Methods and compounds for treating genetic diseases

Abstract

The present disclosure relates to methods and compounds for treating genetic diseases, in particular, compounds and methods for modulating the expression of dmpk, and treating diseases and conditions in which dmpk plays a positive role. The compound may be a transcriptional regulating molecule having a first end, a second end, and an oligomeric backbone, wherein: a) the first end comprises a DNA binding moiety capable of non-covalently binding to a nucleotide repeat CAG or CTG; b) the second terminal comprises a protein binding moiety that binds to a regulatory molecule that modulates the expression of a gene comprising the nucleotide repeat CAG or CTG; and c) the oligomeric backbone comprises a linker between the first end and the second end.

Description

[0001] This application is a divisional application of Chinese patent application No. 202180027594.3, entitled "Method and Compound for Treating Genetic Diseases" (the corresponding PCT application was filed on February 3, 2021, with application number 202180027594.3).

[0002] Cross-references

[0003] This application claims the benefits of U.S. Application No. 62 / 969,644, filed February 3, 2020, and U.S. Application No. 63 / 135,476, filed January 8, 2021, which are incorporated herein by reference in their entirety. Invention Field

[0004] This article discloses novel chimeric heterocyclic polyamide compounds and compositions, as well as their applications as pharmaceuticals for treating diseases. Methods for modulating the expression of fxn in human or animal subjects are also provided for the treatment of diseases such as Friedreich's ataxia. Background Technology

[0005] This disclosure relates to the treatment of hereditary diseases characterized by excessive production of mRNA.

[0006] Friedreich ataxia (FA or FRDA) is an autosomal recessive neurodegenerative disorder caused by mutations in the fxn gene, which encodes the protein frataxin (FXN), an iron-binding mitochondrial protein involved in electron transport and metabolism. In most individuals with FA, the first intron of fxn contains a GAA trinucleotide repeat (approximately 66 to over 1000 trinucleotides), and this overamplification leads to the observed pathology. This overamplification of the GAA repeat results in decreased FXN expression.

[0007] Friedreich's ataxia is characterized by progressive degeneration of the nervous system, particularly sensory neurons. Furthermore, cardiomyocytes and pancreatic β-cells are susceptible to ataxia due to the reduction of ataminants. Symptoms typically appear by age 18; however, late diagnosis of FA is not uncommon. FA patients exhibit neurodegeneration of large sensory neurons and the spinocerebellar tract, as well as cardiomyopathy and diabetes. Clinical symptoms of FA include ataxia; gait ataxia; muscle weakness; loss of upper body strength; loss of balance; lack of reflexes in the lower limbs and tendons; loss of sensation (especially vibration); impaired proprioception; impaired perception of temperature, touch, and pain; hearing and vision impairment, including color vision distortion and involuntary eye movements; abnormal foot morphology, including pes cavus and inversion; hearing impairment; dysarthria; dysphagia; respiratory impairment; scoliosis; diabetes; glucose and carbohydrate intolerance; and cardiac dysfunction, including hypertrophic cardiomyopathy, arrhythmias, myocardial fibrosis, and heart failure. Currently, there is no cure for FA. Medical treatment is limited to surgical interventions targeting the spine and heart, as well as therapies to assist in balance and coordination, movement and speech. Summary of the Invention

[0008] This disclosure utilizes regulatory molecules present in the cell nucleus that control gene expression. Eukaryotic cells provide several mechanisms for controlling gene replication, transcription, and / or translation. Regulatory molecules produced within the cell through various biochemical mechanisms can regulate various processes involved in the conversion of genetic information into cellular components. It is known that several regulatory molecules regulate mRNA production, and that if these molecules target fxn, they will regulate the production of fxn mRNA that leads to Friedreich's ataxia, thus reversing disease progression.

[0009] This disclosure provides compounds and methods for recruiting regulatory molecules to sites immediately adjacent to fxn. The disclosed compounds contain: (a) a recruitment moiety that binds to the regulatory molecule, said recruitment moiety being linked to (b) a DNA-binding moiety that selectively binds to fxn. The compounds will resist the expression of defective fxn in such a manner as:

[0010] (1) The DNA-binding portion will selectively bind to the characteristic GAA trinucleotide repeat sequence of fxn;

[0011] (2) The recruitment portion attached to the DNA binding site will therefore remain adjacent to fxn;

[0012] (3) Currently, the recruitment portion adjacent to fxn recruits regulatory molecules to neighboring genes; and

[0013] (4) Regulatory molecules will regulate expression, thus resisting the generation of defective fxn by interacting directly with genes.

[0014] The mechanisms described above would provide an effective treatment for Friedreich's ataxia caused by defective fxn expression. Therefore, correcting the expression of the defective fxn gene represents a promising approach for the treatment of Friedreich's ataxia.

[0015] This disclosure provides a recruitment motif that binds to a regulatory molecule. Small molecule inhibitors of the regulatory molecule serve as templates for designing the recruitment motif, as these inhibitors typically function by non-covalently binding to the regulatory molecule.

[0016] This disclosure also provides a DNA-binding moiety that selectively binds to one or more copies of the GAA trinucleotide repeat, which is characteristic of the defective fxn gene. The selective binding of the DNA-binding moiety to fxn, made possible by the high GAA count associated with the defective fxn gene, will guide the moiety to be recruited to a neighboring gene and to the appropriate location to upregulate gene transcription.

[0017] The DNA-binding portion will contain a polyamide segment that will selectively bind to the target GAA sequence. Polyamides that selectively bind to selected DNA sequences have been designed by Dervan and others. These polyamides are located in the minor groove of the double-stranded DNA and form hydrogen-bonded interactions with Watson-Crick base pairs. Polyamides that selectively bind to specific DNA sequences can be designed by linking monoamide constructs according to defined chemical rules. A construct is provided for each DNA base pair, where each construct binds non-covalently and selectively to one of the following DNA base pairs: A / T, T / A, G / C, and C / G. Following this guideline, the trinucleotide will bind to a molecule having three amide units, i.e., a triamide. Generally, these polyamides will be oriented in either direction of the DNA sequence such that the 5'-GAA-3' trinucleotide repeat sequence of fxn can be targeted by a polyamide selective for either GAA or AAG. In addition, polyamides that bind to complementary sequences (in this case TTC or CTT) will also bind to the trinucleotide repeat sequence of fxn, and can also be used.

[0018] In theory, longer DNA sequences can be targeted with higher specificity and / or higher affinity by combining a larger number of monoamide building blocks into longer polyamide chains. Ideally, the binding affinity of the polyamide would simply equal the sum of the interactions of each individual monoamide / DNA base pair. However, in reality, due to the rather rigid geometric mismatch between the polyamide and the DNA structure, longer polyamide sequences do not bind as tightly to longer DNA sequences as would be expected from a simple additive contribution. The geometric mismatch between the longer polyamide sequence and the longer DNA sequence induces unfavorable geometric strain, which reduces the originally expected binding affinity.

[0019] Therefore, this disclosure provides a DNA portion comprising a triamide linked by a flexible spacer region. The spacer region mitigates geometric strain that would otherwise reduce the binding affinity of larger polyamide sequences.

[0020] This article discloses polyamide compounds that can bind to one or more copies of the trinucleotide repeat sequence GAA and can modulate the expression of the defective fxn gene. Treatment with these compounds will resist the expression of the defective fxn gene, and this will reduce the occurrence, severity, and / or frequency of symptoms associated with Friedreich ataxia. Some of the compounds disclosed herein will provide higher binding affinity and / or selectivity than those previously observed for compounds in this class.

[0021] Other objectives, features, and advantages of the block copolymers, methods, and compositions described herein will become apparent from the following detailed description. However, it should be understood that while specific embodiments are indicated, the detailed description and specific examples are given by way of illustration only, as various variations and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art from this detailed description.

[0022] Incorporation

[0023] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent or patent application had been specifically and individually indicated to be incorporated herein by reference. Attached Figure Description

[0024] The various aspects of this disclosure are specifically set forth in the appended claims. A fuller understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of illustrative embodiments in which the principles of this disclosure are utilized, and the accompanying drawings.

[0025] Figure 1 The changes in fxn gene expression in cells of patients with Friedreich ataxia were shown after treatment with transcriptional regulatory molecules. Detailed Implementation

[0026] The transcriptional regulatory molecules described herein represent a fusion of chemistry, biology, and precision medicine because they can be programmed to regulate the expression of target genes containing the nucleotide repeat GAA. These transcriptional regulatory molecules contain a DNA-binding moiety that selectively binds to one or more copies of the GAA hexanucleotide repeat, a characteristic feature of the defective fxn gene. They also contain a moiety that binds to a regulatory protein. This selective binding to the target gene causes the regulatory protein to proximity to the target gene, thus downregulating its transcription. The molecules and compounds disclosed herein offer higher binding affinity and selectivity than previously observed with compounds in this class, and thus provide more effective treatment for diseases associated with the defective fxn gene.

[0027] Treatment with these compounds modulates the expression of the defective fxn gene, which reduces the occurrence, severity, or frequency of ALS-related symptoms. The transcriptional regulatory molecules described herein recruit regulatory molecules to modulate the expression of the defective fxn gene and effectively treat and alleviate symptoms associated with diseases such as Friedreich ataxia.

[0028] transcriptional regulatory molecules

[0029] The transcriptional regulatory molecules disclosed herein have activity useful for regulating the transcription of target genes (e.g., fxn) having one or more GAA repeats, and can be used to treat or prevent diseases or conditions in which the target gene (e.g., fxn) plays an active role. Therefore, in a broad aspect, some embodiments also provide pharmaceutical compositions comprising one or more of the compounds disclosed herein and a pharmaceutically acceptable carrier, and methods for preparing and using said compounds and compositions. Some embodiments provide methods for regulating the expression of fxn. Other embodiments provide methods for treating fxn-mediated conditions in patients requiring such treatment, the methods comprising administering a therapeutically effective amount of the disclosed compounds or compositions to said patient. Use of certain compounds disclosed herein in the manufacture of medicaments for treating diseases or conditions improved by regulating the expression of fxn is also provided.

[0030] Some embodiments relate to a transcriptional regulatory molecule or compound having a first end, a second end, and an oligomeric backbone, wherein: a) the first end comprises a DNA-binding portion capable of non-covalently binding to the nucleotide repeat sequence GAA; b) the second end comprises a protein-binding portion binding to a regulatory molecule that regulates the expression of a gene containing the nucleotide repeat sequence GAA; and c) the oligomeric backbone comprises a linker between the first and second ends. In some embodiments, the second end is not a Brd4-binding portion.

[0031] In some embodiments, the compound has structural formula (I):

[0032] XLY

[0033] Formula (I)

[0034] Or its salt, wherein:

[0035] X contains a recruitment portion that can non-covalently bind to the regulatory portion within the core;

[0036] Y contains a DNA recognition portion capable of non-covalently binding to one or more copies of the trinucleotide repeat sequence GAA; and

[0037] L stands for connector.

[0038] Some of the compounds disclosed herein may have activity useful for regulating the transcription of fxn and may be used to treat and / or prevent diseases or conditions in which fxn plays an active role. Therefore, in a broad sense, some embodiments also provide pharmaceutical compositions comprising one or more of the compounds disclosed herein and a pharmaceutically acceptable carrier, and methods for preparing and using said compounds and compositions. Some embodiments provide methods for regulating fxn expression. Other embodiments provide methods for treating fxn-mediated conditions in patients requiring such treatment, said methods comprising administering a therapeutically effective amount of a compound or composition disclosed herein to said patient. Use of some of the compounds disclosed herein in the manufacture of medicaments for treating diseases or conditions improved by regulating fxn expression is also provided.

[0039] In some embodiments, the regulatory molecules are selected from proteins containing bromo domains, nucleosome remodeling factor (NURF), bromo domain PHD finger transcription factor (BPTF), ten-eleven translocation enzyme (TET), methylcytosine dioxygenase (TET1), DNA demethylase, helicase, acetyltransferase, and histone deacetylase (“HDAC”).

[0040] In some implementations, the first end is Y, the second end is X, and the oligomeric backbone is L.

[0041] In some embodiments, the compound has structural formula (II):

[0042] XL-(Y1-Y2-Y3) n -Y0

[0043] Equation (II)

[0044] or its salt, wherein:

[0045] X contains a recruitment portion that can non-covalently bind to regulatory molecules within the nucleus;

[0046] L stands for connector;

[0047] Y1, Y2, and Y3 are internal subunits, each containing components selected from heterocyclic or C-rings. 1-6 A portion of the straight-chain aliphatic segment, and each of them is chemically linked to its two adjacent components;

[0048] Y0 is a terminal subunit that contains a portion selected from heterocyclic or straight-chain aliphatic segments, which is chemically linked to its single neighbor.

[0049] Each subunit can non-covalently bind to a single nucleotide in the GAA repeat sequence;

[0050] n is an integer between 1 and 200, inclusive; and

[0051] (Y1-Y2-Y3) n -Y0 combinations form a DNA recognition portion capable of non-covalently binding to one or more copies of the trinucleotide sequence GAA.

[0052] In some implementations, the compound of structure (II) contains a subunit for each individual nucleotide in the GAA repeat sequence.

[0053] In some embodiments, each internal subunit has an amino (-NH-) and a carboxyl (-CO-) group.

[0054] In some embodiments, compounds of structure (II) contain amide (-NHCO-) ​​bonds between each pair of internal subunits.

[0055] In some embodiments, compounds of structure (II) contain an amide (-NHCO-) ​​bond between L and the leftmost inner subunit.

[0056] In some embodiments, the compound of structure (II) contains an amide bond between the rightmost inner subunit and the terminal subunit.

[0057] In some implementations, each subunit comprises portions independently selected from heterocyclic and aliphatic chains.

[0058] In some embodiments, the heterocycle is a monocyclic heterocycle. In some embodiments, the heterocycle is a monocyclic 5-membered heterocycle. In some embodiments, each heterocycle contains a heteroatom independently selected from N, O, or S. In some embodiments, each heterocycle is independently selected from pyrrole, imidazole, thiazole, oxazole, thiophene, and furan.

[0059] In some implementations, the aliphatic chain is C 1-6 A straight-chain aliphatic chain. In some embodiments, the aliphatic chain has the structural formula -(CH2).m -, m is selected from 1, 2, 3, 4, and 5. In some embodiments, the aliphatic chain is -CH2CH2-.

[0060] In some implementations, each subunit comprises portions independently selected from the following:

[0061] -NH-benzopyrazinyl-CO-, -NH-phenylene-CO-, -NH-pyridyl-CO-, -NH-piperidinyl-CO-, -NH-pyrimidinyl-CO-, -NH-anthrayl-CO-, -NH-quinoline-CO- and Where Z represents H, NH2, and C. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Alkyl-NH2.

[0062] In some implementations, Py is I am HP is Th is Pz is Nt is Tn is Nh is iNt is iIm is HpBi is ImBi is PyBi is Dp is ,-NH-benzopyrazine-CO- is -NH-phenylene-CO- is -NH-pyridyl-CO- is -NH-piperidinyl-CO- is -NH-pyrazinyl-CO- is -NH-anthracene-CO- is And -NH-quinoline-CO- is In some implementations, Py is I am HP is Th is Pz is Nt is Tn is Nh is iNt is And iIm is

[0063] In some embodiments, n is between 1 and 100, including the end value. In some embodiments, n is between 1 and 50, including the end value. In some embodiments, n is between 1 and 20, including the end value. In some embodiments, n is between 1 and 10, including the end value. In some embodiments, n is between 1 and 5, including the end value. In some embodiments, n is an integer between 1 and 3, including the end value. In some embodiments, n is selected from 1 and 2. In some embodiments, n is 1.

[0064] In some implementations, n is an integer between 1 and 5, including end values.

[0065] In some implementations, n is an integer between 1 and 3, including the end value.

[0066] In some implementations, n is an integer between 1 and 2, including end values.

[0067] In some implementations, n is 1.

[0068] In some implementations, L includes C 1-6 Straight-chain aliphatic segment.

[0069] In some implementations, L contains (CH2OCH2). m And m is an integer between 1 and 20, including the end value. In some other embodiments, m is an integer between 1 and 10, including the end value. In some other embodiments, m is an integer between 1 and 5, including the end value.

[0070] In some embodiments, the compound has structural formula (III):

[0071] XL-(Y1-Y2-Y3)-(W-Y1-Y2-Y3) n -Y0

[0072] Equation (III)

[0073] or its salt, wherein:

[0074] X contains a recruitment portion that can non-covalently bind to regulatory molecules within the nucleus;

[0075] L stands for connector;

[0076] Y1, Y2, and Y3 are internal subunits, each containing components selected from heterocyclic or C-rings. 1-6 A portion of the straight-chain aliphatic segment, and each of them is chemically linked to its two adjacent components;

[0077] Y0 is a terminal subunit that contains a portion selected from heterocyclic or straight-chain aliphatic segments, which is chemically linked to its single neighbor.

[0078] Each subunit can non-covalently bind to a single nucleotide in the GAA repeat sequence;

[0079] W is the interval region;

[0080] n is an integer between 1 and 200, inclusive; and

[0081] (Y1-Y2-Y3)-(W-Y1-Y2-Y3) n -Y0 combinations form a DNA recognition portion capable of non-covalently binding to one or more copies of the trinucleotide repeat sequence GAA.

[0082] In some implementations, Y1-Y2-Y3 are:

[0083]

[0084] In some implementations, Y1-Y2-Y3 are:

[0085]

[0086] In some implementations, Y1-Y2-Y3 is Im-Py-β.

[0087] In some implementations, Y1-Y2-Y3 is Im-Im-β.

[0088] In some implementations, each Y1-Y2-Y3 is independently selected from β-Py-Im and β-Im-Im.

[0089] In some implementations, at most one Y1-Y2-Y3 is β-Im-Im.

[0090] In some embodiments of the compound of formula (III), n is between 1 and 100, including the end values. In some embodiments of the compound of formula (III), n is between 1 and 50, including the end values. In some embodiments of the compound of formula (III), n is between 1 and 20, including the end values. In some embodiments of the compound of formula (III), n is between 1 and 10, including the end values. In some embodiments of the compound of formula (III), n is between 1 and 5, including the end values. In some embodiments of the compound of formula (III), n is selected from 1 and 2. In some embodiments of the compound of formula (III), n is 1.

[0091] In some embodiments, the compound has structural formula (IV):

[0092] XL-(Y1-Y2-Y3)-V-(Y4-Y5-Y6)-Y0

[0093] Formula (IV)

[0094] or its salt, wherein:

[0095] X contains a recruitment portion that can non-covalently bind to regulatory molecules within the nucleus;

[0096] Y1, Y2, Y3, Y4, Y5, and Y6 are internal subunits, each containing components selected from heterocyclic or C-rings. 1-6 A portion of the straight-chain aliphatic segment, and each of them is chemically linked to its two adjacent components;

[0097] Y0 is a terminal subunit that contains a portion selected from heterocyclic or straight-chain aliphatic segments, which is chemically linked to its single neighbor.

[0098] Each subunit can non-covalently bind to a single nucleotide in the GAA repeat sequence;

[0099] L stands for connector;

[0100] V is a corner component used to form the hairpin corner;

[0101] n is an integer between 1 and 200, inclusive; and

[0102] (Y1-Y2-Y3)-V-(Y4-Y5-Y6)-Y0 combine to form a DNA recognition portion capable of non-covalently binding to one or more copies of the trinucleotide repeat sequence GAA.

[0103] In some embodiments of the compound of formula (IV), n is between 1 and 100, including the end values. In some embodiments of the compound of formula (IV), n is between 1 and 50, including the end values. In some embodiments of the compound of formula (IV), n is between 1 and 20, including the end values. In some embodiments of the compound of formula (IV), n is between 1 and 10, including the end values. In some embodiments of the compound of formula (IV), n is between 1 and 5, including the end values. In some embodiments of the compound of formula (IV), n is selected from 1 and 2. In some embodiments of the compound of formula (IV), n is 1.

[0104] In some implementations, V is -HN-CH2CH2CH2-CO-.

[0105] In some embodiments, the compound has the structural formula (V):

[0106] XC(=O)-CH2CH2-(Y1-Y2-Y3) n -NH-Y0

[0107] Formula (V)

[0108] or its salt, wherein:

[0109] X contains a recruitment portion that can non-covalently bind to regulatory molecules within the nucleus;

[0110] Each Y1-Y2-Y3 is independently selected from β-Py-Im and β-Im-Im;

[0111] Y0 is a terminal subunit comprising a portion selected from heterocyclic or straight-chain aliphatic segments, chemically linked to its single neighbor; and

[0112] n is an integer between 1 and 200, inclusive.

[0113] In some embodiments of the compound of structural formula (V), at most one of Y1-Y2-Y3 is β-Im-Im.

[0114] In some embodiments of the compound of structural formula (V), Y1-Y2-Y3 is β-Py-Im.

[0115] In some embodiments of the compound of structural formula (V), n is between 1 and 100, including the end values. In some embodiments of the compound of structural formula (V), n is between 1 and 50, including the end values. In some embodiments of the compound of structural formula (V), n is between 1 and 20, including the end values. In some embodiments of the compound of structural formula (V), n is between 1 and 10, including the end values. In some embodiments of the compound of structural formula (V), n is between 1 and 5, including the end values. In some embodiments of the compound of structural formula (V), n is selected from 1 and 2. In some embodiments of the compound of structural formula (V), n is 1.

[0116] In some embodiments, the compound has structural formula (VI):

[0117]

[0118] or its salt, wherein:

[0119] X contains a recruitment portion that can non-covalently bind to regulatory molecules within the nucleus;

[0120] Y0 is a terminal subunit containing a portion selected from heterocyclic or straight-chain aliphatic segments, chemically linked to its single neighbor; and n is an integer between 1 and 200, including terminal values.

[0121] In some embodiments of the compound of structural formula (VI), n is between 1 and 100, including the end values. In some embodiments of the compound of structural formula (VI), n is between 1 and 50, including the end values. In some embodiments of the compound of structural formula (VI), n is between 1 and 20, including the end values. In some embodiments of the compound of structural formula (VI), n is between 1 and 10, including the end values. In some embodiments of the compound of structural formula (VI), n is between 1 and 5, including the end values. In some embodiments of the compound of structural formula (VI), n is selected from 1 and 2. In some embodiments of the compound of structural formula (VI), n is 1.

[0122] In some embodiments, the compound has structural formula (VII):

[0123]

[0124] or its salt, wherein:

[0125] X contains a recruitment portion capable of non-covalently binding to regulatory molecules within the nucleus; and

[0126] W is the interval region;

[0127] Y0 is a terminal subunit comprising a portion selected from heterocyclic or straight-chain aliphatic segments, chemically linked to its single neighbor; and

[0128] n is an integer between 1 and 200, inclusive.

[0129] In some embodiments of the compound of structural formula (VII), n is between 1 and 100, including the end values. In some embodiments of the compound of structural formula (VII), n is between 1 and 50, including the end values. In some embodiments of the compound of structural formula (VII), n is between 1 and 20, including the end values. In some embodiments of the compound of structural formula (VII), n is between 1 and 10, including the end values. In some embodiments of the compound of structural formula (VII), n is between 1 and 5, including the end values. In some embodiments of the compound of structural formula (VII), n is selected from 1 and 2. In some embodiments of the compound of structural formula (VII), n is 1.

[0130] In some embodiments of the compound of structural formula (VII), wherein: W is -NHCH2-(CH2OCH2) p -CH2CO-; and p is an integer between 1 and 4, including the end value.

[0131] In some implementations, (V) is -(CH2). a -NR 1 -(CH2)b -、-(CH2) a -、-(CH2) a -O-(CH2) b -、–(CH2) a- CH(NHR 1 )-、–(CH2) a -CH(NHR 1 )-、–(CR 2 R 3 ) a -or-(CH2) a -CH(NR 1 3) + -(CH2) b - where each a is an independent integer between 2 and 4; R 1 H, or C which is optionally substituted. 1-6 Alkyl groups, optionally substituted C 3-10 cycloalkyl, optionally substituted C 6-10 Aryl, optionally substituted 4-10-membered heterocyclic, or optionally substituted 5-10-membered heteroaryl; each R 2 and R 3 Independently, it is H, halogen, OH, NHAc, or C. 1-4 Alkyl group. In some embodiments, R 1 It is H. In some implementations, R 1 It is a C group optionally substituted with 1-3 substituents selected from -C(O)-phenyl. 1-6 Alkyl group. In some embodiments, (V) is –(CR) 2 R 3 -(CH2)a- or –(CH2)a-(CR) 2 R 3 )-(CH2) b - where each a is independently 1-3, b is 0-3, and each R 2 and R 3 Independently, it is H, halogen, OH, NHAc, or C. 1-4 Alkyl group. In some embodiments, (V) is -(CH2)-CH(NH3). + -(CH2)- or -(CH2)-CH2CH(NH3) + -

[0132] In one aspect, the disclosed compound binds to the GAA of fxn and recruits the regulatory moiety to the vicinity of fxn. Because the regulatory moiety is close to the gene, it is more likely to regulate the expression of fxn.

[0133] Embodiments are also provided in which any of the compounds disclosed above, including those of formulas (I)–(VIII), are deuterated individually, partially, or completely. Methods for achieving deuteration of hydrogen are known in the art.

[0134] It also provides implementation schemes in which any of the above implementation schemes can be combined with any one or more of these implementation schemes, provided that the combinations are not mutually exclusive.

[0135] As used herein, two embodiments are "mutually exclusive" when one embodiment is defined as something different from another embodiment. For example, an embodiment in which two groups are combined to form a cycloalkyl group is mutually exclusive with an embodiment in which one group is ethyl and the other is hydrogen. Similarly, an embodiment in which one group is CH2 is mutually exclusive with an embodiment in which the group is NH.

[0136] In one aspect, the disclosed compound binds to the GAA of fxn and recruits the regulatory moiety to the vicinity of fxn. Because the regulatory moiety is close to the gene, it is more likely to regulate the expression of fxn.

[0137] In one aspect, the compounds of this disclosure provide a polyamide sequence for the interaction of a single polyamide subunit with each base pair in a GAA repeating sequence. In another aspect, the compounds of this disclosure provide a turn component V to enable hairpin binding of the compound with GAA, wherein each nucleotide pair interacts with two subunits of the polyamide.

[0138] In one aspect, the compounds disclosed herein provide more than one copy of a polyamide sequence for non-covalent bonding with fxn, and individual polyamide sequences in this compound are connected by spacer regions W as defined above. The spacer regions W allow the compound to adjust its geometry as needed to mitigate geometric strain that would otherwise affect the non-covalent bonding of longer polyamide sequences.

[0139] First-terminal DNA-binding region

[0140] The first end interacts with and binds to the gene, particularly the minor groove of the GAA sequence. In one aspect, the compounds disclosed herein provide a polyamide sequence for the interaction of a single polyamide subunit with each base pair in a GAA repeat sequence. In another aspect, the compounds disclosed herein provide a turn assembly (e.g., an aliphatic amino acid moiety) to enable hairpin binding of the compound to GAA, wherein each nucleotide pair interacts with both subunits of the polyamide.

[0141] In one respect, because of the high number of GAA repeats associated with fxn, the compounds disclosed herein are more likely to bind to repeating GAAs of fxn than to GAAs that bind to other parts of the DNA of the target.

[0142] In one aspect, the compounds of this disclosure provide more than one copy of a polyamide sequence for non-covalent binding with GAA. In another aspect, the compounds of this disclosure bind to fxn with greater affinity compared to corresponding compounds containing a single polyamide sequence.

[0143] In one aspect, the compounds disclosed herein provide more than one copy of a polyamide sequence for non-covalent bonding with GAA, and the individual polyamide sequences in this compound are connected by spacer regions W as defined above. The spacer regions W allow the compound to adjust its geometry as needed to mitigate geometric strain that would otherwise affect the non-covalent bonding of the longer polyamide sequences.

[0144] In some implementations, the DNA recognition or binding portion binds in the minor groove of the DNA.

[0145] In some implementations, the DNA recognition or binding portion comprises a polymeric sequence of monomers, wherein each monomer in the polymer selectively binds to a specific DNA base pair.

[0146] In some implementations, the DNA recognition or binding portion comprises a polyamide portion.

[0147] In some embodiments, the DNA recognition or binding portion comprises a polyamide portion containing heteroaromatic monomers, wherein each heteroaromatic monomer is non-covalently bound to a specific nucleotide, and each heteroaromatic monomer is attached to one or more of its neighbors via amide bonds.

[0148] In some embodiments, the DNA recognition portion binds to a sequence containing at least 1000 pentanucleotide repeats. In some embodiments, the DNA recognition portion binds to a sequence containing at least 500 trinucleotide repeats. In some embodiments, the DNA recognition portion binds to a sequence containing at least 200 trinucleotide repeats. In some embodiments, the DNA recognition portion binds to a sequence containing at least 100 trinucleotide repeats. In some embodiments, the DNA recognition portion binds to a sequence containing at least 50 trinucleotide repeats. In some embodiments, the DNA recognition portion binds to a sequence containing at least 20 trinucleotide repeats.

[0149] In some embodiments, the compound includes a cell-penetrating ligand portion.

[0150] In some implementations, the cell-penetrating ligand portion is a polypeptide.

[0151] In some implementations, the cell-penetrating ligand portion is a polypeptide containing fewer than 30 amino acid residues.

[0152] In some embodiments, the polypeptide is selected from any one of SEQ ID NO.1 to SEQ ID NO.37, including SEQ ID NO.1 and SEQ ID NO.37.

[0153] The form of the selected polyamide can vary based on the target gene. The first end may include a polyamide selected from linear polyamides, hairpin polyamides, H-pin polyamides, overlapping polyamides, slip polyamides, cyclic polyamides, tandem polyamides, and extended polyamides. In some embodiments, the first end comprises a linear polyamide. In some embodiments, the first end comprises a hairpin polyamide.

[0154] The binding affinity between the polyamide and the target gene can be tuned based on the composition of the polyamide. In some embodiments, the polyamide is capable of binding DNA with an affinity of less than about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 250 nM, about 200 nM, about 150 nM, about 100 nM, or about 50 nM. In some embodiments, the polyamide is capable of binding DNA with an affinity of less than about 300 nM. In some embodiments, the polyamide is capable of binding DNA with an affinity of less than about 500 nM. In some embodiments, the polyamide is capable of binding DNA with an affinity of less than about 300 nM. In some embodiments, the polyamide is capable of binding DNA with an affinity of less than about 200 nM. In some embodiments, the polyamide is capable of binding DNA with an affinity of less than about 100 nM. In some embodiments, the polyamide is capable of binding DNA with an affinity of less than about 50 nM. In some embodiments, the polyamide is capable of binding DNA with an affinity greater than about 200 nM, about 150 nM, about 100 nM, about 50 nM, about 10 nM, or about 1 nM. In some embodiments, the polyamide is capable of binding DNA with an affinity in the range of about 1-600 nM, 10-500 nM, 20-500 nM, 50-400 nM, or 100-300 nM.

[0155] The binding affinity between polyamide and target DNA can be determined using quantitative footprint titration assays. The experiment involves measuring the dissociation constant K of the polyamide against the target sequence at 24°C or 37°C. d And the use of standard polyamide to determine solution conditions or approximate intracellular solution conditions.

[0156] The binding affinity between a regulatory protein and a ligand at the second terminal can be determined using assays suitable for the specific protein. The experiment involves measuring the dissociation constant K of the ligand with respect to the protein. dAnd the use of standard protein assay solution conditions or approximate intracellular solution conditions.

[0157] In some embodiments, the first terminal comprises –NH-QC(O)-, where Q is C that is optionally substituted. 6-10 The arylene, optionally substituted 4-10-membered heterocyclic alkylene, optionally substituted 5-10-membered heteroarylene, or optionally substituted alkylene. In some embodiments, Q is an optionally substituted C. 6-10 The arylene group or optionally substituted 5-10-membered heteroarylene group. In some embodiments, Q is an optionally substituted 5-10-membered heteroarylene group. In some embodiments, the 5-10-membered heteroarylene group is optionally substituted by 1-4 substituents selected from: H, OH, halogen, C. 1-10 Alkyl, NO2, CN, NR′R″, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, (C 1-6 Alkoxy)C 1-6 Alkyl, C 2-10 alkenyl, C 2-10 alkynyl group, C 3-7 Carbocyclic group, 4-10 membered heterocyclic group, C 6-10 Aryl, 5-10 heteroaryl, (C 3-7 (Carbocyclic group)C 1-6 Alkyl, (4-10 membered heterocyclic) C 1-6 Alkyl, (C 6-10 Aryl)C 1-6 Alkyl, (C 6-10 Aryl)C 1-6 Alkoxy, (5-10 heteroaryl)C 1-6 Alkyl, (C 3-7 (Carbocyclic)-amines, (4-10 membered heterocyclic)amines, (C 6-10 (aryl)amine, (5-10 heteroaryl)amine, acyl, C-carboxyl, O-carboxyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, -SR ’ , COOH or CONR′R″; where each R′ and R″ is independently H, C 1-10 Alkyl, C 1-10 Haloalkyl, C 1-10 Alkyl group.

[0158] In some embodiments, the first terminal comprises –NH-QC(O)-, where Q is C that is optionally substituted. 6-10 arylene, optionally substituted 4-10-membered heterocyclic alkylene, optionally substituted 5-10-membered heteroarylene, or optionally substituted alkylene.

[0159] In some embodiments, the first terminal includes at least three aromatic carboxamide moieties selected to correspond to the nucleotide repeat sequence GAA, and at least one aliphatic amino acid residue selected from glycine, β-alanine, γ-aminobutyric acid, 2,4-diaminobutyric acid, and 5-aminovaleric acid. In some embodiments, the first terminal includes at least one β-alanine subunit. In some embodiments, the first terminal includes at least three heteroaromatic carboxamide moieties containing at least one heteroatom selected from O, N, and S, and at least one aliphatic amino acid residue selected from glycine, β-alanine, γ-aminobutyric acid, 2,4-diaminobutyric acid, and 5-aminovaleric acid.

[0160] In some implementations, the heteroaromatic carboxamide moiety is a monocyclic or bicyclic moiety.

[0161] In some embodiments, the monomer element is independently selected from optionally substituted pyrrolic carboxamide monomers, optionally substituted imidazole carboxamide monomers, optionally substituted CC-linked heterocyclic / heterocyclic moieties, and β-alanine. The first end comprises one or more carboxamide moieties selected from optionally substituted pyrrolic carboxamide monomers, optionally substituted imidazole carboxamide monomers, and β-alanine monomers.

[0162] In some embodiments, the first terminal contains a (A-1) structure or a pharmaceutically acceptable salt thereof:

[0163] –L 1a -[AM] p –E1

[0164] (A-1)

[0165] in;

[0166] Each [AM] appears p times, where p is an integer in the range 1 to 10.

