# US 11,896,671 B2 · Conjugation method for carrier-linked prodrugs

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- Number: US 11,896,671 B2
- Document ID: US11896671B2
- Status: Granted
- Kind: B2
- Application number: 16/317,689
- Filed: 2017-07-12
- Published: 2021-12-02
- Granted: 2024-02-13
- Earliest claimed: 2016-07-13
- Assignees: Ascendis Pharma A/S
- Applicants: Ascendis Pharma A/S
- Inventors: Nicola Bisek; Samuel Weisbrod; Kornelia Bigott
- Web page: https://patentgrep.com/patent/US11896671B2

## CPC

- A61K 47/545: {Heterocyclic compounds (A61K47/558 takes precedence)}
- A61K 47/542: {Carboxylic acids, e.g. a fatty acid or an amino acid}
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 47/61: the organic macromolecular compound being a polysaccharide or a derivative thereof
- A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65)
- C07K 17/08: the carrier being a synthetic polymer
- C07K 17/10: the carrier being a carbohydrate
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention relates to reagents comprising a substituted acyl borate or a substituted hydroxylamine, to a method of synthesizing a carrier-linked prodrug using said reagents and to carrier-linked prodrugs obtainable by said method.

## Claims

### Claim 1

A compound of formula (I)
D-(L1-L2-A)a (I),
wherein
-D is drug moiety;
each -L1- is independently a reversible prodrug linker, which is covalently conjugated to -D through a reversible linkage that is cleavable in the absence of enzymes in aqueous buffer at pH 7.4 and 37° C.;
each -L2- is independently a chemical bond or a spacer;
each -A is —X0 or —Y0;
a is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7 and 8;
—X0 is a substituted acyl borate; and
—Y0 is a substituted hydroxylamine.

### Claim 2 (depends on 1)

The compound of claim 1, wherein -D is selected from the group consisting of small molecule drug moieties, oligonucleotide moieties, peptide nucleic acid moieties, peptide moieties and protein moieties.

### Claim 3 (depends on 2)

The compound of claim 2, wherein -L1- is of formula (a-i):
wherein
the dashed line indicates the attachment to a primary or secondary amine of -D by forming an amide bond;
—X— is —C(R4R4a)—; —N(R4)—; —O—; —C(R4R4a)—C(R5R5a)—; —C(R5R5a)—C(R4R4a)—; —C(R4R4a)—N(R6)—; —N(R6)—C(R4R4a)—; —C(R4R4a)—O—; —O—C(R4R4a)—; or —C(R7R7a)—;
>X1═ is C; or S(O);
—X2— is —C(R8R8a)— or —C(R8R8a)—C(R9R9a)—;
═X3 is ═O; ═S; or ═N—CN;
—R1, —R1a, —R2, —R2a, —R4, —R4a, —R5, —R5a, —R6, —R8, —R8a, —R9, —R9a are independently selected from the group consisting of —H and C1-6 alkyl;
—R3, —R3a are independently selected from the group consisting of —H and C1-6 alkyl, provided that in case one of —R3, —R3a or both are other than —H they are connected to N to which they are attached through a sp3-hybridized carbon atom;
—R7 is —N(R10R10a); or —NR10—(C═O)—R11;
—R7a, —R10, —R10a, —R11 are independently of each other —H; or C1-6 alkyl;
optionally, one or more of the pairs —R1a/—R4a, —R1a/—R5a, —R1a/—R7a, —R4a/—R5a, —R8a/R9a form a chemical bond;
optionally, one or more of the pairs —R/—R1a, —R2/—R2a, —R4/—R4a, —R5/—R5a, —R8/—R8a, —R9/—R9a are joined together with the atom to which they are attached to form a C3-10 cycloalkyl; or 3- to 10-membered heterocyclyl;
optionally, one or more of the pairs —R1/—R4, —R1/—R5, —R1/—R6, —R1/—R7a, —R4/—R5, —R4/—R6, —R8/—R9, —R2/—R3 are joined together with the atoms to which they are attached to form a ring A0;
optionally, R3/R3a are joined together with the nitrogen atom to which they are attached to form a 3- to 10-membered heterocycle;
A0 is selected from the group consisting of phenyl; naphthyl; indenyl; indanyl; tetralinyl; C3-10 cycloalkyl; 3- to 10-membered heterocyclyl; and 8- to 11-membered heterobicyclyl; and
wherein -L1- is substituted with 1, 2, 3, 4, 5, 6, 7 or 8 moieties of -L2-A and wherein -L1- is optionally further substituted, provided that the hydrogen marked with the asterisk in formula (a-i) is not replaced by -L2-A or a substituent.

