# US 2019/0169609 A1 · Antisense antibacterial compounds and methods

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- Number: US 2019/0169609 A1
- Document ID: US20190169609A1
- Status: Published application
- Kind: A1
- Application number: 15/540,387
- Filed: 2015-12-23
- Published: 2019-06-06
- Earliest claimed: 2014-12-31
- Assignees: Oregon State University; Board of Regents, The University of Texas System
- Applicants: Oregon State University; Board of Regents, The University of Texas System
- Inventors: Bruce L. Geller; David Greenberg
- Web page: https://patentgrep.com/patent/US20190169609A1

## CPC

- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- A61K 31/7088: Compounds having three or more nucleosides or nucleotides
- A61K 38/12: Cyclic peptides {, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C (A61K38/043 - A61K38/046 take precedence)}
- A61P 31/04: Antibacterial agents
- C07F 9/65583: {each of the hetero rings containing nitrogen as ring hetero atom}
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12N 2310/11: Antisense
- C12N 2310/3233: Morpholino-type ring
- C12N 2320/31: Combination therapy
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07F: ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM (metal-containing porphyrins C07D487/22)
- 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

Provided are antisense oligonucleotides targeted against bacterial genes involved in biochemical pathways and/or cellular processes, and related compositions and methods of using the oligonucleotides and compositions, alone or in combination with other antimicrobial agents, for instance, in the treatment of an infected mammalian subject.

## Claims

### Claim 1

A compound of formula (I):
or a pharmaceutically acceptable salt thereof, wherein:
each Nu is a nucleobase which taken together forms a nucleobase sequence;
Z is an integer from 8 to 38;
T is selected from OH and a moiety of the formula:
wherein:
each R4 is independently C1-C6 alkyl, and
R5 is selected from an electron pair and H, and
R6 is selected from —N(R7)CH2C(O)NH2, and a moiety of the formula:
wherein:
R7 is selected from H and C1-C6 alkyl; and
R8 is selected from G, —C(O)—R9, acyl, trityl, and 4-methoxytrityl,
wherein:
R9 is of the formula —(O-alkyl)y-OH wherein y is an integer from 3 to 10 and each of the y alkyl groups is independently selected from C2-C6 alkyl;
each instance of R1 is —N(R10)2R11 wherein each R10 is independently C1-C6 alkyl, and R11 is selected from an electron pair and H;
R2 is selected from the group consisting of H, G, acyl, trityl, 4-methoxytrityl, and a moiety of the formula:
wherein,
L is selected from —C(O)(CH2)6C(O)— and —C(O)(CH2)2S2(CH2)2C(O)—; and
each R12 is of the formula —(CH2)2OC(O)N(R26)2 wherein each R26 is of the formula (CH2)6NHC(═NH)NH2; and
R3 is selected from the group consisting of an electron pair, H, and C1-C6 alkyl,
wherein G is a cell penetrating peptide (“CPP”) and linker moiety selected from the group consisting of —C(O)(CH2)5NH—CPP, —C(O)(CH2)2NH—CPP, —C(O)(CH2)2NHC(O)(CH2)5NH—CPP, —C(O)CH2NH—CPP, and —C(O)CH(pyrrolidin-2-yl)NH—CPP wherein the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus, with the proviso that one instance of G is present, and
wherein the nucleobase sequence comprises a targeting sequence that is complementary to a Pseudomonas aeruginosa mRNA that encodes RpsJ, LpxC, FabG, AcpP, RpmB, WaaC, MraY, MurC, AccA, LpxA, LpxB, WaaG, WaaA, WaaF, MurB, MurE, AccB, FabZ, MurF, MurG, or AmpR.

### Claim 2 (depends on 1)

The compound of claim 1, wherein the targeting sequence is selected from Table 1, a fragment of at least 10 contiguous nucleotides of a targeting sequence in Table 1, or variant having at least 80% sequence identity to a targeting sequence in Table 1, wherein the thymine bases may be uracil bases.

### Claim 3 (depends on 1)

The compound of claim 1, wherein Z is 9 and the targeting sequence is selected from Table 1.

### Claim 4 (depends on 1)

The compound according to claim 1, wherein the CPP is selected from Table 2, wherein B is β-alanine and X is 6-aminohexanoic acid.

### Claim 5 (depends on 1)

The compound of claim 1, wherein:
T is of the formula:
wherein R6 is of the formula:
and
R2 is G.

### Claim 6 (depends on 1)

The compound of claim 1, wherein T is of the formula:
and
R2 is G.

