Nucleosidic and non-nucleosidic folate conjugates

• US 6,335,434 B1
• Filed: 03/24/1999
• Issued: 01/01/2002
• Est. Priority Date: 06/16/1998
• Status: Expired due to Term

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Bhat, Balkrishen, Manoharan, Muthiah, Guzaev, Andrei P., Cook, Phillip Dan
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/275,505
• Time in Patent Office: 1,014 Days
• Field of Search: 435/6, 536/22.1, 536/23.1, 536/24.3, 536/25.3, 514/44
• US Class Current: 536/23.1
• CPC Class Codes: C07D 475/04 with a nitrogen atom directly attached in position 2
• CPC Class Codes: C07H 15/26 Acyclic or carbocyclic radicals, substituted by hetero rings

Abstract

Oligonucleotide-folate conjugates are described wherein folates are conjugated to one or more sites on an oligonucleotide including the 2′-, 3′-, 5′-, nucleobase and internucleotide linkage sites. The folate can be attached via the α- or γ-carboxylate, optionally through a linking group. Methods for the regiospecific synthesis of the conjugates are diclosed. Also disclosed are nucleosidic and non-nucleosidic linkers conjugated to folic acid and related folates.

Claims

1. a compound having formula XVIA, XVIB, XVIC or XVID:

wherein;

W₁₄ has the formula;

wherein;

X₄ is —

CH(X_4′

) or a group of formula;

X_4′

is the side chain of a naturally-occuning amino acid or analog thereof, or a protected side chain of a naturally-occurring amino acid or analog thereof;

t is 1 or 2;

X₅ is —

N(X_6′

)C(O)—

, —

C(O)NH—

, —

NHC(O)—

, —

OC(O)NH—

, —

C(S)NH—

, —

SC(S)NH—

, —

SC(O)NH—

, —

OC(S)NH—

, —

C(O)O—

, —

C(O)(CH₂)_n—

or a bond;

n is an integer from 1 to 50;

each X₆, X_6′

and X₉ is, independently, a bond, hydrogen or a hydrocarbyl group selected from C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₂₀ alkynyl, C₆-C₁₄ aryl, C₆-C₁₄ aralkyl, C₃-C₁₄ cycloalkyl, C₅-C₁₄ fused cycloalkyl, C₄-C₁₄ heterocycle, C₄-C₁₄ heterocyclylalkyl, C₄-C₁₄ heteroaryl and C₄-C₁₄ heteroarylalkyl;

wherein said hydrocarbyl group is substituted with at least two hydroxyl groups, and is optionally substituted with oxo, acyl, alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, amido, azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo, haloalkyl, haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro, sulfide, disulfide, sulfone, sulfonate, sulfonamide, thiol, and thioalkoxy;

provided that each X₆ and X₉ is not hydrogen and X_6′

is not a bond;

R₁ is hydrogen or a hydroxyl protecting group;

R₄ is a hydroxyl group or a protected hydroxyl group;

each R_5′

and R₄₀ is, independently, hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₂₀ alkynyl, C₆-C₁₄ aryl or an amino-protecting group

R_5″

is hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₂₀ alkynyl, C₆-C₁₄ aryl, C₆-C₁₄ aralkyl, C₃-C₁₄ cycloalkyl, formyl, aminoalkyl or hydroxymethyl;

R₆ is hydrogen or an amino protecting group;

R₂₀ is hydrogen or a group of formula;

R₂ is —

N(R₇)₂, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;

R₇ is straight or branched chain alkyl having from 1 to 10 carbons;

R₃ is a phosphorus protecting group;

R₂₁ is hydrogen, hydroxyl, fluoro or a group of formula Z—

R₂₂—

(R₂₃)_v;

Z is O, S, NH or N—

R₂₂—

(R₂₃)_v;

R₂₂ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

R₂₃ is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, or a polyether;

or R₂₁ has one of the formulas;

wherein;

y1 is 0 or 1;

each y2 is, independently, 0 to 10;

y3 is 1 to 10;

