Enhancing polymerization of nucleic acids by aromatic compounds

Abstract

The invention relates to enhancement of polymerization of nucleic acids by aromatic compounds. The present invention relates to compounds, methods and compositions for improving nucleic acid polymerization, including DNA replication by in vitro primer extension to produce polymers, e.g., for nanopore-based single molecule sequencing of DNA templates. Nucleic acid polymerase reaction compositions have polymerization enhancing moieties that allow for enhancing the activity of DNA polymerases and nucleotide analogs such that the length of primer extension products for sequencing applications is increased.

Description

[0001] This application is a divisional application of the international application PCT / US2020 / 038682 with an international application date of June 19, 2020, entering China, with application number 202080053441.1, entitled "Enhancing Nucleic Acid Polymerization by Aromatic Compounds". Technical Field

[0002] The present invention relates generally to new chemical entities and more particularly to new organic molecules, optionally with inorganic components, including compositions thereof, and methods of making and using them, particularly in affecting enzyme performance.

[0003] Statement regarding sequence listing

[0004] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 870225_425WO_SEQUENCE_LISTING.txt. The text file is 15KB, was created on June 19, 2020, and is being submitted electronically through EFS-Web. Background Art

[0005] The measurement of biomolecules is fundamental to modern medicine and is widely used in medical research, more specifically in diagnostics and therapeutics, as well as in drug development. Nucleic acids encode information necessary for the activity and reproduction of living organisms and are essentially the blueprints of life. Determining such blueprints is useful in both pure research and applied science. In medicine, sequencing can be used to diagnose and develop treatments for a variety of conditions, including cancer, heart disease, autoimmune diseases, multiple sclerosis, and obesity. In industry, sequencing can be used to design improved enzymatic processes or synthetic organisms. In biology, for example, the tool can be used to study the health of ecosystems and therefore has a wide range of uses. Similarly, the measurement of proteins and other biomolecules provides markers and insights into the spread of disease and pathogens.

[0006] An individual's unique DNA sequence provides valuable information about their susceptibility to certain diseases. It also provides patients with the opportunity to be screened for early detection and / or receive preventive treatment. Furthermore, based on a patient's personal blueprint, clinicians will be able to administer personalized therapies to maximize drug efficacy and / or minimize the risk of adverse drug reactions. Similarly, determining the blueprint of a pathogenic organism could provide new treatments for infectious diseases and more robust pathogen surveillance. Low-cost whole-genome DNA sequencing will lay the foundation for modern medicine. To achieve this goal, sequencing technology must continue to advance in throughput, accuracy, and read length.

[0007] In the past decade, a large number of next-generation DNA sequencing technologies have become commercially available, which has greatly reduced the cost of whole genome sequencing. These include sequencing by synthesis ("SBS") platforms (Illumina, Inc., 454 Life Sciences, Ion Torrent, Pacific Biosciences) and similar ligation-based platforms (Complete Genomics, Life Technologies Corporation). Many other technologies are under development, which utilize a variety of sample processing and detection methods. For example, GnuBio (Cambridge, Mass.) uses picoliter reaction vessels to control millions of discreet probe sequencing reactions, and Halcyon Molecular (Redwood City, Calif.) is attempting to develop a technology for direct DNA measurement using a transmission electron microscope.

[0008] Nanopore-based nucleic acid sequencing is an attractive approach that has been extensively studied. Kasianowicz et al. (Proc. Natl. Acad. Sci. USA 93: 13770-13773, 1996) characterized single-stranded polynucleotides as they were electrically translocated through alpha-hemolysin nanopores embedded in lipid bilayers. It has been demonstrated that partial blockage of the nanopore orifice can be measured as a measure of reduced ionic current during polynucleotide translocation. However, polynucleotide sequencing in nanopores suffers from small signal differences immersed in significant background noise due to the need to resolve closely spaced bases (0.34 nm). The measurement challenge of single-base resolution in nanopores becomes more demanding due to the observed rapid translocation rates of polynucleotides, typically on the order of 1 base per microsecond. The translocation rate can be reduced by adjusting operating parameters such as voltage, salt composition, pH, temperature, and viscosity. However, such adjustments cannot reduce the translocation rate to a level that allows single-base resolution.

[0009] Stratos Genomics has developed a method called expanded sequencing ("SBX") that uses a biochemical process to transcribe DNA sequences onto measurable polymers called "Xpandomers" (Kokoris et al., U.S. Patent No. 7,939,259 "High Throughput Nucleic Acid Sequencing by Expansion"). The transcribed sequence is encoded in high signal-to-noise ratio reporters along the Xpandomer backbone, which are separated by approximately 10 nm and designed for high signal-to-noise ratio, well-differentiated reactions. These differences provide significant performance enhancements in sequence read efficiency and accuracy of Xpandomers relative to native DNA. Xpandomers can enable a variety of next-generation DNA sequencing detection technologies and are well suited for nanopore sequencing.

[0010] Xpandomers are produced from non-natural nucleotide analogs called XNTPs, characterized by long substituents that enable the Xpandomer backbone to be extended after synthesis (see Published PCT Application No. WO2016 / 081871 to Kokoris et al., incorporated herein by reference in its entirety). Due to their atypical structures, XNTPs and other nucleotide analogs (e.g., nucleotide analogs partially modified with detectable labels) introduce new challenges as substrates for currently available DNA polymerases. Published PCT Application Nos. WO2017 / 087281 and WO2018 / 204717 to Kokoris et al. (incorporated herein by reference in their entirety) describe engineered DP04 polymerase variants with enhanced primer extension activity that utilize non-natural, large nucleotide analogs as substrates.

[0011] Within the DNA template itself, certain nucleotide sequence motifs are known to present additional replication challenges to DNA polymerases. Of particular importance are runs of homopolymers or short repetitive DNA sequences, which can trigger slipped strand mispairing or "replication slips." Replication slips are thought to encompass the following steps: (i) copying of the first repeat sequence by the replication machinery, (ii) replication pause and dissociation of the polymerase from the newly synthesized end, (iii) unpairing of the newly synthesized strand and its pairing with the second repeat sequence, and (iv) reinitiation of DNA synthesis. Thus, stalling of the replication machinery within the repeat region can lead to misalignment of the primer and template. In vivo, misalignment of the two DNA strands during replication can lead to DNA rearrangements, such as deletions or duplications of different lengths. In vitro, replication slips can lead to replication errors at the site of the slip event. This reduction in polymerase processivity or accuracy significantly impairs specific applications or desired genetic manipulations.

[0012] Therefore, new methods and compositions for enhancing polymerase reactions under conditions involving one or more reagents having atypical structures are needed (e.g., in sequencing by extension (SBX) and other applications in biotechnology and biomedicine (e.g., DNA amplification, conventional sequencing, labeling, detection, cloning, etc.)) and would be of value in the art. The present invention satisfies these needs and provides further related advantages.

[0013] All subject matter discussed in the background section is not necessarily prior art and should not be assumed to be prior art simply because it is discussed in the background section. Along these lines, any recognition of a problem in the prior art discussed in the background section or related to such subject matter should not be considered prior art unless it is explicitly stated to be prior art. Rather, the discussion of any subject matter in the background section should be considered as part of the inventor's approach to solving a particular problem, which may itself be inventive. Summary of the invention

[0014] In short, the present disclosure provides compounds, compositions and uses thereof that enhance nucleic acid polymerase activity. In certain embodiments, polymerase activity is enhanced under conditions that introduce one or more challenges to the polymerase in a polymerization reaction (e.g., conditions that include non-natural nucleotide analog substrates or template motifs that impair the polymerase's processivity). This enhancement is achieved by supplementing the polymerization reaction with one or more compounds of the present disclosure, which may optionally be referred to herein as polymerase enhancing molecules or PEMs.

[0015] In one aspect, the PEM is a compound of formula (I)

[0016]

[0017] or a solvate, hydrate, tautomer, chelate or salt thereof, wherein:

[0018] m is 1, 2 or 3;

[0019] m′ is 1, 2 or 3;

[0020] n is 0, 1 or 2;

[0021] p is 0, 1, or 2;

[0022] When X is C, W is N, or when X is N, W is C;

[0023] is a single bond or a double bond, wherein the double bond starts at any one of W or X that is carbon;

[0024] L is a linking group;

[0025] M is independently selected at each occurrence from hydrogen, halogen and C1-C4 alkyl;

[0026] Ar1 is independently selected at each occurrence from optionally substituted pyridine, pyrazine, pyridazine, furan, thiophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine and carbazole, wherein: the substituents of Ar1 are independently selected at each occurrence from halogen, -OH, -CN, -NO2, C1-C6 alkyl, C 1- C6 haloalkyl, C 1- C6 heteroalkyl, C1-C6 cycloalkyl, -OR 0 、-CONH2、-C(O)NR 1 R 1′ 、-NR 1 R 1′ 、-NR 1 C(O)R 3 、-C(O)SR 3 、-COR 3 、-OC(O)R 3 、-C(O)OR 3 , thiol, -R 4 -H, -SOR 1 、-S(O)2R 1 、-S(O)2NR 1 R 1′ and -NS(O)2R 3 ;

[0027] R 0 is independently selected at each occurrence from C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;

[0028] R 1 and R 1′ is independently selected at each occurrence from H, hydroxy, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl and substituted or unsubstituted heteroaryl, -C(=NH)NH2, -CH2CO2R 0 , -CH2C(O)NHCH2CO2H, -CH2CH2OH, -CH2CH2NHC(O)R 3 、-CH2C(O)NHCH2CO2H、

[0029] and

[0030] Where R 1 and R 1′ can form a heterocyclic ring, including but not limited to azetidine, pyrrolidine, piperidine, piperazine, morpholine,