[0167] L 1a It is key, C 1-6 Alkylene, -NR a -C 1-6 Alkylene-C(O)-, -NR a C(O)-、-NR a -C 1-6 Alkylene, -O- or -OC 1-6 Alkylene;

[0168] Each A is selected from key, C 1-10 Alkylene, optionally substituted C 6-10 arylene, optionally substituted 4-10 heterocyclic arylene, optionally substituted 5-10 heteroarylene, -C 1-10 Alkylene -C(O)-, -C1-10 Alkylene-NR a -、—CO—、—NR a —、—CONR a —、—CONR a C 1-4 Alkylene-, -NR a CO-C 1-4 Alkylene—, —C(O)O—, —O—, —S—, —S(O)—, —S(O)2—, —C(=S)-NH—, —C(O)-NH-NH—, —C(O)-N=N—, —C(O)-CH=CH—, (CH2) 0-4 -CH = CH-(CH2) 0-4 -N(CH3)-C 1-6 Alkylene and -NH-C 1-6 Alkylene -NH-, -OC 1-6 Alkylenes -O-, -NH-N=N-, -NH-C(O)-NH-, and any combination thereof, and at least one A is -CONH-;

[0169] Each M is a C that is optionally replaced. 6-10 arylene, optionally substituted 4-10-membered heterocyclic alkylene, optionally substituted 5-10-membered heteroarylene or optionally substituted alkylene;

[0170] E1 is H or –A E -G;

[0171] A E It does not exist or is –NHCO-;

[0172] G is selected from C, which is arbitrarily replaced. 6-10 Aryl, optionally substituted 4-10 membered heterocyclic, optionally substituted 5-10 membered heteroaryl, optionally substituted C 1-6 Alkyl, C 0-4 Alkylene -NHC(=NH)NH, -CN, -C 0-4 Alkylene-C(=NH)(NR) a R b -C 0-4 Alkylene-C(=N) + H2)(NR a R b C 1-5 Alkylene-NR a R b C 0-4 Alkylene-NHC(=NH)R a and optionally substituted amines; and

[0173] Each R a and R b C, independently selected from H and optionally substituted. 1-6 Alkyl groups, optionally substituted C 3-10 cycloalkyl, optionally substituted C 6-10 Aryl, optionally substituted 4-10 heterocyclic groups and optionally substituted 5-10 heteroaryl groups.

[0174] In some embodiments, the first end comprises a polyamide having the structure of formula (A-2) or a pharmaceutically acceptable salt thereof:

[0175]

[0176] in;

[0177] m1 is 1-4;

[0178] n1 is 0-2;

[0179] Each Y 1 Y 2 Y 3 and Y 4 It can be CH or N independently;

[0180] Each Z 1 Z 2 Z 3 and Z 4 Independently O, S, or NR 1D ;

[0181] W1 is hydrogen, optionally substituted C1-C6 alkyl, -NR 1E -C(O)-NR 1E R 1F -C(O)-NR 1E R 1F Or (AA) 1-10 ;

[0182] W2 is hydrogen, optionally substituted C1-C6 alkyl, -C(O)-NR 1E R 1F Or (AA) 1-10 ;

[0183] in

[0184] AA stands for amino acid residue;

[0185] Each R 1D and R 1E It is independently hydrogen or C1-C6 alkyl; and

[0186] R 1FIt is hydrogen, optionally substituted C1-C 10 Alkyl, C1-C 10 Heteroalkyl, PEG 1-20 Or (AA) 1-10 .

[0187] In some embodiments, W2 is optionally a substituted C1-C6 alkyl group, -C(O)-NR 1E R 1F Or (AA) 1-10 In some implementation schemes, W 2 It is hydrogen.

[0188] In some implementation schemes, W 2 Yes (AA) 1-10 In some embodiments, AA is an amino acid residue selected from β-alanine, lysine, and arginine. In some embodiments, AA is a naturally occurring or non-naturally occurring amino acid. In some embodiments, AA is a naturally occurring amino acid. In some embodiments, AA is β-alanine, lysine, or arginine. In some embodiments, AA is at least one β-alanine.

[0189] In some embodiments, the first end comprises a polyamide having the structure of formula (A-3) or a pharmaceutically acceptable salt thereof:

[0190]

[0191] In some implementation schemes, each Z 1 Z 2 Z 3 and Z 4 Independently is NR 1D , where R 1D It is a C1-C6 alkyl group. In some embodiments, each Z 1 Z 2 Z 3 and Z 4 It is NCH3 on its own.

[0192] In some embodiments, the first end comprises a polyamide having the structure of formula (A-4) or a pharmaceutically acceptable salt thereof:

[0193]

[0194] In some implementation schemes, each Y 1 and Y 3 It is N; and each Y 2 and Y 4 Independently, it is CH or N. In some implementations, each Y... 2 and Y4 Independently, it is CH. In some implementations, each Y... 2 and Y 4 N is independent. In some implementations, Y 2 It is CH, and Y 4 It is N. In some implementations, Y 2 It is N, and Y 4 It is CH.

[0195] In some implementations, m1 is 2 or 3; and n1 is 0 or 1.

[0196] In some implementations, m1 is 2. In some implementations, m1 is 1.

[0197] In some implementations, n1 is 0. In some implementations, n1 is 1.

[0198] In some embodiments, W1 is optionally a substituted C1-C6 alkyl group or a -C(O)-NR group. 1E R 1F In some implementations, W1 is -C(O)-NR 1E R 1F , where R 1E It is hydrogen; and R 1F It is hydrogen, optionally substituted C1-C 10 Alkyl or PEG 1-20 .

[0199] In some implementations, W1 is -C(O)-NR 1E R 1F , where R 1E It is hydrogen; and R 1F Yes (AA) 1-10 In some implementations, AA is an amino acid chain containing 1-4, 1-3, or 1-2 amino acids.

[0200] In some implementation schemes, W 1 Yes (AA) 1-10 In some implementation schemes, W 1It is an amino acid chain. In some embodiments, AA comprises amino acid residues selected from β-alanine, lysine, and arginine. In some embodiments, AA is a naturally occurring or non-naturally occurring amino acid. In some embodiments, AA is a naturally occurring amino acid. In some embodiments, AA is β-alanine, lysine, or arginine. In some embodiments, AA is β-alanine. In some embodiments, AA is arginine. In some embodiments, AA is lysine. In some embodiments, the AA chain length is 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids.

[0201] In some implementations, W1 is hydrogen.

[0202] In some embodiments, the first end comprises a polyamide having the structure of formula (A-6) or a pharmaceutically acceptable salt thereof:

[0203]

[0204] in;

[0205] Each A 1 It is –NH- or –NH-(CH2) m -CH2-C(O)-NH-;

[0206] Each M is a C that is optionally replaced. 6-10 arylene, optionally substituted 4-10-membered heterocyclic alkylene, optionally substituted 5-10-membered heteroarylene or optionally substituted alkylene;

[0207] m is an integer between 1 and 10; and

[0208] n is an integer between 1 and 6.

[0209] In some implementations, [A] of formula (A-6) 1 -M 1 Each M in ] 1 It is C 6-10 arylene, 4-10 heterocyclic, optionally substituted 5-10 heterocyclic or C 1-6 Alkylene; each optionally substituted with 1 to 3 substituents selected from the following: H, OH, halogen, C 1-10 Alkyl, NO2, CN, NR′R″, C 1-6 Halogenated alkyl, -C 1-6 Alkoxy, C 1-6 Haloalkoxy, (C 1-6 Alkoxy)C 1-6 Alkyl, C 2-10 alkenyl, C 2-10alkynyl group, C 3-7 Carbocyclic group, 4-10 membered heterocyclic group, C 6-10 Aryl, 5-10 heteroaryl, -(C 3-7 (Carbocyclic group)C 1-6 Alkyl, (4-10 membered heterocyclic) C 1-6 Alkyl, (C 6-10 Aryl)C 1-6 Alkyl, (C 6-10 Aryl)C 1-6 Alkoxy, (5-10 heteroaryl)C 1-6 Alkyl, -(C 3-7 (Carbocyclic)-amines, (4-10 membered heterocyclic)amines, (C 6-10 (aryl)amine, (5-10 heteroaryl)amine, acyl, C-carboxyl, O-carboxyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, -SR ’ , COOH or CONR′R″; where each R′ and R″ is independently H, C 1-10 Alkyl, C 1-10 Halogenated alkyl, -C 1-10 Alkyl group. In some embodiments, [A] of formula (A-6) 1 -R 1 Each R in ] 1 It is a 5-10 member heteroaryl group or C containing at least one heteroatom selected from O, S, and N. 1-6 Alkylene, and the heteroaryl or the C 1-6 The alkylene group is optionally substituted by 1 to 3 substituents selected from the following: OH, halogen, C 1-10 Alkyl, NO2, CN, NR′R″, C 1-6 Halogenated alkyl, -C 1-6 Alkoxy, C 1-6 Halogenated alkoxy groups, C 3-7 Carbocyclic group, 4-10 membered heterocyclic group, C 6-10 Aryl, 5-10 heteroaryl, -SR ’ , COOH or CONR′R″; where each R′ and R″ is independently H, C 1-10 Alkyl, C 1-10 Halogenated alkyl, -C 1-10 Alkyl group. In some embodiments, [A] of formula (A-6) 1 -R 1 Each R in ] 1 It is a 5-10 membered heteroaryl group containing at least one heteroatom selected from O, S, and N, and said heteroaryl group is optionally substituted by 1-3 substituents selected from: OH, C 1-6 Alkyl, halogen and C1-6 Alkyl group.

[0210] In some embodiments, the first end has a structure of formula (A-7) or a pharmaceutically acceptable salt thereof:

[0211] in:

[0212] E is a terminal subunit that contains a portion selected from heterocyclic groups or straight-chain aliphatic groups, which is chemically linked to its single neighbor.

[0213] Each m 1 X in the unit 1 Y 1 and Z 1 Independently selected from CR 4 , N or NR 5 ;

[0214] Each m 3 X in the unit 2 Y 2 and Z 2 Independently selected from CR 4 , N or NR 5 ;

[0215] Each m 5 X in the unit 3 Y 3 and Z 3 Independently selected from CR 4 , N or NR 5 ;

[0216] Each m 7 X in the unit 4 Y 4 and Z 4 Independently selected from CR 4 , N or NR 5 ;

[0217] Each R 4 Independently, it is H, -OH, halogen, C 1-6 Alkyl, C 1-6 Alkoxy;

[0218] Each R 5 H and C independently 1-6 Alkyl or C 1-6 Alkylamine;

[0219] Each m 1 m 3 m 5 and m 7 Independently, it is an integer between 0 and 5;

[0220] Each m 2 m 4 and m 6 Independently, it is an integer between 0 and 3; and

[0221] m 1 +m 2 +m 3 +m 4 +m 5 +m 6 +m 7 Between 3 and 15.

[0222] In some implementations, m 1 It is 3, and X in the first unit 1 Y 1 and Z 1 They are CH, N(CH3), and CH, respectively; X in the second unit 1 Y 1 and Z 1 They are CH, N(CH3), and N, respectively; and X in the third unit 1 Y 1 and Z 1 These are CH, N(CH3), and N, respectively. In some implementations, m 3 It is 1, and X in the first unit is 1. 2 Y 2 and Z 2 These are CH, N(CH3), and CH, respectively. In some implementations, m 5 It is 2, and X in the first unit 3 Y 3 and Z 3 They are CH, N(CH3), and N, respectively; X in Unit 2 3 Y 3 and Z 3 These are CH, N(CH3), and N, respectively. In some implementations, m 7 It is 2, and X in the first unit 4 Y 4 and Z 4 They are CH, N(CH3), and CH, respectively; X in the second unit 4 Y 4 and Z 4 These are CH, N(CH3), and CH, respectively. In some implementations, each m 2 m 4 and m 6 It is independently 0 or 1. In some implementations, each m 1 X in the unit 1 Y 1 and Z1 Each of them is independently selected from CH, N, or N(CH3). In some implementations, each m 3 X in the unit 2 Y 2 and Z 2 Each of them is independently selected from CH, N, or N(CH3). In some implementations, each m 5 X in the unit 3 Y 3 and Z 3 Each of them is independently selected from CH, N, or N(CH3). In some implementations, each m 7 X in the unit 4 Y 4 and Z 4 Each of them is independently selected from CH, N, or N(CH3). In some implementations, each m 1 Each Z in the unit 1 Independently selected from CR 4 or NR 5 In some implementations, each m 3 Each Z in the unit 2 Independently selected from CR 4 or NR 5 In some implementations, each m 5 Each Z in the unit 3 Independently selected from CR 4 or NR 5 In some implementations, each m 7 Each Z in the unit 4 Independently selected from CR 4 or NR 5 In some implementations, R 4 It is H, CH3, or OH. In some implementations, R 5 It is H or CH3.

[0223] In some implementations, for equation (A-7), m 2 m 4 and m 6 The sum is between 1 and 6. In some implementations, for equation (A-7), m 2 m 4 and m 6 The sum is between 2 and 6. In some implementations, for equation (A-7), m 1 m 3 m 5 and m 7 The sum is between 2 and 10. In some implementations, m 1 m3 m 5 and m 7 The sum is between 3 and 8. In some implementations, for equation (A-7), (m 1 +m 2 +m 3 +m 4 +m 5 +m 6 +m 7 The value is between 3 and 12. In some implementations, (m) 1 +m 2 +m 3 +m 4 +m 5 +m 6 +m 7 () is between 4 and 10.

[0224] In some embodiments, for formulas (A-1) to (A-7), the first end comprises at least one β-alanine moiety. In some embodiments, for formulas (A-1) to (A-7), the first end comprises at least two β-alanine moietyes. In some embodiments, for formulas (A-1) to (A-7), the first end comprises at least three or four β-alanine moietyes.

[0225] DNA recognition or binding may include one or more subunits selected from the following:

[0226] -NH-benzopyrazinyl-CO-, -NH-phenylene-CO-, -NH-pyridyl-CO-, -NH-piperidinyl-CO-, -NH-pyrimidinyl-CO-, -NH-anthrayl-CO-, -NH-quinoline-CO- and Where Z represents H, NH2, and C. 1-6 Alkyl or C 1-6 Alkyl NH2.

[0227] In some implementations, Py is I am HP is Th is Pz is Nt is Tn is Nh is iNt is iIm is HpBi is ImBi is PyBi is Dp is ,-NH-benzopyrazine-CO- is -NH-phenylene-CO- is -NH-pyridyl-CO- is -NH-piperidinyl-CO- is -NH-pyrazinyl-CO- is -NH-anthracene-CO- is And -NH-quinoline-CO- is

[0228] In some embodiments, the first end comprises one or more subunits selected from optionally substituted N-methylpyrrole, optionally substituted N-methylimidazole, and β-alanine (β).

[0229] The first terminal of the molecule described herein exhibits a high binding affinity for multiple repeats of sequences containing GAA and preferentially binds to target nucleotide repeats compared to other nucleotide repeats or sequences. In some embodiments, the first terminal exhibits a higher binding affinity for multiple repeats of sequences containing GAA compared to sequences containing CGG. In some embodiments, the first terminal exhibits a higher binding affinity for multiple repeats of sequences containing GAA compared to sequences containing CCG. In some embodiments, the first terminal exhibits a higher binding affinity for multiple repeats of sequences containing GAA compared to sequences containing CCTG. In some embodiments, the first terminal exhibits a higher binding affinity for multiple repeats of sequences containing GAA compared to sequences containing TGGAA. In some embodiments, the first terminal exhibits a higher binding affinity for multiple repeats of sequences containing GAA compared to sequences containing GGGGCC. In some embodiments, the first terminal exhibits a higher binding affinity for multiple repeats of sequences containing GAA compared to sequences containing CAG. In some implementations, the first terminal has a higher binding affinity for multiple repeats of GAA than for repeats of CTG.

[0230] Due to the preferential binding between the first terminal and the target nucleotide repeat, the transcriptional regulatory molecules described herein become localized around regions with multiple repeats of GAA. In some embodiments, the local concentration of the first terminal or molecule described herein is higher near sequences with multiple repeats of GAA compared to near sequences with repeats of CGG. In some embodiments, the local concentration of the first terminal or molecule described herein is higher near sequences with multiple repeats of GAA compared to near sequences with repeats of CCG. In some embodiments, the local concentration of the first terminal or molecule described herein is higher near sequences with multiple repeats of GAA compared to near sequences with repeats of CCTG. In some embodiments, the local concentration of the first terminal or molecule described herein is higher near sequences with repeats of GAA compared to near sequences with repeats of TGGAA. In some embodiments, the local concentration of the first terminal or molecule described herein is higher near sequences with multiple repeats of GAA compared to near sequences with repeats of GGGGCC. In some embodiments, the local concentration of the first terminal or molecule described herein is higher near sequences with multiple repeats of GAA than near sequences with repeats of CTG. In some embodiments, the local concentration of the first terminal or molecule described herein is higher near sequences with multiple repeats of GAA than near sequences with repeats of CAG.

[0231] The first end is located at a sequence containing multiple repeats of GAA and preferentially binds to the target nucleotide repeat compared to other nucleotide repeats. In some embodiments, the sequence has at least 2, 3, 4, 5, 8, 10, 12, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, or 500 repeats of GAA. In some embodiments, the sequence contains at least 1000 nucleotide repeats of GAA. In some embodiments, the sequence contains at least 500 nucleotide repeats of GAA. In some embodiments, the sequence contains at least 200 nucleotide repeats of GAA. In some embodiments, the sequence contains at least 100 nucleotide repeats of GAA. In some embodiments, the sequence contains at least 50 nucleotide repeats of GAA. In some embodiments, the sequence contains at least 20 nucleotide repeats of GAA.

[0232] In one respect, compared to GAAs that bind to other parts of the DNA of the object, the compounds disclosed herein can bind to repeating GAAs of fxn.

[0233] Polyamides, composed of pre-selected combinations of subunits, can selectively bind to DNA in the minor groove. In their hairpin structures, antiparallel side-by-side pairs of two aromatic amino acids bind to the DNA sequence, with the polyamide rings specifically stacked against each DNA base. N-methylpyrrole (Py) favors T, A, and C bases and excludes G; N-methylimidazole (Im) is a G read; and 3-hydroxy-N-methylpyrrole (Hp) is specific to thymine bases. Nucleotide base pairs can be identified using different pairs of amino acid subunits using the pairing principles shown in Tables 1A and 1B below. For example, the Im / Py pair symmetrically reads G·C, the Py / Im pair reads C·G, the Hp / Py pair can distinguish T·A from A·T, G·C from C·G, and the Py / Py pair nonspecifically distinguishes A·T and T·A from G·C and C·G.

[0234] In some embodiments, the first terminal includes Im corresponding to nucleotide G; Py or β corresponding to nucleotide A; wherein Im is an N-alkylimidazole, Py is an N-alkylpyrrole, and β is β-alanine. In some embodiments, the first terminal includes Im / Py corresponding to nucleotide pair G / C, Py / β or Py / Py corresponding to nucleotide pair A / T, wherein Im is an N-alkylimidazole (e.g., N-methylimidazole), Py is an N-alkylpyrrole (e.g., N-methylpyrrole), and β is β-alanine.

[0235] Table 1A. Base pairing of single amino acid subunits (favored (+), unfavored (-)).

[0236]

[0237]

[0238]

[0239] *Subunits HpBi, ImBi, and PyBi act as conjugates of two monomeric subunits and bind to two nucleotides. The binding properties of HpBi, ImBi, and PyBi correspond to Hp-Py, Im-Py, and Py-Py, respectively.

[0240] Table 1B. Base pairings of hairpin polyamides.

[0241]

[0242]

[0243]

[0244] The monomer subunits of polyamide can be chained together based on the pairing principles shown in Tables 1A and 1B. The monomer subunits of polyamide can be chained together based on the pairing principles shown in Tables 1C and 1D.

[0245] Table 1C shows examples of monomeric subunits that can bind to specific nucleotides. The first end may include the described polyamide having several monomeric subunits chained together, wherein the monomeric subunits are selected from each row. For example, a polyamide may include Im-β-Py that binds to GAA, wherein Im is selected from the first G column, β from the A column, and Py from the second A column. The polyamide may be any combination of subunits that bind to GAA, wherein the subunits are selected from each column in Table 1C, and wherein the subunits are chained together following the GAA order.

[0246] In addition, polyamides may include portions or multiple sets of five subunits, such as sets of 1.5, 2, 2.5, 3, 3.5, or 4 sets of three subunits. Polyamides may include 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, and 16 monomeric subunits. Multiple sets can be bonded together via W. In addition to five or ten subunits, polyamides may also include 1-4 additional subunits that can connect multiple sets of five subunits.

[0247] Polyamides may include monomeric subunits bound to 2, 3, 4, or 5 nucleotides of GAA. For example, polyamides may be bound to GA, AA, GAA, AAG, AGA, GAAG, AAGA, GAAGA, or GAAGAA. Polyamides may include monomeric subunits bound to 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides repeating GAA. Nucleotides may be conjugated via W.

[0248] When a monomeric subunit is positioned as a terminal unit, it does not have an amine, carbonyl, or carboxylic acid group at the end. The amine or carboxylic acid group at the end is replaced by hydrogen. For example, Py, when used as a terminal unit, should be understood to have... Structure (e.g.) Furthermore, when Im is positioned as an end-unit, it should be understood as having... Structure (e.g.) Furthermore, when Py or Im is used as the terminator, Py and Im can be derived from PyT respectively. (For example ) and ImT (For example )replace.

[0249] The linear polyamide may have, but is not limited to, non-limiting examples of β-Py-Im, Im-Py-β-Im-Py-β-Im-Py, Im-Py-β-Im-Py-Py-Im-β, Im-Py-Py-Im-Py-β-Im-β, and any combination thereof.

[0250] Table 1C. Examples of monomer subunits in linear polyamides bound to GAA.

[0251]

[0252] The DNA-binding portion may also include hairpin polyamides having subunits chained together based on the pairing principles shown in Table 1B. Table 1D shows some examples of monomer subunit pairs that selectively bind to nucleotide pairs. Hairpin polyamides may comprise 2n monomer subunits (n being an integer in the range of 2-8), and the polyamide also includes a W at the center of each of the 2n monomer subunits. W may be -(CH2). a -NR 1 -(CH2) b -、-(CH2) a -、-(CH2) a -O-(CH2) b -、–(CH2) a -CH(NHR 1 )-、–(CH2)a-CH(NHR 1 )-、–(CR 2 R 3 ) a -or-(CH2) a -CH(NR 1 3) + -(CH2) b - where each a is an independent integer between 2 and 4; R 1 H, or C which is optionally substituted. 1-6 Alkyl groups, optionally substituted C 3-10 cycloalkyl, optionally substituted C 6-10 Aryl, optionally substituted 4-10-membered heterocyclic, or optionally substituted 5-10-membered heteroaryl; each R 2 and R 3 Independently, it is H, halogen, OH, NHAc, or C. 1-4 Alkyl group. In some embodiments, W is -(CH2)-CH(NH3). + -(CH2)- or -(CH2)-CH2CH(NH3) + -. In some implementations, R 1 It is H. In some implementations, R 1It is a C group optionally substituted with 1-3 substituents selected from -C(O)-phenyl. 1-6 Alkyl group. In some embodiments, W is –(CR 2 R 3 -(CH2)a- or –(CH2) a -(CR 2 R 3 )-(CH2) b - where each a is independently 1-3, b is 0-3, and each R 2 and R 3 Independently, it is H, halogen, OH, NHAc, or C. 1-4 Alkyl group. W can be an aliphatic amino acid residue such as gAB as shown in Table 4.

[0253] Because the target gene may include multiple repeats of GAA, subunits can be strung together to bind at least two, three, four, five, six, seven, eight, nine, or ten nucleotides in one or more GAA repeats (e.g., GAAGAAGAAGAA). For example, polyamides can bind to GAA repeats by binding to partial copies, complete copies, or multiple repeats of GAA (such as GA, AA, GAA, AAG, AGA, GAAG, AAGA, GAAGA, or GAAGAA). For example, polyamides may include Im-Py-β-W-Py-β-Py, which binds to GAA and its complementary nucleotide on double-stranded DNA, wherein the Im / Py pair binds to G·C, the Py / β pair binds to A·T, and the β / Py pair binds to G·A. In another example, Im-Py-β-Im-W-β-Py-β-Py binds to GAAG and its complementary nucleotides on double-stranded DNA, wherein the Im / Py pair binds to G·C, the Py / β pair binds to A·T, the β / Py pair binds to G·A, and the Im / β pair binds to G·C, where W can be an aliphatic amino acid residue such as gAB or other suitable spacer regions as shown in Table 4. In another example, Im-Py-β-Im-gAB-Im-Py binds to a portion of the complementary nucleotides (ACG) on double-stranded DNA, wherein Im binds to G, Py binds to A, β / Py binds to A·T, and Im / Im binds to G·C.

[0254] Additional examples of polyamides include, but are not limited to, Im-Py-Py-Im-gAB-Py-Im-Im-Py; Im-Py-Py-Im-gAB-Py-Im-Im-PyT; Im-Py-Py-Im-gAB-Py-Im-Im-β; Im-Py-Py-Im-gAB-Py-Im-Im-β-G; Im-β-β-Py-Im-gAB-Py-Im-Im-β; Im-β-Py-Im-gAB-Py-Im-Im-β. Im-Im-β-G; Im-β-Py-Im-gAB-Py-Im-Im-Py; Im-β-Py-Im-gAB-Py-Im-Im-PyT; Py-Py-Im-β-gAB-Im-Py-I m-Im; Py-Py-Im-β-gAB-Im-Py-Im-ImT; Py-Py-Im-Py-gAB-Im-Py-Im-Im; T;Py-Py-Im-β-gAB-Im-β-Im-Im;Py-Py-Im-β-gAB-Im-β-Im-ImT;Py-Py-Im-Py-gAB-Im-β-Im-Im;Py-Py -Im-Py-gAB-Im-β-Im-ImT;Im-β-Py-gAB-Im-Im-Py;Im-β-Py-gAB-Im-Im-PyT;Im-β-Py-gAB-Im-Im-β;Im -β-Py-gAB-Im-Im-β-G;Im-Py-Py-gAB-Im-Im-β;Im-Py-Py-gAB-Im-Im-β-G;Im-Py-Py-gAB-Im-Im-Py;Im -Py-Py-gAB-Im-Im-PyT; Im-p-Py-gAB-Im-Im-Py; and Im-β-Py-gAB-Im-Im-PyT; where G can be hydrogen, alkyl, alkenyl, alkynyl or -C(O)-R B And R B It can be hydrogen, C1-C6 alkyl, C1-C6 alkenyl, or C1-C6 alkynyl. In some embodiments, the hairpin polyamide has the structure Im-Py-β-Im-gAB-Im-Py; Im-Py-β-Im-gAB-Im-Py-β-Im; Py-β-Im-gAB-Im-Py-β-Im; or β-Im-gAB-Im-Py-β-Im.

[0255] Table 1D. Examples of monomer pairs in hairpin or H-pin polyamides combined with CAG or CTG.

[0256]

[0257]

[0258] The recognition of nucleotide repeats or DNA sequences by two antiparallel polyamide chains depends on the encoding of side-by-side aromatic amino acid pairs in the minor groove, which are typically N-to-C oriented relative to the 5' to 3' direction of the DNA helix. Enhanced affinity and specificity of polyamide nucleotide binding are achieved through covalently linking the antiparallel chains. "Hairpin motifs" connect the N-termini and C-termini of the two chains with a W (e.g., a γ-aminobutyric acid unit (γ-turn)) to form a folded straight chain. "H-pin motifs" connect the antiparallel chains via short, flexible bridges across or near-center loop / loop pairs.

[0259] The DNA-binding portion may also include H-pin polyamide having subunits chained together based on the pairing principles shown in Tables 1A and / or 1B. Table 1C shows some examples of monomeric subunits that selectively bind to nucleotides, and Table 1D shows some examples of monomeric subunit pairs that selectively bind to nucleotide pairs. H-pin polyamide may comprise two strands, each strand may have a plurality of monomeric subunits (each strand may comprise 2-8 monomeric subunits), and the polyamide may also include a bridge L1 to connect the two strands at or near the center of each strand. Following the pairing principles in Table 1D, at least one or two monomeric subunits on each strand pair with corresponding monomeric subunits on the other strand to facilitate binding to G·C or C·G, A·T or T·A pairs, and these monomeric subunit pairs are often located centrally, near the central region, at or near the bridge connecting the two strands. In some cases, all monomeric subunits of the H-pin polyamide may pair with corresponding monomeric subunits on the antiparallel strands based on the pairing principles in Tables 1B and 1D to bind to nucleotide pairs on the double-stranded DNA. In some cases, a subset of monomeric subunits (2, 3, 4, 5, or 6) of the H-pin polyamide may pair with the corresponding monomeric subunits on the antiparallel strand to bind to nucleotide pairs on the double-stranded DNA, based on the binding principles in Tables 1B and 1D, while the remaining monomeric subunits bind to nucleotides based on the binding principles in Tables 1A and 1C, but do not pair with the monomeric subunits on the antiparallel strand. The H-pin polyamide may have one or more prominent monomeric subunits that bind to nucleotides but do not pair with the monomeric subunits on the antiparallel strand.

[0260] Another polyamide structure derived from the h-pin structure connects two antiparallel chains at the ends via a bridge, but only the two monomer subunits connected by the bridge form a pairing with nucleotide pairs G·C or C·G based on the binding principles in Tables 1B / 1D, while the remaining monomer subunits on the chain form protruding ends that bind to nucleotides based on the binding principles in Tables 1A and / or 1C and do not pair with monomer subunits on the other chain.

[0261] The bridge can be a divalent or trivalent group selected from the following: C 1-10 Alkylene, -NH-C 0-6 Alkylene -C(O)-, -N(CH3)-C 0-6 Alkylene and -(CH2) a -NR 1 -(CH2) b -、-(CH2) a -、-(CH2) a -O-(CH2) b -、–(CH2) a -CH(NHR 1 )-、–(CH2) a -CH(NHR 1 )-、–(CR 2 R 3 ) a -or-(CH2) a -CH(NR 1 3) + -(CH2) b - where m is an integer in the range 0 to 10; n is an integer in the range 0 to 10; each a is independently an integer between 2 and 4; R 1 H, or C which is optionally substituted. 1-6 Alkyl groups, optionally substituted C 3-10 cycloalkyl, optionally substituted C 6-10 Aryl, optionally substituted 4-10-membered heterocyclic, or optionally substituted 5-10-membered heteroaryl; each R 2 and R 3 Independently, it is H, halogen, OH, NHAc, or C. 1-4 Alkyl group. In some embodiments, W is -(CH2)-CH(NH3). + -(CH2)- or -(CH2)-CH2CH(NH3) + -. In some implementations, R 1 It is H. In some implementations, R 1 It is a C group optionally substituted with 1-3 substituents selected from -C(O)-phenyl. 1-6 Alkyl group. In some embodiments, L1 is –(CR 2 R 3 )-(CH2) a -or–(CH2) a -(CR 2 R 3 )-(CH2) b- where each a is independently 1-3, b is 0-3, and each R 2 and R 3 Independently, it is H, halogen, OH, NHAc, or C. 1-4 Alkyl group. L1 can be C 2-9 Alkylene or (PEG) 2-8 .

[0262] Additional examples of polyamides include, but are not limited to, Im-Py-Py-Im (connected at the middle, i.e., position 2 or 3) to Py-Py-Py-Py; Im-Py-Py-Im (connected at the middle, i.e., position 3py and Py) to Im-Py-β-Py-Py; Im-Py-β-Im (connected at the bold position) Im-Py; Im-Py-β-Im (connected at the middle, i.e., position 2 or 3) Im-Py-b-Im; Py-β-Im (connected at the middle of the bold position) Im-Py-β-Im; or β-Im (connected at the bold position) Im-Py-β-Im.

[0263] Second end – Adjustment and connection part

[0264] In some embodiments, the regulatory molecules are selected from nucleosome remodeling factor (NURF), bromo domain PHD finger transcription factor (BPTF), 10-11 translocation enzyme (TET), methylcytosine dioxygenase (TET1), DNA demethylase, helicase, acetyltransferase and histone deacetylase (“HDAC”).

[0265] The binding affinity between the regulatory protein and the second terminus can be tuned based on the molecular composition or the protein type. In some embodiments, the second terminus binds to the regulatory molecule with an affinity of less than about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 250 nM, about 200 nM, about 150 nM, about 100 nM, or about 50 nM. In some embodiments, the second terminus binds to the regulatory molecule with an affinity of less than about 300 nM. In some embodiments, the second terminus binds to the regulatory molecule with an affinity of less than about 200 nM. In some embodiments, the polyamide is capable of binding to DNA with an affinity greater than about 200 nM, about 150 nM, about 100 nM, about 50 nM, about 10 nM, or about 1 nM. In some embodiments, polyamides are capable of binding DNA with an affinity in the range of about 1-600 nM, 10-500 nM, 20-500 nM, 50-400 nM, 100-300 nM or 50-200 nM.

[0266] In some embodiments, the second end includes one or more optionally replaced C's. 6-10 aryl, optionally substituted C 4-10Carbocyclic ring, optionally substituted 4- to 10-membered heterocyclic ring, or optionally substituted 5- to 10-membered heteroaryl ring.

[0267] In some embodiments, the protein-binding moiety binds to a regulatory molecule selected from the following: CREB-binding protein (CBP), P300, O-linked β-N-acetylglucosamine transferase- (OGT-), P300-CBP-associated factor- (PCAF-), histone methyltransferase, histone demethylase, chromatin domain, cyclin-dependent kinase-9- (CDK9-), nucleosome remodeling factor- (NURF-), Bromo domain PHD finger transcription factor- (BPTF-), 10-11 transloase- (TET-), methylcytosine dioxygenase- (TET1-), histone acetyltransferase (HAT), histone deacetylase (HDAC), host cytokine-1 (HCF1-), octamer-binding transcription factor- (OCT1-), P-TEFb-, cyclin-T1-, PRC2-, DNA demethylase, helicase, acetyltransferase, histone deacetylase, and methylated histone lysine protein.

[0268] In some embodiments, the second terminal includes a portion that binds to an O-linked β-N-acetylglucosamine transferase (OGT) or CREB-binding protein (CBP). In some embodiments, the protein-binding portion is a residue of a compound that binds to an O-linked β-N-acetylglucosamine transferase (OGT) or CREB-binding protein (CBP).

[0269] In some embodiments, the second terminal includes JQ1, iBET762, OTX015, RVX208, or AU1. In some embodiments, the second terminal includes JQ1. In some embodiments, the second terminal includes a portion that binds to the bromo domain protein.

[0270] In some embodiments, the second end includes one or more optionally replaced C's. 6-10 aryl, optionally substituted C 4-10 The second terminal may contain a carbocyclic ring, a optionally substituted 4- to 10-membered heterocyclic ring, or an optionally substituted 5- to 10-membered heteroaryl group. In some embodiments, the second terminal comprises a diazine or a diazepine. cyclic ring, wherein the diazine or diazapyridine Ring and C 6-10 The aryl group or a 5-10 membered heteroaryl ring containing one or more heteroatoms selected from S, N, and O is fused. In some embodiments, the second end comprises at least one 5-10 membered heteroaryl group having at least two nitrogen atoms.