E is N(R₄₁)(R₄₂) or N═

C(R₄₁)(R₄₂);

each R₄₁ and each R₄₂ is independently H, C₁-C₁₀ alkyl, a nitogen protecting group, or R₄₁ and R₄₂ taken together form a nitrogen protecting group;

or R₄₁ and R₄₂ taken together with the N or C atom to which they are attached form a ring structure that can include at least one heterotom selected from N and O;

B is a nucleobase;

M is an optionally protected internucleoside linkage;

q is from zero to about 50; and

v is from zero to about 10;

provided that when said compound has formula XVIC, at least one R₂₁ is a group other than hydrogen, and when said compound has formula XVIC or XVID, q is at least 1.

2. A compound having formula XVIA, XVIB, XVIC or XVID:

wherein;

W₁₄ has the formula;

wherein;

is —

CH(X_4′

) or a group of formula;

X_4′

is the side chain of a naturally-occurring amino acid or analog thereof, or a protected side chain of a naturally-occurring amino acid or analog thereof;

t is 1 or 2;

X₅ is —

N(X_6′

)C(O)—

, —

C(O)NH—

, —

NHC(O)—

, —

OC(O)NH—

, —

C(S)NH—

, —

SC(S)NH—

, —

SC(O)NH—

, —

OC(S)NH—

, —

C(O)O—

, —

C(O)(CH₂)_n—

or a bond;

n is an integer from 1 to 50;

each X₆ and X_6′

is, independently, a bond, hydrogen or a hydrocarbyl group selected from C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₂₀ alkynyl, C₆-C₁₄ aryl, C₆-C₁₄ aralkyl, C₃-C₁₄ cycloalkyl, C₅-C₁₄ fused cycloalkyl, C₄-C₁₄ heterocycle, C₄-C₁₄ heterocyclylalkyl, C₄-C₁₄ heteroaryl and C₄-C₁₄ heteroarylalkyl;

wherein said hydrocarbyl group is substituted with at least two hydroxyl groups, and is optionally substituted with oxo, acyl, alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, armido, azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo, haloalkyl, haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro, sulfide, disulfide, sulfone, sulfonate, sulfonamide, thiol, and thioalkoxy;

provided that X₆ is not hydrogen and X_6′

is not a bond;

R₁ is hydrogen or a hydroxyl protecting group;

R₄ is a hydroxyl group or a protected hydroxyl group;

each R_5′

and R₄₀ is, independently, hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₂₀ alkynyl, C₆-C₁₄ aryl or an amino-protecting group

R_5″

is hydrogen, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₂₀ alkynyl, C₆-C₁₄ aryl, C₆-C₁₄ aralkyl, C₃-C₁₄ cycloalkyl, formyl, aminoalkyl or hydroxymethyl;

R₆ is hydrogen or an amino protecting group;

R₂₀ is hydrogen or a group of formula;

R₂ is —

N(R₇)₂, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;

R₇ is straight or branched chain alkyl having from 1 to 10 carbons;

R₃ is a phosphorus protecting group;

R₂₁ is hydrogen, hydroxyl, fluoro or a group of formula Z—

R₂₂—

(R₂₃)_v;

Z is O, S, NH or N—

R₂₂—

(R₂₃)_v;

R₂₂ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

R₂₃ is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, or a polyether;

or R₂₁ has one of the formulas;

wherein;

y1 is 0 or 1;

each y2 is, independently, 0 to 10;

y3 is 1 to 10;

E is N(R₄₁)(R₄₂) or N═

C(R₄₁)(R₄₂);

each R₄₁ and each R₄₂ is independently H, C₁-C₁₀ alkyl, a nitogen protecting group, or R₄₁ and R₄₂ taken together form a nitrogen protecting group;

or R₄₁ and R₄₂ taken together with the N or C atom to which they are attached form a ring structure that can include at least one heterotom selected from N and O;

B is a nucleobase;

M is an optionally protected internucleoside linkage;

q is from zero to about 50; and

v is from zero to about 10;

provided that when said compound has formula XVIC or XVID, q is at least 1.