### Claim 7 (depends on 1)

The compound of claim 1, wherein R2 is selected from H or G, and R3 is selected from an electron pair or H.

### Claim 8 (depends on 7)

The compound of claim 7, wherein R2 is G and G is selected from Table 2.

### Claim 9 (depends on 7)

The compound of claim 7, wherein R2 is H or acyl.

### Claim 10 (depends on 1)

The compound of claim 1, wherein each R1 is —N(CH3)2.

### Claim 11 (depends on 1)

The compound of claim 1, wherein:
T is of the formula:
each R1 is —N(CH3)2, and
R2 is G,
wherein the targeting sequence and corresponding G are selected from Table 3.

### Claim 12 (depends on 1)

The compound of claim 1, wherein:
T is of the formula:
each R1 is —N(CH3)2, and
R2 is —C(O)CH3,
wherein the targeting sequence and corresponding G are selected from Table 4.

### Claim 13 (depends on 1)

The compound of claim 1, wherein the compound is of the formula:
or a pharmaceutically acceptable salt thereof, wherein the targeting sequence, from 5′ to 3′, is selected from the group consisting of:
a)
(SEQ ID NO: 2)
GTT GTT TGA TC;
b)
(SEQ ID NO: 3)
TTC TCT CCT TT;
c)
(SEQ ID NO: 4)
CAT ACC TTG TT;
d)
(SEQ ID NO: 5)
CTC TAG ACA TG;
e)
(SEQ ID NO: 6)
AGC ACC CTC AT;
f)
(SEQ ID NO: 7)
TGA CTC TCC TC;
g)
(SEQ ID NO: 8)
CCA CCT CCA GG;
h)
(SEQ ID NO: 9)
AGG CTT CCG TC;
i)
(SEQ ID NO: 10)
ATC AAA CTC AT;
j)
(SEQ ID NO: 11)
TAA TCC GTC AG;
k)
(SEQ ID NO: 12)
GCC AGG GTC AT;
l)
(SEQ ID NO: 13)
GCA TTT GAC CT;
m)
(SEQ ID NO: 14)
GTA CGG TTC AT;
n)
(SEQ ID NO: 15)
AGA ATT CTC AT;
o)
(SEQ ID NO: 16)
CAG TCG CCC CT;
p)
(SEQ ID NO: 17)
AGG CTC ATA GG;
q)
(SEQ ID NO: 18)
CTA GCA CTC CC;
r)
(SEQ ID NO: 19)
ATG TCC ATC AT;
s)
(SEQ ID NO: 20)
ACC TCC CAG GC;
t)
(SEQ ID NO: 21)
GCA AAG TCC TC;
and
u)
(SEQ ID NO: 35)
CTC ATA CCT TG,
wherein thymine bases may be uracil bases.

### Claim 14 (depends on 1)

The compound of claim 1, wherein the compound is selected from:
or a pharmaceutically acceptable salt thereof,
wherein the targeting sequence, from 5′ to 3′, is CCT CAG ACT CC (SEQ ID NO: 1),
wherein thymine bases may be uracil bases.

### Claim 15 (depends on 1)

The compound of claim 1, wherein the compound is of the formula:
wherein the targeting sequence, from 5′ to 3′, is GTC GAA CCA AT (SEQ ID NO: 22), wherein thymine bases may be uracil bases.

### Claim 16 (depends on 1)

The compound according to claim 1, wherein the thymine bases are uracil bases.

### Claim 17 (depends on 1)

A combination, comprising:
a) the compound of Formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof; and
b) a second compound selected from the group consisting of polymyxin E (PME), polymyxin B (PMB), polymyxin B nonapeptide (PMBN), polymyxin E nonapeptide, a pharmaceutically acceptable salt of any of the foregoing, and combinations thereof.

### Claim 18 (depends on 17)

The combination of claim 17, wherein the second compound is PME.

### Claim 19

(canceled)

### Claim 20 (depends on 17)

The combination of claim 17, wherein the second compound is PMBN.