[0271] In some embodiments, the second end comprises an optionally substituted bicyclic or tricyclic structure. In some embodiments, the optionally substituted bicyclic or tricyclic structure comprises a diazapyridine fused to a thiophene ring. ring.

[0272] In some embodiments, the second end comprises an optionally substituted bicyclic structure, wherein the bicyclic structure comprises a diazapyridine fused with a thiophene ring. A ring. In some embodiments, the second end comprises at least one selected from optionally substituted diazine or optionally substituted diazepine. And optionally substituted phenyl groups.

[0273] In some embodiments, the second end comprises an optionally substituted tricyclic structure, wherein the tricyclic structure is a diazapyridine fused with thiophene and triazole. ring.

[0274] In some embodiments, the second end includes a portion capable of binding to a regulatory protein, and said portion is derived from a compound capable of binding to a regulatory protein.

[0275] In some embodiments, the second end comprises JQ1, JQ-1, OTX015, RVX208 acid, or RVX208 hydroxyl.

[0276] In some embodiments, the second terminal includes a portion that binds to the bromo domain protein. In some embodiments, the regulatory molecule is a protein containing the bromo domain selected from BRD2, BRD3, BRD4, and BRDT.

[0277] In some embodiments, the regulatory molecule is BRD4. In some embodiments, the recruitment moiety is a BRD4 activator. In some embodiments, the BRD4 activator is selected from JQ-1, OTX015, RVX208 acid, and RVX208 hydroxyl.

[0278]

[0279] In some implementations, the molecular regulation regulates histone rearrangements.

[0280] In some implementations, the molecular regulation regulates the glycosylation, phosphorylation, alkylation, or acylation of histones.

[0281] In some implementations, the regulatory molecules are transcription factors.

[0282] In some implementations, the regulatory molecule is RNA polymerase.

[0283] In some implementations, the regulatory molecule is part of the mechanism that regulates the activity of RNA polymerase.

[0284] In some implementations, the regulatory molecule interacts with the TATA-binding protein.

[0285] In some implementations, the regulatory molecule interacts with transcription factor IID.

[0286] In some embodiments, X binds to the regulatory molecule but does not inhibit its activity. In some embodiments, X binds to the regulatory molecule and inhibits its activity. In some embodiments, X binds to the regulatory molecule and increases its activity.

[0287] In some embodiments, X binds to the active site of the regulatory molecule. In some embodiments, X binds to the regulatory site of the regulatory molecule.

[0288] In some embodiments, the second end is a compound of formula 7 or a pharmaceutically acceptable salt thereof:

[0289]

[0290] Wherein; R1 and R3 are each independently selected from alkoxy, alkyl, amino, halogen, and hydrogen; R2 is selected from alkoxy, alkyl, alkenyl, alkynyl, amide, amino, halogen, and hydrogen; R6 and R7 are each independently selected from alkyl, alkoxy, amino, halogen, and hydrogen; R5 is hydrogen; each W is independently selected from C and N, where if W is N, then p is 0 or 1, and if W is C, then p is 1; for W—(R4) p If W is C, then p is 1 and R4 is H, or if W is N, then p is 0; if R1 is hydrogen, then R3 is alkoxy; if R3 is hydrogen, then R1 is selected from amino and alkoxy groups; and at least one of R6 and R7 is independently selected from alkyl, alkoxy, amino, and halogen groups.

[0291] The bromo domain binding portion of Formula 9 or its pharmaceutically acceptable salt:

[0292]

[0293] Among them; R8, R 10 and R 11 Each is independently selected from hydrogen, methyl, ethyl, and halomethyl; R9 is selected from hydrogen, C1-C6 alkyl, and substituted C1-C6 alkyl; R 12 The group is selected from halogen, aryl, substituted aryl, amino, and amide groups; and X is an integer from 1 to 6.

[0294] In some embodiments, the second end comprises a compound having the structure of Formula 7:

[0295]

[0296] in;

[0297] R1 and R3 are each independently selected from alkoxy, alkyl, amino, halogen and hydrogen;

[0298] R2 is selected from alkoxy, alkyl, alkenyl, alkynyl, amide, amino, halogen, and hydrogen;

[0299] R6 and R7 are each independently selected from alkyl, alkoxy, amino, halogen, and hydrogen;

[0300] R5 is hydrogen;

[0301] Each W is independently selected from C and N, where if W is N, then p is 0 or 1, and if W is C, then p is 1; for W—(R4) p If W is C, then p is 1 and R4 is H, or if W is N, then p is 0; if R1 is hydrogen, then R3 is alkoxy; if R3 is hydrogen, then R1 is selected from amino and alkoxy; and at least one of R6 and R7 is independently selected from alkyl, alkoxy, amino and halogen.

[0302] In some embodiments, the second end is a compound of formula 7, a bromo domain binding portion having the structure of formula 9, and a bromo domain binding portion having the structure of formula 20:

[0303]

[0304] Among them; R b It is hydrogen, C1-C6 alkyl, or a substituted C1-C6 alkyl; R a R c and R d Each is independently hydrogen, methyl, ethyl, or halomethyl; R e It is a halogen, aryl, substituted aryl, amino, or amide group; and y is an integer from 1 to 6.

[0305] In some implementations, the bromo domain binding portion has a structure of formula 8 or a pharmaceutically acceptable salt thereof:

[0306]

[0307] In some embodiments, the second end comprises a compound having the structure of formula (9-A) or a pharmaceutically acceptable salt thereof:

[0308]

[0309] in;

[0310] Ring A is absent or is a 6-membered monocyclic aryl or heteroaryl group;

[0311] Y is -NH- or -O-;

[0312] R 8 Is it hydrogen or C? 1-6 alkyl;

[0313] R 9 R 10 and R 11 Each C is independently selected from hydrogen and optionally substituted. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl;

[0314] R 12 Selected from hydrogen, halogen, -NO2, -CN, optionally substituted aryl, optionally substituted C 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl;

[0315] or R 12 Yes -NR A R B ,in

[0316] R A and R B Each is independently hydrogen, and the C atoms are optionally substituted. 1-6 Alkyl or C 1-6 Heteroalkyl; and

[0317] x1 is an integer from 1 to 6.

[0318] In some embodiments, ring A is a 6-membered monocyclic aryl or heteroaryl group. In some embodiments, ring A is a phenyl group. In some embodiments, ring A is a 6-membered monocyclic heteroaryl group. In some embodiments, ring A is pyridine or pyrimidine.

[0319] In some implementations, ring A is absent.

[0320] In some implementations, Y is -NH-. In some implementations, Y is -O-.

[0321] In some implementation schemes, R 8 It is hydrogen.

[0322] In some implementation schemes, R 9 R 10 and R 11 Each is independently selected from the C that is arbitrarily replaced. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl. In some embodiments, R 9 R 10 and R 11Each is independently selected from the C that is arbitrarily replaced. 1-6 Alkyl group. In some embodiments, R 9 R 10 and R 11 Each is independently methyl, ethyl, or propyl. In some embodiments, R 9 R 10 and R 11 Each is a methyl group independently.

[0323] In some implementation schemes, R 12 Selected from hydrogen, halogens, and optionally substituted C 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl. In some embodiments, R 12 It is bromine, chlorine, or fluorine.

[0324] In some implementation schemes, R 12 Yes -NR A R B , where R A and R B Each is independently hydrogen, and the C atoms are optionally substituted. 1-6 alkyl.

[0325] In some implementations, x1 is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some implementations, x1 is 1. In some implementations, x1 is 2.

[0326] In some embodiments, the second end comprises a compound having the structure of formula (9-B) or a pharmaceutically acceptable salt thereof:

[0327]

[0328] in;

[0329] R8, R 10 and R 11 Each is independently selected from hydrogen, methyl, ethyl, and halomethyl;

[0330] R9 is selected from hydrogen, C1-C6 alkyl, and substituted C1-C6 alkyl;

[0331] R 12 Selected from halogen, aryl, substituted aryl, amino, and amide groups; and

[0332] x is an integer from 1 to 6.

[0333] In some embodiments, the second end comprises a compound having the structure of formula (9-C) or a pharmaceutically acceptable salt thereof:

[0334]

[0335] In some embodiments, the second end contains a compound of formula (10-A) or a pharmaceutically acceptable salt thereof:

[0336]

[0337] in,

[0338] Ring B is absent or is either a 5-6 membered monocyclic aryl or heteroaryl or a 4-8 membered heterocyclic ring;

[0339] Y is -NH- or -O-;

[0340] R 13 Selected from hydrogen or optionally substituted C1-C6 alkyl groups;

[0341] R 14 and R 15 Each C is independently selected from hydrogen and optionally substituted. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl;

[0342] R 16 Selected from hydrogen, halogen, -NO2, -CN, optionally substituted aryl, optionally substituted C 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl;

[0343] or R 16 Yes -NR A R B ,in

[0344] R A and R B Each is independently hydrogen, and the C atoms are optionally substituted. 1-6 Alkyl or C 1-6 Heteroalkyl; and

[0345] x2 is an integer from 1 to 6.

[0346] In some embodiments, ring B is a 6-membered monocyclic aryl or heteroaryl group. In some embodiments, ring B is a phenyl group. In some embodiments, ring B is a 6-membered monocyclic heteroaryl group. In some embodiments, ring B is pyridine or pyrimidine.

[0347] In some implementations, ring B is absent.

[0348] In some implementations, Y is -NH-. In some implementations, Y is -O-.

[0349] In some implementation schemes, R 13 It is hydrogen.

[0350] In some implementation schemes, R 14 and R 15 Each is independently selected from the C that is arbitrarily replaced. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl. In some embodiments, R 14 and R 15 Each is independently selected from the C that is arbitrarily replaced. 1-6 Alkyl group. In some embodiments, R 14 and R 15 Each is independently methyl, ethyl, or propyl. In some embodiments, R 14 and R 15 Each is a methyl group independently.

[0351] In some implementation schemes, R 16 Selected from hydrogen, halogens, and optionally substituted C 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl. In some embodiments, R 16 It is bromine, chlorine, or fluorine.

[0352] In some implementation schemes, R 16 Yes -NR A R B , where R A and R B Each is independently hydrogen, and the C atoms are optionally substituted. 1-6 alkyl.

[0353] In some implementations, x2 is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some implementations, x2 is 1. In some implementations, x2 is 2.

[0354] In some embodiments, the second end comprises a compound having the structure of formula (10-B) or a pharmaceutically acceptable salt thereof:

[0355]

[0356] In some embodiments, the second end comprises a compound having the structure of formula (10-C) or a pharmaceutically acceptable salt thereof:

[0357]

[0358] In some embodiments, the second end comprises a compound having the structure of formula (12-A) or a pharmaceutically acceptable salt thereof:

[0359]

[0360] in;

[0361] The ring C is absent; it is a monocyclic 6-membered aryl, heteroaryl, or 6-membered heterocyclic alkylene.

[0362] X 1 It is CH or N;

[0363] L 2 Yes -NR D -or-CR D H-;

[0364] R 23 It is a C1-C6 alkyl or a C3-C6 cycloalkyl; and

[0365] R 24 It is a halogen, alkyl, hydroxyalkyl, or haloalkyl; optionally substituted C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 hydroxyalkyl; and

[0366] R D Is it hydrogen or C? 1-3 alkyl.

[0367] In some embodiments, the ring C is a 6-membered monocyclic aryl or heteroaryl. In some embodiments, the ring C is phenyl. In some embodiments, the ring C is a 6-membered monocyclic heteroaryl. In some embodiments, the ring C is pyridine or pyrimidine. In some embodiments, the ring C is a 6-membered heterocyclic alkylene.

[0368] In some implementations, ring C is

[0369] In some implementations, ring C is absent.

[0370] In some implementation schemes, X 1 It is CH. In some implementations, X 1 It is N.

[0371] In some implementations, L 2 Yes -NR D - In some implementations, L 2 It is -NH-. In some implementations, L 2 Yes -CR D H. In some implementations, L 2 It is -CH2-.

[0372] In some implementation schemes, R 23 It is methyl, ethyl, or propyl. In some embodiments, R 23 It is methyl. In some embodiments, R 23 It is ethyl. In some embodiments, R 23 It is propyl. In some implementations, R 23 It is cyclopropyl.

[0373] In some implementation schemes, R 24 It is an alkyl, hydroxyalkyl, or haloalkyl; optionally substituted C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 hydroxyalkyl. In some embodiments, R 24 It is a hydroxyalkyl group. In some embodiments, R 24 It is a halogen. In some implementations, R 24 It is bromine, chlorine, or fluorine. In some implementations, R 24 It is -CH2OH.

[0374] In some embodiments, the second end comprises a compound having the structure of formula (12-B) or a pharmaceutically acceptable salt thereof:

[0375]

[0376] In some embodiments, the second end comprises a compound having the structure of formula (12-B1) or a pharmaceutically acceptable salt thereof:

[0377]

[0378] In some embodiments, the second end comprises a compound having the structure of formula (12-C) or a pharmaceutically acceptable salt thereof:

[0379]

[0380] In some embodiments, the second end comprises a compound having the structure of formula (12-C1) or a pharmaceutically acceptable salt thereof:

[0381]

[0382] In some embodiments, the second end comprises a compound having the structure of formula (13-A) or a pharmaceutically acceptable salt thereof:

[0383]

[0384] in;

[0385] The ring D is absent, or is optionally substituted with a phenyl group, an optionally substituted 5- or 6-membered heteroaryl group, or a 6-membered heterocyclic alkyl group.

[0386] In some embodiments, ring D is phenyl. In some embodiments, ring D is absent.

[0387] In some implementations, ring D is

[0388] In some embodiments, the second end comprises a compound having the structure of formula (13-B) or a pharmaceutically acceptable salt thereof:

[0389]

[0390] In some embodiments, the second end comprises a compound having the structure of formula (14-A) or a pharmaceutically acceptable salt thereof:

[0391]

[0392] in;

[0393] The ring E is absent, or optionally substituted with a phenyl group, an optionally substituted 5- or 6-membered heteroaryl group, or a 6-membered heterocyclic alkyl group.

[0394] In some embodiments, ring E is phenyl. In some embodiments, ring E is absent.

[0395] In some implementations, ring E is

[0396] In some embodiments, the second end comprises a compound having the structure of formula (14-B) or a pharmaceutically acceptable salt thereof:

[0397]

[0398] In some embodiments, the second end comprises a compound having the structure of formula (15-A) or a pharmaceutically acceptable salt thereof:

[0399]

[0400] In some implementations, the second terminal is:

[0401] Or their pharmaceutically acceptable salts.

[0402] In some implementations, the protein-binding moiety is selected from:

[0403]

[0404] Or their pharmaceutically acceptable salts.

[0405] In some implementations, the protein-binding moiety is Or its pharmaceutically acceptable salt.

[0406] connector

[0407] The oligomeric backbone contains a linker that connects the first and second ends and causes the regulatory molecule to be adjacent to the target gene to regulate gene expression. In some embodiments, the terms "oligomeric backbone" and "backbone" refer to the linker connecting the first and second ends.

[0408] The length of the linker depends on the type of regulatory protein and the target gene. In some embodiments, the linker has a length of less than about 50 angstroms. In some embodiments, the linker has a length of about 15 to 40 angstroms. In some embodiments, the linker contains between 5 and 50 chain atoms. In some embodiments, the linker has a length of about 20 to 30 angstroms.

[0409] In some implementations, the connector contains between 5 and 50 chain atoms.

[0410] In some embodiments, the connector comprises a polymer having 2 to 50 spacer portions, wherein said spacer portions are independently selected from -((CR) 3a R 3b ) x -O) y -、-((CR 3a R 3b ) x -NR 4a ) y -、-((CR 3a R 3b ) x -CH=CH-(CR 3a R 3b ) x -O) y - Optionally replaced -C 1-12 Alkyl groups, optionally substituted C 2-10 Alkenyl, optionally substituted C 2-10 alkynyl group, optionally substituted C 6-10 Aromatic, optionally substituted C 3-7 Cycloalkylene, optionally substituted 5- to 10-membered heteroalkylene, optionally substituted 4- to 10-membered heteroalkylene, amino acid residue, —O—, —C(O)NR 4a —、—NR 4aC(O)—、—C(O)—、—NR 1 —, —C(O)O—, —O—, —S—, —S(O)—, —SO2—, —SO2NR 4a —、—NR 4a SO2— and —P(O)OH—, and any combination thereof;

[0411] Each x is independently 2-4;

[0412] Each y is independently 1-10;

[0413] Each R 3a and R 3b Independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkoxy, optionally substituted amino, carboxyl, carboxylic acid ester, acyl, acyloxy, acylamino, aminoacyl, optionally substituted alkylamide, sulfonyl, optionally substituted thioalkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and optionally substituted heterocyclic; and

[0414] Each R 4a C is either hydrogen or optionally substituted. 1-6 alkyl.

[0415] In some implementations, the connector includes -((CR) 3a R 3b ) x -O) y - In some implementations, the connector contains -((CH2)2-O). y - In some embodiments, the connector comprises PEG. In some embodiments, the connector comprises 1-20 PEG units. In some embodiments, the connector comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 PEG units. In some embodiments, the connector comprises 6 PEG units. In some embodiments, the connector comprises 7 PEG units. In some embodiments, the connector comprises 8 PEG units. In some embodiments, the connector comprises 10 PEG units. In some embodiments, the connector comprises 15 PEG units.

[0416] In some implementations, the oligo-chain contains -(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 —,

[0417] Where a, b, c, d and e are each independently 0 or 1, and the sum of a, b, c, d and e is between 1 and 5;

[0418] T 1 T 2 T 3 T 4 and T 5 Each is independently selected from the arbitrarily replaced (C1-C) 12 Alkylene, optionally substituted alkenylene, optionally substituted alkyneylene, (EA) w (EDA) m (PEG) n (Modified PEG) n (AA) p —(CR) 2a OH) h —Optionally replaced (C6-C) 10 ) aryl, optionally substituted C 3-7 Cycloalkylene groups, optionally substituted 5- to 10-membered heteroalkylene groups, optionally substituted 4- to 10-membered heterocycloalkylene groups, acetal groups, disulfides, hydrazine, carbohydrates, β-lactams, and esters.

[0419] w is an integer from 1 to 20;

[0420] m is an integer from 1 to 20;

[0421] n is an integer from 1 to 30;

[0422] p is an integer from 1 to 20;

[0423] h is an integer from 1 to 12;

[0424] EA has the following structure

[0425]

[0426] EDA has the following structure:

[0427]

[0428] Each q is an integer from 1 to 6, each x is an integer from 1 to 4, and each r is either 0 or 1.

[0429] (PEG) n With –(CR 2a R 2b -CR 2a R 2b -O) n -CR 2a R 2b - structure;

[0430] (Modified PEG) n With –(CH2-CR 2a =CR 2a -CH2-O)- or –(CR 2a R 2b -CR 2a R 2b -S)- Replacement (PEG) n At least one of –(CR) 2a R 2b -CR 2a R 2b The structure of -O)-;

[0431] AA stands for amino acid residue;

[0432] V 1 V 2 V 3 V 4 and V 5 Each is independently selected from the bond, CO-, -NR 1a -、-CONR 1a -、-NR 1a CO-、-CONR 1a C 1-4 Alkyl-, -NR 1a CO-C 1-4 Alkyl-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O-, -SO2-, -SO2NR 1a -、-NR 1a SO2- and -P(O)OH-;

[0433] Each R 1a Independently hydrogen and / or optionally substituted C 1-6 Alkyl; and

[0434] Each R 2a and R 2bIt is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogen, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylic acid ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic.

[0435] In some implementations, a, b, c, d, and e are each independently 0 or 1, where the sum of a, b, c, d, and e is 1. In some implementations, a, b, c, d, and e are each independently 0 or 1, where the sum of a, b, c, d, and e is 2. In some implementations, a, b, c, d, and e are each independently 0 or 1, where the sum of a, b, c, d, and e is 3. In some implementations, a, b, c, d, and e are each independently 0 or 1, where the sum of a, b, c, d, and e is 4. In some implementations, a, b, c, d, and e are each independently 0 or 1, where the sum of a, b, c, d, and e is 5.

[0436] In some implementations, n is 3-9. In some implementations, n is 4-8. In some implementations, n is 5 or 6.

[0437] In some implementations, T 1 T 2 T 3 And T 4 and T 5 Each is independently selected from (C1-C) 12 )alkyl, substituted (C1-C) 12 Alkyl group, (EA) w (EDA) m (PEG) n (Modified PEG) n (AA) p —(CR) 2a OH) h — Phenyl, substituted phenyl, piperidine-4-amino (P4A), p-aminobenzylmethyloxycarbonyl (PABC), m-aminobenzylmethyloxycarbonyl (MABC), p-aminobenzylmethyloxy (PABO), m-aminobenzylmethyloxy (MABO), p-aminobenzyl, acetal group, disulfide, hydrazine, carbohydrate, β-lactam, ester, (AA) p -MABC-(AA) p (AA) p -MABO-(AA) p (AA) p-PABO-(AA) p and (AA) p -PABC-(AA) p In some implementations, piperidine-4-amino (P4A) is Where R 1a Is it H or C? 1-6 alkyl.

[0438] In some implementations, T 1 T 2 T 3 T 4 and T 5 Each is independently selected from (C1-C) 12 )alkyl, substituted (C1-C) 12 Alkyl group, (EA) w (EDA) m (PEG) n (Modified PEG) n (AA) p —(CR) 2a OH) h —Optionally replaced (C6-C) 10 ) arylene, 4-10 membered heterocyclic olefins, optionally substituted 5-10 membered heteroarylene. In some embodiments, EA has the following structure:

[0439] and

[0440] EDA has the following structure:

[0441]

[0442] In some implementations, for EA and EDA, x is 2-3, and q is 1-3. In some implementations, R 1a Is it H or C? 1-6 alkyl.

[0443] In some implementations, T 4 or T 5 It is arbitrarily replaced (C6-C) 10 )Asyl.

[0444] In some implementations, T 4 or T 5 It is a phenylene or a substituted phenylene. In some embodiments, T 4 or T 5 It is a phenylene or composed of 1-3 atoms selected from -C 1-6 A phenylene oxide substituted with an alkyl, halogen, OH, or amine substituent. In some embodiments, T 4or T 5 It is a 5-10 nucleotide heteroaryl group or a substituted heteroaryl group. In some embodiments, T 4 or T 5 It is a 4-10 membered heterocyclic group or a substituted heterocyclic group. In some embodiments, T 4 or T 5 It is optional to be selected from 1-3 of -C 1-6 A heterocyclic or heterocyclic group substituted with an alkyl, halogen, OH or amine substituent.

[0445] In some implementations, T 1 T 2 T 3 T 4 and T 5 and V 1 V 2 V 3 V 4 and V 5 Selected from Table 2 below.

[0446] Table 2.

[0447]

[0448]

[0449] In some implementations, the connector includes Or any combination thereof, where r is an integer between 1 and 10, preferably between 3 and 7; and X is 0, S, or NR. 1a In some implementations, X is O or NR. 1a In some implementations, X is O.

[0450] In some implementations, the connector includes Or any combination thereof; wherein at least one –(CH2-CH2-O)- is –((CR 1a R 1b ) x -CH=CH-(CR 1a R 1b ) x -O)- or any combination thereof; W' does not exist, it is (CH2). 1-5 -(CH2) 1-5 O, (CH2) 1-5- C(O)NH-(CH2) 1-5 -O、(CH2) 1-5- C(O)NH-(CH2) 1-5 -(CH2) 1-5 NHC(O)-(CH2)1-5 -O or -(CH2) 1-5- NHC(O)-(CH2) 1-5 -;E 3 C is arbitrarily replaced 6-10 arylene, optionally substituted 4-10-membered heterocyclic alkylene, or optionally substituted 5-10-membered heteroarylene; X is O, S, or NH; each R 1a and R 1b Independently, it is H or C 1-6 Alkyl; r is an integer between 1 and 10; and x is an integer between 1 and 15. In some embodiments, X is O. In some embodiments, X is NH. In some embodiments, E 3 It is optional to be selected from 1-3 of -C 1-6 C substituents of alkyl, halogen, OH or amine groups 6-10 Alpha-aryl.

[0451] In some implementation schemes, E 3 It is a phenylene or a substituted phenylene.

[0452] In some implementations, the connector includes

[0453] In some implementations, the connector includes –X(CH2). m (CH2CH2O) n – where X is –O–, –NH– or –S–, where m is 0 or greater, and n is at least 1.

[0454] In some implementations, the connector is included after the second end. Where R c Selected from key, –N(R) 1a )–, –O– and –S–;R d Selected from –N(R) 1a )–, –O– and –S–; and R e Independently selected from hydrogen and optionally substituted C 1-6 alkyl.

[0455] In some implementations, the connector comprises one or more structures selected from the following: -C 1-12 Alkyl, arylene, cycloalkylene, heteroalkylene, heteroalkylene, -O-, -C(O)NR 1a -、-C(O)-、-NR 1a -、-(CH2CH2CH2O) y -and-(CH2CH2CH2NR) 1a ) y- where each d and y is independently 1-10, and each R 1a Independently hydrogen or C 1-6 Alkyl group. In some embodiments, d is 4-8.

[0456] In some implementations, the connector includes And each d is independently 3-7. In some implementations, d is 4-6.

[0457] In some implementations, the connector includes –N(R 1a (CH2) x N(R 1b (CH2) x N–, where R 1a and R 1b Each x is independently selected from hydrogen or optionally substituted C1-C6 alkyl groups; and each x is independently an integer in the range of 1-6.

[0458] In some implementations, the connector comprises -(CH2-C(O)N(R”)-(CH2) q -N(R')-(CH2) q -N(R”)C(O)-(CH2) x -C(O)N(R”)-A 2 -、-(CH2) x -C(O)N(R”)-(CH2CH2O) y (CH2) x -C(O)N(R”)-A 2 -、-C(O)N(R”)-(CH2) q -N(R')-(CH2) q -N(R”)C(O)-(CH2) x -A 2 -、-(CH2) x -O-(CH2CH2O) y -(CH2) x -N(R”)C(O)-(CH2) x -A 2 -or -N(R”)C(O)-(CH2)-C(O)N(R”)-(CH2) x -O(CH2CH2O) y (CH2) x -A 2 -; where R' is methyl; R” is hydrogen; each x and y is independently an integer from 1 to 10; each q is independently an integer from 2 to 10; and each A 2 C is independently selected from the key and optionally replaced. 1-12Alkyl groups, optionally substituted C 6-10 Aromatic, optionally substituted C 3-7 Cycloalkylene, optionally substituted 5- to 10-membered heteroalkylene, and optionally substituted 4- to 10-membered heteroalkylene.

[0459] In some embodiments, the connector is coupled to the first end with a group selected from the following: –CO-, -NR 1a -、-CONR 1a -、-NR 1a CO-、-CONR 1a C 1-4 Alkyl-, -NR 1a CO-C 1-4 Alkyl-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O-, -SO2-, -SO2NR 1a -、-NR 1a SO2-, -P(O)OH-, -((CH2) x -O)-、-((CH2) y -NR 1a -, optionally replaced -C 1-12 Alkylene, optionally substituted C 2-10 alkenyl, optionally substituted C 2-10 Ethyne group, optionally substituted C 6-10 Aromatic, optionally substituted C 3-7 Cycloalkylene, optionally substituted 5- to 10-membered heteroalkylene, and optionally substituted 4- to 10-membered heteroalkylene; wherein each x and y is independently 1-4, and each R 1a C is either hydrogen or optionally substituted. 1-6 alkyl.

[0460] In some embodiments, the connector is coupled to the first end with a group selected from the following: –CO-, -NR 1a -、C 1-12 Alkyl, -CONR 1a -and-NR 1a CO-; where each R 1a C is either hydrogen or optionally substituted. 1-6 Alkyl or optionally substituted -C 1-12 Alkylene, optionally substituted C 2-10 alkenyl, optionally substituted C 2-10 Ethyne group, optionally substituted C 6-10 Aromatic, optionally substituted C 3-7 Cycloalkylene, optionally substituted 5- to 10-membered heteroalkylene, and optionally substituted 4- to 10-membered heteroalkylene.

[0461] In some embodiments, the connector is coupled to the first end with a group selected from the following: –CO-, -NR 1a -、C 1-12 Alkyl, -CONR 1a -and-NR 1a CO-; where each R 1a C is either hydrogen or optionally substituted. 1-6 alkyl.

[0462] In some embodiments, the connector is coupled to the first end with a group selected from: —CO—, —NR 1a —、—CONR 1a —、—NR 1a CO—、—CONR 1a C 1-4 Alkyl-, -NR 1a CO-C 1-4 Alkyl group —, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO2—, —SO2NR 1a —、—NR 1 SO2—,—P(O)OH—,—((CH2) x -O)—、—((CH2) y -NR 1a —, optional substitution of -C 1-12 Alkylene, optionally substituted C 2-10 alkenyl, optionally substituted C 2-10 Ethyne group, optionally substituted C 6-10 Aromatic, optionally substituted C 3-7 Cycloalkylene, optionally substituted 5- to 10-membered heteroalkylene, and optionally substituted 4- to 10-membered heteroalkylene, wherein each x is independently 1-4, each y is independently 1-4, and each R 1a C is either hydrogen or optionally substituted. 1-6 alkyl.

[0463] In some embodiments, the connector is coupled to the first end with a group selected from: —CO—, —NR 1a —、C 1-12 Alkyl, -CONR 1a —and—NR 1a CO—.

[0464] In some embodiments, the connector is coupled to the second end with a group selected from the following: —CO—, —NR 1a —、—CONR 1a —、—NR 1aCO—、—CONR 1a C 1-4 Alkyl-, -NR 1a CO-C 1-4 Alkyl group —, —C(O)O—, —OC(O)—, —O—, —S—, —S(O)—, —SO2—, —SO2NR 1a —、—NR 1 SO2—,—P(O)OH—,—((CH2) x -O)—、—((CH2) y -NR 1a —, optional substitution of -C 1-12 Alkylene, optionally substituted C 2-10 alkenyl, optionally substituted C 2-10 Ethyne group, optionally substituted C 6-10 Aromatic, optionally substituted C 3-7 Cycloalkylene, optionally substituted 5- to 10-membered heteroalkylene, and optionally substituted 4- to 10-membered heteroalkylene, wherein each x is independently 1-4, each y is independently 1-4, and each R 1a C is either hydrogen or optionally substituted. 1-6 alkyl.

[0465] In some embodiments, the connector is coupled to the second end with a group selected from the following: —CO—, —NR 1a —、—CONR 1a —、—NR 1a CO—、—((CH2) x -O)—、—((CH2) y -NR 1a -, -O-, and -C (optionally substituted) 1-12 Alkyl groups, optionally substituted C 6-10 Aromatic, optionally substituted C 3-7 Cycloalkylene, optionally substituted 5- to 10-membered heteroalkylene, and optionally substituted 4- to 10-membered heteroalkylene, wherein each x is independently 1-4, each y is independently 1-4, and each R 1 C is either hydrogen or optionally substituted. 1-6 alkyl.

[0466] In some embodiments, the connector is joined to the second end with a group selected from the group consisting of optionally substituted 4- to 10-membered heterocyclic alkyl groups.

[0467] Cell penetration ligand

[0468] In some embodiments, the compound includes a cell-penetrating ligand portion.

[0469] In some implementations, the cell-penetrating ligand portion is a polypeptide.

[0470] In some implementations, the cell-penetrating ligand portion is a polypeptide containing fewer than 30 amino acid residues.

[0471] In some embodiments, the polypeptide is selected from any one of SEQ ID NO.1 to SEQ ID NO.37, including SEQ ID NO.1 and SEQ ID NO.37.

[0472] It also provides implementation schemes in which any of the above implementation schemes can be combined with any one or more of these implementation schemes, provided that the combinations are not mutually exclusive.

[0473] As used herein, two embodiments are "mutually exclusive" when one embodiment is defined as something different from another embodiment. For example, an embodiment in which two groups are combined to form a cycloalkyl group is mutually exclusive with an embodiment in which one group is ethyl and the other is hydrogen. Similarly, an embodiment in which one group is CH2 is mutually exclusive with an embodiment in which the group is NH.

[0474] In some implementations, non-limiting examples of the transcriptional regulatory compounds described herein are presented in Table 3.

[0475] Table 3. Compounds disclosed herein.

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490] How to use

[0491] This disclosure also relates to a method for regulating the transcription of fxn, the method comprising the step of contacting fxn with a compound as described herein. Cell phenotype, cell proliferation, fxn transcription, mRNA production resulting from fxn transcription, fxn translation, changes in biochemical outputs produced by proteins encoded by fxn, or non-covalent binding of proteins encoded by fxn to natural binding partners can be monitored. Such methods can serve as modalities for disease treatment, bioassays, cellular assays, biochemical assays, etc.

[0492] This article also provides a method for treating diseases mediated by fxn transcription, the method comprising administering a therapeutically effective amount of a compound as disclosed herein or a salt thereof to a patient in need.

[0493] In some implementations, the disease is Friedreich ataxia.

[0494] This article also provides a compound as disclosed herein that can be used as a medicine.

[0495] This article also provides a compound disclosed herein that can be used as a medicament for treating diseases mediated by the transcription of fxn.

[0496] The use of compounds such as those disclosed herein as pharmaceuticals is also provided.

[0497] The use of compounds such as those disclosed herein as medicaments for treating diseases mediated by the transcription of fxn is also provided.

[0498] Also provided is a compound disclosed herein for manufacturing a medicament for treating diseases mediated by the transcription of fxn.

[0499] The use of compounds such as those disclosed herein for the treatment of diseases mediated by fxn transcription is also provided.

[0500] This article also provides a method for regulating the transcription of fxn, the method comprising contacting fxn with a compound as disclosed herein or a salt thereof.

[0501] This article also provides a method for achieving an effect in a patient, the method comprising administering a therapeutically effective amount of a compound as disclosed herein or a salt thereof to the patient, wherein the effect is selected from improvements in neurosensory perception, visual acuity, balance, gait, reduced sensitivity to glucose, and reduced sensitivity to carbohydrates.

[0502] Certain compounds of this disclosure are effective in treating subjects with 5 or more duplicates of the GAA genotype. Certain compounds of this disclosure are effective in treating subjects with 10 or more duplicates of the GAA genotype. Certain compounds of this disclosure are effective in treating subjects with 20 or more duplicates of the GAA genotype. Certain compounds of this disclosure are effective in treating subjects with 50 or more duplicates of the GAA genotype. Certain compounds of this disclosure are effective in treating subjects with 100 or more duplicates of the GAA genotype. Certain compounds of this disclosure are effective in treating subjects with 200 or more duplicates of the GAA genotype. Certain compounds of this disclosure are effective in treating subjects with 500 or more duplicates of the GAA genotype.

[0503] A method for modulating fxn-mediated function in a subject is also provided, the method comprising administering a therapeutically effective amount of a compound as disclosed herein.

[0504] A pharmaceutical composition comprising the compounds disclosed herein and a pharmaceutically acceptable carrier is also provided.

