# US 2020/0399634 A1 · Dna-templated macrocycle library

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- Number: US 2020/0399634 A1
- Document ID: US20200399634A1
- Status: Published application
- Kind: A1
- Application number: 16/968,408
- Filed: 2019-02-08
- Published: 2020-12-24
- Earliest claimed: 2018-02-09
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard Co
- Inventors: David R. Liu; Dmitry L. Usanov; Juan Pablo Maianti; Alix I. Chan
- Web page: https://patentgrep.com/patent/US20200399634A1

## CPC

- C12N 15/1068: {Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis}
- A61K 38/12: Cyclic peptides {, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C (A61K38/043 - A61K38/046 take precedence)}
- C07K 7/56: the cyclisation not occurring through 2,4-diamino-butanoic acid
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- 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)
- 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 provides nucleic acid templates (e.g., including orthogonal codon sets (e.g., codons from orthogonal codon sets depicted in Tables 5 or 7)) for DNA-templated methods of synthesizing, selecting, and amplifying compounds (e.g., polymers and/or small molecules) described herein. Also provided are novel macrocyclic compounds of Formula (I), and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, libraries, and compositions thereof. Also provided are methods and kits involving the inventive compounds or compositions for treating and/or preventing a disease (e.g., a disease associated with aberrant enzyme activity (e.g., aberrant protease and/or kinase activity (e.g., aberrant IDE activity)), impaired insulin signaling, or insulin resistance in a subject (e.g., a subject having diabetes). Treatment of a subject with a proliferative disease using a compound or composition of the invention may inhibit aberrant protease activity (e.g., aberrant IDE activity).

## Claims

### Claim 1

A compound of Formula (I):
or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, polymorph, tautomer, isotopically enriched form, or prodrug thereof,
wherein:
is a single or double C—C bond, wherein when is a double C—C bond, then indicates that the adjacent C—C double bond is in a cis or trans configuration;
L1 is substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclyene, substituted or unsubstituted arylene, or substituted or unsubstituted heterocyclylene;
L2 is substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclyene, substituted or unsubstituted arylene, or substituted or unsubstituted heterocyclylene;
L3 is substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclyene, substituted or unsubstituted arylene, or substituted or unsubstituted heterocyclylene;
X is —O—C((R3A)(R3B))s—, —C((R3A)(R3B))s—, optionally substituted carbocyclylene, optionally substituted heterocyclylene, or optionally substituted arylene;
R3A is hydrogen; halogen; substituted or unsubstituted aliphatic; substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl;
R3B is hydrogen; halogen; substituted or unsubstituted aliphatic; substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; or optionally R3A and R3B are joined together to form optionally substituted carbocyclyl or
each instance of RE, RF, RG, RH, and RL is independently hydrogen; halogen; substituted or unsubstituted acyl; a nitrogen protecting group; substituted or unsubstituted aliphatic; substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; or optionally RG and one instance of R3A or R3B are joined together with the intervening atoms to form optionally substituted heterocyclyl;
m1 is 0, 1, or 2;
s is 0, 1, 2, 3, or 4; and
y is 0 or 1.

### Claim 2 (depends on 1)

The compound of claim 1, wherein L1 is substituted or unsubstituted C1-6 aliphatic, substituted or unsubstituted arylene, or substituted or unsubstituted heterocyclylene.

### Claim 3 (depends on 1)

The compound of claim 1, wherein L2 is of the formula:
wherein:
R1 is hydrogen; halogen; substituted or unsubstituted aliphatic; substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; —ORA; —N(RA)2; —SRA; ═O; —CN; —NO2; —SCN; —SORA; or —SO2RA; wherein each occurrence of RA is independently hydrogen; a protecting group; substituted or unsubstituted aliphatic; substituted or unsubstituted heteroaliphatic; substituted or unsubstituted acyl; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl;
R2 is hydrogen; halogen; substituted or unsubstituted aliphatic; substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; —ORB; —N(RB)2; —SRB; ═O; —CN; —NO2; —SCN; —SORB; or —SO2RB; wherein each occurrence of RB independently hydrogen; a protecting group; substituted or unsubstituted aliphatic; substituted or unsubstituted heteroaliphatic; substituted or unsubstituted acyl; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl, or optionally R1 and R2 are joined together to form a spiro-linked, optionally substituted carbocyclyl, or optionally RH and one instance of R1 or R2 are joined together with the intervening atoms to form optionally substituted heterocyclyl; and
x is 0, 1, 2, or 3.