[0031]

[0032] R 2 is independently selected at each occurrence from C2-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy;

[0033] R 3 is independently selected at each occurrence from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine;

[0034] R 4 is independently selected at each occurrence from alkylene interrupted by one or more heteroatoms, wherein the heteroatoms are O, S, NH or a combination thereof;

[0035] Y is independently selected at each occurrence from Ar2, -(CH2)3PO(OEt)2 or -CH2CO2Me;

[0036] Ar2 is independently selected at each occurrence from substituted 5-membered and 6-membered monocyclic aromatic rings and 9-membered and 10-membered fused bicyclic rings comprising two monocyclic rings together, wherein at least one of the two monocyclic rings is aromatic, wherein: Ar2 is replaced by G 1 , G 2 , G 3 , G 4 and G 5 substituted, wherein: when Ar2 is monosubstituted, G 1 is independently selected at each occurrence from oxo, -NH2, -COR 3 、-E-CO2H、-C(O)NR 1 R 1′ 、-E-PO(OR 1 )2 and G 2 , G 3 , G 4 and G 5 Substituted aryl; G 2 , G 3 , G 4 and G 5is independently selected at each occurrence from being absent or from a group comprising: halogen, -CN, C1-C6 alkyl, C1-C6 haloalkyl, -E-CO2H, -E-CHO, -EC(O)R 3 、-EC(O)NH(OH)、-EC(O)NHR 1 、-E-CONR 1 R 1′ 、-E-NR 1 R 1′ AND-E-OR 2 , wherein E at each occurrence is independently selected from a direct bond and a C1-C6 alkylene group.

[0037] In one aspect, the present disclosure provides a method for enhancing a nucleic acid polymerase reaction, the method comprising the steps of forming a nucleic acid polymerase reaction composition, the nucleic acid polymerase reaction composition comprising a template nucleic acid, a nucleic acid polymerase, a mixture of nucleotides and / or nucleotide analogs, and at least one PEM; and incubating the nucleic acid polymerase reaction composition under conditions that allow nucleic acid polymerization. The PEM increases the processivity, rate, and / or fidelity of the nucleic acid polymerase reaction. In one embodiment, the at least one PEM increases the length of the resulting nucleic acid product compared to a nucleic acid polymerase reaction lacking the PEM.

[0038] In other embodiments, the nucleic acid polymerase is a DNA polymerase. In certain embodiments, the DNA polymerase is DPO4 or its variant. In other embodiments, the mixture of the nucleotide or nucleotide analogs is a mixture of nucleotide analogs comprising nucleoside triphosphate aminoesters, wherein each nucleoside triphosphate aminoester comprises a core base and a polymer tethering part selected from the group consisting of adenine, guanine, thymine and cytosine, wherein the first end of the polymer tethering part is connected to the core base, and the second end of the polymer tethering part is connected to the α phosphate of the nucleoside triphosphate aminoester to provide the expansion of the nucleotide analog by the cutting of the aminophosphorothioate bond. In certain embodiments, the nucleic acid polymerization reaction produces the extensible polymer of nucleotide analogs, wherein the extensible polymer encodes the core base sequence information of the template nucleic acid. In other embodiments, the conditions allowing the nucleic acid polymerization reaction include suitable polymerization buffer and oligonucleotide primers. In further embodiments, the suitable buffer includes one or more of the following, such as each of the following: Tris OAc, NH4OAc, PEG, water miscible organic solvents (such as DMF, NMP and acetone), polyphosphate 60 and MnCl2. In other embodiments, the reaction mixture further comprises a nucleic acid intercalator. In other embodiments, the reaction mixture further comprises a polyanion recognition moiety. In further embodiments, the mixture of nucleotides or nucleotide analogs comprises a nucleotide analog containing a detectable label. In yet other embodiments, the detectable label is an optically detectable label selected from the group consisting of luminescent, chemiluminescent, fluorescent, chromogenic or chromogenic labels.

[0039] In another aspect, the present disclosure provides a composition comprising a mixture of at least one PEM and nucleotide analogs. Such a composition is useful, for example, when combined with a polymerase, wherein the at least one PEM increases the number and accuracy of nucleotide analogs incorporated into a daughter strand during a template-dependent polymerization reaction relative to the same polymerization reaction without at least one PEM. In other embodiments, the at least one PEM comprises a plurality of PEMs.

[0040] Optionally, the mixture of the nucleotide analogs comprises nucleoside triphosphate aminoesters, wherein each nucleoside triphosphate aminoester comprises a core base and a polymer tethering portion selected from the group consisting of adenine, guanine, thymine and cytosine, wherein the first end of the polymer tethering portion is connected to the core base, and the second end of the polymer tethering portion is connected to the α phosphate of the nucleoside triphosphate aminoester to provide the expansion of the nucleotide analogs by the cutting of the aminophosphoroester bond. In other embodiments, the composition further comprises at least one of the following, such as two, three, four or each buffer agent: Tris OAc, NH4OAc, PEG, water miscible organic solvents (such as DMF and NMP), polyphosphate 60, N-methylsuccinimide (NMS) and MnCl2. In other embodiments, the composition further comprises a single-stranded binding protein (SSB). In other embodiments, the composition further comprises urea. In certain embodiments, the mixture of the nucleotide analogs comprises a nucleotide analog containing a detectable label. In some embodiments, the detectable label is an optically detectable label selected from the group consisting of luminescent, chemiluminescent, fluorescent, fluorescent, chromogenic or chromogenic labels.

[0041] On the other hand, the present invention provides a method for sequencing a DNA or RNA template, the method comprising the step of forming a DNA polymerase reaction composition, the reaction composition comprising a DNA or RNA template, a replication primer complexed with the template, a DNA polymerase, a mixture of nucleotides or nucleotide analogs and at least one PEM, incubating the DNA polymerase reaction composition under conditions that allow DNA polymerization, wherein at least one PEM improves the rate, fidelity or continuous synthesis ability of the DNA polymerase reaction. The method further comprises determining the sequence of nucleotides or nucleotide analogs in the resulting nucleotide or nucleotide analog polymer. The PEM can be described as a compound of formula (I). In some embodiments, the at least one PEM is selected from a compound of formula (II). In other embodiments, the mixture of nucleotide analogs comprises nucleoside triphosphate amino esters, wherein each nucleoside triphosphate amino ester comprises a nucleobase and a polymer tethering portion selected from the group consisting of adenine, guanine, thymine and cytosine, wherein the first end of the polymer tethering portion is connected to the nucleobase, and the second end of the polymer tethering portion is connected to the alpha phosphate of the nucleoside triphosphate amino ester to provide an extension of the nucleotide analog by cutting of the amino phosphorothioate bond. In other embodiments, the DNA polymerase is DPO4 or its variant. In other embodiments, the polymer of the nucleotide analogue gained is an expandable polymer. In other embodiments, the method further comprises contacting the expandable polymer with the phosphoramidate cutting agent to produce the step of the expanded polymer of the nucleotide analogue. In certain embodiments, the polymer tethering part of each nucleotide analogue comprises a report part unique to the core base of the analogue. In other embodiments, the report part produces a characteristic electronic signal. In other embodiments, the step of determining the nucleotide analogue sequence comprises the step of transferring the expandable polymer of the nucleotide analogue through the nanopore.

[0042] Thus, in one embodiment, the present disclosure provides a composition comprising a PEM and a polynucleotide. In another embodiment, the present disclosure provides a composition comprising a PEM and a polypeptide (eg, a polypeptide such as an enzyme, wherein the enzyme may be a nucleic acid polymerase).

[0043] The following are some exemplary specific and numbered embodiments of the present disclosure. In addition, unless otherwise specifically stated, each atom identified in a chemical formula may be any isotope of that atom. For example, the name C (carbon) includes 12 C. 13 C or 14 C and mixtures thereof, especially mixtures of natural abundance isotopes, while H (hydrogen) includes 1 H. 2 H and 3 H and mixtures thereof, and O (oxygen) including 16 O and18 O and mixtures thereof, and N (nitrogen) including 14 N and 15 N and its mixtures, etc. Other atoms are similar:

[0044] 1) A compound of formula (I)

[0045]

[0046] or a solvate, hydrate, tautomer, chelate or salt thereof, wherein:

[0047] m is 1, 2 or 3;

[0048] m′ is 1, 2 or 3;

[0049] n is 0, 1 or 2;

[0050] p is 0, 1, or 2;

[0051] When X is C, W is N, or when X is N, W is C;

[0052] is a single bond or a double bond, wherein the double bond starts at any one of W or X that is carbon;

[0053] L is a linking group;

[0054] M is independently selected at each occurrence from hydrogen, halogen and C1-C4 alkyl;

[0055] Ar1 is independently selected at each occurrence from optionally substituted pyridine, pyrazine, pyridazine, furan, thiophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine and carbazole, wherein: the substituents of Ar1 are independently selected at each occurrence from halogen, -OH, -CN, -NO2, C1-C6 alkyl, C1-C6 haloalkyl, C 1- C6 heteroalkyl, C1-C6 cycloalkyl, -OR 0 、-CONH2、-C(O)NR 1 R 1′ 、-NR 1 R 1′ 、-NR 1 C(O)R 3 、-C(O)SR 3 、-COR 3 、-OC(O)R 3 、-C(O)OR 3 , thiol, -R 4 -H, -SOR 1 、-S(O)2R 1 、-S(O)2NR 1 R1′ and -NS(O)2R 3 ;

[0056] R 0 is independently selected at each occurrence from C1-C6 alkyl, C 1- C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;