### Claim 4

A compound of formula (II)
Z-(L2′-B0)b (II),
wherein
—Z is a carrier comprising a C8-24 alkyl moiety or a polymeric moiety;
-L2′- is a chemical bond or a spacer;
—B0 is —Y0;
b is an integer of at least 1; and
—Y0 is a substituted hydroxylamine.

### Claim 5 (depends on 4)

The compound of claim 4, wherein —Z comprises a PEG-based polymer.

### Claim 6 (depends on 4)

The compound of claim 4, wherein —Z comprises a hyaluronic acid-based polymer.

### Claim 7 (depends on 4)

The compound of claim 4, wherein b is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.

### Claim 8 (depends on 1, 4)

The compound claim 1 or 4, wherein -L2- and -L2′- are selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(Ry1)—, —S(O)2N(Ry1)—, —S(O)N(Ry1)—, —S(O)2—, —S(O)—, —N(Ry1)S(O)2N(Ry1a)—, —S—, —N(Ry1)—, —OC(ORy1)(Ry1a)—, —N(Ry1)C(O)N(Ry1a)—, —OC(O)N(Ry1)—, C1-50 alkyl, C2-50 alkenyl, and C2-50 alkynyl; wherein -T-, C1-50 alkyl, C2-50 alkenyl, and C2-50 alkynyl are optionally substituted with one or more —Ry2, which are the same or different and wherein C1-50 alkyl, C2-50 alkenyl, and C2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(Ry3)—, —S(O)2N(Ry3)—, —S(O)N(Ry3)—, —S(O)2—, —S(O)—, —N(Ry3)S(O)2N(Ry3a)—, —S—, —N(Ry3)—, —OC(OR3)(Ry3a)—, —N(Ry3)C(O)N(Ry3a)—, and —OC(O)N(Ry3)—;
—Ry1 and —Ry1a are independently of each other selected from the group consisting of —H, -T, C1-50 alkyl, C2-50 alkenyl, and C2-50 alkynyl; wherein -T, C1-50 alkyl, C2-50 alkenyl, and C2-50 alkynyl are optionally substituted with one or more —Ry2, which are the same or different, and wherein C1-50 alkyl, C2-50 alkenyl, and C2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, —C(O)O—, —O—, —C(O)—, —C(O)N(Ry4)—, —S(O)2N(Ry4)—, —S(O)N(Ry4)—, —S(O)2—, —S(O)—, —N(Ry4)S(O)2N(Ry4a)—, —S—, —N(Ry4)—, —OC(OR4)(Ry4a)—, —N(Ry4)C(O)N(Ry4a)— and —OC(O)N(Ry4)—;
each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; wherein each T is independently optionally substituted with one or more —Ry2, which are the same or different;
each —Ry2 is independently selected from the group consisting of halogen, —CN, oxo(═O), —COORy5, —ORy5, —C(O)Ry5, —C(O)N(Ry5Ry5a), —S(O)2N(Ry5Ry5a), —S(O)N(R5Ry5a), —S(O)2Ry5, —S(O)Ry5, —N(Ry5)S(O)2N(Ry5aRy5b), —SRy5, —N(Ry5Ry5a), —NO2, —OC(O)Ry5, —N(Ry5)C(O)Ry5a, —N(Ry5)S(O)2Ry5a, —N(Ry5)S(O)Ry5a, —N(Ry5)C(O)ORy5a, —N(Ry5)C(O)N(Ry5aRy5b), —OC(O)N(Ry5Ry5a), and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and
each —Ry3, —Ry3a, —Ry4, —Ry4a, —Ry5 —Ry5a and —Ry5b is independently selected from the group consisting of —H, and C1-6 alkyl, wherein C1-6 alkyl is optionally substituted with one or more halogen, which are the same or different.

### Claim 9 (depends on 8)

The compound of claim 8, wherein -L2- and -L2′- are selected from the group consisting of C1-10 alkyl, phenyl, naphthyl, azulenyl, indenyl, indanyl, C3-10 cycloalkyl, 3- to 10-membered heterocyclyl and -A is —X0.

### Claim 10 (depends on 9)

The compound of claim 9, wherein -L2- and -L2′- are selected from C1-10 alkyl and phenyl.