### Claim 4 (depends on 1)

The compound of claim 1, wherein L2 is of the formula:
wherein
R5 is substituted or unsubstituted aliphatic; substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted amino; —C(═O)—N(RJ)2; —C(═O)—ORJ; or —C(═O)—SRJ, or —CH2—C(═O)N(RJ)2, wherein each occurrence of RJ is independently hydrogen; a protecting group; substituted or unsubstituted aliphatic; substituted or unsubstituted heteroaliphatic; substituted or unsubstituted acyl; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl; or two RJ groups are joined to form a substituted or unsubstituted heterocyclic group; optionally wherein R5 further comprises a label, resin, or therapeutic agent attached thereto.

### Claim 5 (depends on 1)

The compound of claim 1, wherein L2 is of the formula:
wherein
w is 0, 1, or 2; and
x1 is 1 or 2.

### Claim 6 (depends on 1)

The compound of claim 1, wherein L2 is of the formula:

### Claim 7 (depends on 1)

The compound of claim 1, wherein L2 is substituted or unsubstituted 5- to 7-membered carbocyclyene, substituted or unsubstituted arylene, or substituted or unsubstituted, 4- to 10-membered heterocyclylene.

### Claim 8 (depends on 3)

The compound of claim 3, wherein R1 and R2 are joined together to form a spiro-linked, optionally substituted carbocyclyl.

### Claim 9 (depends on 3)

The compound of claim 3, wherein R1 is substituted or unsubstituted aryl.

### Claim 10 (depends on 3)

The compound of claim 3, wherein R2 is substituted or unsubstituted aryl.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The compound of any one of claims 1-10, wherein y is 1.

### Claim 12 (depends on 1)

The compound of claim 1, wherein L3 is substituted or unsubstituted C1-6 aliphatic, substituted or unsubstituted carbocyclyene, substituted or unsubstituted arylene, or substituted or unsubstituted heterocyclylene.

### Claim 13 (depends on 1)

The compound of claim 1, wherein L3 is of the formula:
wherein
R4A is hydrogen; halogen; substituted or unsubstituted aliphatic; substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl;
R4B is hydrogen; halogen; substituted or unsubstituted aliphatic; substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; or optionally RF and one instance of R4A or R4B are joined together with the intervening atoms to form optionally substituted heterocyclyl, and
r is 0, 1, or 2.

### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The compound of any one of claims 1-13, wherein R4A is hydrogen, substituted or unsubstituted C1-6 aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted heteroaryl.

### Claim 15 (depends on 13)

The compound of claim 13, wherein R4B is hydrogen, substituted or unsubstituted C1-6 aliphatic, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclyl, or substituted or unsubstituted heteroaryl.

### Claim 16 (depends on 13)

The compound of claim 13, wherein R4A and R4B are joined together with the intervening atoms to form optionally substituted carbocyclyl, or optionally substituted heterocyclyl.

### Claim 17 (depends on 13)

The compound of claim 13, wherein RF and one instance of R4A are joined together with the intervening atoms to form optionally substituted heterocyclyl.

### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

The compound of any one of claims 1-17, wherein X is —O—(CH2)2—, —C((R3A)(R3B))s—, optionally substituted carbocyclylene, optionally substituted heterocyclylene, or optionally substituted arylene.

### Claim 19 (depends on 18)

The compound of claim 18, wherein R3A is hydrogen; substituted or unsubstituted C1-6 aliphatic; substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; or substituted or unsubstituted acyl.