[0057] R 1 and R 1′ is independently selected at each occurrence from H, hydroxy, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, and substituted or unsubstituted heteroaryl, -C(=NH)NH2, -CH2CO2R 0 , -CH2C(O)NHCH2CO2H, -CH2CH2OH, -CH2CH2NHC(O)R 3 、-CH2C(O)NHCH2CO2H、

[0058] and

[0059] Where R 1 and R 1′ can form a heterocyclic ring, including but not limited to azetidine, pyrrolidine, piperidine, piperazine, morpholine,

[0060]

[0061] R 2 is independently selected at each occurrence from C2-C6 alkyl, C 1- C6 haloalkyl, C1-C6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy;

[0062] R 3 independently selected at each occurrence from H, C l -C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine;

[0063] R 4is independently selected at each occurrence from alkylene interrupted by one or more heteroatoms, wherein the heteroatoms are O, S, NH or a combination thereof;

[0064] Y is independently selected at each occurrence from Ar2, -(CH2)3PO(OEt)2 or -CH2CO2Me;

[0065] Ar2 is independently selected at each occurrence from substituted 5-membered and 6-membered monocyclic aromatic rings and 9-membered and 10-membered fused bicyclic rings comprising two monocyclic rings together, wherein at least one of the two monocyclic rings is aromatic, wherein: Ar2 is replaced by G 1 , G 2 , G 3 , G 4 and G 5 substituted, wherein: when Ar2 is monosubstituted, G 1 is independently selected at each occurrence from oxo, -NH2, -COR 3 、-E-CO2H、-C(O)NR 1 R 1′ 、-E-PO(OR 1 )2 and G 2 , G 3 , G 4 and G 5 Substituted aryl; G 2 , G 3 , G 4 and G 5 is independently selected at each occurrence from being absent or from a group comprising: halogen, -CN, C1-C6 alkyl, C1-C6 haloalkyl, -E-CO2H, -E-CHO, -EC(O)R 3 、-EC(O)NH(OH)、-EC(O)NHR 1 、-E-CONR 1 R 1′ 、-E-NR 1 R 1′ AND-E-OR 2 , where E is independently selected at each occurrence from a direct bond and C 1- C6 alkylene.

[0066] 2) The compound according to Embodiment 1, wherein Ar1 is a monocyclic heteroaryl group.

[0067] 3) A compound according to embodiment 2, wherein Ar1 is selected from:

[0068]

[0069] The triazole ring is located at position k on Ar1.

[0070] 4) The compound according to Embodiment 1, wherein Ar1 is a bicyclic aryl group.

[0071] 5) A compound according to embodiment 4, wherein Ar1 is a bicyclic carbocyclic aromatic group selected from:

[0072]

[0073] The triazole ring is located at position k on Ar1.

[0074] 6-) A compound according to Embodiment 4, wherein Ar1 is a bicyclic heteroaryl group selected from:

[0075]

[0076] The triazole ring is located at position k on Ar1.

[0077] 7) The compound according to Embodiment 1, wherein Ar1 is a tricyclic aryl group.

[0078] 8) A compound according to Embodiment 7, wherein Ar1 is a tricyclic carbocyclic aromatic group selected from:

[0079]

[0080] 9) A compound according to Embodiment 7, wherein Ar1 is a tricyclic heteroaryl selected from:

[0081]

[0082] The triazole ring is located at position k on Ar1.

[0083] 10) A compound according to Example 7, wherein Ar1 is a tricyclic heteroaryl group selected from

[0084]

[0085] The triazole ring is located at position k on Ar1.

[0086] 11) A compound according to embodiment 1, wherein Ar2 is a substituted 5-membered monocyclic aromatic ring selected from the group consisting of thiophene, 1,2-thiazole, 1,3-thiazole, furan, 1,2-oxazole, 1,3-oxazole, 1H-pyrrole, 1H-pyrazole, oxadiazole, thiadiazole, 1,2,4-triazole, 1,2,3-triazole and 1H-imidazole.

[0087] 12) The compound according to embodiment 1, wherein Ar2 is a 6-membered monocyclic aromatic ring selected from the group consisting of benzene, pyridine, pyridazine, pyrimidine and pyrazine.

[0088] 13) A compound according to embodiment 1, wherein Ar2 is a 9-membered fused bicyclic aromatic ring system selected from the group consisting of benzofuran, 1,3-benzoxazole, furano[3,2-b]pyridine, furano[3,2-c]pyridine, furano[2,3-c]pyridine, furano[2,3-b]pyridine, indole, 1H-benzimidazole, 1H-pyrrolo[3,2-b]pyridine , 1H-pyrrolo[3,2-c]pyridine, 1H-pyrrolo[2,3-c]pyridine, 1H-pyrrolo[2,3-b]pyridine, benzothiophene, 1,3-benzothiazole, thieno[3,2-b]pyridine, thieno[3,2-c]pyridine, thieno[2,3-c]pyridine, benzoxadiazole, benzothiadiazole, benzisoxazole, benzotriazole and thieno[2,3-b]pyridine.

[0089] 14) A compound according to embodiment 1, wherein Ar2 is a 10-membered fused bicyclic aromatic ring system selected from the group consisting of naphthylene, quinoline, quinazoline, quinoxaline, 1,5-naphthyridine, 1,6-naphthyridine, 1,7-naphthyridine, 1,8-naphthyridine, isoquinoline, phthalazine, 2,6-naphthyridine and 2,7-naphthyridine.

[0090] 15) A compound according to embodiment 1, wherein Ar2 is a pyridyl ring selected from the following:

[0091]

[0092] wherein the substituent G(G 1 , G 2 , G 3 , G 4 and G 5 as defined above) occurs 0, 1 or 2 times on the pyridyl ring.

[0093] 16) A compound according to embodiment 1, wherein Ar2 is of the formula

[0094] A phenyl ring, wherein the substituent G (G 1 , G 2 , G 3 , G 4 and G 5 As defined above) occurs 0, 1 or 2 times on the phenyl ring. In one embodiment, G is replaced by G 2 , G 3 , G 4 and G 5 Substituted aryl (eg, phenyl).

[0095] 17) A compound according to embodiment 1, wherein Ar2 is selected from

[0096] of the phenyl ring.

[0097] 18) A compound according to embodiment 1 wherein the substitution on Ar2 comprises amino.

[0098] 19) A compound according to embodiment 1 wherein the substitution on Ar2 comprises methoxy.

[0099] 20) The compound according to embodiment 1, wherein the substitution on Ar2 comprises formic acid.

[0100] 21) A compound according to Embodiment 1, wherein the substitution on Ar2 comprises -CH2-CO2-CH3.

[0101] 22) A compound according to Embodiment 1 wherein the substitution on Ar2 comprises trifluoromethyl.

[0102] 23) A compound according to Embodiment 1 wherein the substitution on Ar2 comprises hydroxy.

[0103] 24) A compound according to embodiment 1, wherein the substitution on Ar2 is one carboxylic acid and one hydroxyl group.

[0104] 25) A compound according to embodiment 1, wherein the substitution on Ar2 is one formic acid and one trifluoromethyl.

[0105] 26) The compound according to embodiment 1 is in the form of a chelate.

[0106] 27) The compound according to Embodiment 26, wherein the chelate is a copper chelate.

[0107] 28) The compound according to Embodiment 1, which has a logP of at least 4.9.

[0108] 29) A compound according to Example 1, wherein n is 0 and m is 2, having one of the following structures (III) or (IV):

[0109]

[0110] 30) The compound according to embodiment 1, which has substitutions on Ar2, said substitutions comprising at least two of hydroxy, carboxamide and trifluoromethyl.

[0111] 31) A compound according to Embodiment 1, wherein the compound has one of the following structures (V), (VI), (VII), (VIII) or (IX):

[0112]

[0113] 32) The compound according to Embodiment 1, wherein the compound has one of the following structures (X), (XI) or (XII):

[0114]

[0115]

[0116] 33) The compound according to Embodiment 1, wherein the compound has one of the following structures (XIII), (XIV) or (XV):

[0117]

[0118]

[0119] 34) A compound according to Example 1, wherein the compound has one of the following structures (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) or (XXII):

[0120]

[0121]

[0122] 35) The compound according to Embodiment 1, wherein the compound has one of the following structures (XXIII), (XXIV) or (XXV):

[0123]

[0124] 36) The compound according to Embodiment 1, wherein the compound has one of the following structures (XXVI), (XXVII) or (XXVIII):

[0125]

[0126]

[0127] 37) The compound according to Embodiment 1, wherein the compound has one of the following structures (XXIX), (XXX), (XXXI) or (XXXII):

[0128]

[0129]

[0130] 38) The compound according to embodiment 1, which is selected from any one of the following:

[0131] 4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0132] 4,4′-(pyridine-3,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0133] 4,4′-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0134] 4,4′-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))dibenzoic acid;

[0135] 4,4′-((4-methoxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0136] 4,4′-((4-carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0137] 4,4′-((4-nitropyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0138] 5,5′-((4-cyanopyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0139] 4,4′-((4-methylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0140] 4,4′-((4-(ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid-);

[0141] 5,5′-((4-(ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0142] 4,4′-((4-(methoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0143] 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0144] 4,4′-((4-(methylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0145] 4,4′-((4-carbamoylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0146] 4,4′-(pyrazine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0147] 4,4′-(1,3-phenylenebis(1H-1,2,3-triazole-4,1-diyl))diphenylamine;

[0148] 4,4′-(1,3-phenylenebis(1H-1,2,3-triazole-4,1-diyl))dibenzoic acid;

[0149] 4-(4-(3-(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)benzoic acid;