### Claim 21

(canceled)

### Claim 22

A method of treating a Pseudomonas aeruginosa infection, comprising administering to a patient in need thereof an effective amount of a composition comprising a compound of formula (I):
or a pharmaceutically acceptable salt thereof, wherein:
each Nu is a nucleobase which taken together forms a nucleobase sequence;
Z is an integer from 8 to 38;
T is selected from OH and a moiety of the formula:
wherein:
each R4 is independently selected from H and C1-C6 alkyl, and
R5 is selected from an electron pair and H, and
R6 is selected from —N(R7)CH2C(O)NH2, and a moiety of the formula:
wherein:
R7 is selected from H and C1-C6 alkyl; and
R8 is selected from G, —C(O)—R9, acyl, trityl, and 4-methoxytrityl,
wherein:
R9 is of the formula —(O-alkyl)y-OH wherein y is an integer from 3 to 10 and each of the y alkyl groups is independently selected from C2-C6 alkyl optionally containing one or more intervening oxygen radicals;
each instance of R1 is —N(R10)2R11 wherein each R10 is independently C1-C6 alkyl, and R11 is selected from an electron pair and H;
R2 is selected from the group consisting of H, G, acyl, trityl, 4-methoxytrityl, and a moiety of the formula:
wherein,
L is selected from —C(O)(CH2)6C(O)— and —C(O)(CH2)2S2(CH2)2C(O)—; and
each R12 is of the formula —(CH2)2OC(O)N(R26)2 wherein each R26 is of the formula (CH2)6NHC(═NH)NH2; and
R3 is selected from the group consisting of an electron pair, H, and C1-C6 alkyl,
wherein G is a cell penetrating peptide (“CPP”) and linker moiety selected from the group consisting of —C(O)(CH2)5NH—CPP, —C(O)(CH2)2NH—CPP, —C(O)(CH2)2NHC(O)(CH2)5NH—CPP, —C(O)CH2NH—CPP, and —C(O)CH(pyrrolidin-2-yl)NH—CPP wherein the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus, with the proviso that up to one instance of G is present, and
wherein the nucleobase sequence comprises a targeting sequence that is complementary to a Pseudomonas aeruginosa mRNA that encodes RpsJ, LpxC, FabG, AcpP, RpmB, WaaC, MraY, MurC, AccA, LpxA, LpxB, WaaG, WaaA, WaaF, MurB, MurE, AccB, FabZ, MurF, MurG, or AmpR; and
a pharmaceutically acceptable carrier.

### Claim 23

A pharmaceutical combination therapy for the treatment or prevention of a Pseudomonas aeruginosa infection in a patient in need thereof, comprising:
a compound of formula (I):
or a pharmaceutically acceptable salt thereof, wherein:
each Nu is a nucleobase which taken together forms a nucleobase sequence;
Z is an integer from 8 to 38;
T is selected from OH and a moiety of the formula:
wherein:
each R4 is independently selected from H and C1-C6 alkyl, and
R5 is selected from an electron pair and H, and
R6 is selected from —N(R7)CH2C(O)NH2, and a moiety of the formula:
wherein:
R7 is selected from H and C1-C6 alkyl; and
R8 is selected from G, —C(O)—R9, acyl, trityl, and 4-methoxytrityl,
wherein:
R9 is of the formula —(O-alkyl)y-OH wherein y is an integer from 3 to 10 and each of the y alkyl groups is independently selected from C2-C6 alkyl optionally containing one or more intervening oxygen radicals;
each instance of R1 is —N(R10)2R11 wherein each R10 is independently C1-C6 alkyl, and R11 is selected from an electron pair and H;
R2 is selected from the group consisting of H, G, acyl, trityl, 4-methoxytrityl, and a moiety of the formula:
wherein,
L is selected from —C(O)(CH2)6C(O)— and —C(O)(CH2)2S2(CH2)2C(O)—; and
each R12 is of the formula —(CH2)2OC(O)N(R26)2 wherein each R26 is of the formula (CH2)6NHC(═NH)NH2; and
R3 is selected from the group consisting of an electron pair, H, and C1-C6 alkyl,
wherein G is a cell penetrating peptide (“CPP”) and linker moiety selected from the group consisting of —C(O)(CH2)5NH—CPP, —C(O)(CH2)2NH—CPP, —C(O)(CH2)2NHC(O)(CH2)5NH—CPP, —C(O)CH2NH—CPP, and —C(O)CH(pyrrolidin-2-yl)NH—CPP wherein the CPP is attached to the linker moiety by an amide bond at the CPP carboxy terminus, with the proviso that up to one instance of G is present, and
wherein the nucleobase sequence comprises a targeting sequence that is complementary to a Pseudomonas aeruginosa mRNA that encodes RpsJ, LpxC, FabG, AcpP, RpmB, WaaC, MraY, MurC, AccA, LpxA, LpxB, WaaG, WaaA, WaaF, MurB, MurE, AccB, FabZ, MurF, MurG, or AmpR; and
a second compound selected from the group consisting of polymyxin E (PME), polymyxin B (PMB), polymyxin B nonapeptide (PMBN), polymyxin E nonapeptide, a pharmaceutically acceptable salt of any of the foregoing, and combinations thereof