[0505] In some embodiments, the pharmaceutical composition is formulated for oral administration.

[0506] In some embodiments, the pharmaceutical composition is formulated for intravenous injection and / or infusion.

[0507] In some embodiments, the oral pharmaceutical composition is selected from tablets and capsules.

[0508] In some embodiments, ex vivo treatment methods are provided. Ex vivo methods typically involve removing cells, organs, and / or tissues from a subject. The cells, organs, and / or tissues may be incubated with the pharmaceutical agent under appropriate conditions, for example. The contacted cells, organs, and / or tissues are typically returned to the donor, placed in the recipient, or stored for future use. Therefore, the compound is typically in a pharmaceutically acceptable carrier.

[0509] In some embodiments, administration of the pharmaceutical composition modulates fxn expression within 6 hours of treatment. In some embodiments, administration of the pharmaceutical composition modulates fxn expression within 24 hours of treatment. In some embodiments, administration of the pharmaceutical composition modulates fxn expression within 72 hours of treatment.

[0510] In some embodiments, administration of the pharmaceutical composition results in a 2-fold increase in fxn expression. In some embodiments, administration of the pharmaceutical composition results in a 5-fold increase in fxn expression. In some embodiments, administration of the pharmaceutical composition results in a 10-fold increase in fxn expression. In some embodiments, administration of the pharmaceutical composition results in a 20-fold increase in fxn expression.

[0511] In some embodiments, administration of the pharmaceutical composition results in a 20% reduction in fxn expression. In some embodiments, administration of the pharmaceutical composition results in a 50% reduction in fxn expression. In some embodiments, administration of the pharmaceutical composition results in an 80% reduction in fxn expression. In some embodiments, administration of the pharmaceutical composition results in a 90% reduction in fxn2 expression. In some embodiments, administration of the pharmaceutical composition results in a 95% reduction in fxn2 expression. In some embodiments, administration of the pharmaceutical composition results in a 99% reduction in fxn expression.

[0512] In some embodiments, administration of the pharmaceutical composition results in fxn expression falling within 25% of the expression level observed in healthy individuals. In some embodiments, administration of the pharmaceutical composition results in fxn expression falling within 50% of the expression level observed in healthy individuals. In some embodiments, administration of the pharmaceutical composition results in fxn expression falling within 75% of the expression level observed in healthy individuals. In some embodiments, administration of the pharmaceutical composition results in fxn expression falling within 90% of the expression level observed in healthy individuals.

[0513] Pharmaceutical composition and administration

[0514] A method for modulating fxn-mediated function in a subject is also provided, the method comprising administering a therapeutically effective amount of a compound as disclosed herein.

[0515] A pharmaceutical composition comprising the compounds disclosed herein and a pharmaceutically acceptable carrier is also provided.

[0516] In some embodiments, the pharmaceutical composition is formulated for oral administration.

[0517] In some embodiments, the pharmaceutical composition is formulated for intravenous injection or infusion.

[0518] In some embodiments, the oral pharmaceutical composition is selected from tablets and capsules.

[0519] In some embodiments, ex vivo treatment methods are provided. Ex vivo methods typically involve removing cells, organs, or tissues from a subject. The cells, organs, or tissues may be incubated with the pharmaceutical agent under appropriate conditions, for example. The contacted cells, organs, or tissues are typically returned to the donor, placed in the recipient, or stored for future use. Therefore, the compound is typically in a pharmaceutically acceptable carrier.

[0520] In some embodiments, the compound is effective at concentrations less than about 5 μM. In some embodiments, the compound is effective at concentrations less than about 1 μM. In some embodiments, the compound is effective at concentrations less than about 400 nM. In some embodiments, the compound is effective at concentrations less than about 200 nM. In some embodiments, the compound is effective at concentrations less than about 100 nM. In some embodiments, the compound is effective at concentrations less than about 50 nM. In some embodiments, the compound is effective at concentrations less than about 20 nM. In some embodiments, the compound is effective at concentrations less than about 10 nM.

[0521] Abbreviations and Definitions

[0522] As used herein, the following terms have indicative meanings.

[0523] It should be understood that, depending on the context, certain group naming conventions may include monovalent or divalent groups. For example, when a substituent requires two connection points with the rest of the molecule, it should be understood that the substituent is a divalent group. For instance, substituents identified as alkyl that require two connection points include divalent groups, such as –CH2–, –CH2CH2–, –CH2CH(CH3)CH2–, etc. Other group naming conventions explicitly indicate that the group is a divalent group, such as “alkylene,” “alkenylene,” “arylene,” and “heteroarylene.”

[0524] When two R groups are referred to as forming a ring (e.g., a carbocyclic, heterocyclic, aryl, or heteroaryl ring) "together with the atoms they are attached to," this means that the unit of assembly of the atoms and the two R groups is the described ring. The ring is not further limited by the definition of each R group when used individually. For example, when the following substructure exists:

[0525]

[0526] And R 1 and R 2 Defined as selected from hydrogen and alkyl, or R 1 and R 2 When they form heterocyclic groups together with the nitrogen atoms they are attached to, this means R 1 and R 2 It may be selected from hydrogen or alkyl groups, or the substructure has the following structure:

[0527]

[0528] Ring A is a heteroaryl ring containing the nitrogen described.

[0529] Similarly, when two “adjacent” R groups are said to form a ring together with the atoms they are attached to, this means that the unit of the atom, the intercalation bond, and the two R groups is the described ring. For example, this occurs when the following substructure exists:

[0530]

[0531] And R 1 and R 2 Defined as selected from hydrogen and alkyl, or R 1 and R 2 When R forms an aryl or carbocyclic group together with the atoms they are attached to, this means that R 1 and R 2 It may be selected from hydrogen or alkyl groups, or the substructure has the following structure:

[0532]

[0533] Where A is an aryl ring or carbocyclic group containing the described double bond.

[0534] Whenever a substituent is described as a divalent group (i.e., having two connection points with the rest of the molecule), it should be understood that unless otherwise indicated, the substituent can be configured in any direction. Thus, for example, described as –AE– or The substituents include those oriented such that A is attached at the leftmost connection point of the molecule, and those attached at the rightmost connection point of the molecule.

[0535] When a numerical range is disclosed and the notation "n1...to n2" or "between n1...and n2" is used, where n1 and n2 are numbers, this notation is intended to include the numbers themselves and the range between them, unless otherwise specified. This range may be integers or consecutive between the endpoints and includes the endpoints. For example, the range "2 to 6 carbons" is intended to include two, three, four, five, and six carbons, since carbon appears in integer units. By comparison, the range "1 to 3 μM (microvolume molar concentration)" is intended to include 1 μM, 3 μM, and every value between them with any number of significant figures (e.g., 1.255 μM, 2.1 μM, 2.9999 μM, etc.).

[0536] As used herein, the term “about” is intended to define the numerical value it modifies, thus representing the value as a variable within a certain error limit. When no specific error limit, such as standard deviation, is stated for the average value given in a data graph or table, the term “about” should be understood to mean the range that will cover the stated value and the range that will be included by rounding up or down to that number, taking into account significant figures.

[0537] The term "polyamide" refers to a polymer of connectable units chemically linked by amide (i.e., CONH) bonds; optionally, the polyamide includes a chemical probe conjugated thereto. Polyamides can be synthesized using methods known in the art, via the stepwise condensation of a carboxylic acid (COOH) with an amine (RR'NH). Alternatively, polyamides can be formed using in vitro enzymatic reactions or by employing microbial fermentation.

[0538] The term "linkable unit" refers to methylimidazolium, methylpyrrole, and linear and branched aliphatic functional groups (e.g., methylene, ethylene, propylene, butylene, etc.), and their chemical derivatives, optionally containing nitrogen substituents. The aliphatic functional groups of the linkable unit can be provided, for example, by condensing β-alanine or dimethylaminopropylamine during the synthesis of polyamides using methods well known in the art.

[0539] The term "joint" refers to a chain of at least 10 consecutive atoms. In some embodiments, the joint contains no more than 20 non-hydrogen atoms. In some embodiments, the joint contains no more than 40 non-hydrogen atoms. In some embodiments, the joint contains no more than 60 non-hydrogen atoms. In some embodiments, the joint contains atoms selected from C, H, N, O, and S. In some embodiments, each non-hydrogen atom is chemically bonded to two adjacent atoms in the joint, or to one adjacent atom in the joint and the end of the joint. In some embodiments, the joint forms an amide bond with at least one of the two other groups to which it is attached. In some embodiments, the joint forms an ester or ether bond with at least one of the two other groups to which it is attached. In some embodiments, the joint forms a thioester or thioether bond with at least one of the two other groups to which it is attached. In some embodiments, the joint forms a direct carbon-carbon bond with at least one of the two other groups to which it is attached. In some embodiments, the joint forms an amine or amide bond with at least one of the two other groups to which it is attached. In some embodiments, the joint comprises a –(CH2OCH2)- unit. In some embodiments, the connector comprises –(CH(CH3)OCH2)- units. In some embodiments, the connector comprises –(CH2NR)- units. N CH2) unit, R N =C 1-4 Alkyl group. In some embodiments, the connector comprises an arylene, cycloalkyl, or heteroalkyl moiety.

[0540] The term "spacer region" refers to a chain of at least five consecutive atoms. In some embodiments, the spacer region contains no more than 10 non-hydrogen atoms. In some embodiments, the spacer region contains atoms selected from C, H, N, O, and S. In some embodiments, the spacer region forms an amide bond with the two other groups to which it is attached. In some embodiments, the spacer region contains –(CH2OCH2)- units. In some embodiments, the spacer region contains –(CH2NR)- units. N CH2)-unit, R N =C 1-4 Alkyl group. In some embodiments, the spacer region contains at least one positive charge at physiological pH.

[0541] The term "corner assembly" refers to a chain of approximately 4 to 10 consecutive atoms. In some embodiments, the corner assembly contains atoms selected from C, H, N, O, and S. In some embodiments, the corner assembly forms amide bonds with two other groups to which it is attached. In some embodiments, the corner assembly contains at least one positive charge at physiological pH.

[0542] The terms “nucleic acid” and “nucleotide” refer to ribonucleotides and deoxyribonucleotides, as well as their analogues, as are well known in the art.

[0543] The term "oligonucleotide sequence" refers to multiple nucleic acids having a defined sequence and length (e.g., 2, 3, 4, 5, 6, or even more nucleotides). The term "oligonucleotide repeat sequence" refers to the sequential amplification of an oligonucleotide sequence.

[0544] The term "transcription," as well known in the art, refers to the synthesis of RNA (i.e., ribonucleic acid) by DNA-directed RNA polymerase. The term "regulated transcription" refers to changes in transcriptional levels that can be measured by methods well known in the art, such as the determination of the transcription product mRNA. In some embodiments, regulation is an increase in transcription. In other embodiments, regulation is a decrease in transcription.

[0545] As used alone or in combination herein, the term "acyl" refers to a carbonyl group in which the atom attached to an alkenyl, alkyl, aryl, cycloalkyl, heteroaryl, heterocyclic, or any other part of the molecule where the atom attached to the carbonyl group is carbon. "Acetyl" refers to a –C(O)CH3 group. "Alkyl carbonyl" or "alkanoyl" refers to an alkyl group attached to a portion of the parent molecule via a carbonyl group. Examples of such groups include methyl carbonyl and ethyl carbonyl. Examples of acyl groups include formyl, alkanoyl, and aromatic acyl groups.

[0546] As used herein, the term "alkenyl" alone or in combination refers to a straight-chain or branched hydrocarbon group having one or more double bonds and containing 2 to 20 carbon atoms. In some embodiments, the alkenyl group will contain 2 to 6 carbon atoms. The term "alkenylene" refers to a carbon-carbon double bond system linked at two or more positions, such as vinylene [(-CH=CH-), (-C::C-)]. Suitable examples of alkenyl groups include vinyl, propenyl, 2-methylpropenyl, 1,4-butadienyl, etc. Unless otherwise specified, the term "alkenyl" may include "alkenylene".

[0547] As used alone or in combination herein, the term "alkoxy" refers to an alkyl ether group, wherein the term alkyl is defined as follows. Examples of suitable alkyl ether groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, etc.

[0548] As used herein, the term "alkyl" alone or in combination means a straight-chain or branched alkyl group containing 1 to 20 carbon atoms. In some embodiments, the alkyl group will contain 1 to 10 carbon atoms. In other embodiments, the alkyl group will contain 1 to 8 carbon atoms. Alkyl groups may optionally be substituted as defined herein. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, octyl, nonyl, etc. As used herein, the term "alkylene" alone or in combination means a saturated aliphatic group derived from a straight-chain or branched saturated hydrocarbon and linked at two or more positions, such as methylene (-CH2-). Unless otherwise specified, the term "alkyl" may include "alkylene".

[0549] As used alone or in combination herein, the term "alkylamino" refers to an alkyl group attached to a parent molecule via an amino group. Suitable alkylamino groups can be monoalkylated or dialkylated, thereby forming groups such as N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-ethylmethylamino, etc.

[0550] As used alone or in combination in this article, the term "alkylidene" refers to an alkenyl group in which one carbon atom of the carbon-carbon double bond is part of the alkenyl group to which it is attached.

[0551] As used alone or in combination herein, the term "alkyl thio" refers to an alkyl thioether (R–S–) group, wherein the term alkyl is as defined above, and wherein the sulfur may be single or double-oxidized. Examples of suitable alkyl thioether groups include methyl thio, ethyl thio, n-propyl thio, isopropyl thio, n-butyl thio, isobutyl thio, sec-butyl thio, tert-butyl thio, methanesulfonyl, ethanesulfinyl, etc.

[0552] As used herein, the term "alkynyl" alone or in combination refers to a straight-chain or branched hydrocarbon group having one or more triple bonds and containing 2 to 20 carbon atoms. In some embodiments, the alkynyl group contains 2 to 6 carbon atoms. In other embodiments, the alkynyl group contains 2 to 4 carbon atoms. The term "ynynyl" refers to a carbon-carbon triple bond connected at two positions, such as ethynylene (-C:::C-, -C≡C-). Examples of alkynyl groups include ethynyl, propynyl, hydroxypropynyl, butyn-1-yl, butyn-2-yl, pentyyn-1-yl, 3-methylbutyn-1-yl, hexyn-2-yl, etc. Unless otherwise specified, the term "alkynyl" may include "ynynyl".

[0553] As used alone or in combination herein, the terms “amide group” and “carbamoyl group” refer to an amino group attached to the parent molecule moiety via a carbonyl group, as described below, or vice versa. As used alone or in combination herein, the term “C-amide group” refers to a -C(O)N(RR') group, where R and R' are as defined herein or as defined by a specifically listed designated “R” group. As used alone or in combination herein, the term “N-amide group” refers to an RC(O)N(R')- group, where R and R' are as defined herein or as defined by a specifically listed designated “R” group. As used alone or in combination herein, the term “acylamino” includes an acyl group attached to the parent moiety via an amino group. An example of “acylamino” is acetylamino (CH3C(O)NH-).

[0554] As used alone or in combination in this article, the term "amide" refers to -C(O)NRR', where R and R ’ The amide is independently selected from hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, any of which may optionally be substituted. Additionally, R and R' may be combined to form a heterocycloalkyl group, either of which may optionally be substituted. The amide can be formed by the direct condensation of a carboxylic acid with an amine or by using an acyl chloride. Furthermore, coupling agents are known in the art, including carbodiimide-based compounds such as DCC and EDCI.

[0555] As used alone or in combination in this article, the term "amino" refers to -NRR. ’ , where R and R ’ R is independently selected from hydrogen, alkyl, acyl, heteroalkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, any of which may be optionally substituted. Additionally, R and R' may be combined to form a heterocycloalkyl group, any of which may be optionally substituted.

[0556] As used alone or in combination herein, the term "aryl" refers to a carbocyclic aromatic system containing one, two, or three rings, wherein such polycyclic systems are fused together. The term "aryl" includes aromatic groups such as phenyl, naphthyl, anthracene, and phenanthryl. The term "arylene" includes aromatic groups such as phenylene, naphthylene, anthracene, and phenanthryl.

[0557] As used alone or in combination in this article, the terms "aryl-alkenyl" or "aryl-alkenyl" refer to an aryl group that is attached to a portion of the parent molecule via an alkenyl group.

[0558] As used alone or in combination herein, the terms “arylalkoxy” or “arylalkoxy” refer to an aryl group attached to a parent molecule via an alkoxy group.

[0559] As used alone or in combination herein, the terms “arylalkyl” or “arylalkyl” refer to an aryl group attached to a portion of the parent molecule by an alkyl group.

[0560] As used alone or in combination in this article, the terms "arylynyl" or "aryynyl" refer to an aryl group that is attached to a part of the parent molecule via an ynyl group.

[0561] As used alone or in combination herein, the terms “arylalkylacyl” or “aryl acyl” refer to an acyl group derived from an aryl-substituted alkyl carboxylic acid, such as benzoyl, naphthoyl, phenylacetyl, 3-phenylpropionyl (hydrogenated cinnamoyl), 4-phenylbutyryl, (2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, etc.

[0562] As used alone or in combination in this article, the term aryloxyl refers to an aryl group that is attached to the parent molecule via an oxygen group.

[0563] As used alone or in combination herein, the term "benzo / benz" refers to the divalent group C6H4= derived from benzene. Examples include benzothiophene and benzimidazole.

[0564] As used alone or in combination herein, the term "carbamate" refers to an ester of carbamic acid (-NHCOO-) that may be attached to the parent molecule from the nitrogen or acid terminus and may optionally be substituted as defined herein.

[0565] As used alone or in combination herein, the term “O-carbamoyl” refers to the -OC(O)NRR' group – where R and R' are as defined herein.

[0566] As used alone or in combination herein, the term “N-carbamoyl” refers to the ROC(O)NR'- group, where R and R' are as defined herein.

[0567] As used herein, the term "carbonyl" includes, when alone, a formyl group [-C(O)H], and when combined, a -C(O)- group.

[0568] As used herein, the term "carboxyl" refers to -C(O)OH or the corresponding "carboxylate" anion, such as in carboxylates. "O-carboxyl" refers to the RC(O)O- group, where R is as defined herein. "C-carboxyl" refers to the -C(O)OR group, where R is as defined herein.

[0569] As used alone or in combination in this article, the term "cyano" refers to -CN.

[0570] As used herein, the term "cycloalkyl" or alternatively "carbocyclic" refers to a saturated or partially saturated monocyclic, bicyclic, or tricyclic alkyl group, wherein each cyclic moiety contains 3 to 12 carbon atom ring members, and which may optionally be a benzo[a]-fused ring system as defined herein. In some embodiments, the cycloalkyl group will contain 5 to 7 carbon atoms. Examples of such cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl, indanyl, octahydronaphthyl, 2,3-dihydro-1H-indanyl, adamantyl, etc. As used herein, "bicyclic" and "tricyclic" are intended to include both fused ring systems (such as decahydronaphthyl, octahydronaphthyl) and polycyclic (multicentric) saturated or partially unsaturated types. Isomers of the latter type are typically exemplified by bicyclic [1,1,1]pentane, camphor, adamantane, and bicyclic [3,2,1]octane.

[0571] As used alone or in combination in this article, the term "ester" refers to the carboxyl group that bridges the two parts connected at the carbon atom.

[0572] As used alone or in combination in this article, the term "ether" refers to the oxygen group that bridges the two parts connected at the carbon atom.

[0573] As used alone or in combination in this article, the terms “halogen” or “halogen” refer to fluorine, chlorine, bromine, or iodine.

[0574] As used alone or in combination in this article, the term "haloalkoxy" refers to a haloalkyl group that is attached to a portion of the parent molecule by an oxygen atom.

[0575] As used alone or in combination herein, the term "haloalkyl" refers to an alkyl group having the meaning as defined above, wherein one or more hydrogen atoms are replaced by a halogen. Specifically, this includes monohaloalkyl, dihaloalkyl, and polyhaloalkyl. As an example, a monohaloalkyl group may have an iodine, bromine, chlorine, or fluorine atom within the group. Dihaloalkyl and polyhaloalkyl groups may have two or more identical halogen atoms or combinations of different halogen groups. Examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. "Haloalkylene" refers to a haloalkyl group linked at two or more positions. Examples include fluoromethylene (-CFH-), difluoromethylene (-CF2-), chloromethylene (-CHCl-), etc.

[0576] As used alone or in combination herein, the term "heteroalkyl" refers to a stable straight or branched chain, or a combination thereof, fully saturated or containing 1 to 3 degrees of unsaturation, consisting of the stated number of carbon atoms and one to three heteroatoms selected from N, O, and S, wherein the N and S atoms may optionally be oxidized, and the N heteroatom may optionally be quaternized. One or more heteroatoms may be placed at any internal position of the heteroalkyl group. Up to two heteroatoms may be sequential, such as -CH2-NH-OCH3.

[0577] As used individually or in combination herein, the term "heteroaryl" refers to a fused monocyclic, bicyclic, or tricyclic ring system of 3 to 15 members, where at least one fused ring is aromatic, containing at least one atom selected from N, O, and S. In some embodiments, the heteroaryl group will contain 1 to 4 heteroatoms as ring members. In other embodiments, the heteroaryl group will contain 1 to 2 heteroatoms as ring members. In some embodiments, the heteroaryl group will contain 5 to 7 atoms. The term also includes fused polycyclic groups, wherein the heterocycle is fused to an aryl ring, wherein the heteroaryl ring is fused to other heteroaryl rings, wherein the heteroaryl ring is fused to a heterocyclic alkyl ring, or wherein the heteroaryl ring is fused to a cycloalkyl ring. Examples of heteroaryl groups include pyrroloyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, pyranyl, furanyl, thiophene, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, thiazolyl, isothiazolyl, indoleyl, isoindoleyl, indoleazinyl, benzimidazolyl, quinolinyl, isoquinolinyl, quinoxolinyl, quinazolinyl, inzolyl, benzotriazolyl, benzom-dioxacyclopentenyl, benzopyranyl, benzooxazolyl, benzooxadiazolyl, benzothiazolyl, benzothiazolyl, benzofuranyl, benzothiophene, cronone, coumarinyl, benzopyranyl, tetrahydroquinolinyl, tetrazopyridazinyl, tetrahydroisoquinolinyl, thiophene-pyridinyl, furan-pyridinyl, pyrrolopyridinyl, etc. Exemplary tricyclic heterocyclic groups include carbazole, benzoindolyl, phenanthrolinyl, dibenzofuranyl, acridineyl, phenanthridineyl, xanthonyl, etc.

[0578] As used individually or in combination herein, the terms "heterocyclic alkyl" and the interchangeable term "heterocycle" each refer to a saturated, partially unsaturated, or fully unsaturated (but non-aromatic) monocyclic, bicyclic, or tricyclic heterocyclic group containing at least one heteroatom as a ring member, wherein each said heteroatom may be independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the heterocyclic alkyl group will contain 1 to 4 heteroatoms as ring members. In other embodiments, the heterocyclic alkyl group will contain 1 to 2 heteroatoms as ring members. In some embodiments, the heterocyclic alkyl group will contain 3 to 8 ring members in each ring. In other embodiments, the heterocyclic alkyl group will contain 3 to 7 ring members in each ring. In other embodiments, the heterocyclic alkyl group will contain 5 to 6 ring members in each ring. "Heterocyclic alkyl" and "heterocycle" are intended to include sulfones, sulfoxides, N-oxides of tertiary nitrogen ring members, and carbocyclic fused ring and benzo[a]-fused ring systems; additionally, both terms also include systems in which the heterocycle is fused with an aryl or another heterocyclic group as defined herein. Examples of heterocyclic groups include tetrahydroisoquinoline, aziridine, aziridine, 1,3-benzodioxacyclopentenyl, dihydroisoindolyl, dihydroisoquinoline, dihydrozolinyl, dihydrobenzodioxacyclohexadienyl, dihydro[1,3]oxazolo[4,5-b]pyridyl, benzothiazolyl, dihydroindolyl, dihydropyridyl, 1,3-dioxacyclohexyl, 1,4-dioxacyclohexyl, 1,3-dioxacyclopentenyl, isoindolinyl, morpholinyl, piperazine, pyrrolidinyl, tetrahydropyridyl, piperidinyl, thiomorpholinyl, etc. Unless expressly prohibited, heterocyclic groups may optionally be substituted.

[0579] As used alone or in combination in this article, the term "hydrazine" refers to two amino groups bonded by a single bond, namely -NN-.

[0580] As used alone or in combination in this article, the term "hydroxyl group" refers to -OH.

[0581] As used alone or in combination in this article, the term "hydroxyalkyl" refers to a hydroxyl group attached to a portion of the parent molecule via an alkyl group.

[0582] As used alone or in combination in this article, the term "imino" refers to =N-.

[0583] As used alone or in combination in this article, the term "iminohydroxyl" refers to =N(OH) and =NO-.

[0584] The phrase “in the main chain” refers to the longest continuous or adjacent chain of carbon atoms that begins at the junction of a group with a compound or molecule having any of the structural formulas disclosed herein.

[0585] The term "isocyanate group" refers to the -NCO group.

[0586] The term "isothiocyanate" refers to the -NCS group.

[0587] The phrase “a straight chain of atoms” refers to the longest straight chain of atoms independently selected from carbon, nitrogen, oxygen, and sulfur.

[0588] As used alone or in combination herein, the term “lower” means, unless otherwise explicitly defined, to contain 1 to (and including) 6 carbon atoms (i.e., C1-C6 alkyl).

[0589] As used alone or in combination herein, the term “lower aryl” means phenyl or naphthyl, either of which may be optionally substituted as provided.

[0590] As used individually or in combination herein, the term “lower heteroaryl” means 1) a monocyclic heteroaryl containing five or six ring members, wherein said members, between one and four, may be heteroatoms selected from N, O, and S, or 2) a bicyclic heteroaryl, wherein each of the fused rings contains five or six ring members, between which one to four heteroatoms selected from N, O, and S.

[0591] As used alone or in combination herein, the term "lower cycloalkyl" means a monocyclic cycloalkyl group (i.e., C3-C6 cycloalkyl) having between three and six ring members. Lower cycloalkyl groups may be unsaturated. Examples of lower cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0592] As used alone or in combination herein, the term "lower heterocyclic alkyl" means a monocyclic heterocyclic alkyl group having between three and six ring members, wherein the heteroatoms between one and four may be selected from N, O, and S (i.e., C3-C6 heterocyclic alkyl). Examples of lower heterocyclic alkyl groups include pyrrolidinyl, imidazoalkyl, pyrazolyl, piperidinyl, piperazinyl, and morpholinyl. Lower heterocyclic alkyl groups may be unsaturated.

[0593] As used alone or in combination in this article, the term "lower amino" refers to -NRR. ’ , where R and R ’ It is independently selected from hydrogen and lower alkyl groups, either of which may optionally be substituted.

[0594] As used alone or in combination herein, the term “thiol” refers to an RS- group, where R is as defined herein.

[0595] As used alone or in combination in this article, the term "nitro" refers to –NO2.

[0596] As used alone or in combination in this article, the terms “oxygen group” or “oxo-” refer to –O–.

[0597] As used alone or in combination in this article, the term "oxo" refers to =O.

[0598] The term "perhaloalkoxy" refers to an alkoxy group in which all hydrogen atoms are replaced by halogen atoms.

[0599] As used alone or in combination herein, the term "fully halogenated alkyl" refers to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0600] As used individually or in combination in this article, the terms “sulfonate,” “sulfonic acid,” and “sulfonic acid group” refer to the –SO3H group and its anion (when sulfonic acid is used to form salts).

[0601] As used alone or in combination in this article, the term "thioalkyl" refers to –S–.

[0602] As used alone or in combination in this article, the term "sulfinyl" refers to –S(O)–.

[0603] As used alone or in combination in this article, the term "sulfonyl" refers to –S(O)2–.

[0604] The term “N-sulfonamide group” refers to the RS(=O)2NR'- group, where R and R' are as defined herein.

[0605] The term “S-sulfonamide group” refers to the -S(=O)2NRR' group, where R and R' are as defined herein.

[0606] As used individually or in combination herein, the terms “thia” and “thio” refer to an –S– group or an ether in which oxygen is replaced by sulfur. Oxidized derivatives of the thio group, namely sulfinyl and sulfonyl groups, are included in the definitions of thia and thio.

[0607] As used alone or in combination in this article, the term "thiol" refers to the –SH group.

[0608] As used herein, the term "thiocarbonyl" includes, when alone, a thioformyl group –C(S)H, and when combined, a –C(S)– group.

[0609] The term “N-thiocarbamoyl” refers to the ROC(S)NR'– group, where R and R' are as defined herein.

[0610] The term “O-thiocarbamoyl” refers to the –OC(S)NRR’ group, where R and R’ are as defined herein.

[0611] The term "cyanothio" refers to the -CNS group.

[0612] The term “trihalomethanesulfonamide” refers to the X3CS(O)2NR– group, where X is a halogen and R is as defined herein.

[0613] The term "trihalomethanesulfonyl" refers to the X3CS(O)2– group, where X is a halogen.

[0614] The term "trihalomethoxy" refers to the X3CO– group, where X is a halogen.

[0615] As used alone or in combination herein, the term "trisubstituted silyl" refers to a silicone group whose three free valences are replaced by groups listed herein under the definition of substituted amino groups. Examples include trimethylsilyl, tert-butyldimethylsilyl, triphenylsilyl, etc.

[0616] Any definition herein may be combined with any other definition to describe complex structural groups. By convention, the tail component of any such definition is a component attached to the parent part. For example, the complex group alkylamide will represent an alkyl group attached to the parent molecule via an amide group, and the term alkoxyalkyl will represent an alkoxy group attached to the parent molecule via an alkyl group.

[0617] When a group is defined as "blank", it means that the group does not exist.

[0618] The term "optionally substituted" means that the preceding group may be substituted or not substituted. When substituted, the substituents of the "optionally substituted" group may include, but are not limited to, one or more substituents independently selected from, alone or in combination, the following groups or a group of specifically designated groups: lower alkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl, lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lower haloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl, phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, Lower acyloxy groups, carbonyl groups, carboxyl groups, lower alkyl carbonyl groups, lower carboxylic acid esters, lower carboxamide groups, cyano groups, hydrogen, halogens, hydroxyl groups, amino groups, lower alkylamino groups, arylamino groups, amide groups, nitro groups, thiols, lower alkylthio groups, lower haloalkylthio groups, lower perhaloalkylthio groups, arylthio groups, sulfonates, sulfonic acids, trisubstituted silyl groups, N3, SH, SCH3, C(O)CH3, CO2CH3, CO2H, pyridyl, thiophene, furanyl, lower carbamates, and lower ureas. When structurally feasible, two substituents may join together to form a fused five-, six-, or seven-membered carbon ring or heterocycle consisting of zero to three heteroatoms, such as forming methylenedioxy or ethylenedioxy. Optionally substituted groups may be unsubstituted (e.g., -CH2CH3), fully substituted (e.g., -CF2CF3), monosubstituted (e.g., -CH2CH2F), or substituted at any level between fully substituted and monosubstituted (e.g., -CH2CF3). When a substituent is described without specifying substitution, both substituted and unsubstituted forms are included. When a substituent is specified as "substituted," the substituted form is explicitly intended. Additionally, different groups of optional substituents for specific parts may be defined as needed; in these cases, optional substitution will, as defined, often immediately follow the phrase "optionally substituted...".

[0619] As used herein, a substituted group is derived from an unsubstituted parent group in which one or more hydrogen atoms have been replaced by another atom or group. Unless otherwise indicated, when a group is considered "substituted," this means that the group is substituted by one or more substituents independently selected from: C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocycloyl (optionally substituted by a halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), C3-C7-carbocyclo-C1-C6-alkyl (optionally substituted by a halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkyl). Alkoxy-substituted), 3-10 membered heterocyclic groups (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3-10 membered heterocyclic -C1-C6-alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl(C1-C6)alkyl (optionally substituted with halogen). C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy substituted, 5-10 heteroaryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5-10 heteroaryl (C1-C6)alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), halogen, cyano, hydroxyl, C1-C6 alkoxy, C1-C6 alkoxy (C1-C6) alkyl The groups include alkyl (i.e., ether), aryloxy, hydrogen sulfide (mercapto), halo(C1-C6)alkyl (e.g., –CF3), halo(C1-C6)alkoxy (e.g., –OCF3), C1-C6 alkylthio, arylthio, amino, amino(C1-C6)alkyl, nitro, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxyl, O-carboxyl, acyl, cyanoxy, isocyanate, cyanothio, isothiocyanate, sulfinyl, sulfonyl, and oxo (=O). Whenever a group is described as "optionally substituted," the group can be substituted with any of the above substituents.

[0620] Unless otherwise defined, the term R or R', whether used alone or in the absence of a numerical designation, refers to a subset selected from: hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl, heteroaryl, and heterocycloalkyl, any of which may optionally be substituted. Such R and R' groups should be understood as optionally substituted as defined herein. Regardless of whether the R group is numerically labeled, for the purpose of selection from the group, each R group (including R, R', and R...) n Where n = (1, 2, 3, ... n)), each substituent and each term should be understood as independent of each other. If any variable, substituent, or term (e.g., aryl, heterocyclic, R, etc.) appears more than once in the structural formula or general structure, its definition for each occurrence is independent of its definition for each subsequent occurrence. Those skilled in the art will further recognize that certain groups may be attached to the parent molecule from either end as written or may occupy a position in the elemental chain. For example, asymmetric groups such as -C(O)N(R)- may be attached to the parent moiety at a carbon or nitrogen position.

[0621] Asymmetric centers are present in the compounds or molecules disclosed herein. These centers are designated by the symbols “R” or “S”, depending on the configuration of the substituents surrounding the chiral carbon atom. It should be understood that this disclosure covers all stereochemical isomers, including diastereomers, enantiomers, and epiomers, as well as d-isomers and l-isomers, and mixtures thereof. Individual stereoisomers of compounds or molecules may be prepared synthetically from commercially available starting materials containing chiral centers, or by preparing mixtures of enantiomers followed by separation, such as by conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic techniques, direct separation of enantiomers on a chiral column, or any other suitable method known in the art. Starting compounds or molecules having a particular stereochemistry are commercially available or may be prepared and resolved by techniques known in the art. Additionally, the compounds or molecules disclosed herein may exist in geometrically isomeric forms. This disclosure includes all cis, trans, syn, anti, engegen (E), and zisammen (Z) isomers, and suitable mixtures thereof. Additionally, the compounds or molecules may exist in tautomeric forms; all tautomeric isomers are provided in this disclosure. Furthermore, the compounds or molecules disclosed herein may exist in unsolvated forms as well as in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc. Generally, the solvated form is considered equivalent to the unsolvated form.

[0622] The term "bond" refers to a covalent bond between two atoms or two parts, where the atoms bound by the bond are considered part of the larger substructure. Unless otherwise specified, a bond can be a single, double, or triple bond. In a molecular drawing, a dashed line between two atoms indicates the location where another bond may or may not be present.