[0150] 4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))dibenzoic acid;

[0151] 4-(4-(3-(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)benzoic acid;

[0152] 4,4′-((3,5-dimethylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0153] 4,4′-((4l3-pyridine-2,6-diyl)bis(5-iodo-1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0154] 4,4′-((4-acetylaminopyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0155] 4,4′-((9-acetyl-9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid-);

[0156] 4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(N,2-dihydroxybenzamide);

[0157] 4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzamide);

[0158] 4,4′-((4-carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0159] 4,4′-((1,10-phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0160] 4,4′-((4-(trifluoromethyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0161] 4,4′-((3-cyanopyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0162] 4,4′-((3-nitropyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0163] 3,3′-((4-cyanopyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0164] 4,4′-((4-(tert-butoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0165] 4-(4-(4-cyanopyridin-2-yl)-1H-1,2,3-triazol-1-yl)-2-hydroxybenzoic acid;

[0166] 5-(4-(6-(4-(3-carboxy-4-hydroxy-5-methylphenyl)-1H-1,2,3-triazol-1-yl)-4-(methoxycarbonyl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)-2-hydroxy-3-methylbenzoic acid;

[0167] 4,4′-((4-(dimethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0168] 4,4′-((4-(cyclopropylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0169] 4,4′-((4-(but-3-yn-1-ylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0170] 4,4′-((4-(butylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0171] 4,4′-((4-(diethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0172] 4,4′-((4-(tert-butylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0173] 4,4′-((4-(morpholine-4-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid-);

[0174] 4,4′-((4-(propylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0175] 4,4′-((4-(phenylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0176] 4,4′-((4-((2-acetylaminoethyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0177] 4,4′-((4-(4-cyclopropylpiperazine-1-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0178] 4,4′-((4-(carbamimidoylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0179] 4,4′-((4-(piperidine-1-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0180] 4,4′-((4-(cyclobutylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0181] 4,4′-((1,10-phenanthroline-3,8-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0182] 4,4′-((4-(cyclopentylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0183] 4,4′-((4-(dipropylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0184] 4,4′-((4-(di-sec-butylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0185] 4,4′-(naphthalene-2,7-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0186] 4,4′-(naphthalene-2,3-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0187] 4,4′-((4-(dibutylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0188] 4,4′-((4-((2-hydroxyethyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0189] 4,4′-((4-(cyclohexylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0190] 4,4′-((4-(Benzylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0191] 4,4′-((4-(4-methylpiperazine-1-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0192] 4-(4-(3-(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)benzoic acid;

[0193] 4,4′,4″,4″′-((((butane-1,4-diylbis(azanediyl))bis(carbonyl))bis(pyridine-4,2,6-triyl))tetrakis(1H-1,2,3-triazole-4,1-diyl))tetrakis(2-hydroxybenzoic acid);

[0194] 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3,5,6-trichloropicolinic acid);

[0195] 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0196] 7,7′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxy-1,8-naphthyridine-4-carboxylic acid);

[0197] 5,5′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0198] 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-fluorobenzoic acid);

[0199] 5,5′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-fluorobenzoic acid);

[0200] 4,4′-((4-(methylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0201] 4,4′-((4-(morpholine-4-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0202] 4,4′-((4-(diethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0203] 4,4′-((4-carbamoylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0204] 4,4′-((4-(ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0205] 4,4′-((4-(azetidine-1-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0206] 4,4′-((4-(ethyl(methyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0207] N-ethyl-2,6-bis(1-(4-(2,2,2-trifluoroacetyl)phenyl)-1H-1,2,3-triazol-4-yl)isonicotinamide;

[0208] 4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0209] 4,4′-((4-(cyclopropylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0210] 4,4′-((4-(butylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0211] 5,5′-((4-(diethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0212] 5,5′-((4-(morpholine-4-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0213] 4,4′-(pyridazine-3,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0214] 5,5′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-(trifluoromethyl)benzoic acid);

[0215] 3,3′-(((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(4,1-phenylene))dipropionic acid;

[0216] 4,4′-(((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(4,1-phenylene))dibutyric acid;

[0217] 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))diphthalic acid;

[0218] 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-methoxybenzoic acid);

[0219] 5,5′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))diisophthalic acid;

[0220] 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-hydroxybenzoic acid);

[0221] Diethyl (3-(4-(6-(1-(3-(diethoxyphosphoryl)propyl)-1H-1,2,3-triazol-4-yl)-4-(ethylcarbamoyl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)propyl)phosphonate;

[0222] 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-methylbenzoic acid);

[0223] 2,2′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-(trifluoromethyl)benzoic acid);

[0224] 4,4′-((4-((2-hydroxyethyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0225] 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-nitrobenzoic acid);

[0226] 4,4′-((4-((3,3,3-trifluoropropyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0227] (4-(4-(4-(ethylcarbamoyl)-6-(1-(4-phosphonophenyl)-1H-1,2,3-triazol-4-yl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)phenyl)phosphonic acid;

[0228] 4,4′,4″,4″′-((((butane-1,4-diylbis(azanediyl))bis(carbonyl))bis(pyridine-4,2,6-triyl))tetrakis(1H-1,2,3-triazole-4,1-diyl))tetrakis(2-(trifluoromethyl)benzoic acid);

[0229] 2,2′-((4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))diacetic acid;

[0230] Dimethyl 2,2′-((4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))diacetate;

[0231] (2S,2′S)-2,2′-((4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))disuccinic acid;

[0232] 2,2′-((2,2′-((4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))bis(acetyl))bis(azanediyl)-)diacetic acid;

[0233] 2,6-Bis(1-(4-cyano-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-N-ethylisonicotinamide;

[0234] 4,4′-(thiophene-2,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); and

[0235] 4,4′-(Furan-2,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid,

[0236] 39) A compound selected from any one of the following:

[0237] 4,4′-(1,3-phenylenebis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0238] 4,4′-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))diphenylamine;

[0239] 3,6-Bis(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)-9H-carbazole;

[0240] 4,4′-(1,4-phenylenebis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0241] 4,4′-(1,3-phenylenebis(1H-1,2,3-triazole-4,1-diyl))diphenylamine;

[0242] 4,4′-(1,3-phenylenebis(1H-1,2,3-triazole-4,1-diyl))dibenzoic acid;

[0243] 1,3-Bis(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)benzene;

[0244] 4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))diphenylamine;

[0245] 4-(4-(3-(1-(4-carboxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxybenzoic acid;

[0246] 4-(4-(3-(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)

[0247] benzoic acid;

[0248] 4,4′-((4-carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid);

[0249] 2-(1-(1H-benzo[d]imidazol-4-yl)-1H-1,2,3-triazol-4-yl)-6-(1-(1H-benzo[d]imidazol-7-yl)-1H-1,2,3-triazol-4-yl)-N-ethylisonicotinamide;

[0250] 4,4′-((4-carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0251] 4,4′-((5-carboxy-1,3-phenylene)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid-);

[0252] 4,4′-((5-carboxy-1,3-phenylene)bis(1H-1,2,3-triazole-1,4-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0253] 4,4′-((5-(ethylcarbamoyl)-1,3-phenylene)bis(1H-1,2,3-triazole-1,4-diyl))bis(2-(trifluoromethyl)benzoic acid);

[0254] 3′-(4-(4-(ethylcarbamoyl)-6-(1-(3′-(trifluoromethyl)-[1,1′-biphenyl]-3-yl)-1H-1,2,3-triazol-4-yl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)-3-(trifluoromethyl)-[1,1′-biphenyl]-4-carboxylic acid;

[0255] 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-cyanobenzoic acid); and

[0256] 4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-chlorobenzoic acid).

[0257] 40) A composition comprising a compound according to any one of embodiments 1-39 and a molecular crowding agent.

[0258] 41) A composition according to embodiment 41, wherein the molecular crowding agent is polyalkylene glycol.

[0259] 42) A composition comprising a compound according to any one of Examples 1-39, and an aqueous buffer.

[0260] 43) The composition according to embodiment 43, wherein the aqueous buffer is Tris HCl.

[0261] 44) A composition comprising the compound according to any one of embodiments 1-39, and a polynucleotide.

[0262] 45) A composition according to embodiment 44, wherein the polynucleotide is a 20-60mer oligonucleotide.

[0263] 46) A composition comprising a compound according to any one of embodiments 1-39, and a protein.

[0264] 47) A composition according to Example 46, wherein the protein is a DNA polymerase.

[0265] 48) A composition comprising a compound according to any one of embodiments 1-39, and a mixture of nucleotides or nucleotide analogs.

[0266] 49) A composition for enhancing the processivity, fidelity or rate of a DNA polymerase reaction, comprising at least one compound according to any one of embodiments 1-39, and a mixture of nucleotide analogs.

[0267] 50) A composition comprising a mixture of at least one compound according to any one of embodiments 1-39 and nucleotide analogs, wherein the at least one compound according to any one of embodiments 1-39 increases the number and accuracy of nucleotide analogs incorporated into daughter chains during a template-dependent polymerization reaction relative to the same polymerization reaction in the absence of the at least one compound according to any one of embodiments 1-39.

[0268] 51) A composition according to embodiment 50, wherein the mixture of nucleotide analogs comprises nucleoside triphosphate amino esters, wherein each nucleoside triphosphate amino ester comprises a nucleobase selected from the group consisting of adenine, guanine, thymine and cytosine and a polymer tether portion, wherein a first end of the polymer tether portion is connected to the nucleobase and a second end of the polymer tether portion is connected to the alpha phosphate of the nucleoside triphosphate amino ester to provide extension of the nucleotide analog by cleavage of the phosphoramidite bond.