### Claim 11 (depends on 8)

The compound of claim 8, wherein -L2- and -L2′- are C1-10 alkyl and -A is —Y0.

### Claim 12 (depends on 11)

The compound of claim 11, wherein -L2- and -L2′- are C6 alkyl.

### Claim 13 (depends on 10)

The compound of claim 10, wherein —X0 is of formula (III)
wherein
the dashed line indicates attachment to -L2- or -L2′-, respectively;
—X1, —X2 and —X3 are independently of each other selected from the group consisting of —F, —OR, —N+R3, —N+R2OR, —N+R2SR and —N+R2NR2;
each —R is independently selected from the group consisting of —H, -T0, C1-50 alkyl, C2-50 alkenyl, and C2-50 alkynyl; wherein -T0, C1-50 alkyl, C2-50 alkenyl, and C2-50 alkynyl are optionally substituted with one or more —Rx1, which are the same or different and
wherein C1-50 alkyl, C2-50 alkenyl, and C2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T0-, —C(O)O—, —O—, —C(O)—, —C(O)N(Rx2)—, —S(O)2N(Rx2)—, S(O)N(Rx2), —S(O)2—, —S(O)—, —N(Rx2)S(O)2N(Rx2a)—, —S—, —N(Rx2)—, —OC(ORx2)(Rx2a), —N(Rx2)C(O)N(Rx2a)—, and —OC(O)N(Rx2)—;
each T0 is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 30-membered heteropolycyclyl; wherein each T0 is independently optionally substituted with one or more —Rx1, which are the same or different;
each —Rx1 is independently selected from the group consisting of halogen, -T0, —CN, oxo (═O), —COORx3, —ORx3, —C(O)Rx3, —C(O)N(Rx3Rx3a), —S(O)2N(Rx3Rx3a), —S(O)N(Rx3Rx3a), —S(O)2Rx3, —S(O)Rx3, —N(Rx3)S(O)2N(Rx3aRx3b), —SRx3, —N(Rx3Rx3a), —NO2, —OC(O)Rx3, —N(Rx3)C(O)Rx3a, —N(Rx3)S(O)2Rx3a, —N(Rx3)S(O)Rx3a, —N(Rx3)C(O)ORx3a, —N(Rx3)C(O)N(Rx3aRx3b), —OC(O)N(Rx3Rx3a), and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogen, which are the same or different;
each —Rx2, —Rx2a, —Rx3, —Rx3a, Rx3b is independently selected from the group consisting of —H and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogen, which are the same or different;
optionally, two or three of —X1, —X2 and —X3 are joined together with the atom to which they are attached to form a ring A0;
A0 is selected from the group consisting of 3- to 10-membered heterocyclyl and 8- to 30-membered heteropolycyclyl.

### Claim 14 (depends on 13)

The compound of claim 13, wherein —X1, —X2 and —X3 are —F.