### Claim 20 (depends on 18)

The compound of claim 18, wherein R3B is hydrogen; substituted or unsubstituted C1-6 aliphatic; substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; or substituted or unsubstituted acyl.

### Claim 21 (depends on 18)

The compound of claim 18, wherein R3A and R3B are joined together to form optionally substituted carbocyclyl or

### Claim 22 (depends on 18)

The compound of claim 18, wherein RG and one instance of R3A or R3B are joined together with the intervening atoms to form optionally substituted heterocyclyl.

### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

The compound of any one of claims 1-22, wherein the compound is of the formula:
or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, polymorph, tautomer, isotopically enriched form, or prodrug thereof,
wherein R is —(CH2)2O(CH2)2NH2.

### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

The compound of any one of claims 1-23, wherein the compound is synthesized using the scaffold depicted in FIG. 1 and the building blocks depicted in FIGS. 3B and 3C.

### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

The compound of any one of claims 1-23, wherein the compound is synthesized using a template based on an orthogonal codon set depicted in Table 5.

### Claim 26 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

The compound of any one of claims 1-23, wherein the compound is synthesized using a template with one or more anticodons and/or reagents depicted in Table 6.

### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)

The compound of any one of claims 1-23, wherein the compound is synthesized using a template based on an orthogonal codon set including one or more codons depicted in Table 7.

### Claim 28

A method of synthesizing a DNA-templated compound library, wherein each compound is encoded by a covalently associated DNA tag, comprising:
creating a template of a combination of orthogonal codons interspaced by constant regions wherein the template is based on codons from the orthogonal codon sets depicted in FIG. 5 or 7; and
running templated coupling reactions to assemble a compound based on the orthogonal codons.

### Claim 29 (depends on 28)

The method of claim 28, wherein the template of the combination of orthogonal codons comprises:
multiple-residue primer-binding sites;
at least three building block codons that determine the identity of three building blocks; and
at least a fourth building block codon that identifies a compound scaffold at the 5′ end of the template.

### Claim 30 (depends on 28)

The method of claim 28, wherein the method further comprises:
capping unreacted templates by acetylation.

### Claim 31 (depends on 30)

The method of claim 30, wherein the method further comprises:
purifying the unreacted templates from the reacted templates.

### Claim 32 (depends on 31)

The method of claim 31, wherein the purifying comprises capturing reacted templates with streptavidin-linked beads.

### Claim 33 (depends on 31)

The method of claim 31, wherein the purifying comprises using a PAGE purification protocol depicted in FIG. 26.

### Claim 34 (depends on 31)

The method of claim 31, wherein the method further comprises:
macrocyclizing DNA-templated products from the reacted templates.

### Claim 35 (depends on 28)

The method of claim 28, wherein the template of the combination of orthogonal codons comprises:
multiple-residue primer-binding sites;
at least three building block codons that determine the identity of three macrocycle building blocks; and
at least a fourth building block codon that identifies a bis-amino acid scaffold at the 5′ end of the template.

### Claim 36 (depends on 35)

The method of claim 35, wherein the fourth building block codon is a codon that determines the identity of a building block selected from the group consisting of 4A through 4ZZ depicted in FIG. 3B.

### Claim 37 (depends on 35)

The method of claim 35, wherein a first one of the at least three building block codons is
selected from the codons that determines the identity of building blocks 1A-1T depicted in FIG. 3C;
a second one of the at least three building block codons is selected from the codons that determines the identity of building blocks 2A-2T depicted in FIG. 3C; and
a third one of the at least three building block codons is selected from the codons that determines the identity of building blocks 3A-3T depicted in FIG. 3C.

### Claim 38 (depends on 28)

The method of claim 28, wherein the DNA-templated compound library is synthesized using a chaotropic buffer.

### Claim 39 (depends on 38)

The method of claim 38, wherein synthesized template-linked macrocycles and/or intermediates of the DNA-templated compound library are isolated from dilute solution using a chaotropic buffer.

### Claim 40 (depends on 28, 38)

The method of any one of claim 28 or 38, wherein the chaotropic buffer comprises saturated aqueous guanidine hydrochloride (4:6 ratio with isopropanol).