[0623] As used herein, the term “disease” is intended to be generally synonymous with the terms “symptom,” “syndrome,” and “condition” (as in a medical condition) and may be used interchangeably with these terms, since all of these terms reflect an abnormal condition of a person or animal’s body or a part thereof, which impairs normal function, is usually manifested by identifiable signs and symptoms, and results in a reduction in the person or animal’s lifespan or quality of life.

[0624] The term "combination therapy" refers to the administration of two or more therapeutic agents to treat the condition or symptom described in this disclosure. Such administration encompasses the combined administration of these therapeutic agents in a substantially simultaneous manner, such as using a single capsule containing a fixed ratio of active ingredients or using multiple separate capsules for each active ingredient. Furthermore, such administration also encompasses the sequential use of each type of therapeutic agent. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0625] The phrase “effective treatment” is intended to limit the amount of active ingredient used to treat a disease or condition or to achieve a clinical endpoint.

[0626] The term "therapeuticly acceptable" means that compounds or molecules (or salts, prodrugs, tautomers, zwitterionic forms, etc.) are suitable for contact with a patient's tissues without excessive toxicity, irritation, or allergic response, in proportion to a reasonable benefit / risk ratio, and effectively achieve their intended use.

[0627] As used herein, the term "treatment" in the context of patient care includes prevention. Treatment can also be preemptive in nature, meaning it can include disease prevention. Disease prevention can involve complete protection from disease, such as in the case of preventing infection by a pathogen, or it can involve preventing disease progression. For example, disease prevention may not mean completely blocking any disease-related effects at any level, but rather may mean preventing the symptoms of disease at a clinically significant or detectable level. Disease prevention can also mean preventing disease progression to a later stage of the disease.

[0628] The term "patient" is generally synonymous with the term "object" and includes all mammals, including humans. Examples of patients include humans; livestock such as cows, goats, sheep, pigs, and rabbits; and companion animals such as dogs, cats, rabbits, and horses. Preferably, the patient is a human.

[0629] The term "prodrug" refers to a compound or molecule that becomes more active in vivo. Some of the compounds or molecules disclosed herein may also exist in prodrug form, as described in Hydrolysis in Drug and Prodrug Metabolism: Chemistry, Biochemistry, and Enzymology (Testa, Bernard and Mayer, Joachim M. Wiley-VHCA, Zurich, Switzerland 2003). The prodrugs of the compounds described herein are structurally modified forms of the compounds that readily undergo chemical changes under physiological conditions to provide the compounds. Additionally, prodrugs can be converted into the compounds in an in vitro environment by chemical or biochemical methods. For example, when placed in a transdermal patch reservoir containing suitable enzymes or chemical reagents, a prodrug can be slowly converted into the compound. Prodrugs are often useful because, in some cases, they can be more readily administered than the compound or the parent drug. They can achieve bioavailability, for example, through oral administration, which is not possible with the parent drug. Prodrugs may also have improved solubility in pharmaceutical compositions compared to the parent drug. A wide variety of prodrug derivatives are known in the art, such as those that depend on the hydrolytic cleavage or oxidative activation of the prodrug. A non-limiting example of a prodrug would be a compound administered in ester form (i.e., "prodrug") but subsequently metabolized and hydrolyzed into a carboxylic acid (i.e., the active entity). Additional examples include peptide derivatives of compounds.

[0630] The compounds or molecules disclosed herein may exist in therapeutically acceptable salt forms. This disclosure includes the compounds or molecules listed above in salt forms comprising acid addition salts. Suitable salts include those formed from both organic and inorganic acids. Such acid addition salts will generally be pharmaceutically acceptable. However, non-pharmaceutically acceptable salts may be useful in the preparation and purification of the compounds or molecules discussed. Base addition salts may also be formed and may be pharmaceutically acceptable. For a more comprehensive discussion of the preparation and selection of salts, refer to Pharmaceutical Salts: Properties, Selection, and Use (Stahl, P. Heinrich, Wiley-VCHA, Zurich, Switzerland, 2002).

[0631] Base addition salts can be prepared during the final separation and purification of compounds or molecules by reacting the carboxyl group with a suitable base (such as a hydroxide, carbonate, or bicarbonate of a metal cation) or with ammonia or an organic primary, secondary, or tertiary amine. Thermo-acceptable cations for salts include lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as non-toxic quaternary ammonium cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, diphenylmethylamine, N,N-diphenylmethylphenethylamine, 1-N-methyl-1,2-diphenyl-2-hydroxyethylamine (1-ephenamine), and N,N'-diphenylmethylethylenediamine. Other representative organic amines that can be used to form base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine.

[0632] Other carrier materials and administration methods known in the pharmaceutical field may also be used. The pharmaceutical compositions disclosed herein can be prepared by any of the well-known pharmaceutical techniques, such as effective formulation and administration procedures. Preferred unit-dose formulations are those containing an effective dose or appropriate fraction of the active ingredient as described herein.

[0633] In addition to the ingredients specifically mentioned above, the formulations described above may also include other agents conventional in the art, taking into account the types of formulations discussed, such as flavoring agents, for example, formulations suitable for oral administration.

[0634] The amount of active ingredient that can be combined with carrier materials to produce a single dosage form will vary depending on the subject being treated and the specific mode of administration.

[0635] Compounds or molecules can be administered in various modes, such as orally, topically, or by injection. The precise amount of compound administered to a patient is the responsibility of the attending physician. The specific dose level for any particular patient will depend on a number of factors, including the activity of the particular compound used, age, weight, general health condition, sex, diet, time of administration, route of administration, rate of excretion, combination of drugs, the exact condition being treated, and the severity of the indication or condition. Furthermore, the route of administration may vary depending on the condition and its severity. The above considerations concerning effective formulation and administration procedures are well known in the art and are described in standard textbooks.

[0636] Combination and combination therapy

[0637] In some cases, it may be appropriate to administer at least one of the compounds described herein (or a pharmaceutically acceptable salt thereof) in combination with another therapeutic agent. For example, if a patient experiences hypertension as a side effect after receiving a compound described herein, it may be appropriate to administer an antihypertensive agent in combination with the initial therapeutic agent. Alternatively, for example, the therapeutic efficacy of a compound described herein may be enhanced by the administration of an adjuvant (i.e., an adjuvant alone may have only minimal therapeutic benefit, but when combined with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced). Alternatively, for example, the benefit experienced by the patient may be increased by administering a compound described herein together with another therapeutic agent (which also includes treatment regimens) that also has a therapeutic benefit. For example, in the treatment of diabetes involving the administration of a compound described herein, the therapeutic benefit may be increased by also providing the patient with another diabetes therapeutic agent. In any case, regardless of the disease, condition, or illness being treated, the overall benefit experienced by the patient may simply be the sum of the two therapeutic agents, or the patient may experience a synergistic benefit.

[0638] Specific, non-limiting examples of possible combination therapies include certain compounds disclosed herein used in conjunction with ACE inhibitors.

[0639] In any case, multiple therapeutic agents (at least one of which is a compound disclosed herein) may be administered in any order or even simultaneously. If simultaneous, the multiple therapeutic agents may be provided in a single combined form or in multiple forms (for example only, in a single pill or in two separate pills). One of the therapeutic agents may be administered in multiple doses, or both may be administered in multiple doses. If not simultaneous, the timing between multiple doses may be any duration ranging from several minutes to four weeks.

[0640] Therefore, in another aspect, some embodiments provide a method for treating an FXN-mediated condition in a human or animal subject requiring such treatment, the method comprising administering to the subject, in combination with at least one additional agent known in the art for treating said condition, an amount of the disclosed compound that effectively reduces or prevents said condition in the subject. In a related aspect, some embodiments provide a therapeutic composition comprising a combination of at least one disclosed compound and one or more additional agents for treating FXN-mediated conditions.

[0641] In addition to their use in human treatment, some of the compounds and formulations disclosed herein can also be used in veterinary treatment of companion animals, exotic animals, and farm animals, including mammals and rodents. More preferred animals include horses, dogs, and cats.

[0642] Compound Synthesis

[0643] The compounds disclosed herein can be prepared using the methods described in the general synthetic schemes and experimental procedures detailed below. The general synthetic schemes and experimental procedures are provided for illustrative purposes and are not intended to be limiting. Starting materials used to prepare the compounds of this disclosure are commercially available or can be prepared using conventional methods known in the art.

[0644] Abbreviation list

[0645] Ac2O = Acetic anhydride; AcCl = Acetyl chloride; AcOH = Acetic acid; AIBN = Azobisisobutyronitrile; aq. = Aqueous; Bu3SnH = Tributyltin hydride; CD3OD = Deuterated methanol; CDCl3 = Deuterated chloroform; CDI = 1,1′-Carbonyldiimidazole; DBU = 1,8-Dazabicyclo[5.4.0]undecyl-7-ene; DCM = Dichloromethane; DEAD = Diethyl azodicarboxylate; DIBAL-H = Diisobutylaluminum hydride; DIEA = DIPEA = N,N-Diisopropylethylamine; DMAP = 4-Dimethylaminopyridine; DMF = N,N-Dimethylformamide; DMSO-d6 = Deuterated dimethyl sulfoxide; DMSO = Dimethyl sulfoxide; DPPA = Diphenyl azidophosphate; EDC.HCl = EDCI.HCl = 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; Et2O = Diethyl ether; EtOAc = Ethyl acetate; EtOH = Ethanol; h = hours; HATU = 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylureon hexafluorophosphate methylammonium; HMDS = Hexamethyldisilazane; HOBT = 1-hydroxybenzotriazole; i-PrOH = Isopropanol; LAH = Lithium aluminum hydride; LiHMDS = Bis(trimethylsilyl)aminolithium; MeCN = Acetonitrile; MeOH = Methanol; MP-carbonate resin = Macroporous triethylammonium methyl polystyrene carbonate Salt resin; MsCl = methanesulfonyl chloride; MTBE = methyl tert-butyl ether; MW = microwave irradiation; n-BuLi = n-butyllithium; NaHMDS = sodium bis(trimethylsilyl)amino; NaOMe = sodium methoxide; NaOtBu = sodium tert-butoxide; NBS = N-bromosuccinimide; NCS = N-chlorosuccinimide; NMP = N-methyl-2-pyrrolidone; Pd(Ph3)4 = tetrakis(triphenylphosphine)palladium(0); Pd2(dba)3 = tris(diphenylmethyleneacetone)dipalladium(0); PdCl2(PPh3)2 = bis(triphenylphosphine)palladium(II); PG = protecting group; preparative HPLC = preparative high performance liquid chromatography; PyBop = ( Benztriazol-1-yloxy)tripyrrolidinephosphonium hexafluorophosphate; Pyr = pyridine; RT = room temperature; RuPhos = 2-dicyclohexylphosphino-2′,6′-diisopropyloxybiphenyl; sat. = saturated; ss = saturated solution; t-BuOH = tert-butanol; T3P = propylphosphonic anhydride; TBS = TBDMS = tert-butyldimethylsilyl; TBSCl = TBDMSCl = tert-butyldimethylchlorosilane; TEA = Et3N = triethylamine; TFA = trifluoroacetic acid; TFAA = trifluoroacetic anhydride; THF = tetrahydrofuran; Tol = toluene; TsCl = toluenesulfonyl chloride; XPhos = 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl. General synthetic methods for preparing the compound.

[0646] Generally, the polyamides of this disclosure can be synthesized via solid-support synthesis methods using compounds such as Boc-protected linear aliphatic and heteroaromatic amino acids and their alkylated derivatives, which are then cleaved from the support by ammonolysis, deprotected (e.g., with sodium thiophene), and purified by reversed-phase HPLC, as is well known in the art. The identity and purity of the polyamides can be verified using any of a variety of analytical techniques available to those skilled in the art, such as... 1 Determination by ¹H-NMR, analytical HPLC, or mass spectrometry.

[0647] The following schemes can be used to implement this disclosure:

[0648] Scheme I: Synthesis of Polyamide

[0649]

[0650] The compounds disclosed herein can be synthesized using Scheme I. For clarity and brevity, the scheme describes the synthesis of a diamide comprising subunits “C” and “D”, both represented as unspecified five-membered rings having amino and carboxyl moieties. The amino group of subunit “D” is protected with a protecting group “PG” such as Boc or CBz carbamate to produce 101. The free carboxylic acid is then reacted with a solid support using a coupling agent such as EDC to produce a supported compound 103. PG is removed under acidic conditions to produce a free amine 104, which is coupled with a nitrogen-protected carboxylic acid 105 to produce an amide 106. PG is removed under acidic conditions to produce a free amine 107. In this example, the free amine is reacted with acetic anhydride to form an acetamide (not shown). The molecule is then cleaved from the solid support under basic conditions to produce a carboxylic acid 108. The method for connecting the linker L and the recruitment portion X is disclosed below.

[0651] Technicians will understand that many variations of the above schemes can be used to provide a wide range of compounds:

[0652] 1) The sequence 104–106–107 can be repeated frequently as needed to form a longer polyamine sequence.

[0653] 2) A variety of aminoheterocyclic carboxylic acids can be used to form different subunits. Table 4 provides several heterocyclic amino acids considered for the synthesis of the compounds disclosed herein, but is not intended to be limiting. The carbamate protecting group PG can be incorporated using techniques well-established in the art.

[0654] Table 4. Heterocyclic amino acids.

[0655]

[0656]

[0657]

[0658] 3) Heterocyclic amino acids containing hydroxyl groups may be incorporated into Scheme I in their TBS ether form. Scheme II provides for the synthesis of TBS-protected heterocyclic amino acids considered for use in the synthesis of the molecules disclosed herein, but is not intended to be limiting.

[0659] Scheme II: Synthesis of TBS-protected heterocyclic amino acids

[0660]

[0661] 4) Aliphatic amino acids can be used in the above synthesis to form the spacer unit "W" and the subunit for recognizing DNA nucleotides. Table 5 provides several aliphatic amino acids considered for use in the synthesis of the molecules disclosed herein, but is not intended to be limiting.

[0662] Table 5. Aliphatic amino acids

[0663]

[0664]

[0665] Scheme III: Synthesis of polyamide / recruiting agent / connector conjugate.

[0666]

[0667] The connection between connector L and recruitment portion X can be achieved using the method disclosed in Scheme III, which uses the triethylene glycol moiety for connector L. Under Mitsunobu conditions, the triethylene glycol mono-TBS ether 301 is converted to a brominated compound 302. Recruitment portion X is then connected by replacing the bromine with a hydroxyl moiety, thereby providing ether 303. The TBS groups are then removed by fluorination to provide alcohol 304, which will be suitable for coupling with the polyamide moiety. Other methods for incorporating alternative connection points for connector L (including, but not limited to, propylene glycol or polyamine connectors) or recruitment portion X are apparent to those skilled in the art, including, but not limited to, the use of amines and thiols.

[0668] Scheme IV: Synthesis of polyamide / recruiting agent / connector conjugate.

[0669]

[0670] The synthesis of the XLY molecule can be accomplished using the method described in Scheme IV. Carboxylic acid 108 is converted to acyl chloride 401. Reaction with the alcohol functional group of 301 under basic conditions provides the coupling product 402. Other methods for performing the coupling procedure are apparent to those skilled in the art, including but not limited to the use of carbodiimide reagents. For example, amide coupling reagents that can be used are combinations of carbodiimides such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and ethyl-(N',N'-dimethylamino)propylcarbodiimide hydrochloride (EDC) with reagents such as 1-hydroxybenzotriazole (HOBt), 4-(N,N-dimethylamino)pyridine (DMAP), and diisopropylethylamine (DIEA), but are not limited thereto. Other reagents frequently used, depending on the specific coupling reaction, include (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidinephosphonium hexafluorophosphate (PyBOP), (7-azabenzotriazol-1-yloxy)tripyrrolidinephosphonium hexafluorophosphate (PyAOP), bromotripyrrolidinephosphonium hexafluorophosphate (PyBrOP), bis(2-oxo-3-oxazolyl)phosphinic acid chloride (BOP-Cl), and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylureonium hexafluorophosphate (HBTU). O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylureon tetrafluoroborate (TBTU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureon hexafluorophosphate (HATU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureon tetrafluoroborate (TATU), O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethylureon hexafluorophosphate (HCTU), carbonyl diimidazole (CDI), and N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH).

[0671] Linking protein-binding molecules to the oligomeric backbone

[0672] Typically, the oligomeric backbone is functionalized to suit the type of chemical reaction that allows for the attachment of the oligomer to the protein-binding moiety. Suitable typical reactions are amide coupling, ether formation (O-alkylation), amine formation (N-alkylation), and sometimes carbon-carbon coupling, but are not limited to these. General reactions for linking oligomers to protein binders are shown in the following schemes (VIII to X). The compounds and structures shown in Table 2 can be attached to the oligomeric backbone described herein at any chemically feasible location without interfering with the hydrogen bonds between the compound and the regulatory protein.

[0673] Scheme V. Amide Coupling

[0674]

[0675] Oligomers or protein binders can be functionalized to have a carboxylic acid, and another coupling counterpart can be functionalized with an amino group, so that these moieties can be conjugated together by an amide coupling agent. Applicable amide coupling agents are combinations of carbodiimides such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and ethyl-(N',N'-dimethylamino)propylcarbodiimide hydrochloride (EDC) with agents such as 1-hydroxybenzotriazole (HOBt), 4-(N,N-dimethylamino)pyridine (DMAP), and diisopropylethylamine (DIEA), but are not limited to these. Other reagents frequently used, depending on the specific coupling reaction, include (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidinephosphonium hexafluorophosphate (PyBOP), (7-azabenzotriazol-1-yloxy)tripyrrolidinephosphonium hexafluorophosphate (PyAOP), bromotripyrrolidinephosphonium hexafluorophosphate (PyBrOP), bis(2-oxo-3-oxazolyl)phosphinic acid chloride (BOP-Cl), and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylureonium hexafluorophosphate (HBTU). O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylureon tetrafluoroborate (TBTU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureon hexafluorophosphate (HATU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylureon tetrafluoroborate (TATU), O-(6-chlorobenzotriazol-1-yl)-N,N,N',N'-tetramethylureon hexafluorophosphate (HCTU), carbonyl diimidazole (CDI), and N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH).

[0676] Scheme VI. Ether Formation Reaction (O-alkylation Reaction)

[0677]

[0678] In ether formation reactions, oligomers or protein binders can be functionalized to have hydroxyl groups (phenols or alcohols), and another coupling counterpart can be functionalized with leaving groups such as halogens, toluenesulfonates, and methanesulfonates, so that these moieties can be conjugated together by a base or catalyst. The base can be selected from, but is not limited to, sodium hydride, potassium hydride, sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate. The catalyst can be selected from silver oxide, phase transfer reagents, iodide salts, and crown ethers.

[0679] Scheme VII. Amine formation reaction (N-alkylation reaction)

[0680]

[0681] In N-alkylation reactions, oligomers or protein binders can be functionalized to have an amino group (arylamine or alkylamine), and another coupling counterpart can be functionalized with a leaving group such as a halogroup, toluenesulfonate, or methanesulfonate, so that these moieties can be conjugated together directly or in the presence of a base or catalyst. The base can be selected from, but is not limited to, sodium hydride, potassium hydride, sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate. The catalyst can be selected from silver oxide, phase transfer reagents, iodide salts, and crown ethers. Alkylation of amines can also be achieved via reductive amination reactions, in which oligomers or protein binders can be functionalized to have an amino group (arylamine or alkylamine), and another coupling counterpart can be functionalized with an aldehyde or ketone group, so that these moieties can be conjugated together directly or in combination with a dehydrating agent, in the presence of a reducing agent (hydride source). The reducing agent can be selected from, but is not limited to, NaBH4, NaHB(OAc)3, and NaBH3CN, and the dehydrating agent is usually Ti(iPrO)4, Ti(OEt)4, Al(iPrO)3, orthoformate esters, and activated molecular sieves.

[0682] Cell penetration ligand

[0683] In one aspect, the molecules disclosed herein include a cell-penetrating ligand moiety. The cell-penetrating ligand moiety facilitates the transport of the compound across the cell membrane. In some embodiments, the cell-penetrating ligand moiety is a polypeptide. Several peptide sequences that facilitate movement into the cell include polycationic sequences such as polyR; and arginine-rich sequences interspersed with spacer regions, such as (RXR). n (X = 6-aminohexanoic acid) and (RXRRBR) n (B = β-alanine); sequences derived from penetrantin peptides; and sequences derived from PNA / PMO internalization peptides (Pip). The Pip5 series is characterized by the sequence ILFQY.

[0684] In some embodiments, the cell-penetrating polypeptide contains the N-terminal cationic sequence H2N-(R). n -CO-, where n = 5-10, including the terminal value. In some embodiments, the N-terminal cation sequence contains 1, 2, or 3 substitutions of R with amino acid residues independently selected from β-alanine and 6-aminohexanoic acid.

[0685] In some embodiments, the cell-penetrating polypeptide comprises an ILFQY sequence. In some embodiments, the cell-penetrating polypeptide comprises a QFLY sequence. In some embodiments, the cell-penetrating polypeptide comprises a QFL sequence.

[0686] In some embodiments, the cell-penetrating polypeptide contains a C-terminal cation sequence -HN-(R). n -COOH, where n = 5-10, including the terminal value. In some embodiments, the C-terminal cation sequence contains 1, 2, or 3 substitutions of R with amino acid residues independently selected from β-alanine and 6-aminohexanoic acid. In some embodiments, the C-terminal cation sequence is substituted with amino acid residues independently selected from β-alanine and 6-aminohexanoic acid at every other position. In some embodiments, the C-terminal cation sequence is -HN-RXRBRXRB-COOH.

[0687] Table 6. Cell-penetrating peptides.

[0688]

[0689]

[0690]

[0691]

[0692] Ac = Acetyl group; Bpg = L-bis-homopropylglycine = B = β-alanine; X = 6-aminohexanoic acid; dK / dR = corresponding D-amino acid.

[0693] This invention provides, but is not limited to, the following embodiments:

[0694] 1. A transcriptional regulatory molecule having a first terminal, a second terminal, and an oligomeric backbone, wherein:

[0695] a) The first end includes a DNA-binding portion capable of non-covalently binding to the nucleotide repeat sequence GAA;

[0696] b) The second end includes a protein-binding portion that binds to a regulatory molecule that regulates the expression of a gene containing the nucleotide repeat sequence GAA; and

[0697] c) The oligomeric backbone includes a joint between the first end and the second end.

[0698] 2. The transcriptional regulatory molecule as described in Embodiment 1, wherein the first end comprises a polyamide selected from linear polyamide, hairpin polyamide, H-pin polyamide, overlapping polyamide, slip polyamide, cyclic polyamide, tandem polyamide, and extended polyamide.

[0699] 3. The transcriptional regulatory molecule as described in embodiment 1 or 2, wherein the first end comprises a linear polyamide.

[0700] 4. The transcriptional regulatory molecule as described in any one of embodiments 1-3, wherein the polyamide is capable of binding the DNA with an affinity of less than 500 nM.

[0701] 5. The transcriptional regulatory molecule as described in any one of embodiments 1-4, wherein the first end comprises –NH-QC(O)-, wherein Q is an optionally substituted C. 6-10 arylene, optionally substituted 4-10-membered heterocyclic alkylene, optionally substituted 5-10-membered heteroarylene, or optionally substituted alkylene.

[0702] 6. The transcriptional regulatory molecule of any one of embodiments 1-5, wherein the first end comprises at least three heteroaromatic carboxamide moieties containing at least one heteroatom selected from O, N and S, and at least one aliphatic amino acid residue selected from glycine, β-alanine, γ-aminobutyric acid, 2,4-diaminobutyric acid and 5-aminovaleric acid.

[0703] 7. The transcriptional regulatory molecule as described in Embodiment 6, wherein the heteroaromatic carboxamide moiety is a monocyclic or bicyclic moiety.

[0704] 8. The transcriptional regulatory molecule of embodiment 6, wherein the first end comprises one or more carboxamide moieties selected from optionally substituted pyrrolic carboxamide monomers, optionally substituted imidazole carboxamide monomers, and β-alanine monomers.

[0705] 9. The transcriptional regulatory molecule of any one of embodiments 1-8, wherein the first terminal contains a structure of formula (A-1) or a pharmaceutically acceptable salt thereof:

[0706] –L 1a -[AM] p –E1

[0707] Equation (A-1),

[0708] in:

[0709] Each [AM] appears p times, and p is an integer in the range of 1 to 10;

[0710] L 1a It is key, C 1-6 Alkylene, -NR a -C 1-6 Alkylene-C(O)-, -NR a C(O)-、

[0711] -NR a -C 1-6 Alkylene, -O- or -OC 1-6 Alkylene;

[0712] Each A is selected from key, C 1-10 Alkylene, optionally substituted C 6-10 arylene, optionally substituted 4-10 heterocyclic arylene, optionally substituted 5-10 heterocyclic arylene,

[0713] -C 1-10 Alkylene -C(O)-, -C 1-10 Alkylene-NR a -、—CO—、—NR a —、

[0714] —CONR a —、—CONR a C 1-4 Alkylene-, -NR a CO-C 1-4 Alkylene-

[0715] —C(O)O—, —O—, —S—, -S(O)-, -S(O)2-, —C(=S)-NH—,

[0716] —C(O)-NH-NH—, —C(O)-N=N—, —C(O)-CH=CH—,

[0717] (CH2) 0-4 -CH = CH-(CH2) 0-4 -N(CH3)-C 1-6 Alkylene -NH-C 1-6 Alkylene -NH-, -OC 1-6 Alkylenes -O-, -NH-N=N-, -NH-C(O)-NH-, and any combination thereof, and at least one A is -CONH-;

[0718] Each M is a C that is optionally replaced. 6-10 arylene, optionally substituted 4-10-membered heterocyclic alkylene, optionally substituted 5-10-membered heteroarylene or optionally substituted alkylene;

[0719] E1 is H or -A E —G;

[0720] A E It does not exist or is -NHCO-;

[0721] G is selected from H and C, which are optionally substituted. 6-10 Aryl, optionally substituted 4-10 membered heterocyclic, optionally substituted 5-10 membered heteroaryl, optionally substituted C 1-6 Alkyl, C 0-4Alkylene -NHC(=NH)NH, -CN, -C 0-4 Alkylene-C(=NH)(NR) a R b -C 0-4 Alkylene-C(=N) + H2)-(NR a R b -C 1-5 Alkylene-NR a R b C 0-4 Alkylene

[0722] -NHC(=NH)R a and optionally substituted amines; and

[0723] Each R a and R b C, independently selected from H and optionally substituted. 1-6 Alkyl groups, optionally substituted C 3-10 cycloalkyl, optionally substituted C 6-10 Aryl, optionally substituted 4-10 heterocyclic groups and optionally substituted 5-10 heteroaryl groups.

[0724] 10. The transcriptional regulatory molecule of any one of embodiments 1-8, wherein the first terminal contains a structure of formula (A-2) or a pharmaceutically acceptable salt thereof:

[0725]

[0726] in:

[0727] Each Y 1 Y 2 Y 3 and Y 4 It can be CH or N independently;

[0728] Each Z 1 Z 2 Z 3 and Z 4 Independently O, S, or NR 1D ;

[0729] W1 is hydrogen, optionally substituted C1-C6 alkyl, -C(O)-NR 1E R 1F ,

[0730] -NR 1E -C(O)-NR 1E R 1F Or (AA) 1-10 ;

[0731] W2 is hydrogen, optionally substituted C1-C6 alkyl, -C(O)-NR 1E R 1F Or (AA) 1-10 ;

[0732] m1 is 1, 2, 3 or 4;

[0733] n1 is 0, 1, or 2; and

[0734] AA is an amino acid residue selected from β-alanine, lysine, and arginine.

[0735] Each R 1D and R 1E It is independently hydrogen or C1-C6 alkyl; and

[0736] R 1F It is hydrogen, optionally substituted C1-C 10 Alkyl, C1-C 10 Heteroalkyl, PEG 1-20 Or one or more AA.

[0737] 11. The transcriptional regulatory molecule of embodiment 10, wherein the first terminal contains a structure of formula (A-3) or a pharmaceutically acceptable salt thereof:

[0738]

[0739] 12. A transcriptional regulatory molecule as described in embodiment 10 or 11, wherein each Z 1 Z 2 Z 3 and Z 4 Independently is NR 1D , where R 1D It is a C1-C6 alkyl group.

[0740] 13. A transcriptional regulatory molecule as described in embodiment 10 or 11, wherein each Z 1 Z 2 Z 3 and Z 4 It is NCH3 on its own.

[0741] 14. The transcriptional regulatory molecule as described in any one of embodiments 10-13, wherein each Z 1 and Z 3 It is N; and each Z 2 and Z 4 It can be CH or N independently.

[0742] 15. The transcriptional regulatory molecule as described in Embodiment 14, wherein each Z 2 and Z 4 It is CH.

[0743] 16. The transcriptional regulatory molecule of any one of embodiments 10-15, wherein the first end comprises the structure of formula (A-4):

[0744]

[0745] 17. The transcriptional regulatory molecule as described in any one of embodiments 10-16, wherein W1 is optionally a substituted C1-C6 alkyl group or -C(O)-NR. 1E R 1F .

[0746] 18. The transcriptional regulatory molecule as described in Embodiment 17, wherein W1 is -C(O)-NR 1E R 1F , where R 1E It is hydrogen; and R 1F It is hydrogen, optionally substituted C1-C 10 Alkyl or PEG 1-20 .

[0747] 19. The transcriptional regulatory molecule as described in any one of embodiments 10-16, wherein W1 is hydrogen.

[0748] 20. The transcriptional regulatory molecule as described in any one of embodiments 10-19, wherein m1 is 2 or 3; and n1 is 0 or 1.

[0749] 21. The transcriptional regulatory molecule as described in embodiment 20, wherein n1 is 0.

[0750] 22. The transcriptional regulatory molecule as described in embodiment 20, wherein n1 is 1.

[0751] 23. The transcriptional regulatory molecule as described in any one of embodiments 1-22, wherein the linker has a length of less than about 50 angstroms.

[0752] 24. The transcriptional regulatory molecule as described in any one of embodiments 1-22, wherein the linker has a length of about 15 to 40 angstroms.

[0753] 25. The transcriptional regulatory molecule of any one of embodiments 1-22, wherein the linker comprises between 5 and 50 chain atoms.

[0754] 26. The transcriptional regulatory molecule of any one of embodiments 1-22, wherein the linker comprises a polymer having 2 to 50 spacer portions, and wherein the spacer portions are independently selected from -((CR 3a R 3b ) x -O) y -、-((CR3a R 3b ) x -NR 4a ) y -、-((CR 3a R 3b ) x -CH=CH-(CR 3a R 3b ) x -O) y - Optionally replaced -C 1-12 Alkyl groups, optionally substituted C 2-10 Alkenyl, optionally substituted C 2-10 alkynyl group, optionally substituted C 6-10 Aromatic, optionally substituted C 3-7 Cycloalkylene, optionally substituted 5- to 10-membered heteroalkylene, optionally substituted 4- to 10-membered heteroalkylene, amino acid residue, -O-, -C(O)NR 4a -、-NR 4a C(O)-、-C(O)-、-NR 4a -, -C(O)O-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 4a -、-NR 4a SO2- and -P(O)OH-, and any combination thereof;

[0755] Each x is independently 2-4;

[0756] Each y is independently 1-10;

[0757] Each R 3a and R 3b Independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkoxy, optionally substituted amino, carboxyl, carboxylic acid ester, acyl, acyloxy, acylamino, aminoacyl, optionally substituted alkylamide, sulfonyl, optionally substituted thioalkoxy, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and optionally substituted heterocyclic; and

[0758] Each R 4a C is either hydrogen or optionally substituted. 1-6 alkyl.

[0759] 27. The transcriptional regulatory molecule of any one of embodiments 1-26, wherein the oligomeric backbone comprises -(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 -,

[0760] Where a, b, c, d, and e are each independently 0 or 1, and where a, b, c, d, and e are all 0 or 1, respectively.

[0761] The sum of c, d, and e is between 1 and 5;

[0762] T 1 T 2 T 3 T 4 and T 5 Each is independently selected from the arbitrarily replaced (C1-C) 12 Alkylene, optionally substituted alkenylene, optionally substituted alkyneylene, (EA) w ,

[0763] (EDA) m (PEG) n (Modified PEG) n (AA) p —(CR) 2a OH) h —Optionally replaced (C6-C) 10 ) aryl, optionally substituted C 3-7 Cycloalkylene, optionally substituted 5- to 10-membered heteroalkylene, optionally substituted 4- to 10-membered heteroalkylene, disulfide, hydrazine, carbohydrate, β-lactam and ester;

[0764] Each m, p, and w is an integer from 1 to 20 independently;

[0765] n is an integer from 1 to 30;

[0766] h is an integer from 1 to 12;

[0767] EA has the following structure:

[0768]

[0769] EDA has the following structure:

[0770]

[0771] Each q is an independent integer from 1 to 6;

[0772] Each x is an independent integer from 2 to 4, and

[0773] Each r is independently 0 or 1;

[0774] (PEG) n With –(CR 2a R 2b -CR 2a R 2b -O) n -CR 2a R 2b - structure;

[0775] (Modified PEG) n With –(CH2-CR 2a =CR 2a -CH2-O)- or –(CR 2a R 2b -CR 2a R 2b -S)- Replacement (PEG) n At least one of –(CR) 2a R 2b -CR 2a R 2b The structure of -O)-;

[0776] AA stands for amino acid residue;

[0777] V 1 V 2 V 3 V 4 and V 5 Each is independently selected from the bond, -CO-, -NR 1a -、

[0778] -CONR 1a -、-NR 1a CO-、-CONR 1a C 1-4 Alkyl-, -NR 1a CO-C 1-4 alkyl-,

[0779] -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 1a -、

[0780] -NR 1a SO2- and -P(O)OH-;

[0781] Each R 1a C is independently hydrogen and / or optionally substituted.1-6 Alkyl; and each R 2a and R 2b It is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, halogen, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylic acid ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic and substituted heterocyclic.

[0782] 28. The transcriptional regulatory molecule as described in embodiment 27, wherein T 1 T 2 T 3 T 4 and T 5 Each is independently selected from (C1-C) 12 )alkyl, substituted (C1-C) 12 Alkyl group, (EA) w (EDA) m (PEG) n (Modified PEG) n (AA) p —(CR) 2a OH) h —Optionally substituted phenyl groups, piperidine-4-amino (P4A), piperidine-3-amino, piperazine, pyrrolidine-3-amino, azacyclobutane-3-amino, p-aminobenzyloxycarbonyl (PABC), m-aminobenzyloxycarbonyl (MABC), p-aminobenzyloxy (PABO), m-aminobenzyloxy (MABO), p-aminobenzyl, acetal groups, disulfides, hydrazine, carbohydrates, β-lactams, esters, (AA) p -MABC-(AA) p (AA) p -MABO-(AA) p (AA) p -PABO-(AA) p and (AA) p -PABC-(AA) p .