[0269] 52) The composition according to Example 51 further comprises a buffer component selected from the group consisting of: TrisOAc, NH4OAc, PEG, a water-miscible organic solvent, polyphosphate 60, NMS and MnCl2.

[0270] 53) The composition according to Example 51 further comprises a single-chain binding protein.

[0271] 54) The composition according to embodiment 51 further comprises urea.

[0272] 55) A composition according to Example 51, wherein the mixture of nucleotide analogs comprises nucleotide analogs containing a detectable label.

[0273] 56) A composition according to embodiment 55, wherein the detectable marker is an optically detectable marker selected from the group consisting of luminescent, chemiluminescent, fluorescent, fluorescent, chromogenic or chromogenic markers.

[0274] 57) A kit for sequencing a nucleic acid template, comprising at least one composition according to any one of Examples 40-56.

[0275] 58) A method for enhancing a nucleic acid polymerase reaction, the method comprising:

[0276] a. forming a nucleic acid polymerase reaction composition comprising:

[0277] i. template nucleic acid,

[0278] ii. Nucleic acid polymerase,

[0279] iii. a mixture of nucleotides or nucleotide analogs, and

[0280] iv. at least one compound according to any one of embodiments 1-39; and

[0281] b. incubating the nucleic acid polymerase reaction composition under conditions permissive for nucleic acid polymerization, wherein the at least one compound according to any one of embodiments 1-39 increases the processivity, rate or fidelity of the nucleic acid polymerase reaction.

[0282] 59) A method according to Example 58, wherein the compound according to any one of Examples 1-39 increases the length of the resulting nucleic acid product compared to a nucleic acid polymerase reaction lacking the compound according to any one of Examples 1-39.

[0283] 60) The method according to embodiment 58, wherein the at least one compound according to any one of embodiments 1-39 comprises a plurality of compounds according to any one of embodiments 1-39.

[0284] 61) A method according to Example 58, wherein the nucleic acid polymerase is a DNA polymerase.

[0285] 62) A method according to Example 61, wherein the DNA polymerase is DPO4 or a variant thereof.

[0286] 63) A method according to Example 58, wherein the mixture of nucleotides or nucleotide analogs is a mixture of nucleotide analogs comprising nucleoside triphosphate amino esters, wherein each nucleoside triphosphate amino ester comprises a nucleobase selected from the group consisting of adenine, guanine, thymine and cytosine and a polymer tether portion, wherein the first end of the polymer tether portion is connected to the nucleobase and the second end of the polymer tether portion is connected to the alpha phosphate of the nucleoside triphosphate amino ester to provide extension of the nucleotide analog by cleavage of the phosphoramidite bond.

[0287] 64) A method according to Example 58, wherein the nucleic acid polymerization reaction produces an expandable polymer of nucleotide analogs, wherein the expandable polymer encodes the nucleobase sequence information of the template nucleic acid.

[0288] 65) A method according to Example 58, wherein the conditions allowing nucleic acid polymerization reaction include a suitable polymerization buffer and oligonucleotide primers.

[0289] 66) The method according to Example 58, wherein the suitable buffer comprises a component selected from the group consisting of TrisOAc, NH4OAc, PEG, a water-miscible organic solvent, polyphosphate 60, NMS and MnCl2.

[0290] 67) A method according to Example 58, wherein the reaction mixture further comprises a single-stranded binding protein.

[0291] 68) The method according to embodiment 58, wherein the reaction mixture further comprises urea.

[0292] 69) A method according to Example 58, wherein the mixture of nucleotides or nucleotide analogs contains nucleotide analogs containing a detectable label.

[0293] 70) A method according to Example 69, wherein the detectable marker is an optically detectable marker selected from the group consisting of luminescent, chemiluminescent, fluorescent, fluorescent, chromogenic or chromogenic markers.

[0294] 71) A method for sequencing a DNA or RNA template, the method comprising the following steps:

[0295] a. forming a DNA polymerase reaction composition comprising:

[0296] i. DNA or RNA template,

[0297] ii. a replication primer complexed with the template,

[0298] iii. DNA polymerase,

[0299] iv. a mixture of nucleotides or nucleotide analogs,

[0300] v. at least one compound according to any one of embodiments 1-39,

[0301] b. incubating the DNA polymerase reaction composition under conditions that allow DNA polymerization, wherein the at least one compound according to any one of embodiments 1-39 increases the rate, fidelity or processivity of the DNA polymerase reaction; and

[0302] c. Determine the sequence of the nucleotides or nucleotide analogs in the resulting nucleotide or nucleotide analog polymer.

[0303] 72) A method according to embodiment 71, wherein the mixture of nucleotide analogs comprises nucleoside triphosphate amino esters, wherein each nucleoside triphosphate amino ester comprises a nucleobase selected from the group consisting of adenine, guanine, thymine and cytosine and a polymer tether portion, wherein a first end of the polymer tether portion is connected to the nucleobase and a second end of the polymer tether portion is connected to the alpha phosphate of the nucleoside triphosphate amino ester to provide extension of the nucleotide analog by cleavage of the amino phosphoroester bond.

[0304] 73) A method according to embodiment 71 or 72, wherein the DNA polymerase is DPO4 or a variant thereof.

[0305] 74) A method according to Example 71 or 72, wherein the resulting polymer of nucleotide analogs is an expandable polymer.

[0306] 75) The method according to Example 74 further comprises the step of contacting the extendable polymer with a phosphoramidate cleavage agent to produce an extended polymer of nucleotide analogs.

[0307] 76) A method according to embodiment 71 or 72, wherein the polymer tether portion of each nucleotide analog comprises a reporter portion specific to the nucleobase of the analog.

[0308] 77) A method according to embodiment 72, wherein the reporter portion generates a characteristic electronic signal.

[0309] 78) A method according to Example 72, wherein the step of determining the sequence of nucleotide analogs includes the step of transferring an expandable polymer of nucleotide analogs through a nanopore.

[0310] The above and additional features of the invention and the manner in which they are obtained will become apparent and will best be understood by reference to the following more detailed description.All references disclosed herein are incorporated herein by reference in their entirety as if each were individually incorporated.

[0311] This brief summary is provided to introduce some concepts in a simplified form that are further described in the detailed description below. Unless otherwise expressly stated, this brief summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to limit the scope of the claimed subject matter.

[0312] Details of one or more embodiments are set forth in the following description. Features shown or described in conjunction with one exemplary embodiment may be combined with features of other embodiments. Thus, any of the various embodiments described herein may be combined to provide further embodiments. If necessary, various aspects of the embodiments may be modified to provide other further embodiments employing concepts from the various patents, applications, and publications identified herein. Other features, objects, and advantages will be apparent from the description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0313] The exemplary features of the present disclosure, its nature and various advantages will be apparent from the following detailed description of the accompanying drawings and various embodiments. Non-limiting and non-exhaustive embodiments are described with reference to the accompanying drawings, wherein the same labels or reference numerals refer to the same parts in the various views unless otherwise indicated. The sizes and relative positions of the elements in the drawings are not necessarily drawn to scale. For example, the shapes of the various elements are selected, enlarged and positioned to improve the legibility of the drawings. The specific shapes of the drawn elements have been selected for ease of identification in the drawings.

[0314] Figure 1A , 1B , 1C and 1D are concise schematic diagrams illustrating the main features of the generalized XNTPs and their use in extended sequencing (SBX-).

[0315] Figure 2 is a schematic diagram illustrating further details of one embodiment of XNTP.

[0316] Figure 3 is a schematic diagram illustrating one embodiment of an Xpandomer passing through a biological nanopore.

[0317] Figure 4 is a gel showing primer extension products.

[0318] Figure 5 is a gel showing primer extension products.

[0319] Fig. 6A and Figure 6B is a histogram display of the aligned read population of the nanopore-derived sequences.

[0320] Fig. 7A and Figure 7B is a histogram display of the aligned read population of the nanopore-derived sequences.

[0321] Figure 8 is a gel showing primer extension products.

[0322] Fig. 9 is a gel showing primer extension products.

[0323] Fig.10 is a gel showing primer extension products.

[0324] Fig.11 is a gel showing primer extension products.

[0325] Fig.12 is a gel showing primer extension products.

[0326] Fig.13 is a gel showing primer extension products.

[0327] Fig.14 is a gel showing primer extension products. DETAILED DESCRIPTION

[0328] By referring to the following detailed description of the preferred embodiments of the present invention and the examples included herein, the present invention can be more easily understood. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by ordinary technicians in the field to which the present invention belongs.