### Claim 41

A template for DNA-templated synthesis of compounds comprising:
multiple-residue primer-binding sites;
at least one building block codon that determines the identity of a compound building block; and
at least a one building block codon that identifies a scaffold at the 5′ end of the template, wherein the template comprises codons from the orthogonal codon sets depicted in FIG. 5 or 7.

### Claim 42 (depends on 41)

The template of claim 41, wherein the template comprises a combination of orthogonal codons interspaced by constant regions comprising:
multiple-residue primer-binding sites;
at least three building block codons that determine the identity of three compound building blocks; and
at least a fourth building block codon that identifies a bis-amino acid scaffold at the 5′ end of the template.

### Claim 43 (depends on 41)

The template of claim 41, wherein the building block codons determine the identity of macrocycle building blocks.

### Claim 44 (depends on 41)

The template of claim 41, wherein the template is an orthogonal codon set comprising: at least one codon selected from the group consisting of 1A-T, 2A-2T, 3A-3T, 4A-4ZZ, and 4A2 through 4P4 depicted in Table 5 or 7.

### Claim 45 (depends on 44)

The template of claim 44, wherein the template is an orthogonal codon set comprising:
a codon selected from the group consisting of 4A-4ZZ depicted in Table 5;
a codon selected from the group consisting of 1A-1T depicted in Table 5;
a codon selected from the group consisting of 2A-2T depicted in Table 5; and
a codon selected from the group consisting of 3A-3T depicted in Table 5.

### Claim 46 (depends on 41)

The template of claim 41, wherein the template is an orthogonal codon set comprising: a codon selected from the group consisting of 4A2 through 4P4 depicted in Table 7.

### Claim 47 (depends on 41)

The template of claim 41, wherein the template is synthesized using one or more reagents 1, 2, or 3 depicted in Table 6.

### Claim 48 (depends on 41)

The template of claim 41, wherein the template is synthesized using anticodons 1A-1T for DTS reagent 5′-3′ depicted in Table 6.

### Claim 49 (depends on 41)

The template of claim 41, wherein the template is synthesized using anticodons 2A-2T for DTS reagent 5′-3′ depicted in Table 6.

### Claim 50 (depends on 41)

The template of claim 41, wherein the template is synthesized using anticodons 3A-3T for DTS reagent 5′-3′ depicted in Table 6.

### Claim 51 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)

A pharmaceutical composition comprising a compound of any one of claims 1-27, and optionally a pharmaceutically acceptable excipient.

### Claim 52 (depends on 51)

The pharmaceutical composition of claim 51 further comprising an additional pharmaceutical agent.

### Claim 53 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)

A method of treating a disease, impaired insulin signaling, or insulin resistance, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-27.

### Claim 54 (depends on 53)

The method of claim 53, wherein the disease is associated with aberrant protease activity.

### Claim 55 (depends on 54)

The method of claim 54, wherein the aberrant protease activity is aberrant IDE activity.

### Claim 56 (depends on 53)

The method of claim 53, wherein the compound is administered in an amount effective to reduce IDE activity in the subject to less than about 50% as compared to the IDE activity in the absence of the compound.

### Claim 57 (depends on 56)

The method of claim 56, wherein the IDE activity is plasma IDE activity and/or pancreas IDE activity.

### Claim 58 (depends on 53)

The method of claim 53, wherein the subject exhibits impaired insulin signaling or insulin resistance.

### Claim 59 (depends on 53)

The method of claim 53, wherein the disease is diabetes.

### Claim 60 (depends on 53)

The method of claim 53, wherein the disease is metabolic syndrome.

### Claim 61 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 51, 52)

Use of a compound of any one of claims 1-27 or a pharmaceutical composition of claim 51 or 52 to treat a disease in a subject in need thereof.

### Claim 62 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 51, 52)

A kit comprising:
a compound of any one of claims 1-27 or a pharmaceutical composition of claim 51 or 52; and
instructions for administering to a subject or contacting a biological sample with the compound or pharmaceutical composition.