[0783] 29. The transcriptional regulatory molecule as described in embodiment 27, wherein T 1 T 2 T 3 T 4 and T 5 Each is independently selected from (C1-C) 12 )alkyl, substituted (C1-C)12 Alkyl group, (EA) w (EDA) m (PEG) n (Modified PEG) n (AA) p —(CR) 2a OH) h —Optionally replaced (C6-C) 10 ) arylene, 4-10 membered heterocyclic alkenes and optionally substituted 5-10 membered heteroarylene.

[0784] 30. The transcriptional regulatory molecule as described in Embodiment 27, wherein T 4 or T 5 It is arbitrarily replaced (C6-C) 10 )Asyl.

[0785] 31. The transcriptional regulatory molecule as described in Embodiment 27, wherein T 4 or T 5 It is a phenylene oxide that is optionally substituted.

[0786] 32. The transcriptional regulatory molecule of any one of embodiments 1-28, wherein the linker comprises –N(R 1a (CH2) x N(R 1b (CH2) x N–, where R 1a and R 1b Each x is independently selected from hydrogen or optionally substituted C1-C6 alkyl groups; and each x is independently an integer in the range of 1-6.

[0787] 33. The transcriptional regulatory molecule of any one of embodiments 1-32, wherein the linker comprises -(CH2-C(O)N(R”)-(CH2) q -N(R')-(CH2) q -N(R”)C(O)-(CH2) x -C(O)N(R”)-A-、-(CH2) x -C(O)N(R”)-(CH2CH2O) y (CH2) x -C(O)N(R”)-A-, -C(O)N(R”)-(CH2) q -N(R')-(CH2) q -N(R”)C(O)-(CH2) x -A-、-(CH2) x -O-(CH2CH2O) y -(CH2)x -N(R”)C(O)-(CH2) x -A- or -N(R”)C(O)-(CH2)-C(O)N(R”)-(CH2) x -O(CH2CH2O) y (CH2) x -A-; where R' is methyl; R” is hydrogen; each x and y is independently an integer from 1 to 10; each q is independently an integer from 2 to 10; and each A is independently selected from the C bonds, optionally substituted. 1-12 Alkyl groups, optionally substituted C 6-10 Aromatic, optionally substituted C 3-7 Cycloalkylene, optionally substituted 5- to 10-membered heteroalkylene, and optionally substituted 4- to 10-membered heteroalkylene.

[0788] 34. The transcriptional regulatory molecule as described in any one of embodiments 1-33, wherein the linker comprises PEG (polyethylene glycol).

[0789] 35. The transcriptional regulatory molecule as described in embodiment 34, wherein the adapter comprises 2-20 PEG units.

[0790] 36. The transcriptional regulatory molecule as described in embodiment 34, wherein the adapter comprises 6, 8, 10 or 15 PEG units.

[0791] 37. The transcriptional regulatory molecule of any one of embodiments 1-36, wherein the linker is coupled to the first end with a group selected from: –CO-, -NR 1a -、-CONR 1a -、-NR 1a CO-、-CONR 1a C 1-4 Alkyl-, -NR 1a CO-C 1-4 Alkyl-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O-, -SO2-, -SO2NR 1a -、-NR 1a SO2-, -P(O)OH-, -((CH2) x -O)-、-((CH2) y -NR 1a -, optionally replaced -C 1-12 Alkylene, optionally substituted C 2-10 alkenyl, optionally substituted C 2-10 Ethyne group, optionally substituted C 6-10 Aromatic, optionally substituted C 3-7Cycloalkylene, optionally substituted 5- to 10-membered heteroalkylene, and optionally substituted 4- to 10-membered heteroalkylene; wherein each x and y is independently 1-4, and each R 1a C is either hydrogen or optionally substituted. 1-6 alkyl.

[0792] 38. The transcriptional regulatory molecule of any one of embodiments 1-36, wherein the linker is coupled to the first end with a group selected from: –CO-, -NR 1a -、C 1-12 Alkyl, -CONR 1a -and-NR 1a CO-; where each R 1a C is either hydrogen or optionally substituted. 1-6 alkyl.

[0793] 39. The transcriptional regulatory molecule of any one of embodiments 1-35, wherein the second end comprises one or more optionally substituted C-termini. 6-10 aryl, optionally substituted C 4-10 Carbocyclic ring, optionally substituted 4- to 10-membered heterocyclic ring, or optionally substituted 5- to 10-membered heteroaryl ring.

[0794] 40. The transcriptional regulatory molecule of any one of embodiments 1-39, wherein the second end comprises at least one 5-10 heteroaryl group having at least two nitrogen atoms.

[0795] 41. The transcriptional regulatory molecule of any one of embodiments 1-40, wherein the second end comprises a portion capable of binding to the regulatory protein, and the portion is derived from a compound capable of binding to the regulatory protein.

[0796] 42. The transcriptional regulatory molecule of any one of embodiments 1-40, wherein the second end comprises at least one selected from optionally substituted diazine or optionally substituted diazepine. And optionally substituted phenyl groups.

[0797] 43. The transcriptional regulatory molecule of any one of embodiments 1-40, wherein the second end comprises a portion that binds to a bromo domain protein.

[0798] 44. The transcriptional regulatory molecule of any one of embodiments 1-43, wherein the second end comprises a diazine or a diazepine. cyclic ring, wherein the diazine or diazapyridine Ring and C 6-10 A 5-10 membered heteroaryl ring fused together, consisting of an aryl group or a heteroatom selected from S, N, and O.

[0799] 45. The transcriptional regulatory molecule of any one of embodiments 1-43, wherein the second end comprises an optionally substituted bicyclic or tricyclic structure.

[0800] 46. ​​The transcriptional regulatory molecule of embodiment 45, wherein the optionally substituted bicyclic or tricyclic structure comprises a diazapyridine fused to a thiophene ring. ring.

[0801] 47. The transcriptional regulatory molecule of embodiment 46, wherein the second end comprises an optionally substituted bicyclic structure, wherein the bicyclic structure comprises a diazapyridine fused to a thiophene ring. ring.

[0802] 48. The transcriptional regulatory molecule of embodiment 45, wherein the second end comprises an optionally substituted tricyclic structure, wherein the tricyclic structure is a diazaphosphonate fused with thiophene and triazole. ring.

[0803] 49. The transcriptional regulatory molecule of any one of embodiments 1-43, wherein the second end comprises a compound having the structure of formula 7:

[0804]

[0805] in;

[0806] R1 and R3 are each independently selected from alkoxy, alkyl, amino, halogen and hydrogen;

[0807] R2 is selected from alkoxy, alkyl, alkenyl, alkynyl, amide, amino, halogen, and hydrogen;

[0808] R6 and R7 are each independently selected from alkyl, alkoxy, amino, halogen, and hydrogen;

[0809] R5 is hydrogen;

[0810] Each W is independently selected from C and N, where if W is N, then p is 0 or 1, and if W is C, then p is 1; for W—(R4) p If W is C, then p is 1 and R4 is H, or if W is N, then p is 0; if R1 is hydrogen, then R3 is alkoxy; if R3 is hydrogen, then R1 is selected from amino and alkoxy; and at least one of R6 and R7 is independently selected from alkyl, alkoxy, amino and halogen.

[0811] 50. The transcriptional regulatory molecule of any one of embodiments 1-43, wherein the second end comprises a compound of formula 8:

[0812]

[0813] 51. The transcriptional regulatory molecule of any one of embodiments 1-40, wherein the second end comprises a compound having the structure of formula (9-A) or a pharmaceutically acceptable salt thereof:

[0814]

[0815] in;

[0816] Ring A is absent or is a 6-membered monocyclic aryl or heteroaryl group;

[0817] Y is -NH- or -O-;

[0818] R 9 R 10 and R 11 Each C is independently selected from hydrogen and optionally substituted. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl;

[0819] R 12 Selected from hydrogen, halogen, -NO2, -CN, optionally substituted aryl, optionally substituted C 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl;

[0820] or R 12 Yes -NR A R B ,in

[0821] R A and R B Each is independently hydrogen, and the C atoms are optionally substituted. 1-6 Alkyl or C 1-6 Heteroalkyl; and

[0822] x1 is an integer from 1 to 6.

[0823] 52. The transcriptional regulatory molecule of embodiment 51, wherein the second end comprises a compound having the structure of formula (9-C) or a pharmaceutically acceptable salt thereof:

[0824]

[0826] 53. The transcriptional regulatory molecule of any one of embodiments 1-43, wherein the second end comprises a compound having the structure of formula (10-A) or a pharmaceutically acceptable salt thereof:

[0827]

[0828] in;

[0829] Ring B is absent or is either a 5-6 membered monocyclic aryl or heteroaryl or a 4-8 membered heterocyclic ring;

[0830] Y is -NH- or -O-;

[0831] R 13 Selected from hydrogen or optionally substituted C1-C6 alkyl groups;

[0832] R 14 and R 15 Each C is independently selected from hydrogen and optionally substituted. 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl;

[0833] R 16 Selected from hydrogen, halogen, -NO2, -CN, optionally substituted aryl, optionally substituted C 1-6 Alkyl, C 1-6 Halogenated alkyl or C 1-6 Hydroxyalkyl;

[0834] or R 16 Yes -NR A R B ,in

[0835] R A and R B Each is independently hydrogen, and the C atoms are optionally substituted. 1-6 Alkyl or C 1-6 Heteroalkyl; and

[0836] x2 is an integer from 1 to 6.

[0837] 54. The transcriptional regulatory molecule of embodiment 53, wherein the second end comprises a compound having the structure of formula (10-B) or a pharmaceutically acceptable salt thereof:

[0838]

[0839] 55. The transcriptional regulatory molecule of any one of embodiments 1-43, wherein the second end comprises a compound having the structure of formula (12-A):

[0840]

[0841] in;

[0842] The ring C is absent or is a monocyclic 6-membered aryl or heteroaryl ring;

[0843] X 1 It is CH or N;

[0844] L 2 Yes -NR D -or-CR D H-;

[0845] R 23 It is a C1-C6 alkyl or a C3-C6 cycloalkyl; and

[0846] R 24 It is a halogen, alkyl, hydroxyalkyl, or haloalkyl; optionally substituted C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 hydroxyalkyl; and

[0847] R D Is it hydrogen or C? 1-3 alkyl.

[0848] 56. The transcriptional regulatory molecule as described in embodiment 55, wherein the loop C is:

[0849]

[0850] 57. The transcriptional regulatory molecule as described in embodiment 55, wherein the ring C is a phenyl group.

[0851] 58. A transcriptional regulatory molecule as described in embodiment 55 or 57, wherein the second end comprises a compound having the structure of formula (12-B) or a pharmaceutically acceptable salt thereof:

[0852]

[0853] 59. A transcriptional regulatory molecule as described in embodiment 52 or 54, wherein the second end comprises a compound having the structure of formula (12-C) or a pharmaceutically acceptable salt thereof:

[0854]

[0855] 60. The transcriptional regulatory molecule of any one of embodiments 1-43, wherein the second end is selected from:

[0856]

[0857] Or their pharmaceutically acceptable salts.

[0858] 61. The transcriptional regulatory molecule as described in any one of embodiments 1-43, wherein the protein-binding moiety is:

[0859] Or its pharmaceutically acceptable salt.

[0860] 62. The transcriptional regulatory molecule described in any of the foregoing embodiments is used as a drug.

[0861] 63. The transcriptional regulatory molecule described in any of the foregoing embodiments, used to manufacture a medicament for preventing or treating a disease or condition improved by overexpression of fxn.

[0862] 64. The transcriptional regulatory molecule described in any of the foregoing embodiments, for the treatment of Friedreich ataxia (FA).

[0863] 65. A pharmaceutical composition comprising a transcriptional regulatory molecule as described in any of the foregoing embodiments and a pharmaceutically acceptable carrier.

[0864] 66. A method for regulating the expression of fxn, the method comprising contacting fxn with a transcriptional regulatory molecule as described in any one of embodiments 1-61.

[0865] 67. A method for treating a disease caused by defective expression of fxn, the method comprising administering a therapeutically effective amount of a transcriptional regulatory molecule as described in any one of embodiments 1-61 to a patient in need.

[0866] 68. The method as described in embodiment 67, wherein the disease is Friedreich ataxia.

[0867] 69. A method for treating a disease caused by the expression of a defective fxn, the method comprising administering:

[0868] Therapeutic effective amounts of transcriptional regulation as described in any of embodiments 1-61

[0869] Molecules; and

[0870] Another treatment.

[0871] 70. A method for achieving an effect in a patient, the method comprising administering a therapeutically effective amount of a transcriptional regulatory molecule as disclosed herein or a salt thereof to the patient, wherein the effect is selected from muscle atrophy, ataxia, fasciculations and dementia.

[0872] Example

[0873] The following examples are given for the purpose of illustrating various embodiments of the invention and are not intended to limit the invention in any way. The embodiments of the invention and the methods described herein represent preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Variations and other uses thereof will be apparent to those skilled in the art, and are covered by the spirit of the invention as defined by the claims.

[0874] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous changes, variations, and substitutions will now be apparent to those skilled in the art without departing from the invention. Various alternatives to the embodiments described herein should be understood. The following claims are intended to define the scope of the invention, and methods and structures within the scope of these claims, as well as their equivalents, are covered by the claims.

[0875] Example 1. Synthesis of the compound

[0876] The following examples are intended to illustrate, and not limit, the disclosed implementations.

[0877] Scheme A describes the steps involved in preparing polyamides by linking a polyamide to an oligomeric backbone, followed by linking a ligand to the other end of the oligomeric backbone. Transcriptional regulatory molecules, such as those listed in Table 8 below, can be prepared using the synthetic schemes shown below.

[0878] Option A: Synthesis of the first end / second connector / connector conjugate.

[0879]

[0880] Ligands or protein binding agents can be attached to the oligomeric backbone using the schemes described below. The oligomeric backbone can be attached to the protein binding agent at any chemically feasible location on the protein binding agent without interfering with the binding between the protein binding agent and the regulatory protein. Protein binding agents often bind to regulatory proteins via hydrogen bonds; therefore, attaching the oligomeric backbone and the regulatory protein should not interfere with hydrogen bond formation.

[0881] Example 1A Synthesis of polyamide intermediates (first end) and derivatives

[0882] Synthesis of 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido)propionic acid (PAO1-OH)

[0883] Option 1.

[0884]

[0885] Step 1: Synthesis of ethyl 4-amino-1-methylimidazolium-2-carboxylate

[0886] Pd / C (8.01 g, 27% w / w) was added to a solution of ethyl 1-methyl-4-nitroimidazole-2-carboxylate (30.00 g, 150.63 mmol, 1.00 equivalent) in EtOH (120.00 mL) and EA (120.00 mL). The reaction was then stirred for 17 h at room temperature under a H2 atmosphere. The solid was filtered off, and the filtrate was concentrated to provide ethyl 4-amino-1-methylimidazole-2-carboxylate (22.30 g, 75.20%) as a yellow solid. LC / MS: C7H 11 The calculated mass of N3O2 is 169.09, while the measured mass is 170.10 [M+H]. + . 1 H NMR (400MHz, DMSO-d6) δ: 7.37 (s, 1H), 4.29-4.34 (m, 2H), 3.94 (s, 3H), 1.31 (t, J = 7.2Hz, 3H).

[0887] Step 2: Synthesis of ethyl 4-[3-[(tert-butoxycarbonyl)amino]propionamido]-1-methylimidazolium-2-carboxylate

[0888] Add 3-[(tert-butoxycarbonyl)amino]propionic acid (22.45 g, 118.65 mmol, 0.90 equivalent) and DMF (180.00 mL) to a 500 mL flask. Cool the mixture to 0 °C, then add HATU (75.18 g, 197.71 mmol, 1.50 equivalent) and DIEA (51.11 g, 395.43 mmol, 3.00 equivalent), and stir the mixture for 10 min. Then add ethyl 4-amino-1-methylimidazolium-2-carboxylate (22.30 g, 131.81 mmol, 1.00 equivalent) in portions. Stir the reaction at room temperature for 1 h. Quench the reaction with ice water (600 mL) and stir the solution for 15 min. Collect the precipitated solid by filtration, wash with water (3 × 50 mL), and dry under vacuum. This produced ethyl 4-[3-[(tert-butoxycarbonyl)amino]propionamido]-1-methylimidazolium-2-carboxylate (34.50 g, 76.90%), a pale yellow solid. LC / MS: C 15 H 24 The calculated mass of N4O5 was 340.17, while the measured mass was 341.20 [M+H]. + . 1H NMR (400MHz, DMSO-d6) δ: 10.63 (s, 1H), 7.52 (s, 1H), 6.80 (t, J = 5.6Hz, 1H), 4.23-4.28 (m, 2H ), 3.90 (s, 3H), 3.15-3.20 (m, 2H), 2.42 (t, J = 7.2Hz, 2H), 1.37 (s, 9H), 1.29 (t, J = 7.2Hz, 3H).

[0889] Step 3: Synthesis of 4-[3-[(tert-butoxycarbonyl)amino]propionamido]-1-methylimidazol-2-carboxylic acid

[0890] At room temperature, ethyl 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazolium-2-carboxylic acid (34.50 g, 101.36 mmol, 1.00 equivalent) was added dropwise to a stirred solution of 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazolium-2-carboxylic acid in MeOH (200.00 mL). The resulting mixture was stirred at 45 °C for 2 h. The mixture was concentrated under reduced pressure. The residue was dissolved in H₂O (50 mL). The mixture was acidified to pH 3–5 with 2 M HCl. The precipitated solid was collected by filtration, washed with H₂O (3 × 30 mL), and dried under vacuum. 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazolium-2-carboxylic acid (30.00 g, 94.77%) was obtained as a white solid. LC / MS: C 13 H 20 The calculated mass of N4O5 is 312.14, while the measured mass is 313.15 [M+H]. + . 1 H NMR (300MHz, DMSO-d6) δ: 10.53 (s, 1H), 7.48 (s, 1H), 6.79 (t, J = 5.4Hz, 1H), 3.89 (s, 3H), 3.15 -3.22 (m, 2H), 2.43 (t, J = 7.2Hz, 2H), 1.37 (s, 9H).

[0891] Step 4: Synthesis of methyl 4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylate

[0892] At 0 °C, TCFH (21.56 g, 76.84 mmol, 1.50 equivalent), NMI (12.62 g, 153.69 mmol, 3.00 equivalent), and methyl 4-amino-1-methylpyrrole-2-carboxylic acid hydrochloride (10.74 g, 56.34 mmol, 1.10 equivalent) were added in portions to a stirred solution of 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazolium-2-carboxylic acid (16.00 g, 51.23 mmol, 1.00 equivalent) in CH3CN (150.00 mL). The resulting mixture was stirred at room temperature for 2.0 h. The precipitated solid was collected by filtration, washed with CH3CN (3 × 20 mL), and dried under vacuum. A white solid, methyl 4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylate (19.00 g, 82.70%), was obtained. LC / MS: C 20 H 28 The calculated mass of N6O6 is 448.21, and the measured mass is 449.25 [M+H]. 1 H NMR(300MHz,DMSO-d6)δ:10.24(s,1H),10.11(s,1H),7.52(s,1H),7.33(s,1H),6.99(s,1H),6.82(t,J=5 .1Hz,1H),3.94(s,3H),3.85(s,3H),3.74(s,3H),3.16-3.23(m,2H),2.47(t,J=6.9Hz,2H),1.38(s,9H).

[0893] Step 5: Synthesis of methyl 4-[4-(3-aminopropionamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylate hydrochloride

[0894] A solution of methyl 4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylate (19.00 g, 42.37 mmol, 1.00 equivalent) in HCl / 1,4-dioxane (4 M, 200.00 mL) was stirred for 2 h at room temperature. The resulting mixture was concentrated under vacuum. A yellow solid of methyl 4-[4-(3-aminopropamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylate hydrochloride (19.00 g crude) was obtained. LC / MS: C 15 H 21 C l The calculated mass of N6O4 is 348.15, while the measured mass is 349.05 [M+H]. + . 1H NMR (300MHz, CD3OD) δ: 7.37 (s, 2H), 6.91 (s, 1H), 4.03 (s, 3H), 3.88 (s, 3H), 3.79 (s, 3H), 3.09 (t, J = 6.6Hz, 2H), 2.64 (t, J = 6.6Hz, 2H).

[0895] Step 6: Synthesis of methyl 3-[(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-yl)formamido]propionate

[0896] Add 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-carboxylic acid (11.00 g, 35.22 mmol, 1.00 equivalent) and DMF (300.00 mL) to a 1000 mL flask. Cool the mixture to 0 °C, then add HATU (20.09 g, 52.83 mmol, 1.50 equivalent), followed by dropwise addition of DIEA (18.21 g, 140.88 mmol, 4.00 equivalent). Stir the mixture for 10 min, then add methyl 3-aminopropionate (3.63 g, 35.22 mmol, 1.00 equivalent) in portions. Stir the reaction mixture at room temperature for 1 h. Pour the reaction mixture into ice / water (600 mL), filter out the solids, and dry under vacuum. The aqueous phase was extracted with EA (3 × 200 mL). The organic phases were combined and washed with H₂O (1 × 200 mL) and NaCl (1 × 200 mL), and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with pure EA. The fractions were combined and concentrated. Methyl 3-[(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-yl)formamido]propionate (13.00 g, 87.95%) was obtained as a yellow solid. LC / MS: C 17 H 27 The calculated mass of N5O6 is 397.20, while the measured mass is 398.20 [M+H]. + . 1 H NMR(400MHz, DMSO-d6)δ:10.28(s,1H),7.92(t,J=6.0Hz,1H),7.37(s,1H),6.77(t,J=6.0Hz,1H),3.88(s,3H) ,3.59(s,3H),3.42-3.47(m,2H),3.13-3.18(m,2H),2.56(t,J=6.0Hz,2H),2.42(t,J=6.0Hz,2H),1.35(s,9H).

[0897] Step 7: Synthesis of methyl 3-[[4-(3-aminopropionamido)-1-methylimidazol-2-yl]formamido]propionate hydrochloride

[0898] A solution of methyl 3-[(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-yl)formamido]propionate (11.00 g, 27.678 mmol, 1.00 equivalent) in HCl / 1,4-dioxane (4 M, 110.00 mL) was stirred for 1.0 h at room temperature. The resulting mixture was concentrated under vacuum to provide methyl 3-[[4-(3-aminopropamido)-1-methylimidazol-2-yl]formamido]propionate hydrochloride (11.00 g, crude product) as a yellow oil. LC / MS: C 12 H 19 The calculated mass of N5O4 is 297.14, while the measured mass is 298.20 [M+H]. + . 1 H NMR (400MHz, DMSO-d6) δ: 10.57 (s, 1H), 7.92 (t, J = 6.0Hz, 1H), 7.37 (s, 1H), 3.89 (s, 3H), 3. 59(s,3H),3.43-3.47(m,2H),2.97-3.05(m,2H),2.57-2.71(m,2H),2.56(t,J=6.0Hz,2H).

[0899] Step 8: Synthesis of methyl 1-methyl-4-(1-methylimidazol-2-amido)pyrrole-2-carboxylate

[0900] At 0 °C, TBTU (38.19 g, 118.94 mmol, 1.50 equivalent), methyl 4-amino-1-methylpyrrole-2-carboxylic acid hydrochloride (16.63 g, 87.24 mmol, 1.10 equivalent), and DIEA (30.74 g, 237.88 mmol, 3.00 equivalent) were added in portions to a stirred solution of 1-methylimidazolium-2-carboxylic acid (10.00 g, 79.29 mmol, 7.00 equivalent) in DMF (150.00 mL). The resulting mixture was stirred for 17 h at room temperature. The reaction mixture was poured into ice / water (450 mL). The precipitated solid was collected by filtration, washed with H2O (3 × 50 mL), and dried under vacuum. Methyl 1-methyl-4-(1-methylimidazolium-2-amido)pyrrole-2-carboxylic acid (16.50 g, 78.37%) was given as a white solid. LC / MS: C 12 H 14 The calculated mass of N4O3 is 262.11, while the measured mass is 263.15 [M+H]. + . 1H NMR(300MHz, DMSO-d6)δ:10.54(s,1H),7.54(s,1H),7.40(s,1H),7.04(s,2H),3.99(s,3H),3.85(s,3H),3.74(s,3H).

[0901] Step 9: Synthesis of 1-methyl-4-(1-methylimidazol-2-amido)pyrrole-2-carboxylic acid

[0902] At room temperature, LiOH solution (2M, 158.00 mL, 5.00 equivalent) was added dropwise to a stirred solution of methyl 1-methyl-4-(1-methylimidazol-2-amido)pyrrole-2-carboxylic acid (16.50 g, 62.91 mmol, 1.00 equivalent) in MeOH (100.00 mL). The resulting mixture was stirred at 45 °C for 2 h. The mixture was concentrated under reduced pressure. The residue was dissolved in H₂O (50 mL). The mixture was acidified to pH 3–5 with 2M HCl. The precipitated solid was collected by filtration, washed with H₂O (3 × 30 mL), and dried under vacuum. 1-Methyl-4-(1-methylimidazol-2-amido)pyrrole-2-carboxylic acid (12.00 g, 76.84%) was obtained as a white solid. LC / MS: C 11 H 12 The calculated mass of N4O3 is 248.09, while the measured mass is 249.10 [M+H]. + .

[0903] 1 H NMR(300MHz, DMSO-d6)δ:10.52(s,1H),7.48(s,1H),7.41(s,1H),7.06(s,1H),6.99(s,1H),3.99(s,3H),3.82(s,3H).

[0904] Step 10: Synthesis of methyl 1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrole-2-yl]formamido]propamido)imidazol-2-amido]pyrrole-2-carboxylic acid ester

[0905] At 0 °C, HATU (20.68 g, 54.38 mmol, 1.50 equivalent), DIEA (14.06 g, 108.77 mmol, 3.00 equivalent), and methyl 4-[4-(3-aminopropamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylate (13.89 g, 39.872 mmol, 1.10 equivalent) were added in portions to a stirred solution of 1-methyl-4-(1-methylimidazol-2-amido)pyrrole-2-carboxylic acid (9.00 g, 36.255 mmol, 1.00 equivalent) in DMF (150.00 mL) at a stirring temperature. The resulting mixture was stirred for 17 h at room temperature. The reaction mixture was then decanted into water / ice (450 mL) at 0 °C. The precipitated solid was collected by filtration, washed with H2O (3 × 50 mL), and dried under vacuum. Methyl 1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-carboxylate (14.00 g, 63.54%) was obtained as a yellow solid. LC / MS: C 26 H 30 N 10 Computational quality of O6: 578.23, Measured value: 579.10 [M+H] + . 1 HNMR(300MHz, DMSO-d6)δ:10.53(s,1H),10.29(s,1H),10.11(s,1H),8.10(t,J=5.4Hz,1H),7.52(s,1H),7.47(s,2H),7.25(s,1H),7.17(s ,1H),6.99(s,1H),6.97(s,1H),3.99(s,3H),3.95(s,3H),3.84(s,3H ), 3.82 (s, 3H), 3.69 (s, 3H), 3.42-3.49 (m, 2H), 2.60 (t, J = 7.2Hz, 2H).

[0906] Step 11: Synthesis of 1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-carboxylic acid]

[0907] LiOH (2M, 72.00 mL, 6.00 equivalents) was added to a solution of methyl 1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-yl]carbamoylformate (14.00 g, 24.20 mmol, 1.00 equivalent) in MeOH (70.00 mL). The mixture was stirred at 45 °C for 2 h. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in H2O (50 mL). The mixture was acidified to pH 3–5 with 2M HCl. The precipitated solid was collected by filtration, washed with H2O (3 × 20 mL), and dried under vacuum. A yellow solid of 1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-carboxylic acid (12.00 g, 81.49%) was obtained. LC / MS: C 25 H 28 N 10 Computational quality of O6: 564.22, Measured value: 565.15 [M+H] + . 1 H NMR(300MHz, DMSO-d6)δ:10.72(s,1H),10.32(s,1H),10.08(s,1H),8.14(t,J=6.0Hz,1H),7.51(s,1H),7.47(s,2H),7.27(s, 1H),7.23(s,1H),6.98(s,1H),6.94(s,1H),4.00(s,3H),3.95(s,3H),3.82(s,6H),3.44-3.46(m,2H),2.60(t,J=6.6Hz,2H).

[0908] Step 12: Synthesis of methyl 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido)propionate)

[0909] At 0 °C, HATU (12.12 g, 31.88 mmol, 1.50 equivalent), DIEA (8.24 g, 63.77 mmol, 3.00 equivalent), and methyl 3-[[4-(3-aminopropamido)-1-methylimidazol-2-yl]formamido]propionate (6.95 g, 23.38 mmol, 1.10 equivalent) were added in portions to a stirred solution of 1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido]formamido]propionate in DMF (100.00 mL). The resulting mixture was stirred for 2 h at room temperature. The reaction mixture was then poured into ice / water (300 mL) at 0 °C. The precipitated solid was collected by filtration and washed with H2O (3 × 30 mL), then dried under vacuum. Methyl 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-yl]carbamoyl]propamido]imidazol-2-yl]carbamoyl)propamido ... 37 H 45 N 15 O9's computational quality: 843.35, measured value: 844.55 [M+H] + . 1 H NMR(300MHz,DMSO-d6)δ:10.41(s,1H),10.37(s,1H),10.32(s,1H),9.96(s,1H),8.08(s,2H),7.96(s,1H),7.46(s,1H),7.42(s,1H),7.38(s, 1H),7.24(s,2H),7.03(s,1H),6.98(s,1H),6.93(s,1H),4.13(s,3H), 3.98(s,3H),3.95(s,3H),3.81(s,9H),3.60(s,6H),2.57-2.69(m,6H).

[0910] Step 13: Synthesis of 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido)propionic acid)

[0911] 2M LiOH (21.20 mL, 42.40 mmol, 4.00 equivalence) was added to a solution of methyl 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-yl]carbamoyl)propamido ...the(21 This produced 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-yl]carbamoyl]propamido]imidazol-2-yl]carbamoyl)propionic acid (8.70 g, 84.14%), as a brown solid. LC / MS: C 36 H 43 N 15 O9's computational quality: 829.34, measured value: 830.25 [M+H] + . 1 HNMR(300MHz,DMSO-d6)δ:10.46(s,1H),10.39(s,1H),10.31(s,1H),9.93(s,1H),8.05 -8.10(m,2H),7.87(t,J=6.0Hz,1H),7.42-7.46(m,3H),7.20-7.23(m,2H),7.07(s,1H),6.90-6.95(m,2H ),3.95(s,3H),3.92(s,3H),3.89(s,3H),3.79(s,3H),3.78(s,3H),3.38-3.41(m,6H),2.44-2.59(m,6H).

[0912] Synthesis of N-(3-((3-(((3-aminopropyl)(methyl)amino)propyl)amino)-3-oxopropyl)-1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrolo-2-carboxamido)propamido)-1H-imidazol-2-carboxamide (PA01-TRA)

[0913] Option 2.

[0914]

[0915] Step 1: Synthesis of tert-butyl (3-((3-aminopropyl)(methyl)amino)propyl)carbamate

[0916] At 0 °C, over 4.0 h, a solution of Boc₂O (22.40 g, 102.64 mmol, 1.00 equivalent) in DCM (100.00 mL) was added dropwise to a solution of bis(3-aminopropyl)(methyl)amine (30.00 g, 206.54 mmol, 2.00 equivalent) in DCM (100.00 mL). The mixture was then stirred overnight at room temperature. The solid was filtered off, and the filtrate was concentrated. The crude product was dissolved in CH₃CN (10 mL) and purified by reverse-phase column chromatography: column, C18 column; mobile phase, CH₃CN / water (0.05% NH₄HCO₃), gradient from 5% to 30% over 2.0 h, detector UV 220 nm. The fractions were combined and concentrated. 18.00 g, 64.00% of tert-butyl (3-((3-aminopropyl)(methyl)amino)propyl)carbamate was obtained as a colorless oil. LC / MS: C 12 H 27 The calculated mass of N3O2 is 245.21, while the measured mass is 246.15 [M+H]. + . 1 H NMR (400MHz, DMSO) δ: 6.80 (t, J = 5.2 Hz, 1H), 3.57-3.60 (m, 2H), 2.87-2.92 (m, 2H), 2.19-2.26 (m, 4H), 2.06 (s, 3H), 1.41-1.52 (m, 4H), 1.35 (s, 9H).

[0917] Step 2: Synthesis of tert-butyl carbamate (3-(methyl(3-(3-(1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carbamate)-1H-pyrrole-2-carbamate)propionamido)-1H-imidazol-2-carbamate)-1H-pyrrole-2-carbamate)propionamido)-1H-imidazol-2-carbamate)propionamido)propyl)amino)propyl)propyl)carbamate)

[0918] To a solution of 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-yl]carbamoyl]carbamoyl]carbamoyl]propamido]carbamoyl]propamido)propionic acid (2.50 g, 3.37 mmol, 1.00 equivalent) in DMF (100.00 mL), N-[3-[(3-aminopropyl)(methyl)amino]propyl]carbamate tert-butyl ester (1.11 g, 4.52 mmol, 1.50 equivalent) and HATU (1.72 g, 4.52 mmol, 1.50 equivalent) were added. Then DIEA (1.17 g, 9.04 mmol, 3.00 equivalent) was added. The mixture was stirred at room temperature for 2.0 h. The mixture was poured into 200 mL of ice / water, the solid was filtered off, and the filter cake was washed with H2O (20 mL) and dried under high vacuum. A yellow solid of tert-butyl (3-(3-(3-(1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propionamido)-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propionamido)propyl)amino)propyl)propyl)carbamate (2.80 g, 79.12%) was obtained. LC / MS: C 48 H 68 N 18 O 10 The calculated quality is 1056.54, while the measured quality is 1057.80 [M+H]. + .

[0919] Step 3: Synthesis of N-(3-((3-((3-aminopropyl)(methyl)amino)propyl)amino)-3-oxopropyl)-1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamide (PA01-TRA)

[0920] TFA (10.00 mL) was added to a solution of N-[3-[methyl([3-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido]pyrrolo-2-yl]formamido]propamido]propyl])amino]propyl]carbamate (2.80 g, 2.65 mmol, 1.00 equivalent) in DCM (30.00 mL). The reaction mixture was stirred for 1.0 h at room temperature. The solvent was then removed and the residue was lyophilized. A deep yellow solid, N-(3-((3-(((3-aminopropyl)(methyl)amino)propyl)amino)-3-oxopropyl)-1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrolo-2-carboxamido)propamido)-1H-imidazol-2-carboxamide (2.80 g, 93.89%), was obtained. LC / MS: C 43 H 60 N 18 O8's computational quality: 956.48, measured value: 957.70 [M+H] + .

[0921] Synthesis of N-[5-[(2-[[2-([2-[(2-aminoethyl)carbamoyl]ethyl]carbamoyl)-1-methylimidazol-4-yl]carbamoyl]ethyl)carbamoyl]-1-methylpyrrolo-3-yl]-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-carboxamide (PA01-EDA)

[0922] Option 3.

