) p

—

—

—.

7. The method of claim 1 , wherein L is selected from the group consisting of the following structures:

wherein:

each f is independently 0 or an integer from 1 to 12;

each w is independently 0 or an integer from 1 to 20;

each n is independently 0 or an integer from 1 to 30;

each p is independently 0 or an integer from 1 to 20;

each h is independently 0 or an integer from 1 to 12;

each R is independently hydrogen, alkyl, substituted alkyl, a polyethylene glycol moiety, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; and

each R′ is independently H, a sidechain group of an amino acid, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acyl amino, amino acyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

8. The method of claim 1 , wherein Q 3 is —(CH 2 ) m NR 3 NHR 2 and Q 4 is Y 4 .

9. The method of claim 1 , wherein m is 1.

10. The method of claim 1 , wherein R 2 and R 3 are each independently selected from alkyl and substituted alkyl.

11. The method of claim 1 , wherein Y 1 , Y 2 , Y 3 and Y 4 are each H.

12. The method of claim 1 , wherein the compound is a compound of formula (VIII):

13. The method of claim 1 , wherein the compound is a compound of formula (IX):

14. The method of claim 1 , wherein W 1 is a drug.

15. The method of claim 14 , wherein the drug comprises a maytansinoid.

16. The method of claim 15 , wherein the maytansinoid comprises deacyl maytansine.