[0329] In one aspect, the PEM of the present disclosure is a compound of formula (I)

[0330]

[0331] or a solvate, hydrate, tautomer, chelate or salt thereof, wherein:

[0332] m is 1, 2 or 3;

[0333] m′ is 1, 2 or 3;

[0334] n is 0, 1 or 2;

[0335] p is 0, 1, or 2;

[0336] When X is C, W is N, or when X is N, W is C;

[0337] is a single bond or a double bond, wherein the double bond starts at any one of W or X that is carbon;

[0338] L is a linking group;

[0339] M is independently selected at each occurrence from hydrogen, halogen and C1-C4 alkyl;

[0340] Ar1 at each occurrence is independently selected from optionally substituted pyridine, pyrazine, pyridazine, furan, thiophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine and carbazole, wherein:

[0341] The substituents of Ar1 are independently selected at each occurrence from halogen, -OH, -CN, -NO2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C1-C6 cycloalkyl, -OR 0 、-CONH2、-C(O)NR 1 R 1′ 、-NR 1 R 1′ 、-NR 1 C(O)R 3 、-C(O)SR 3 、-COR 3 、-OC(O)R 3 、-C(O)OR 3 , thiol, -R 4 -H, -SOR 1 、-S(O)2R 1 、-S(O)2NR 1 R 1′ and -NS(O)2R 3 ; and among them

[0342] R 0 is independently selected at each occurrence from C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;

[0343] R 1 and R 1′ is independently selected at each occurrence from H, hydroxy, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, arylalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl and substituted or unsubstituted heteroaryl, -C(=NH)NH2, -CH2CO2R 0 , -CH2C(O)NHCH2CO2H, -CH2CH2OH, -CH2CH2NHC(O)R 3 、-CH2C(O)NHCH2CO2H、

[0344] and

[0345] Where R 1 and R 1′ can form a heterocyclic ring, including but not limited to azetidine, pyrrolidine, piperidine, piperazine, morpholine,

[0346]

[0347] R 2 is independently selected at each occurrence from C2-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy;

[0348] R 3 is independently selected at each occurrence from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine;

[0349] R 4 is independently selected at each occurrence from alkylene interrupted by one or more heteroatoms, wherein the heteroatoms are O, S, NH or a combination thereof;

[0350] Y is independently selected at each occurrence from Ar2, -(CH2)3PO(OEt)2 or -CH2CO2Me;

[0351] Ar2 is independently selected at each occurrence from substituted 5-membered and 6-membered monocyclic aromatic rings and 9-membered and 10-membered fused bicyclic rings comprising two monocyclic rings taken together, wherein at least one of the two monocyclic rings is aromatic, wherein:

[0352] Ar2 by G 1 , G 2 , G 3 , G 4 and G 5 Replace, where:

[0353] When Ar2 is monosubstituted, G 1 is independently selected at each occurrence from oxo, -NH2, -COR 3 、-E-CO2H、-C(O)NR 1 R 1′ 、-E-PO(OR 1 )2 and G 2 , G 3 , G 4 and G 5 substituted aryl; and

[0354] G 2 , G 3 , G 4 and G 5 is independently selected at each occurrence from being absent or from a group comprising: halogen, -CN, C1-C6 alkyl, C1-C6 haloalkyl, -E-CO2H, -E-CHO, -EC(O)R 3 、-EC(O)NH(OH)、-EC(O)NHR 1 、-E-CONR 1 R 1′ 、-E-NR 1 R 1′ AND-E-OR 2 ;

[0355] wherein E at each occurrence is independently selected from a direct bond and C1-C6 alkylene.

[0356] As used in the specification and the appended claims, the following terms have the meanings indicated unless otherwise indicated.

[0357] Certain chemical groups named herein are preceded by an abbreviated notation indicating the total number of carbon atoms found in the specified chemical group. For example; C1-C4 alkyl, which can alternatively be written as C 1-4Alkyl describes an alkyl group having at least one and up to 4 carbon atoms, while C4-C 12 Cycloalkylalkyl (also referred to as C 4-12 Cycloalkyl) describes a cycloalkylalkyl group having a total of 4 to 12 carbon atoms. The total number of carbons in the shorthand notation does not include carbons that may be present in substituents of the group. For example, C1-C6 alkyl refers to an alkyl group containing one to six carbon atoms; C1-C6 haloalkyl refers to a haloalkyl group containing one to six carbon atoms; and C1-C6 alkylene refers to an alkylene diradical containing one to six carbon atoms.

[0358] In addition to the foregoing, as used in the specification and the appended claims, the following terms have the meanings indicated unless otherwise indicated:

[0359] "Alkyl " refers to the straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, does not contain unsaturation, and optionally has a specified number of carbon atoms, for example, has one to twelve carbon atoms, one to eight carbon atoms or one to six carbon atoms or one to four carbon atoms, and is connected to the rest of the molecule by a single bond. Examples are such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl etc. When unsaturation is introduced in an alkyl group, the resulting group may be referred to as an unsaturated alkyl group, wherein the unsaturated alkyl group is generally referred to as an alkenyl group (having at least one carbon-carbon double bond) and an alkynyl group (having at least one carbon-carbon triple bond). In one embodiment, and when specified, the alkyl group in the compound of the present disclosure may be or include an unsaturated alkyl group.

[0360] "Alkenyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing at least one double bond, optionally having a specified number of carbons, e.g., two to twelve carbon atoms or two to eight carbon atoms or two to six carbon atoms or two to four carbon atoms, and attached to the rest of the molecule by a single bond, e.g., ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like.

[0361] "Alkynyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing at least one triple bond, optionally having a specified number of carbons, e.g., two to twelve carbon atoms, or two to eight carbon atoms, or two to six carbon atoms, or two to four carbon atoms, and attached to the rest of the molecule by a single bond, e.g., ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like.

[0362] "Halo" refers to bromo, chloro, fluoro or iodo.

[0363] "Haloalkyl" refers to an alkyl group as defined above substituted with one or more halo groups as defined above, for example, trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl, 1-bromomethyl-2-bromoethyl, etc. Likewise, "haloalkenyl" refers to an alkenyl group as defined herein substituted with one or more halo groups as defined herein, and "haloalkynyl" refers to an alkynyl group as defined herein substituted with one or more halo groups as defined herein.

[0364] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a group consisting only of carbon and hydrogen, not containing unsaturation and optionally having a specified number of carbon atoms. Examples are such as methylene, ethylene, propylene, n-butene, etc. The alkylene chain is connected to the rest of the molecule by a single bond and is connected to the group by a single bond. The connection point between the alkylene chain and the rest of the molecule and the group can be through one carbon or any two carbons in the chain. Similar to the alkyl group, unsaturation can be introduced into the alkylene chain to provide an unsaturated alkylene chain. When unsaturation is introduced into the alkylene chain, the resulting group can be referred to as an unsaturated alkylene group or chain, wherein the unsaturated alkylene chain is generally referred to as an alkenylene group (having at least one carbon-carbon double bond) and an alkynylene group (having at least one carbon-carbon triple bond). In one embodiment, and when specified, the alkylene chain in the compound of the present disclosure can be or include an unsaturated alkylene chain.

[0365] "Alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the remainder of the molecule to a group consisting only of carbon and hydrogen, containing at least one double bond and optionally having a specified number of carbon atoms (e.g., two to twelve carbon atoms). Examples of alkenylene groups include vinylene, propenylene, n-butenylene, etc. The alkenylene chain is connected to the remainder of the molecule by a single bond and to the group by a double bond or a single bond. The point of attachment of the alkenylene chain to the remainder of the molecule and to the group can be through one carbon or any two carbons within the chain.

[0366] "Aryl" refers to a ring system radical containing at least 5 ring atoms, optionally containing 1-6 heteroatoms selected from O, S and N and at least one aromatic ring. A 5-membered monocyclic aromatic ring contains 5 ring atoms selected from carbon and heteroatoms, while a 6-membered monocyclic aromatic ring contains 6 ring atoms selected from carbon and heteroatoms. An exemplary monocyclic aromatic ring with 5 members is pyrrole and an exemplary monocyclic aromatic ring with 6 members is pyridine. Aryl groups can be, for example, monocyclic, bicyclic, tricyclic or tetracyclic ring systems, which can include fused or bridged ring systems. Carbocyclic aryl groups contain only carbon on the ring atoms, examples of which include, but are not limited to, those derived from aceanthrene, acenaphthylene, acephenanthrenes, anthracene, azulene, benzene, The aryl groups of fluoranthene, fluorene, asymmetric indacene, symmetric indacene, indane, indene, naphthalene, phenanthrenes, phenanthrenes, pyrenes and triphenylene. In one embodiment, the aryl group is phenyl or naphthyl, and in another embodiment, it is phenyl. When the aryl group includes non-carbon ring atoms, such as oxygen, sulfur and nitrogen, the aryl group can be referred to as a heteroaryl group. The heteroaryl group can be, for example, a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include a fused or bridged ring system. The nitrogen, carbon or sulfur atom in the heteroaryl group can be optionally oxidized; The nitrogen atom can be optionally quaternized.

[0367] An "arylalkyl" group is an alkyl, alkenyl or alkynyl group as defined above, wherein its hydrogen atoms are replaced by aryl groups as defined above. Exemplary aralkyl groups include benzyl (-CH2 phenyl), phenylethyl (-CH2CH2 phenyl) and phenylethylene (-CH=CH phenyl) groups and fused (cycloalkylaryl) alkyl groups such as 4-ethyl-indanyl. Aralkyl groups can be substituted on the aryl moiety, the alkyl, alkenyl or alkynyl moiety, or both.

[0368] "Fused" refers to a ring system containing fusion between rings, where fusion refers to rings that share two adjacent ring atoms. A fused ring containing two 5-membered and / or 6-membered monocyclic rings fused together refers to a bicyclic ring system, where each ring is a monocyclic ring and independently has 5 or 6 ring atoms, and the two rings are fused because they share two ring atoms. For example, naphthalene is a 10-membered fused ring system formed by two 6-membered monocyclic rings (benzene) fused together. Naphthalene is bicyclic because it contains two (bi=2) rings. As another example, 1,3-benzothiazole is a 9-membered fused ring system formed by a 6-membered ring (benzene) and a 5-membered ring (1,3-thiazole) fused together. 1,3-Benzothiazole is bicyclic because it contains two rings.

[0369] "Carbocyclic group" refers to a stable 3- to 18-membered aromatic or non-aromatic ring group consisting of 3 to 18 carbon atoms. Unless otherwise specifically stated in the specification, the carbocyclic group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems and can be partially or fully saturated. Non-aromatic carbocyclic groups include cycloalkyl, while aromatic carbocyclic groups include aryl.