[0923]

[0924] Step 1: Synthesis of N-[2-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido)propamido]imidazol-2-yl]formamido)propamido]ethyl]tert-butyl carbamate

[0925] The procedure was the same as that for methyl 1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-yl]carbamoyl]imidazol-2-amido]pyrrolo-2-carboxylic acid. Using 150.00 mg of 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-yl]carbamoyl]propamido]imidazol-2-yl]carbamoyl)propionic acid, 120.00 mg of a yellow oily substance was obtained. N-[2-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamate]propamido)imidazol-2-amido]pyrrolo-2-yl]carbamate]imidazol-2-yl]carbamate]ethyl]tert-butyl carbamate (66.73% yield). LC / MS: C 43 H 57 N 17 O 10 Calculated quality: 971.45, Measured value: 487.05 [1 / 2M+H] + .

[0926] Step 2: Synthesis of N-[5-[(2-[[2-([2-[(2-aminoethyl)carbamoyl]ethyl]carbamoyl)-1-methylimidazol-4-yl]carbamoyl]ethyl)carbamoyl]-1-methylpyrrolo-3-yl]-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-carboxamide (PA01-EDA)

[0927] The procedure is the same as that for N-(3-((3-(((3-aminopropyl)(methyl)amino)propyl)amino)-3-oxopropyl)-1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamide. Using 110.00 mg of N-[2-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]ethyl]tert-butyl carbamate, 100.00 mg was obtained. N-[5-[(2-[[2-([2-[(2-aminoethyl)carbamoyl]ethyl]carbamoyl)-1-methylimidazol-4-yl]carbamoyl]ethyl)carbamoyl]-1-methylpyrrolo-3-yl]-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-carboxamide (81.08% yield) was found as a yellow oil. LC / MS: C 38 H 49 N 17 Computational mass of O8: 871.40, measured mass: 894.60 [M+Na] + .

[0928] Synthesis of N-[5-([2-[(2-[[2-([3-[(3-aminopropyl)(2,2,2-trifluoroethyl)amino]propyl]carbamoyl]ethyl]carbamoyl]-1-methylimidazol-4-yl)carbamoyl]ethyl]carbamoyl)-1-methylpyrrole-3-yl]-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrole-2-yl]carbamoyl]propamido)imidazol-2-carboxamide (PA01-TRA(CH2CF3))

[0929] Option 4

[0930]

[0931] Step 1: Synthesis of N-[3-[(3-aminopropyl)amino]propyl]carbamate

[0932] Add DIEA (5.91 g, 45.72 mmol, 1.00 equivalent) to a stirred solution of norsemidine (6.00 g, 45.72 mmol, 1.00 equivalent) in THF (50.00 mL). Add 2.28 g, 9.15 mmol, 0.20 equivalent) of methyl benzoate 2,5-dioxopyrrolidine-1-yl ester in THF (30.00 mL) dropwise at -30 °C. Stir the resulting mixture at room temperature for 17 h. Filter the resulting mixture and wash the filter cake with THF (3 × 10 mL). Concentrate the filtrate under reduced pressure. Purify the residue by reversed-phase rapid chromatography under the following conditions: column, C18 column; mobile phase, CH3CN / water (0.05% TFA), 15% to 30% gradient over 10 min; detector, UV 254 nm. Combine the fractions and concentrate. N-[3-[(3-aminopropyl)amino]propyl]carbamate (1.50 g, 60.00%) was obtained as a yellow oil. LC / MS: C 14 H 23 The calculated mass of N3O2 is 265.18, while the measured mass is 266.15 [M+H]. + .

[0933] Step 2: Synthesis of N-[3-([3-[(tert-butoxycarbonyl)amino]propyl]amino)propyl]carbamate

[0934] At -60 °C, a solution of (Boc)₂O (0.58 g, 2.66 mmol, 0.50 equivalent) in DCM (10.00 mL) was added dropwise to a stirred solution of N-[3-[(3-aminopropyl)amino]propyl]carbamate (1.40 g, 5.28 mmol, 1.00 equivalent) in DCM (20.00 mL). The resulting mixture was stirred at -60 °C for 5 h, then heated to room temperature and stirred again at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 column; mobile phase, CH₃CN / water (0.05% NH₄HCO₃), 30% to 50% gradient over 20 min; detector, UV 220 nm. The fractions were combined and concentrated. N-[3-([3-[(tert-butoxycarbonyl)amino]propyl]amino)propyl]carbamate (500 mg, 23.63%) was obtained as a yellow oil. LC / MS: C 19 H 31 The calculated mass of N3O4 is 365.23, while the measured mass is 366.10 [M+H]. + .

[0935] Step 3: Synthesis of N-[3-([3-[(tert-butoxycarbonyl)amino]propyl](2,2,2-trifluoroethyl)amino)propyl]benzoate

[0936] At room temperature, methyl benzoate N-[3-([3-[(tert-butoxycarbonyl)amino]propyl]amino)propyl]carbamate (280.00 mg, 0.77 mmol, 1.00 equivalent) and K2CO3 (317.65 mg, 2.30 mmol, 3.00 equivalent) were added fractionally to a stirred solution of N-[3-([3-[(tert-butoxycarbonyl)amino]propyl]amino)propyl]carbamate in CH3CN (8.00 mL). The resulting mixture was stirred at 50 °C for 17 h. The solid was filtered off, and the filtrate was concentrated. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, ACN / water (0.05% NH4HCO3), 20% to 30% gradient over 100 min; detector, UV 254 nm. The fractions were combined and concentrated. N-[3-([3-[(tert-butoxycarbonyl)amino]propyl](2,2,2-trifluoroethyl)amino)propyl]carbamate (150 mg, 43.75%) was obtained as a colorless oil. LC / MS: C 21 H 32 The calculated mass of F3N3O4 is 447.23, while the measured mass is 448.20 [M+H]. + .

[0937] Step 4: Synthesis of N-[3-[(3-aminopropyl)(2,2,2-trifluoroethyl)amino]propyl]tert-butyl carbamate

[0938] Pd / C (30.00 mg, 20% w / w) was added to a solution of N-[3-([3-[(tert-butoxycarbonyl)amino]propyl](2,2,2-trifluoroethyl)amino)propyl]carbamate (150.00 mg, 0.34 mmol, 1.00 equivalent) in MeOH (8.00 mL). The reaction was then stirred for 17 h at room temperature under a H2 atmosphere. The resulting mixture was filtered, and the filter cake was washed with MeOH (3 × 5 mL). The filtrate was concentrated under reduced pressure. N-[3-[(3-aminopropyl)(2,2,2-trifluoroethyl)amino]propyl]carbamate tert-butyl ester (90.00 mg, 85.68%) was obtained as a colorless oil. LC / MS: C 13 H 26 The calculated mass of F3N3O2 is 313.20, while the measured mass is 314.15 [M+H]. + .

[0939] Step 5: Synthesis of N-[3-([3-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]propyl](2,2,2-trifluoroethyl)amino)propyl]tert-butyl carbamate

[0940] The procedure was the same as that for ethyl 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]carbamoyl]propamido)-1-methylimidazol-2-carboxylate. Using 212.00 mg of 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-yl]carbamoyl]propamido]imidazol-2-yl]carbamoyl)propamido)propionic acid (PA01-OH), 150.00 mg of N-[3-( [3-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]propyl](2,2,2-trifluoroethyl)amino)propyl]tert-butyl carbamate (42.94% yield). LC / MS: C 49 H 67 F3N 18 O 10 Calculated mass: 1124.52, measured mass: 1125.45 [M+H] + .

[0941] Step 6: Synthesis of N-[5-([2-[(2-[[2-([3-[(3-aminopropyl)(2,2,2-trifluoroethyl)amino]propyl]carbamoyl]ethyl]carbamoyl]-1-methylimidazol-4-yl)carbamoyl]ethyl]carbamoyl)-1-methylpyrrole-3-yl]-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrole-2-yl]carbamoyl]propamido)imidazol-2-carboxamide

[0942] The procedure is the same as that for N-(3-((3-(((3-aminopropyl)(methyl)amino)propyl)amino)-3-oxopropyl)-1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamide (PA01-TRA). Using 150.00 mg of N-[3-([3-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]propyl](2,2,2-trifluoroethyl)amino)propyl]carbamate, 150.0 mg was obtained. Crude N-[5-([2-[(2-[[2-([3-[(3-aminopropyl)(2,2,2-trifluoroethyl)amino]propyl]carbamoyl]ethyl]carbamoyl]-1-methylimidazol-4-yl)carbamoyl]ethyl]carbamoyl)-1-methylpyrrolo-3-yl]-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-carboxamide, present as a yellow oil. LC / MS: C 44 H 59 F3N 18 O8's computational quality: 1024.47, measured value: 1025.35 [M+H] + .

[0943] Synthesis of 1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)-N-[1-methyl-5-[(2-[[1-methyl-2-([2-[(3-[methyl[3-(methylamino)propyl]amino]propyl)carbamoyl]ethyl]carbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrolo-3-yl]imidazol-2-carboxamide (PA01-TRA(CH3))

[0944] Option 5

[0945]

[0946] Step 1: Synthesis of N-methyl-N-[3-(methylamino)propyl] tert-butyl carbamate

[0947] At 0 °C, (Boc)₂O (2135.89 mg, 9.79 mmol, 0.50 equivalent) was added dropwise to a stirred solution of methyl[3-(methylamino)propyl]amine (2000.00 mg, 19.57 mmol, 1.00 equivalent) in DCM (4.00 mL). The resulting mixture was stirred for 17 h at room temperature. Afterward, the reaction was quenched with water (30 mL) and extracted with DCM (3 × 50 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Tert-butyl N-methyl-N-[3-(methylamino)propyl]carbamate (2500.00 mg, 63.14%) was given as a yellow oil. LC / MS: C 10 H 22 The calculated mass of N₂O₂ is 202.18, while the measured mass is 203.20 [M+H]. + .

[0948] Step 2: Synthesis of N-(3-[[3-(1,3-dioxoisoindol-2-yl)propyl](methyl)amino]propyl)-N-methylcarbamate tert-butyl ester

[0949] At room temperature, N-(3-bromopropyl)phthalimide (3313.31 mg, 12.36 mmol, 1.00 equivalent) and K₂CO₃ (5123.83 mg, 37.07 mmol, 3.00 equivalent) were added in portions to a stirred solution of N-methyl-N-[3-(methylamino)propyl]carbamate (2500.00 mg, 12.36 mmol, 1.00 equivalent) in CH₃CN (100.00 mL). The resulting mixture was stirred at 70 °C for 17 h. After cooling to room temperature, the solid was filtered off, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel chromatography (DCM:MeOH = 10:1) to provide N-(3-[[3-(1,3-dioxoisoindol-2-yl)propyl](methyl)amino]propyl)-N-methylcarbamate tert-butyl ester (2600.00 mg, 54.02%) as a yellow oil. LC / MS: C 21 H 31 The calculated mass of N3O4 is 389.23, while the measured mass is 390.20 [M+H]. + .

[0950] Step 3: Synthesis of N-[3-[(3-aminopropyl)(methyl)amino]propyl]-N-methylcarbamate tert-butyl ester

[0951] Hydrazine (1028.44 mg, 32.09 mmol, 5.00 equivalent) was added dropwise to a stirred solution of N-(3-[[3-(1,3-dioxoisoindol-2-yl)propyl](methyl)amino]propyl)-N-methylcarbamate (2500.00 mg, 6.42 mmol, 1.00 equivalent) in MeOH (30.00 mL). The resulting mixture was stirred at 60 °C for 3 h. Afterward, the reaction was quenched with water (40 mL) and extracted with ethyl acetate (3 × 60 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. N-[3-[(3-aminopropyl)(methyl)amino]propyl]-N-methylcarbamate (2.30 g, crude) was given as a yellow oil. LC / MS: C 13 H 29 The calculated mass of N3O2 is 259.23, while the measured mass is 260.20 [M+H]. + .

[0952] Step 4: Synthesis of N-methyl-N-[3-[methyl([3-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]propyl])amino]propyl]tert-butyl carbamate

[0953] The procedure is the same as that for (3-(methyl(3-(3-(1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carbamoyl)-1H-pyrrole-2-carbamoyl)propamido)-1H-imidazol-2-carbamoyl)-1H-pyrrole-2-carbamoyl)propamido)-1H-imidazol-2-carbamoyl)propamido)propyl)amino)propyl)tert-butyl carbamate. Using 300.00 mg of 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido)propionic acid (PA01-OH), 200.00 mg of a yellow oily N-methyl -N-[3-[methyl([3-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]propyl])amino]propyl]tert-butyl carbamate (42.86% yield). LC / MS: C 49 H 70 N 18 O 10 The calculated mass is 1070.55, and the measured mass is 1071.75 [M+H]. + .

[0954] Step 5: Synthesis of 1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)-N-[1-methyl-5-[(2-[[1-methyl-2-([2-[(3-[methyl[3-(methylamino)propyl]amino]propyl)carbamoyl]ethyl]carbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrolo-3-yl]imidazol-2-carboxamide

[0955] The procedure is the same as that for N-(3-((3-(((3-aminopropyl)(methyl)amino)propyl)amino)-3-oxopropyl)-1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamide (PA01-TRA). Using 90.00 mg of N-methyl-N-[3-[methyl([3-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]propyl])amino]propyl]carbamate tert-butyl carbamate, 100 0.00 mg of a yellow oily substance, 1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)-N-[1-methyl-5-[(2-[[1-methyl-2-([2-[(3-[methyl[3-(methylamino)propyl]amino]propyl)carbamoyl]ethyl]carbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrolo-3-yl]imidazol-2-carboxamide. LC / MS: C 44 H 62 N 18 O8's computational quality: 970.50, measured value: 971.75 [M+H] + .

[0956] Synthesis of 1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)-N-[1-methyl-5-[(2-[[1-methyl-2-([2-[methyl(3-[methyl[3-(methylamino)propyl]amino]propyl)carbamoyl]ethyl]carbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrolo-3-yl]imidazol-2-carboxamide (PA01-TRA(diCH3))

[0957] Option 6

[0958]

[0959] Step 1: Synthesis of N-methyl-N-(3-[methyl[3-(methylamino)propyl]amino]propyl)carbamate tert-butyl ester

[0960] The procedure was the same as for tert-butyl (3-((3-aminopropyl)(methyl)aminopropyl)carbamate. Using 1.40 g of methyl (3-[methyl[3-(methylamino)propyl]amino]propyl)amine, 350.00 mg of N-methyl-N-(3-[methyl[3-(methylamino)propyl]amino]propyl)carbamate (15.85% yield) as a yellow oil was obtained. LC / MS: C 14 H 31 The calculated mass of N3O2 is 273.24, while the measured mass is 274.10 [M+H]. + .

[0961] Step 2: Synthesis of N-methyl-N-[3-[methyl([3-[N-methyl-3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido])imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]propyl])amino]propyl]tert-butyl carbamate

[0962] The procedure was the same as that for ethyl 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]carbamoyl]propamido)-1-methylimidazol-2-carboxylate. Using 300.00 mg of 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-yl]carbamoyl]propamido]imidazol-2-yl]carbamoyl)propionic acid (PA01-OH), 150.00 mg of N-methyl-N- [3-[methyl([3-[N-methyl-3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]propyl])amino]propyl]tert-butyl carbamate (38.10% yield). LC / MS: C 50 H 72 N 18 O 10 The calculated quality is 1084.57, and the measured value is 543.70 [M / 2+H]. + .

[0963] Step 3: Synthesis of 1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)-N-[1-methyl-5-[(2-[[1-methyl-2-([2-[methyl(3-[methyl[3-(methylamino)propyl]amino]propyl)carbamoyl]ethyl]carbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrolo-3-yl]imidazol-2-carboxamide]

[0964] The procedure is the same as that for N-(3-((3-(((3-aminopropyl)(methyl)amino)propyl)amino)-3-oxopropyl)-1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamide (PA01-TRA). Using 150.00 mg of tert-butyl N-methyl-N-[3-[methyl([3-[N-methyl-3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]propyl])amino]propyl]carbamate, 15 0.00 mg of crude 1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)-N-[1-methyl-5-[(2-[[1-methyl-2-([2-[methyl(3-[methyl[3-(methylamino)propyl]amino]propyl)carbamoyl]ethyl]carbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrolo-3-yl]imidazol-2-carboxamide, a yellow oil. LC / MS: C 45 H 64 N 18 O8's computational quality: 984.52, measured value: 985.40 [M+H] + .

[0965] Synthesis of N-(5-[[2-([2-[(2-[[3-(3-aminopropoxy)propyl]carbamoyl]ethyl)carbamoyl]-1-methylimidazol-4-yl]carbamoyl)ethyl]carbamoyl]-1-methylpyrrolo-3-yl)-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-carboxamide (PA01-TRA(O))

[0966] Option 7

[0967]

[0968] Step 1: Synthesis of N-[3-(3-aminopropoxy)propyl]tert-butyl carbamate

[0969] The procedure was the same as for tert-butyl (3-((3-aminopropyl)(methyl)amino)propyl)carbamate. Using 1.00 g of 3-(3-aminopropoxy)propyl-1-amine, 430.00 mg of N-[3-(3-aminopropoxy)propyl]carbamate (24.47% yield) as a yellow oil was obtained. LC / MS: C 11 H 24 The calculated mass of N₂O₃ is 232.18, while the measured mass is 233.15 [M+H]. + .

[0970] Step 2: Synthesis of N-(3-[3-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propoxy]propyl)tert-butyl carbamate

[0971] The procedure is the same as that for (3-(methyl(3-(3-(1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carbamoyl)-1H-pyrrole-2-carbamoyl)propamido)-1H-imidazol-2-carbamoyl)-1H-pyrrole-2-carbamoyl)propamido)-1H-imidazol-2-carbamoyl)propamido)propyl)amino)propyl)tert-butyl carbamate. Using 100.00 mg of 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido)propionic acid (PA01-OH), 90.00 mg of a yellow oily substance was obtained. N-(3-[3-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]propoxy]propyl)tert-butyl carbamate (64.37% yield). LC / MS: C 47 H 65 N 17 O 11 Calculated mass: 1043.50, measured mass: 1044.75 [M+H] + .

[0972] Step 3: Synthesis of N-(5-[[2-([2-[(2-[[3-(3-aminopropoxy)propyl]carbamoyl]ethyl)carbamoyl]-1-methylimidazol-4-yl]carbamoyl)ethyl]carbamoyl]-1-methylpyrrolo-3-yl)-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-carboxamide (PA01-TRA(O))

[0973] The procedure is the same as that for N-(3-((3-(((3-aminopropyl)(methyl)amino)propyl)amino)-3-oxopropyl)-1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamide (PA01-TRA). Using 90.00 mg of N-(3-[3-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propoxy]propyl)tert-butyl carbamate, 90.00 mg was obtained. Crude N-(5-[[2-([2-[(2-[[3-(3-aminopropoxy)propyl]carbamoyl]ethyl)carbamoyl]-1-methylimidazol-4-yl]carbamoyl]ethyl]carbamoyl]-1-methylpyrrolo-3-yl)-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-carboxamide, present as a yellow oil. LC / MS: C 42 H 57 N 17 O9 computational quality: 943.45, measured value: 944.70 [M+H] + .

[0974] Synthesis of N-[5-[(2-[[2-([2-[(7-aminoheptyl)carbamoyl]ethyl]carbamoyl)-1-methylimidazol-4-yl]carbamoyl]ethyl)carbamoyl]-1-methylpyrrolo-3-yl]-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-carboxamide (PA01-TRA(CH2))

[0975] Option 8

[0976]

[0977] Step 1: Synthesis of N-[7-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido)propamido]imidazol-2-yl]formamido)propamido]heptyl]tert-butyl carbamate

[0978] The procedure is the same as that for (3-(methyl(3-(3-(1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carbamoyl)-1H-pyrrole-2-carbamoyl)propamido)-1H-imidazol-2-carbamoyl)-1H-pyrrole-2-carbamoyl)propamido)-1H-imidazol-2-carbamoyl)propamido)propyl)amino)propyl)tert-butyl carbamate. Using 150.00 mg of 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido)propionic acid (PA01-OH), 100.00 mg of a yellow-colored product was obtained. N-[7-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]heptyl]tert-butyl carbamate (33.23% yield). LC / MS: C 48 H 67 N 17 O 10 The calculated mass is 1041.53, and the measured mass is 1042.85 [M+H]. + .

[0979] Step 2: Synthesis of N-[5-[(2-[[2-([2-[(7-aminoheptyl)carbamoyl]ethyl]carbamoyl)-1-methylimidazol-4-yl]carbamoyl]ethyl)carbamoyl]-1-methylpyrrole-3-yl]-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrole-2-yl]carbamoyl]propamido)imidazol-2-carboxamide

[0980] The procedure is the same as that for N-(3-((3-(((3-aminopropyl)(methyl)amino)propyl)amino)-3-oxopropyl)-1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamide (PA01-TRA). Using 90.00 mg of N-[7-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]heptyl]tert-butyl carbamate, 90.00 mg was obtained. Crude N-[5-[(2-[[2-([2-[(7-aminoheptyl)carbamoyl]ethyl]carbamoyl)-1-methylimidazol-4-yl]carbamoyl]ethyl)carbamoyl]-1-methylpyrrolo-3-yl]-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-carboxamide. LC / MS: C 43 H 59 N 17 O8's computational quality: 941.47, measured value: 942.75 [M+H] + .

[0981] Synthesis of N-[5-([2-[(2-[[2-([3-[(3-aminopropyl)(methyl)amino]propyl]carbamoyl]ethyl]carbamoyl]-1-methylimidazol-4-yl)carbamoyl]ethyl]carbamoyl)-1-methylpyrrole-3-yl]-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrole-2-yl]carbamoyl]propamido)imidazol-2-carboxamide (PA05-TRA)

[0982] Option 9

[0983]

[0984] Step 1: Synthesis of methyl 1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrole-2-yl]formamido]propamido)imidazol-2-amido]pyrrole-2-yl]formamido)propamido]imidazol-2-amido]pyrrole-2-carboxylic acid ester

[0985] NMI (727.14 mg, 8.86 mmol, 5.00 equivalent), TCFH (546.68 mg, 1.95 mmol, 1.10 equivalent), and methyl 4-[4-(3-aminopropamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylic acid (748.19 mg, 1.95 mmol, 1.10 equivalent) were added to a solution of 1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxylic acid (1.00 g, 1.77 mmol, 1.00 equivalent) in DMF (10.00 mL). The reaction was then stirred at room temperature for 2 h. The reaction mixture was purified by reversed-phase rapid chromatography under the following conditions: column, C18 column; mobile phase, MeOH / H2O (0.05% TFA), in a 5% to 75% gradient over 70 min; detector, UV 254 nm. The fractions were combined and concentrated to provide methyl 1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]imidazol-2-amido]pyrrolo-2-yl]carbamoyl]imidazol-2-amido]pyrrolo-2-yl]carbamoyl]imidazol-2-amido]pyrrolo-2-carboxylate as a white solid (1.00 g, 63.09% yield). LC / MS: C 40 H 46 N 16 O9 computational quality: 894.36, measured value: 895.55 [M+H] + .

[0986] Step 2: Synthesis of 1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido)propamido]imidazol-2-amido]pyrrolo-2-carboxylic acid]

[0987] The procedure was the same as for 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrole-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrole-2-yl]carbamoyl]propamido]imidazol-2-yl]carbamoyl)propionic acid (PA01-OH). Using 1.00 g of methyl 1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrole-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrole-2-yl]carbamoyl]imidazol-2-amido]pyrrole-2-carboxylic acid, 800.00 g was obtained. mg of 1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-yl]carbamoyl]imidazol-2-amido]pyrrolo-2-carboxylic acid as a white solid (81.27% yield). LC / MS: C 39 H 44 N 16 O9 computational quality: 880.35, measured value: 881.45 [M+H] + .

[0988] Step 3: Synthesis of N-[3-[methyl([3-[(1-methyl-4-[1-methyl-4-[3([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl)formamido]propyl])amino]propyl]tert-butyl carbamate

[0989] The procedure is the same as that for N-(3-((3-(((3-aminopropyl)(methyl)amino)propyl)amino)-3-oxopropyl)-1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamide. Using 800.00 mg of 1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]imidazol-2-amido]pyrrolo-2-yl]carbamoyl]imidazol-2-amido]pyrrolo-2-yl]carbamoyl]imidazol-2-amido]pyrrolo-2-carboxylic acid, 800.00 mg of N-[3-[methyl ([3-[(1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl)formamido]propyl])amino]propyl]tert-butyl carbamate (79.49% yield). LC / MS: C 51 H 69 N 19 O 10 Calculated quality: 1107.55, measured value: 1108.80 [M+H] + .

[0990] Step 4: Synthesis of N-[5-([2-[(2-[[2-([3-[(3-aminopropyl)(methyl)amino]propyl]carbamoyl]ethyl]carbamoyl]-1-methylimidazol-4-yl)carbamoyl]ethyl]carbamoyl)-1-methylpyrrole-3-yl]-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrole-2-yl]carbamoyl]propamido)imidazol-2-carboxamide

[0991] The procedure is the same as that for N-(3-((3-(((3-aminopropyl)(methyl)amino)propyl)amino)-3-oxopropyl)-1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamide (PA01-TRA). Using 800.00 mg of N-[3-[methyl([3-[3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido]propamido]propyl])amino]propyl]carbamate tert-butyl carbamate, 800 0.00 mg of N-[5-([2-[(2-[[2-([3-[(3-aminopropyl)(methyl)amino]propyl]carbamoyl]ethyl]carbamoyl]-1-methylimidazol-4-yl)carbamoyl]ethyl]carbamoyl)-1-methylpyrrolo-3-yl]-1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-carbamoyl. LC / MS: C 46 H 61 N 19 O8's computational quality: 1007.50, measured value: 1008.80 [M+H] + .

[0992] Synthesis of 3-[(1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl)formamido]propionic acid (PA14-OH)

[0993] Option 10

[0994]

[0995] Step 1: Synthesis of 4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylic acid

[0996] The procedure was the same as for 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-carboxylic acid. Using 2.50 g of methyl 4-(3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylic acid, 2.12 g of 4-(3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylic acid as a white solid was obtained (85.94% yield). LC / MS: C 19 H 26 The calculated mass of N6O6 is 434.19, while the measured mass is 435.20 [M+H]. + .

[0997] Step 2: Synthesis of methyl 3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]formamido]propionate

[0998] The procedure was the same as for methyl 3-[(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-yl)carbamate]propionate. Using 2.12 g of 4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylic acid, 2.30 g of methyl 3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamate]propionate as a yellow oil was obtained (87.33% yield). LC / MS: C 23 H 33 The calculated mass of N7O7 is 519.24, while the measured mass is 520.35 [M+H]. + .

[0999] Step 3: Synthesis of methyl 3-([4-[4-(3-aminopropionic-amido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl]carbamate)propionate

[1000] At room temperature, methyl 3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamate]propionate (1.50 g, 2.89 mmol, 1.00 equivalent) was added dropwise to a stirred solution in DCM (10.00 mL). The resulting mixture was stirred for 1 h at room temperature. The mixture was then concentrated under vacuum. Methyl 3-([4-[4-(3-aminopropamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl]carbamate)propionate (1.70 g, crude product) was obtained as a red oil. LC / MS: C 18 H 25 The calculated mass of N7O5 is 419.19, while the measured mass is 420.15 [M+H]. + .

[1001] Step 4: Synthesis of methyl 3-[(1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl)formamido]propamido]methyl propionate]

[1002] The procedure is the same as that for methyl propionate 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido)propionate. Using 800.00 mg of 1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-carboxylic acid, 800.00 mg of methyl 3-[(1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido]imidazol-2-amido] ... 42 H 49 N 17 O 10 Computational quality: 965.40, Measured quality: 966.40 [M+H] + .

[1003] Step 5: Synthesis of 3-[(1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl)formamido]propionic acid]

[1004] The procedure is the same as that for 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido)propionic acid (PA01-OH). Using 800.00 mg of methyl 3-[(1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl]formamido]propionate, 650 mg of 3-[(1-methyl-4-[1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-amido]pyrrolo-2-yl)formamido]propionic acid (68.67% yield), a yellow solid. LC / MS: C 42 H 49 N 17 O 10 Computational quality: 951.38, Measured value: 952.35 [M+H] + .

[1005] Synthesis of 4-(3-[[4-(4-amino-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]carbamoyl]propamido)-1-methyl-N-[1-methyl-5-([2-[(1-methyl-2-[[2-(propylcarbamoyl)ethyl]carbamoyl]imidazol-4-yl)carbamoyl]ethyl]carbamoyl)pyrrolo-3-yl]imidazol-2-carboxamide (PA15)

[1006] Option 11

[1007]

[1008]

[1009] Step 1: Synthesis of tert-butyl (3-oxo-3-(propylamino)propyl)carbamate

[1010] At room temperature, EDC (114.48 mg, 5.81 mmol, 1.10 equivalent), HOBt (785.56 mg, 5.81 mmol, 1.10 equivalent), propylamine (312.41 mg, 5.23 mmol, 1.00 equivalent), and DIEA (2732.26 mg, 21.14 mmol, 4.00 equivalent) were added in portions to a stirred solution of 3-[(tert-butoxycarbonyl)amino]propionic acid (1000.00 mg, 5.29 mmol, 1.00 equivalent) in DCM (30.00 mL). The resulting mixture was stirred for 17 h at room temperature. The reaction was quenched with cold water (30 mL). The resulting mixture was extracted with dichloromethane (3 × 50 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. A white solid, tert-butyl (3-oxo-3-(propylamino)propyl)carbamate (600.00 mg, crude) was obtained. LC / MS: C 11 H 22 The calculated mass of N₂O₃ is 230.16, while the measured mass is 231.15 [M+H]. + .

[1011] Step 2: Synthesis of 3-amino-N-propylpropionamide

[1012] TFA (2.00 mL) was added dropwise to a stirred solution of N-[2-(propylcarbamoyl)ethyl]carbamate (500.00 mg, 2.17 mmol, 1.00 equivalent) in DCM (10.00 mL) at room temperature. The resulting mixture was stirred for 1 h at room temperature. The mixture was then concentrated under vacuum. 3-Amino-N-propylpropionamide (500.00 mg, crude product) was obtained as a yellow oil. LC / MS: C6H 14 Computed mass of N₂O: 130.11, measured mass: 131.20 [M+H] + .

[1013] Step 3: Synthesis of ethyl 4-(3-aminopropionamido)-1-methylimidazolium-2-carboxylate

[1014] The procedure was the same as that for methyl 4-[4-(3-aminopropamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylate hydrochloride. Using 4.00 g of ethyl 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-carboxylate, 4.00 g of crude ethyl 4-(3-aminopropamido)-1-methylimidazol-2-carboxylate as a white solid was obtained. LC / MS: C 10 H 16 The calculated mass of N4O3 is 240.12, and the measured mass is 241.15[M+H]+.

[1015] Step 4: Synthesis of methyl 4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-imidazol-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate

[1016] The procedure was the same as for ethyl 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-carboxylate. Using 1.00 g of 4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-carboxylic acid, 1.34 g of methyl 4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-imidazol-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylate as a brown solid was obtained (85.00% yield). LC / MS: C 17 H 23 The calculated mass of N5O5 is 377.17, and the measured mass is 378.25[M+H]+.

[1017] Step 5: Synthesis of 4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-imidazol-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylic acid

[1018] The procedure was the same as for 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-carboxylic acid. Using 1.33 g of methyl 4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-imidazol-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylic acid, 946 mg of 4-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-imidazol-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxylic acid (74.00% yield) as a white solid was obtained. LC / MS: C 16 H 21 The calculated mass of N5O5 is 363.15, while the measured mass is 364.25 [M+H]. + .

[1019] Step 6: Synthesis of ethyl 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-carboxylate

[1020] At 0 °C, NMI (2.04 g, 24.86 mmol, 3.00 equivalent), TCFH (3.49 g, 12.43 mmol, 1.50 equivalent), and ethyl 4-(3-aminopropamido)-1-methylimidazolium-2-carboxylate (2.19 g, 9.12 mmol, 1.10 equivalent) were added in portions to a stirred solution of 4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazolium-2-carboxylate in 50.00 mL of DMF. The resulting mixture was stirred for 1 h at room temperature. The reaction was quenched with ice / water (150 mL) at 0 °C. The precipitated solid was collected by filtration, washed with H2O (3 × 30 mL), and dried under vacuum. Ethyl 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]carbamoyl]propamido)-1-methylimidazol-2-carboxylate (5.00 g, 90.08%) was obtained as a yellow solid. LC / MS: C 29 H 40 N 10 O8's computational quality: 656.30, measured value: 657.50 [M+H] + .

[1021] Step 7: Synthesis of ethyl 4-[3-([4-[4-(3-aminopropamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl]carbamate]-1-methylimidazol-2-carboxylate

[1022] The procedure was the same as for 3-amino-N-propylpropionamide. Using 4.90 g of ethyl 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propionamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamoyl]propionamido]-1-methylimidazol-2-carboxylate, 4.90 g of crude ethyl 4-[3-([4-[4-(3-aminopropionamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl]carbamoyl]propionamido]-1-methylimidazol-2-carboxylate as a yellow oil, LC / MS: C 24 H 32 N 10Computational quality of O6: 556.25, Measured value: 557.45 [M+H] + .

[1023] Step 8: Synthesis of ethyl 4-(3-[[4-(4-[3-[(4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl)formamido]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-carboxylate

[1024] The procedure is the same as that for ethyl 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-carboxylate. Using 2.80 g of 4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylic acid, 5.00 g of ethyl 4-(3-[[4-(4-[3-[(4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl)carbamoyl]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamoyl]propamido)-1-methylimidazol-2-carboxylic acid was obtained as a yellow solid (71.94% yield). LC / MS: C 40 H 51 N 15 O 10 Computational quality: 901.39, Measured value: 902.70 [M+H] + .

[1025] Step 9: 4-(3-[[4-(4-[3-[(4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl)carbamoyl]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]carbamoyl]propamido)-1-methylimidazol-2-carboxylic acid

[1026] The procedure is the same as that for 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido)propionic acid (PA01-OH). Using 2.00 g of ethyl 4-(3-[[4-(4-[3-[(4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl)carbamate]propamido]-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]carbamate]propamido)-1-methylimidazol-2-carboxylic acid, 1.60 g of 4-(3-[[4-(4-[3-[(4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl)carbamate]propamido]propamido)-1-methylimidazol-2-carboxylic acid as a yellow solid (71.60% yield). LC / MS: C 38 H 47 N 15 O 10 Computational quality: 873.36, Measured value: 874.55 [M+H] + .