### Claim 15 (depends on 12)

The compound of claim 12, wherein each —Y0 is independently of formula (IV)
wherein
the dashed line indicates attachment to -L2- or -L2′-, respectively;
—Ra1 is selected from the group consisting of —COORx1, —ORx1, —C(O)Rx1, —C(O)N(Rx1Rx1a), —S(O)2N(Rx1Rx1a), —S(O)N(Rx1Rx1a), —S(O)2Rx1, —S(O)Rx1, —S(O)2ORx1, —N(Rx1Rx1a), -T0, C1-20 alkyl, C2-20 alkenyl, and C2-20 alkynyl; wherein -T0, C1-20 alkyl, C2-20 alkenyl, and C2-20 alkynyl are optionally substituted with one or more —Rx2, which are the same or different and wherein C1-20 alkyl, C2-20 alkenyl, and C2-20 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T0-, —C(O)O—, —O—, —C(O)—, —C(O)N(Rx3)—, —S(O)2N(Rx3)—, —S(O)N(Rx3)—, —S(O)2—, —S(O)—, —N(Rx3)S(O)2N(Rx3a)—, —S—, —N(Rx3)—, —OC(ORx3)(Rx3a)—, —N(Rx3)C(O)N(Rx3a)—, and —OC(O)N(Rx3)—;
—Rx1, —Rx1a, —Rx1b are independently of each other selected from the group consisting of —H, -T0, C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl; wherein -T0, C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl are optionally substituted with one or more —Rx2, which are the same or different and wherein C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T0-, —C(O)O—, —O—, —C(O)—, —C(O)N(Rx3)—, —S(O)2N(Rx3)—, —S(O)N(Rx3)—; —S(O)2—, —S(O)—, —N(Rx3)S(O)2N(Rx3a)—, —S—, —N(Rx3)—, —OC(ORx3)(Rx3a)—, —N(Rx3)C(O)N(Rx3a)—, and —OC(O)N(Rx3)—;
each T0 is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each T0 is independently optionally substituted with one or more —Rx2, which are the same or different;
each —Rx2 is independently selected from the group consisting of halogen, -T0, —CN, oxo(═O), —COORx4, —ORx4, —C(O)Rx4, —C(O)N(Rx4Rx4a), —S(O)2N(Rx4Rx4a), —S(O)N(Rx4Rx4a), —S(O)2Rx4, —S(O)Rx4, —N(Rx4)S(O)2N(Rx4aRx4b), —SRx4, —N(Rx4Rx4a), —NO2, —OC(O)Rx4, —N(Rx4)C(O)Rx4a, —N(Rx4)S(O)2Rx4a, —N(Rx4)S(O)Rx4a, —N(Rx4)C(O)ORx4a, —N(Rx4)C(O)N(Rx4aRx4b), —OC(O)N(Rx4Rx4a), and C1-4 alkyl; wherein C1-4 alkyl is optionally substituted with one or more halogen, which are the same or different;
each —Rx3, —Rx3a, —Rx4, —Rx4a, —Rx4b is independently selected from the group consisting of —H and C1-4 alkyl; wherein C1-4 alkyl is optionally substituted with one or more halogen, which are the same or different.

### Claim 16 (depends on 15)

The compound of claim 15, wherein —Ra1 of formula (IV) is selected from the group consisting of methyl, ethyl, propyl,
wherein
—Rc1 and —Rc1a are independently of each other selected from the group consisting of —H, -T0 and C1-6 alkyl;
—Rd is selected from the group consisting of C1-6 alkyl and —NO2, —CN, —C(O)ORe, —S(O)2ORe;
each -T0 is independently of each other selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3- to 10-membered heterocyclyl, and 8- to 11-membered heterobicyclyl; wherein each -T0 is independently optionally substituted with one or more —Re, which are the same or different;
each —Re is independently C1-6 alkyl;
n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;
m is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; and
p is selected from the group consisting of 0, 1, 2, 3, 4 and 5.

### Claim 17 (depends on 16)

The compound of claim 16, wherein —Ra1 is

### Claim 18

A method of synthesizing a carrier-linked prodrug, the method comprising the step of reacting a compound of formula (I)
D-(L1-L2-A)a (I),
wherein
-D is a drug moiety;
each -L1- is independently a reversible prodrug linker, which is covalently conjugated to -D through a reversible linkage that is cleavable in the absence of enzymes in aqueous buffer at pH 7.4 and 37° C.;
each -L2- is independently a chemical bond or a spacer;
each -A is —X0 or —Y0;
a is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7 and 8;
—X0 is a substituted acyl borate; and
—Y0 is a substituted hydroxylamine;
under aqueous conditions with a compound of formula (II)
Z-(L2′-B0)b (II),
wherein
—Z is a carrier comprising a C8-24 alkyl moiety or a polymeric moiety;
-L2′- is a chemical bond or a spacer;
—B0 is —Y0;
b is an integer of at least 1; and
—Y0 is a substituted hydroxylamine;
with -A being —X0 and —B0 being —Y0, and forming an amide bond between moiety -A and a moiety —B0.

### Claim 19 (depends on 18)

The method of claim 18, wherein the method is performed at a pH ranging from and including 1 to 8.

### Claim 20 (depends on 19)

The method of claim 19, wherein the method is performed at a pH ranging from and including 3 to 5.

### Claim 21 (depends on 18)

The method of claim 18, wherein the method is conducted in an aqueous buffer.

### Claim 22 (depends on 18)

The method of claim 18, wherein the reaction is conducted in a solvent mixture selected from the group consisting of THF/H2O, CH3CN/H2O, tBuOH/H2O and DMSO/tBuOH/H2O.

### Claim 23 (depends on 22)

The method of claim 22, wherein the method of the present invention is performed for 1 minute to 5 hours.

### Claim 24 (depends on 21)

The method of claim 21, wherein the aqueous buffer comprises additives.