[0370] "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon group consisting only of carbon and hydrogen atoms, which may include a fused or bridged ring system, having three to fifteen carbon atoms, preferably three to ten carbon atoms, and which is saturated or unsaturated and connected to the remainder of the molecule by a single bond. Monocyclic groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Polycyclic groups include, for example, adamantyl, norbornyl, decahydronaphthyl, 7,7-dimethyl-bicyclo-[2.2.1]heptanyl, etc.

[0371] "Heterocyclyl" refers to a stable 3- to 18-membered aromatic or non-aromatic ring group consisting of two to twelve carbon atoms and one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless otherwise specifically stated in the specification, the heterocyclyl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include a fused or bridged ring system; the nitrogen, carbon or sulfur atoms in the heterocyclyl may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl group may be partially or fully saturated. Examples of non-aromatic heterocyclyl groups include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinyl, pyrrolidinyl, pyrazolidinyl, pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl, thiomorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.

[0372] Optionally, but only in detail, each of the alkyl, alkenyl, alkylene, alkenylene, carbocyclyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl groups in the PEM compounds of the present disclosure may be substituted with one or more unsubstituted (e.g., an alkyl substituent on an alkyl group is not further substituted, i.e., the alkyl substituent is an unsubstituted alkyl) substituents selected from the group consisting of alkyl, alkenyl, halo, haloalkyl, haloalkenyl, cyano, oxo, thio, nitro, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, --R b -OR a 、--R b --OC(O)-R a 、--R b --N(R a )2. --R b --C(O)R a 、--R b --C(O)OR a 、--R b --C(O)N(R a )2. --R b --N(R a )C(O)OR c 、--R b --N(R a )C(O)R c 、--R b--N(R a )S(O) t R c (where t is 1 to 2), --R b -N=C(OR a )R a , --R b --S(O) t OR c (where t is 1 to 2), --R b --S(O) s R c (where s is 0 to 2) and -R b --S(O) t N(R a )2 (where t is 1 to 2), where each R a R is independently hydrogen, alkyl, alkenyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl; R is each independently a direct bond or a straight or branched alkylene or alkenylene chain; and each R c is alkyl, alkenyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl.

[0373] "Amino" refers to the --NH2 group. "Cyano" refers to the --CN group. "Hydroxy" refers to the --OH group. "Nitro" refers to the --NO2 group. "Oxo" refers to the =O substituent. "Thio" refers to the =S substituent. "Trifluoromethyl" refers to the --CF3 group. "Trifluoromethoxy" refers to the --OCF3 group. Thiol, also known as sulfhydryl, refers to the -SH group.

[0374] "Acyl" refers to the group --C(O)R, which can also be written as -C(=O)R, where R is alkyl, aralkyl, carbocyclyl, aryl, heteroaryl or heterocyclyl. For example, when R is methyl, the acyl group can be referred to as acetyl.

[0375] "Alkoxy" refers to a group of formula --OR, wherein R is an alkyl or haloalkyl group. In one embodiment, the alkoxy group contains up to six carbon atoms. Exemplary alkoxy groups include methoxy and ethoxy. Alkoxy substituted with halogen may be referred to herein as haloalkoxy, including, for example, trifluoromethoxy, trichloromethoxy, etc.

[0376] "Heteroalkenylene" or "heteroalkenylene chain" refers to a straight or branched divalent hydrocarbon chain connecting the rest of the molecule to a group consisting of carbon and hydrogen and at least one heteroatom selected from N, O and S.

[0377] "Haloalkoxy" refers to an alkoxy group substituted with one or more halo groups as defined above, for example, trifluoromethoxy, difluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy, 3-bromo-2-fluoropropoxy, etc. The alkoxy portion of the haloalkoxy group may be optionally substituted as defined above for an alkoxy group.

[0378] "N-heterocyclyl" refers to a heterocyclyl group that contains at least one nitrogen. An N-heterocyclyl group may be optionally substituted as described above for a heterocyclyl group.

[0379] "Heterocyclylalkyl" refers to a group of the formula --R b R h The group wherein Rb is an alkylene chain as defined above and Rh is a heterocyclic group as defined above, and if the heterocyclic group is a nitrogen-containing heterocyclic group, the heterocyclic group can be connected to the alkyl group on the nitrogen atom. The alkylene chain of the heterocyclic alkyl group can be optionally substituted, as defined above for the alkylene chain. The heterocyclic moiety of the heterocyclic alkyl group can be optionally substituted, as defined above for the heterocyclic group.

[0380] "N-heteroaryl" refers to a heteroaryl group as defined above containing at least one nitrogen, wherein the point of attachment of the heteroaryl group to the rest of the molecule is through the nitrogen atom in the heteroaryl group. N-heteroaryl groups may be optionally substituted as described above for heteroaryl groups.

[0381] "Heteroarylalkyl" refers to a group of the formula --R b R i A group in which R b is an alkylene chain as defined above and R i is a heteroaryl group as defined herein. The heteroaryl portion of the heteroarylalkyl group may be optionally substituted as defined herein for heteroaryl groups. The alkylene chain portion of the heteroarylalkyl group may be optionally substituted as defined herein for alkylene chains. Similarly, an arylalkyl group refers to a heteroarylalkyl group in which the heteroaryl portion is replaced by a corresponding carbocyclic aryl group, i.e., a heteroatom is replaced by a carbon, and adjusted as needed for hydrogen substitution.

[0382] "Hydroxyalkyl" refers to a group of the formula --R b OH group, where R b is an alkylene chain as defined herein. The --OH (hydroxy, also known as hydroxy) group may be attached to any carbon atom in the alkylene chain. The alkylene chain portion of the heteroarylalkyl group may further be optionally substituted as defined above for an alkylene chain.

[0383] The PEM compounds with acidic or basic groups described herein can generally be used as free acids or free bases. Alternatively, the PEM compounds with acidic or basic groups can be used in the form of salts, such as acid or base addition salts. The acid addition salts of free amino compounds can be prepared by methods well known in the art, and can be formed by organic and inorganic acids. Suitable organic acids include maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, methanesulfonic acid, acetic acid, trifluoroacetic acid, oxalic acid, propionic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, mandelic acid, cinnamic acid, aspartic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid and benzenesulfonic acid. Suitable inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and nitric acid. Base addition salts include those formed with formate anions and include salts formed with organic and inorganic cations, such as those selected from alkali metals and alkaline earth metals (e.g., lithium, sodium, potassium, magnesium, barium and calcium), as well as ammonium ions and substituted derivatives thereof (e.g., dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, etc.). Thus, the term "salt" of the PEM compounds described herein is intended to encompass any and all salt forms.

[0384] The PEM compounds of the present disclosure may be in the form of chelates. Chelates refer to compounds containing organic ligands (eg, triazole-Ar groups) bonded to a central metal atom at two or more points.

[0385] With regard to stereoisomers, the PEM compounds described herein may have one or more chiral (or asymmetric) centers, so enantiomers, diastereomers, and other stereoisomeric forms that can be defined as (R)- or (S)- according to absolute stereochemistry may be produced. When the compounds described herein contain olefinic double bonds or other geometric asymmetric centers, unless otherwise indicated, it is intended to represent that these compounds contain both E and Z geometric isomers (e.g., cis or trans) simultaneously. Similarly, unless otherwise indicated, all possible isomers, as well as their racemic and optically pure forms, and are also intended to include all tautomeric forms. It is therefore contemplated that various stereoisomers and mixtures thereof include "enantiomers", which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other. Therefore, compounds may exist in any isomeric form, including racemates, racemic mixtures, and as separate enantiomers or diastereomers.

[0386] In addition, some crystalline forms of PEM compounds may exist in the form of polymorphs, which are also considered herein. In addition, some PEM compounds may also form solvates with water or other organic solvents. Such solvates are similarly included in the scope of the compounds described herein.

[0387] As will be appreciated by those skilled in the art, any of the above compounds may be incorporated with radioactive isotopes. Thus, isotopically labeled compounds identical to those described herein are also contemplated, wherein one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Examples of isotopes that may be incorporated into these compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine. Thus, reference to an element, such as hydrogen (H) or carbon (C), is intended to encompass all isotopes thereof. For example, the name C (carbon) includes 12 C. 13 C or 14 C and mixtures thereof, and H (hydrogen) includes 1 H. 2 H and 3 H and mixtures thereof, and O (oxygen) including 16 O and 18 O and mixtures thereof, and N (nitrogen) including 14 N and 15 N and mixtures thereof, etc., and similarly for other atoms. Isotopically labeled PEM compounds can be used to track the PEM compounds or portions thereof in assays and the like.

[0388] In the PEM compound of formula (I), Ar1 is an aryl group, also referred to as an aromatic moiety. The aromatic moiety can be a carbocyclic or heterocyclic aromatic moiety, wherein each aromatic ring atom is a carbon in a carbocyclic aromatic moiety and at least one of the aromatic ring atoms is a nitrogen, oxygen or sulfur in a heterocyclic aromatic moiety.

[0389] In one embodiment, Ar1 can comprise 1-6 rings, wherein six ring atoms at most can be selected from oxygen, sulphur and nitrogen, and the rest are carbon atoms.Optionally, Ar1 part can comprise 1-5 rings, wherein five ring atoms at most can be selected from oxygen, sulphur and nitrogen.As another selection, Ar1 group can comprise 1-4 rings, wherein four ring atoms at most can be selected from oxygen, sulphur and nitrogen.As another selection again, Ar1 part can comprise 1-3 rings, wherein three ring atoms at most can be selected from oxygen, sulphur and nitrogen.As another example, Ar1 can comprise 1-2 rings, wherein three ring atoms at most can be selected from oxygen, sulphur and nitrogen.In any case, each ring can be independently five-membered ring (i.e. five ring atoms form ring) or six-membered ring or seven-membered ring, and in a selection, each ring is five-membered ring or six-membered ring.