[1027] Step 10: Synthesis of N-(1-methyl-2-[[1-methyl-5-([2-[(1-methyl-2-[[1-methyl-5-([2-[(1-methyl-2-[[2-(propylcarbamoyl)ethyl]carbamoyl]imidazol-4-yl)carbamoyl]ethyl]carbamoyl)pyrrolo-3-yl]carbamoyl]imidazol-4-yl)carbamoyl]ethyl]carbamoyl)pyrrolo-3-yl]carbamoyl]imidazol-4-yl)carbamate tert-butyl carbamate

[1028] The procedure was the same as that for ethyl 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamate]propamido)-1-methylimidazol-2-carboxylic acid. Using 550.00 mg of 4-(3-[[4-(4-[3-[(4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl)carbamate]propamido]propamido]-1-methylimidazol-2-carboxylic acid, 310.00 mg of N-(1-methyl-2-[ [1-Methyl-5-([2-[(1-Methyl-2-[[1-Methyl-5-([2-[(1-Methyl-2-[[2-(propylcarbamoyl)ethyl]carbamoyl]imidazol-4-yl)carbamoyl]ethyl]carbamoyl)pyrrolo-3-yl]carbamoyl]imidazol-4-yl)carbamoyl]ethyl]carbamoyl)pyrrolo-3-yl]carbamoyl]imidazol-4-yl)tert-butyl carbamate (44.96% yield). LC / MS: C 44 H 59 N 17 O 10 Computational quality: 985.46, Measured value: 986.35 [M+H] + .

[1029] Step 11: Synthesis of 4-(3-[[4-(4-amino-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamoyl]propamido)-1-methyl-N-[1-methyl-5-([2-[(1-methyl-2-[[2-(propylcarbamoyl)ethyl]carbamoyl]imidazol-4-yl)carbamoyl]ethyl]carbamoyl)pyrrole-3-yl]imidazol-2-carboxamide (PA15)

[1030] The procedure is the same as for 3-amino-N-propylpropionamide. Use 140.00 mg of N-(1-methyl-2-[[1-methyl-5-([2-[(1-methyl-2-[[1-methyl-5-([2-[(1-methyl-2-[[2-(propylcarbamoyl)ethyl]carbamoyl]imidazol-4-yl)carbamoyl]ethyl]carbamoyl)pyrrole-3-yl]carbamoyl]imidazol-4-yl)carbamoyl]ethyl]carbamoyl)pyrrole-3-yl]carbamoyl]imidazol-4-yl)amino tert-butyl formate was used to obtain 140.00 mg of crude 4-(3-[[4-(4-amino-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]carbamoyl]propamido)-1-methyl-N-[1-methyl-5-([2-[(1-methyl-2-[[2-(propylcarbamoyl)ethyl]carbamoyl]imidazol-4-yl)carbamoyl]ethyl]carbamoyl)pyrrolo-3-yl]imidazol-2-carboxamide. LC / MS: C 39 H 51 N 17 O8's computational quality: 885.41, measured value: 886.60 [M+H] + .

[1031] Synthesis of 4-[3-[(4-[4-[3-([4-[4-(3-aminopropamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]formamido)propamido]-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl)formamido]propamido]-1-methyl-N-propylimidazol-2-carboxamide (PA16-CO-NH-n-Pr)

[1032] Option 12

[1033]

[1034] Step 1: Synthesis of N-[1-methyl-2-([1-methyl-5-[(2-[[1-methyl-2-([1-methyl-5-[(2-[[1-methyl-2-(propylcarbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrolo-3-yl]carbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrolo-3-yl]carbamoyl)imidazol-4-yl]tert-butyl carbamate

[1035] The procedure was the same as that for ethyl 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-carboxylate. Using 1.00 g of 4-(3-[[4-(4-[3-[(4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl)carbamoyl]propamido]-1-methylimidazol-2-carboxylic acid, 680.00 mg of N-[1-methylimidazol-2-carboxylic acid as a brown solid was obtained.] tert-butyl carbamate (57.86% yield). LC / MS: C 41 H 54 N 16 O9's computational quality: 914.43, measured value: 915.65 [M+H] + .

[1036] Step 2: Synthesis of 4-[3-([4-[4-(3-[[4-(4-amino-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]formamido]propamido]-1-methyl-N-propylimidazol-2-carboxamide]

[1037] The procedure is the same as for 3-amino-N-propylpropionamide. 657.00 mg of N-[1-methyl-2-([1-methyl-5-[(2-[[1-methyl-2-([1-methyl-5-[(2-[[1-methyl-2-(propylcarbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrole-3-yl]carbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrole-3-yl]carbamoyl]imidazol- 4-[3-([4-[4-(3-[[4-(4-(1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]carbamate]propamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]carbamate]-1-methyl-N-propylimidazol-2-carbamate was obtained as a crude product of 630.00 mg as a yellow oil. LC / MS: C 36 H 46 N 16 O7's calculated quality: 814.37, measured value: 815.60 [M+H]+ .

[1038] Step 3: Synthesis of N-(2-[[1-methyl-2-([1-methyl-5-[(2-[[1-methyl-2-([1-methyl-5-[(2-[[1-methyl-2-(propylcarbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrolo-3-yl]carbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrolo-3-yl]carbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]tert-butyl carbamate

[1039] The procedure was the same as for ethyl 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazolium-2-carboxylate, but the product was purified by reverse-phase column chromatography in an NH3·H2O system. Using 630.00 mg of 4-[3-([4-[4-(3-[[4-(4-amino-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamoyl]propamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl]carbamoyl]-N-ethyl-1-methylimidazolium-2-carboxamide, 200.00 mg of N-(2-[[1-methyl-2-]propamido]propamido]-N-ethyl-1-methylimidazolium-2-carboxamide as a yellow solid was obtained. 1-([1-methyl-5-[(2-[[1-methyl-2-([1-methyl-5-[(2-[[1-methyl-2-(propylcarbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrolo-3-yl]carbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrolo-3-yl]carbamoyl)imidazol-4-yl]carbamoyl]ethyl)tert-butyl carbamate (25.80% yield). LC / MS: C 44 H 59 N 17 O 10 Computational quality: 985.46, Measured value: 986.70 [M+H] + .

[1040] Step 4: Synthesis of 4-[3-[(4-[4-[3-([4-[4-(3-aminopropamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]formamido)propamido]-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl)formamido]propamido]-1-methyl-N-propylimidazol-2-carboxamide (PA16-CO-NH-n-Pr)

[1041] The procedure is the same as for 3-amino-N-propylpropionamide. Use 100.00 mg of N-(2-[[1-methyl-2-([1-methyl-5-[(2-[[1-methyl-2-([1-methyl-5-[(2-[[1-methyl-2-(propylcarbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrole-3-yl]carbamoyl)imidazol-4-yl]carbamoyl]ethyl)carbamoyl]pyrrole-3-yl]carbamoyl)imidazol-4-yl]aminomethyl tert-butyl 4-[3-[(4-[4-[3-([4-[4-(3-aminopropionamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]carbamate]-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]carbamate]-1-methyl-N-propylimidazol-2-carbamate was obtained as a crude product of 100.00 mg as a yellow oil. LC / MS: C 39 H 51 N 17 O8's computational quality: 885.41, measured value: 886.70 [M+H] + .

[1042] Synthesis of 3-([4-[3-([4-[4-(3-[[4-(4-hexadecamido-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]formamido)propamido]-1-methylimidazol-2-yl]formamido)propionic acid (PA17-OH)

[1043] Option 13

[1044]

[1045] Step 1: Synthesis of methyl propionate 3-([4-[3-([4-[4-(3-[[4-(4-hexadecamido-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]formamido)propamido]-1-methylimidazol-2-yl]formamido)propamido]-1-methylimidazol-2-yl]formamido)propamido]

[1046] The procedure was the same as that for ethyl 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]formamido]propamido)-1-methylimidazol-2-carboxylate. Using 500.00 mg of methyl 3-([4-[3-([4-[4-(3-[[4-(4-amino-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl]formamido]propamido]propamido]-1-methylimidazol-2-yl]formamido)propamido)propamido]methylimidazol-2-yl]formamido)propamido)propamido]methylimidazol-2-yl]formamido)propamido)propamido), 510.00 mg was obtained. g of methyl 3-([4-[3-([4-[4-(3-[[4-(4-hexadecamido-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]formamido]propamido]-1-methylimidazol-2-yl]formamido)propamido]-1-methylimidazol-2-yl]formamido)propamido] 53 H 76 N 16 O 10 Computational quality: 1096.59, Measured value: 1097.50 [M+H] + .

[1047] Step 2: Synthesis of 3-([4-[3-([4-[4-(3-[[4-(4-hexadecamido-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]formamido)propamido]-1-methylimidazol-2-yl]formamido)propionic acid

[1048] The procedure is the same as that for 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido)propionic acid (PA01-OH). Using 270.00 mg of methyl 3-([4-[3-([4-[4-(3-[[4-(4-(4-hexadecamido-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamoyl]propamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl]carbamoyl)propamido]-1-methylimidazol ... LC / MS: C 52 H 74 N 16 O 10 The calculated quality is 1082.57, and the measured quality is 1083.90 [M+H]. + .

[1049] Synthesis of N-[5-([2-[(2-[5-([2-[(2-[[2-([3-[(3-aminopropyl)(methyl)amino]propyl]carbamoyl]ethyl]carbamoyl]-1-methylimidazol-4-yl)carbamoyl]ethyl]carbamoyl)-1-methylpyrrole-3-yl]carbamoyl]-1-methylimidazol-4-yl)carbamoyl]ethyl]carbamoyl)-1-methylpyrrole-3-yl]-4-hexadecanoyl-1-methylimidazol-2-carboxamide (PA17-TRA)

[1050] Option 14

[1051]

[1052] Step 1: Synthesis of N-[3-([3-[3-([4-[3-([4-[4-(3-[[4-(4-(3-[[4-(4-(4-(4-(4-hexadecamido-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]formamido]propamido]-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl]formamido]propamido]-1-methylimidazol-2-yl]formamido]propamido]propyl](methyl)amino)propyl]tert-butyl carbamate

[1053] The procedure is the same as that for (3-(methyl(3-(3-(1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carbamoyl)-1H-pyrrole-2-carbamoyl)propamido)-1H-imidazol-2-carbamoyl)-1H-pyrrole-2-carbamoyl)propamido)-1H-imidazol-2-carbamoyl)propamido)propyl)amino)propyl)tert-butyl carbamate. Using 70.00 mg of 3-([4-[3-([4-[4-(3-[[4-(4-hexadecamido-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl]formamido]propamido]-1-methylimidazol-2-yl]formamido)propionic acid, 50.00 mg of N-[3-([3 -[3-([4-([4-(4-(3-[[4-(4-(4-hexadecamido-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]formamido)propamido]-1-methylimidazol-2-yl]formamido)propamido]propyl](methyl)amino)propyl]tert-butyl carbamate (59.04% yield). LC / MS: C 64 H 99 N 19 O 11 The calculated quality is 1309.78, while the measured quality is 1311.20 [M+H]. + .

[1054] Step 2: Synthesis of N-[5-([2-[(2-[5-([2-[(2-[[2-([3-[(3-aminopropyl)(methyl)amino]propyl]carbamoyl]ethyl]carbamoyl]-1-methylimidazol-4-yl)carbamoyl]ethyl]carbamoyl)-1-methylpyrrole-3-yl]carbamoyl]-1-methylimidazol-4-yl)carbamoyl]ethyl]carbamoyl)-1-methylpyrrole-3-yl]-4-hexadecanoyl-1-methylimidazol-2-carboxamide (PA17-TRA)

[1055] The procedure is the same as that for N-(3-((3-(((3-aminopropyl)(methyl)amino)propyl)amino)-3-oxopropyl)-1-methyl-4-(3-(1-methyl-4-(1-methyl-4-(3-(1-methyl-4-(1-methyl-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamido)-1H-pyrrole-2-carboxamido)propamido)-1H-imidazol-2-carboxamide (PA01-TRA). Using 50.00 mg of N-[3-([3-[3-([4-[3-([4-[4-(3-[[4-(4-(4-(4-(4-(4-(4-(4-(4-(4-(4-hexadecamido-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]formamido]propamido)-1-methylimidazol-2-yl]propamido]propamido]propyl](methyl)amino)propyl]carbamate, 40.00 mg of yellow tert-butyl carbamate was obtained. Crude N-[5-([2-[(2-[5-([2-[(2-[[2-([3-[(3-aminopropyl)(methyl)amino]propyl]carbamoyl]ethyl]carbamoyl]-1-methylimidazol-4-yl)carbamoyl]ethyl]carbamoyl)-1-methylpyrrolo-3-yl]carbamoyl]-1-methylimidazol-4-yl)carbamoyl]ethyl]carbamoyl)-1-methylpyrrolo-3-yl]-4-hexadecanoyl-1-methylimidazol-2-carboxamide. LCMS: C 59 H 91 N 19 O9's calculated quality: 1209.72, measured value: 1211.05 [M+H] + .

[1056] Synthesis of 4-[4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-carboxylic acid (PA18-OH)

[1057] Option 15

[1058]

[1059] Step 1: Synthesis of 4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylic acid

[1060] The procedure was the same as for 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-carboxylic acid. Using 10.00 g of methyl 4-(3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylic acid, 8.50 g of 4-(3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylic acid (87.74% yield) as a white solid was obtained. LC / MS: C 19 H 26 The calculated mass of N6O6 is 434.19, while the measured mass is 435.20 [M+H]. + .

[1061] Step 2: Synthesis of methyl 4-[4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylate

[1062] The procedure was the same as that for methyl 4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylate. Using 1.00 g of 4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylic acid, 1.65 g of methyl 4-[4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylate as a white solid was obtained (80.11% yield). LCMS: C 34 H 44 N 12 O9's computational quality: 764.34, measured value: 765.50 [M+H] + .

[1063] Step 3: Synthesis of 4-[4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylic acid]

[1064] The procedure was the same as for 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-carboxylic acid. Using 100.00 mg of 4-[4-(3-[[4-(3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamoyl]propamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylic acid ester, 80.00 mg of 4-[4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamoyl]propamido]-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylic acid (77.42% yield) as a yellow solid was obtained. LC / MS: C 33 H 42 N 12 O9's calculated quality: 750.32, measured value: 751.30 [M+H] + .

[1065] Synthesis of methyl 4-[4-[3-([4-[4-(3-aminopropamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl]carbamate]-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylate (PA18-NH2)

[1066] Option 16

[1067]

[1068] The procedure was the same as for 3-amino-N-propylpropionamide, but the reaction time was 2.0 h. Using 500.00 mg of methyl 4-[4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propionamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamate]propionamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylate, 500.00 mg of methyl 4-[4-[3-([4-[4-(3-aminopropionamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl]carbamate]-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylate as a yellow solid, HRMS: C 29 H 36 N 12 O7's calculated quality: 664.2830, measured value: 665.2891 [M+H] + .

[1069] Synthesis of 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-carboxylic acid (PA19-OH)

[1070] Option 17

[1071]

[1072] Step 1: Synthesis of ethyl 4-(3-aminopropionamido)-1-methylimidazolium-2-carboxylate

[1073] The procedure was the same as that for methyl 4-[4-(3-aminopropamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylate hydrochloride. Using 2.00 g of ethyl 4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-carboxylate, 2.00 g of crude ethyl 4-(3-aminopropamido)-1-methylimidazol-2-carboxylate as a grayish-white solid was obtained. LCMS: C 10 H 16 The calculated mass of N4O3 is 240.12, while the measured mass is 241.10 [M+H]. + .

[1074] Step 2: Synthesis of ethyl 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-carboxylate

[1075] The procedure was the same as that for ethyl 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]carbamoyl]propamido)-1-methylimidazol-2-carboxylate. Using 900.00 mg of 4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-carboxylic acid, 1.10 g of ethyl 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]carbamoyl]propamido)-1-methylimidazol-2-carboxylate as a grayish-white solid was obtained (75.80% yield). LCMS: C 29 H 40 N 10 O8's computational quality: 656.30, measured value: 657.50 [M+H] + .

[1076] Step 3: Synthesis of 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-carboxylic acid

[1077] The procedure is the same as that for 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido)propionic acid (PA01-OH). Using 600 mg of ethyl 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamoyl]propamido)-1-methylimidazol-2-carboxylic acid, 500.00 mg of 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamoyl]propamido)-1-methylimidazol-2-carboxylic acid was obtained as a yellow solid (78.35% yield). LCMS: C 27 H 36 N 10 O8's calculated quality: 628.27, measured value: 629.45[M+H]+.

[1078] Synthesis of ethyl 4-(3-(4-(4-(3-aminopropamido)-1-methyl-1H-imidazol-2-carboxamido)-1-methyl-1H-pyrrolo-2-carboxamido)propamido)-1-methyl-1H-imidazol-2-carboxylate (PA19-NH2)

[1079] Option 18

[1080]

[1081] The procedure was the same as for 3-amino-N-propylpropionamide. Using 900.00 mg of ethyl 4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propionamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamoyl]propionamido]-1-methylimidazol-2-carboxylate, 900.00 mg of crude ethyl 4-[3-([4-[4-(3-aminopropionamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl]carbamoyl]propionamido]-1-methylimidazol-2-carboxylate as a white solid, LCMS: C 24 H 32 N 10Computational quality of O6: 556.25, Measured value: 557.50 [M+H] + .

[1082] Synthesis of 4-(3-(4-(4-(3-(4-(4-(3-((tert-butoxycarbonyl)amino)propamido)-1-methyl-1H-imidazol-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)propamido)-1-methyl-1H-imidazol-2-carboxamido)-1-methyl-1H-pyrrole-2-carboxamido)propamido)-1-methyl-1H-imidazol-2-carboxylic acid (PA16-OH)

[1083] Option 19

[1084]

[1085] Step 1: Synthesis of ethyl 4-[3-([4-[4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]formamido]-1-methylimidazol-2-carboxylate

[1086] Ethyl 4-[3-([4-[4-(3-aminopropamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylic acid (120.00 mg, 0.276 mmol, 1.00 equivalent) and EDCI (134.00 mg, 0.70 mmol, 2.53 equivalent), DMAP (86.00 mg, 0.70 mmol, 2.55 equivalent) in a stirred solution of 4-(4-(3-aminopropamido)-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl]carboxamido]-1-methylimidazol-2-carboxylic acid (199.85 mg, 0.36 mmol, 1.30 equivalent) in DMF (5.00 mL) was added. The resulting solution was stirred for 16 h at room temperature. The resulting mixture was poured into ice / water (20 mL), and the precipitated solid was collected by filtration and washed with water (3 × 10 mL). The solid was dried under reduced pressure to provide ethyl 4-[3-([4-[4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-yl]carbamate]-1-methylimidazol-2-carboxylate (240.00 mg, 80.37%) as a yellow solid. LCMS: C 43 H 56 N16 O 11 The calculated quality is 972.43, and the measured quality is 973.45 [M+H]. + .

[1087] Step 2: Synthesis of 4-[3-([4-[4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]formamido]-1-methylimidazol-2-carboxylic acid]

[1088] The procedure was the same as for 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]carbamoyl]propamido)imidazol-2-amido]pyrrolo-2-yl]carbamoyl]propamido]imidazol-2-yl]carbamoyl)propamido)propionic acid (PA01-OH). Using 400.00 mg of ethyl 4-[3-([4-[4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]propamido]-1-methylimidazol-2-carboxylate, 260 was obtained. 00 mg of 4-[3-([4-[4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]carbamate]-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]carbamate]-1-methylimidazol-2-carboxylic acid (66.93% yield) was obtained as a white solid. LC / MS: C 41 H 52 N 16 O 11 Computational quality: 944.40, Measured quality: 945.40 [M+H] + .

[1089] Synthesis of 4-(4-[3-[(4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl)carbamate]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-carboxylic acid (PA20-OH)

[1090] Option 20

[1091]

[1092] Step 1: Synthesis of 4-(4-[3-[(4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl)carbamate]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylate

[1093] The procedure is the same as that for ethyl 4-[3-([4-[4-(3-[[4-(4-[3-[(tert-butoxycarbonyl)amino]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-yl]formamido]propamido)-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl]formamido]-1-methylimidazol-2-carboxylate. Using 1.00 g of 4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrole-2-carboxylic acid, 1.10 g of methyl 4-(4-[3-[(4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrole-2-yl)carbamoyl]propamido]-1-methylimidazol-2-amido)-1-methylpyrrole-2-carboxylic acid (51.28% yield) as a deep yellow solid was obtained. LC / MS: C 31 H 39 N 11 O8's computational quality: 693.30, measured value: 694.15 [M+H] + .

[1094] Step 2: Synthesis of 4-(4-[3-[(4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl)carbamoyl]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-carboxylic acid)

[1095] The procedure is the same as that for 3-([1-methyl-4-[3-([1-methyl-4-[1-methyl-4-(3-[[1-methyl-4-(1-methylimidazol-2-amido)pyrrolo-2-yl]formamido]propamido)imidazol-2-amido]pyrrolo-2-yl]formamido]propamido]imidazol-2-yl]formamido)propionic acid (PA01-OH). Using 500.00 mg of methyl 4-(4-[3-[(4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl)carbamoyl]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-carboxylic acid, 400.00 mg of 4-(4-[3-[(4-[4-[(tert-butoxycarbonyl)amino]-1-methylimidazol-2-amido]-1-methylpyrrolo-2-yl)carbamoyl]propamido]-1-methylimidazol-2-amido)-1-methylpyrrolo-2-carboxylic acid (81.65% yield) as a deep yellow solid was obtained. LCMS: C 30 H 37 N 11 O8's computational quality: 679.28, measured value: 680.25 [M+H] + .

[1096] Example 1B Synthesis of Protein Binding Agent (Second Binding Domain)

[1097] (S)-2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diaza -4-yl)-N-(4-hydroxyphenyl)acetamide (SM10) and (R)-2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diaza Synthesis of 4-yl)-N-(4-hydroxyphenyl)acetamide (SM20)

[1098] Option 21

[1099]

[1100] Step 1: Synthesis of 6-methoxy-2-methyl-4H-benzo[d][1,3]oxazin-4-one

[1101] Add 55.00 g (0.33 mol, 1.00 equivalent) of 2-amino-5-methoxybenzoic acid and 700.00 mL of acetic anhydride to a 1000 mL round-bottom flask. Stir the mixture at 145 °C for 5.0 h. Then remove the solvent and wash the residue with Et₂O (100 mL). Filter the solid and dry it. This yielded 57.00 g (90% yield) of 6-methoxy-2-methyl-4H-benzo[d][1,3]oxazin-4-one as a pale yellow solid. LC / MS: C 10 The calculated mass of H9NO3 was 191.06, while the measured mass was 192.20 [M+H]. + .

[1102] Step 2: Synthesis of (2-amino-5-methoxyphenyl)(4-chlorophenyl) ketone

[1103] At 0 °C, magnesium 4-chlorophenyl bromide (1.0 M, in THF, 141.00 mL, 141.00 mmol, 0.90 equivalence) was added dropwise to a solution of 6-methoxy-2-methyl-3,1-benzoxazin-4-one (30.00 g, 156.92 mmol, 1.00 equivalence) in toluene (300.00 mL) and Et₂O (150.00 mL). The mixture was then stirred at 0 °C for 2.0 h and quenched with 2 M HCl (100 mL). The mixture was extracted with EA (3 × 200 mL), the organic phases were combined, washed with saturated NaCl solution (300 mL), and dried over anhydrous Na₂SO₄. The solid was filtered off and concentrated. The crude product was dissolved in EtOH (300 mL) and 6 M HCl (100 mL) was added. The mixture was stirred at 85 °C for 2.0 h. Remove the solvent, dissolve the residue in water, and adjust the pH to 9 with 2N NaOH. Extract the mixture by EA (3 × 300 mL), combine the organic phases, wash with saturated NaCl solution (1 × 300 mL), and dry over anhydrous Na₂SO₄. Filter off the solid and concentrate the filtrate. Purify the residue by passing it through a silica gel column using PE / EA = 3:1. This yielded 30.00 g of the desired product as an orange solid (58% yield). LC / MS: C 14 H 12 The calculated mass of ClNO2 was 261.06, while the measured mass was 262.00 [M+H]. + .

[1104] Step 3: Synthesis of methyl (S)-3-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-4-((2-(4-chlorobenzoyl)-4-methoxyphenyl)amino)-4-oxobutyrate

[1105] HATU (7.55 g, 19.87 mmol, 1.30 equivalence) and DIEA (5.93 g, 45.85 mmol, 3.00 equivalence) were added to a solution of (2S)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]-4-methoxy-4-oxobutyric acid (6.21 g, 16.81 mmol, 1.10 equivalence) in DMF (50.00 mL). Then, 2-(4-chlorobenzoyl)-4-methoxyaniline (4.00 g, 15.28 mmol, 1.00 equivalence) was added. The mixture was stirred at room temperature for 17 h. The mixture was poured into 300 mL of ice / water, and the solid was filtered off. The solid was then dissolved in DCM and purified by silica gel column chromatography using PE:EA = 3:1. Methyl (S)-3-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-4-((2-(4-chlorobenzoyl)-4-methoxyphenyl)amino)-4-oxobutyrate (5.00 g, 53.00%) was obtained as a yellow solid. LC / MS: C 34 H 29 The calculated mass of ClN₂O₇ is 612.17, while the measured mass is 613.15 [M+H]. + .

[1106] Step 4: (S)-2-(5-(4-chlorophenyl)-7-methoxy-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diaza) Synthesis of methyl 3-yl)acetate

[1107] Add Et3N (15.00 mL) to a solution of methyl (3S)-3-[[2-(4-chlorobenzoyl)-4-methoxyphenyl]carbamoyl]-3-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]propionate (5.00 g, 8.16 mmol, 1.00 equivalent) in DCM (15.00 mL). Stir the mixture at 50 °C for 5.0 h. Remove the solvent and dissolve the residue in DCE (50.00 mL) with AcOH (4.50 g, 73.404 mmol, 9.00 equivalent). Stir the mixture at 65 °C for 2.0 h. Remove the solvent and purify the residue by silica gel column chromatography using PE:EA = 1:1. (S)-2-(5-(4-chlorophenyl)-7-methoxy-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diaza) was obtained as a yellow solid. 3-yl)methyl acetate (2.80 g, 78.00% yield). LC / MS: C 19 H 17 The calculated mass of ClN₂O₄ is 372.09, while the measured mass is 373.15 [M+H]. + .

[1108] Step 5: (S)-2-(5-(4-chlorophenyl)-7-methoxy-2-thio-2,3-dihydro-1H-benzo[e][1,4]diaza) Synthesis of methyl 3-yl)acetate

[1109] Add P2S5 (4.51 g, 20.28 mmol, 3.60 equivalence), Na2CO3 (1.07 g, 0.01 mmol, 1.80 equivalence), and ClCH2CH2Cl (60.00 mL) to a 250 mL flask. Stir the mixture at room temperature for 2 h, then add 2-[(3S)-5-(4-chlorophenyl)-7-methoxy-2-oxo-1,3-dihydro-1,4-benzodiazepine]. Methyl 2-[(3S)-5-(4-chlorophenyl)-7-methoxy-2-thionyl-1,3-dihydro-1,4-benzodiazepine]acetate (2.10 g, 5.63 mmol, 1.00 equivalent). The reaction was stirred at 65 °C for 3 h, followed by filtration of the solid. The organic phase was washed with water (50 mL) and dried over Na₂SO₄. The solid was filtered off and the filtrate was concentrated. This yielded a yellow solid of 2-[(3S)-5-(4-chlorophenyl)-7-methoxy-2-thionyl-1,3-dihydro-1,4-benzodiazepine]acetate. Methyl methyl acetate (1.70 g, 63.53%). LC / MS: C 19 H 17 The calculated mass of ClN₂O₃S is 388.06, while the measured mass is 389.10 [M+H]. + .

[1110] Step 6: 2-[(2Z,3S)-5-(4-chlorophenyl)-2-(acetamidoimino)-7-methoxy-1,3-dihydro-1,4-benzodiazepine] Synthesis of methyl 3-yl]acetate

[1111] Add 2-[(3S)-5-(4-chlorophenyl)-7-methoxy-2-thionyl-1,3-dihydro-1,4-benzodiazepine] to a 250 mL flask Methyl methyl acetate (1.50 g, 3.86 mmol, 1.00 equivalent) and THF (40.00 mL) were added. The mixture was cooled to 0 °C, and then NH₂NH₂·H₂O (0.58 g, 0.01 mmol, 3.00 equivalent) was added dropwise. The mixture was stirred at 0–5 °C for 1 h. Then Et₃N (1.17 g, 0.01 mmol, 3.00 equivalent) was added, and AcCl (0.91 g, 0.01 mmol, 3.00 equivalent) was added dropwise at 0–5 °C. The reaction was stirred at room temperature for 2 h. The reaction was quenched with H₂O at room temperature, extracted with EA (3 × 40 mL), the organic layers were combined, washed with brine (2 × 20 mL), and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. This produces a yellow solid, 2-[(2Z,3S)-5-(4-chlorophenyl)-2-(acetamidoimino)-7-methoxy-1,3-dihydro-1,4-benzodiazepine] Methyl methyl acetate (1.70 g, crude product). LC / MS: C 21 H 21 The calculated mass of ClN4O4 is 428.13, while the measured mass is 429.15 [M+H]. + .

[1112] Step 7: (S)-2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diaza) Synthesis of 4-yl)methyl acetate

[1113] Add 2-[(2Z,3S)-5-(4-chlorophenyl)-2-(acetamidoimino)-7-methoxy-1,3-dihydro-1,4-benzodiazepine] to a 100 mL flask Methyl methyl acetate (1.77 g crude), THF (10.00 mL), and AcOH (10.00 mL) were reacted and stirred at room temperature for 3 h. The reaction mixture was then concentrated. A NaHCO3 / DCM mixture (20 mL / 20 mL) was added to the residue to separate the organic phase, and the aqueous phase was extracted with DCM (2 × 20 mL). The organic phases were combined and washed with water (20 mL), NaCl solution (20 mL), and dried over anhydrous Na2SO4. The solid was filtered off and the filtrate was concentrated. The residue was purified by silica gel column chromatography, eluting with CH2Cl2:MeOH = 20:1 to provide (S)-2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diaza) as a yellow solid. 4-yl)methyl acetate (1.58 g, two-step, 75.0% yield). LC / MS: C 21 H 19 The calculated mass of ClN4O3 is 410.11, while the measured mass is 411.25 [M+H]. + .

[1114] Step 8: (S)-2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diaza) Synthesis of 4-yl)acetic acid (SM29)

[1115] Add (S)-2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diaza to a 100 mL flask Methyl 4-(4-yl)acetate (1.58 g, 3.85 mmol, 1.00 equivalent), LiOH solution (2 M, 6.00 mL, 12.00 mmol, 3.12 equivalent), MeOH (20.00 mL), and THF (10.00 mL) were prepared. The reaction was stirred at room temperature for 2 h. The mixture was then concentrated, the residue was dissolved in 30 mL of water, cooled to 0 °C, and the pH was adjusted to 3–5 with 2 M HCl. The purified solid was collected by filtration, washed with water (3 × 20 mL), and dried under vacuum. This yielded a yellow solid (S)-2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diaza -4-yl)acetic acid (1.30 g, 80.62% yield). If the crude product is purified by chiral preparative HPLC under the following conditions: column: Reg-AD, 3*25 cm, 5 μm; mobile phase A: CO2, mobile phase B: MEOH (0.1% 2M NH3-MEOH); flow rate: 100 mL / min; gradient: 30% B; UV: 220 nm; RT1: 3.05; RT2: 4.08, pure SM29 can be obtained. LC / MS: C 20 H 17 The calculated mass of ClN4O3 is 396.10, while the measured mass is 397.15 [M+H]. + .

[1116] Step 9: (S)-N-(4-((tert-butyldimethylsilyl)oxy)phenyl)-2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diaza Synthesis of 4-yl)acetamide

[1117] Aminophenol (2.00 g, 18.33 mmol, 1.00 equivalent), THF (60.00 mL), and imidazole (1.62 g, 0.02 mmol, 1.30 equivalent) were added to a 250 mL flask. The mixture was cooled to 0 °C, and then TBDMSCl (4.14 g, 0.03 mmol, 1.50 equivalent) was slowly added. The reaction was stirred at room temperature for 1 h. The reaction was quenched with water (100 mL), extracted with EA (3 × 50 mL), and the organic phase was washed with NaCl solution (50 mL), dried over Na2SO4, filtered off the solid, and concentrated the filtrate. The residue was purified by silica gel column chromatography, eluting with PE:EA = 1:1 to provide 4-[(tert-butyldimethylsilyl)oxy]aniline (3.80 g, 92.82%) as a brown oil. LC / MS: C 12 H 21 The calculated quality of NOSi is 223.14, while the measured quality is 224.15 [M+H]. + .

[1118] Add (S)-2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-4H-benzo[f][1,2,4]triazolo[4,3-a][1,4]diaza to a 100 mL flask 4-[(tert-butyldimethylsilyl)oxy]aniline (500.00 mg, 1.26 mmol, 1.00 equivalent), DMF (10.00 mL), HATU (718.63 mg, 1.89 mmol, 1.50 equivalent), and DIEA (651.38 mg, 5...

Claims

1. A transcriptional regulatory molecule having a first terminal, a second terminal, and an oligomeric backbone, wherein: a) The first end includes a DNA-binding portion capable of non-covalently binding to the nucleotide repeat sequence GAA; b) The second end includes a protein-binding portion that binds to a regulatory molecule that regulates the expression of a gene containing the nucleotide repeat sequence GAA; and c) The oligomeric backbone includes a joint between the first end and the second end.

2. The transcriptional regulatory molecule of claim 1, wherein the first end comprises a polyamide selected from linear polyamide, hairpin polyamide, H-pin polyamide, overlapping polyamide, slip polyamide, cyclic polyamide, tandem polyamide, and extended polyamide.

3. The transcriptional regulatory molecule of claim 1 or 2, wherein the first end comprises a linear polyamide.

4. The transcriptional regulatory molecule according to any one of claims 1-3, wherein the polyamide is capable of binding the DNA with an affinity of less than 500 nM.

5. The transcriptional regulatory molecule of any one of claims 1-4, wherein the first terminal comprises –NH-QC(O)-, wherein Q is an optionally substituted C. 6-10 arylene, optionally substituted 4-10-membered heterocyclic alkylene, optionally substituted 5-10-membered heteroarylene, or optionally substituted alkylene.

6. A pharmaceutical composition comprising a transcriptional regulatory molecule as described in any of the preceding claims and a pharmaceutically acceptable carrier.

7. A method for regulating the expression of fxn, the method comprising contacting fxn with a transcriptional regulatory molecule as described in any one of claims 1-5.

8. A method for treating a disease caused by defective expression of fxn, the method comprising administering a therapeutically effective amount of the transcriptional regulatory molecule as described in any one of claims 1-5 to a patient in need.

9. A method for treating a disease caused by the expression of a defective fxn, the method comprising administering: A therapeutically effective amount of the transcriptional regulatory molecule as described in any one of claims 1-5; and Another treatment.

10. A method for achieving an effect in a patient, the method comprising administering a therapeutically effective amount of a transcriptional regulatory molecule as disclosed herein or a salt thereof to the patient, wherein the effect is selected from muscle atrophy, ataxia, fasciculations and dementia.

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