3. A compound having the formula XIIIA, XIIIB, XIIIC or XIIID:

wherein;

W₁₃ has the formula;

R₁ is H or a hydroxyl protecting group;

B is a nucleobase;

each R₂₁ is H, OH, F, or a group of formula Z—

R₂₂—

(R₂₃)_v;

Z is O, S, NH or N—

R₂₂—

(R₂₃)_v;

R₂₂ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

R₂₃ is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, or a polyether;

or R₂₁ has one of the formulas;

wherein;

y1 is 0 or 1;

y2 is 0 to 10;

y3 is 1 to 10;

E is N(R₄₁)(R₄₂) or N═

C(R₄₁)(R₄₂);

each R₄₁ and each R₄₂ is independently H, C₁-C₁₀ alkyl, a nitogen protecting group, or R₄₁ and R₄₂ taken together form a nitrogen protecting group;

or R₄₁ and R₄₂ taken together with the N or C atom to which they are attached form a ring structure that can include at least one heterotom selected from N and O;

v is from 0 to about 10;

q is from 0 to about 50;

M is an optionally protected intemucleoside linkage;

W₁ is a linking group, O, NH or S;

R₂₀ is H or a group of Formula;

R₂ is —

N(R₇)₂, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;

R₇ is straight or branched chain alkyl having from 1 to 10 carbons;

R₃ is a phosphorus protecting group;

n is from 1 to about 10;

R₅ is H or an amino protecting group;

R₆ is H or an amino protecting group;

X₃ is —

CH(Z₁)—

or a group of Formula XI;

Z₁ is the sidechain of a naturally occuning amino acid or analog thereof, or a protected sidechain of a naturally occurring amino acid or analog thereof;

p is 1 or 2; and

R₄ is a hydroxyl group, or a protected hydroxy group;

provided that when said compound has formula XIIIC, at least one R₂₁ is a group other than hydrogen, and when said compound has formula XIIIC or XIIID, q is at least 1.

4. A compound having the formula XIIIA, XIIIB, XIIIC or XIIID:

wherein;

W₁₃ has the formula;

R₁ is H or a hydroxyl protecting group;

B is a nucleobase;

each R₂₁ is H, OH, F, or a group of formula Z—

R₂₂—

(R₂₃)_v;

Z is O, S, NH or N—

R₂₂ —

(R₂₃)_v;

R₂₂ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

R₂₃ is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, or a polyether;

or R₂₁ has one of the formulas;

wherein;

y1 is 0 or 1;

y2 is 0 to 10;

y3 is 1 to 10;

E is N(R₄₁)(R₄₂) or N═

C(R₄₁)(R₄₂);

each R₄₁ and each R₄₂ is independently H, C₁-C₁₀ alkyl, a nitogen protecting group, or R₄₁ and R₄₂ taken together form a nitrogen protecting group;

or R₄₁ and R₄₂ taken together with the N or C atom to which they are attached form a ring structure that can include at least one heterotom selected from N and O;

v is from 0 to about 10;

q is from 0 to about 50;

M is an optionally protected intemucleoside linkage;

W₁ is a linking group, O, NH or S;

R₂₀ is H or a group of Formula;

R₂ is —

N(R₇)₂, or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;

R₇ is straight or branched chain alkyl having from 1 to 10 carbons;

R₃ is a phosphorus protecting group;

R₅ is H or an amino protecting group;

R₆ is H or an amino protecting group;

X₃ is —

CH(Z₁)—

or a group of Formula XI;

Z₁ is the sidechain of a naturally occuning amino acid or analog thereof, or a protected sidechain of a naturally occurring amino acid or analog thereof;

p is 1 or 2; and

R₄ is a hydroxyl group, or a protected hydroxy group;

provided that when said compound has formula XIIIC, at least one R₂₁ is a group other than hydrogen, and when said compound has formula XIIIC or XIIID, q is at least 1.