[0390] Exemplary aromatic moieties are carbocyclic aromatic moieties. The carbocyclic moiety may contain one (e.g., benzene) or two (e.g., naphthalene, azulene) or three (e.g., acenaphthylene, fluorene) or four (e.g., fluoranthene, acetothracene) or five (e.g., pentacene, pyrrolidine) or six (e.g., hexacene) aromatic rings, wherein for convenience, the Ar1 group may be exemplified herein by naming its unsubstituted form (e.g., benzene), although in the compounds disclosed herein the Ar1 group is a corresponding group, e.g., when m is 2 and Ar1 is otherwise unsubstituted, two ring hydrogens are replaced by triazole groups. For example, the aromatic moiety may be a monocyclic carbocyclic moiety, i.e., phenyl, also referred to as a C6 aromatic moiety. As another example, the aromatic moiety may be a bicyclic carbocyclic moiety, e.g., naphthyl, which is C 10 Aromatic part.

[0391] Exemplary Ar1 aromatic moieties are heterocyclic aromatic moieties, which may also be referred to as heteroaryl groups. The heterocyclic moiety may contain one or two or three or four or five or six aromatic rings in addition to containing 1 or 2 or 3 or 4 or 5 or 6 heteroatoms (i.e., atoms selected from nitrogen, sulfur and oxygen other than carbon). Optionally, the heteroatoms (if present) are nitrogen. For example, the aromatic moiety may be a monocyclic heterocyclic moiety, such as pyridyl (which is a six-membered C5 aromatic moiety) or pyrazinyl (which is a six-membered C4 aromatic moiety). As another example, the aromatic moiety may be a bicyclic heterocyclic moiety, such as quinolyl or isoquinolyl (which are ten-membered C9 aromatic moieties) or 1,5-naphthyridinyl, 2,6-naphthyridinyl or 2,7-naphthyridinyl (which are exemplary ten-membered C8 aromatic moieties).

[0392] Thus, a heteroaryl group is an aromatic ring compound containing 5 or more ring members, one or more of which are heteroatoms, such as, but not limited to, N, O, and S. A heteroaryl group designated as a C2-heteroaryl group can be a 5-membered ring with two carbon atoms and three heteroatoms, a 6-membered ring with two carbon atoms and four heteroatoms, etc. Similarly, a C4-heteroaryl group can be a 5-membered ring with one heteroatom, a 6-membered ring with two heteroatoms, etc. The sum of the number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridyl, phenylthio, benzophenylthio, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridyl, isoxazolpyridyl, thianaphthalenyl, purinyl, xanthosyl, adeninyl, guaninyl, quinolyl, isoquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, quinoxalinyl, and quinazolinyl groups. Thus, the terms "heteroaryl" and "heteroaryl groups" include fused ring compounds, such as where at least one ring but not necessarily all rings are aromatic, including tetrahydroquinolyl, tetrahydroisoquinolyl, indolyl, and 2,3-dihydroindole.

[0393] When m is 2, so that Ar1 must be substituted with two triazole-Ar2 moieties, any two carbons of the aromatic portion of Ar1 can be substituted with one of the two triazole-Ar2 moieties. For example, when Ar1 is a substituted benzene, Ar1 can be substituted at the ortho, meta or para position, as shown below, where k represents the position where the substitution may occur on the aromatic portion:

[0394] (adjacent), (meta) and (Counterpoint).

[0395] As another example, when Ar1 is a substituted naphthalene and m is 2, Ar1 may be substituted on any two naphthyl carbon atoms, where the following structure shows the substitution options, and k shows where the triazole substitution provided by (triazole-Ar2) may occur on the aromatic portion:

[0396]

[0397] The foregoing examples illustrate triazole substitution on Ar1 using carbocyclic aromatic Ar1 groups as illustrative Ar1 moieties. However, the same principles apply to triazole substitution on heterocyclic aromatic Ar1 groups. For example, when Ar1 is a substituted pyridine and m is 2, the two triazole groups of (triazole-Ar2) can be located at any of the following positions on the pyridine ring, where k is used to specify the position at which the triazole group can be located:

[0398]

[0399] Therefore, in one exemplary embodiment, Ar1 is selected from A monocyclic heteroaromatic structure wherein the triazole ring is substituted at position k on Ar1. In another exemplary embodiment, Ar1 is selected from In another exemplary embodiment, Ar1 is selected from In another embodiment, Ar1 is a polycyclic heterocyclic structure having two six-membered rings and one five-membered ring and one nitrogen ring atom, and is selected from wherein the triazole ring is substituted at position k on Ar1. In yet another exemplary embodiment, Ar1 is a polycyclic heterocyclic structure having three six-membered rings and two nitrogen ring atoms, and is selected from The triazole ring is substituted at position k on Ar1.

[0400] Ar1 includes substituted and unsubstituted aromatic moieties as described herein. In one embodiment, Ar1 is a substituted aromatic moiety. In one embodiment, Ar1 is an unsubstituted aromatic moiety, which may also be referred to as an unsubstituted aromatic moiety. In a substituted aromatic moiety, one or more hydrogen atoms that would otherwise be bonded to a ring atom have been replaced by a substituent, for example, optionally 1 or 2 or 3 or 4 or 5 or 6 hydrogen atoms may be replaced by a substituent. The substituents on Ar1 do not refer to the triazole-Ar2 moiety that necessarily exists when m is equal to 1, or the two triazole-Ar2 moieties that necessarily exist when m is equal to 2, or the three triazole Ar2 moieties that necessarily exist when m is equal to 3.

[0401] In one embodiment, Ar1 substituent will be composed of atoms selected from deuterium, halogen (F, Cl, Br, I), carbon, nitrogen, oxygen and sulphur, and optionally will also contain hydrogen, and will also contain other atoms forming counter ions (if present). Deuterium and halides are considered to be monovalent atoms, while carbon, nitrogen, oxygen and sulphur are considered to be polyvalent atoms because they can form more than one covalent bond simultaneously. In addition to monovalent atoms, Ar1 substituent can have multiple polyvalent atoms, such as 1-25 polyvalent atoms or 1-22 polyvalent atoms or 1-15 polyvalent atoms or 1-10 polyvalent atoms or 1-5 polyvalent atoms, and these atoms are optionally selected from carbon, nitrogen, oxygen and sulphur. The following provides an explanation of the substituent with up to 10 polyvalent atoms. Other substituents, including substituents with up to 25 polyvalent atoms, are known to those of ordinary skill in the art.

[0402] In one embodiment, the substituents on Ar1 contain zero polyvalent atoms. In this embodiment, a hydrogen bonded to a ring atom is replaced by another monovalent atom, such as deuterium, fluorine, chlorine, bromine, or iodine.

[0403] In one embodiment, the substituent on Ar1 contains 1 polyvalent atom. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by a single polyvalent atom, wherein the open valence on the polyvalent atom is filled by one or more monovalent atoms, such as hydroxyl (OH), thiol (SH), amino (NH2), methyl (CH3) and methylene (=CH2), including fully or partially halogenated and deuterated forms thereof, such as CF3.

[0404] In one embodiment, the substituent on Ar1 contains 2 polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by a first polyvalent atom, which in turn is bonded to a second polyvalent atom, thereby providing a substituent formed by two polyvalent atoms, wherein the open valences on the polyvalent atoms are filled by one or more monovalent atoms. Examples of these substituents are well known to those of ordinary skill in the art. Specific examples include ethyl (CH2CH3), ethylene (CH=CH2), ethynyl (C≡CH), ethylene (=CHCH3), aminomethyl (CH2NH2), aminomethylene (=CHNH2), thiomethylene (=CHSH), hydroxymethylene (=CHOH), hydroxymethyl (CH2OH), mercaptomethyl (CH2SH), N-methylamine (NHCH3), methylthio (SCH3), methoxy (OCH3), nitrile (CN), formyl (C(O)H), thialdehyde (C(S)H), N-hydroxy (N-OH), hydroxylamine (ONH2), hydrazine (NH2NH2), diazine (N=NH), diazo compounds (N≡N), including fully or partially halogenated and deuterated versions thereof, such as OCF3 and CH2CD3.

[0405] In one embodiment, the substituent on Ar1 contains 3 polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by a first polyvalent atom, which in turn is directly or indirectly bonded to each of the second and third polyvalent atoms; therefore, the first polyvalent atom is bonded to the second polyvalent atom, and the third polyvalent atom is bonded to one or both of the first and second polyvalent atoms, thereby providing a substituent formed by three polyvalent atoms, wherein the open valence on the polyvalent atom is filled by one or more monovalent atoms. Examples of these substituents are well known to those of ordinary skill in the art and are provided herein, such as nitro, methyl ketone, carboxyl.

[0406] In one embodiment, the substituent on Ar1 contains 4 polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by the first polyvalent atom, which in turn directly or indirectly bonds to each of the second, third and fourth polyvalent atoms, thereby providing a substituent formed by four polyvalent atoms, wherein the open valence on the polyvalent atom is filled by one or more monovalent atoms. The examples of these substituents are well known to those of ordinary skill in the art and are provided herein, such as methyl esters (CO2CH3), N-methylformamide (C(O)NHCH3) and acetamide (NHC(O)CH3).

[0407] In one embodiment, Ar1 substituent contains 5 polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by the first polyvalent atom, and the first polyvalent atom is directly or indirectly bonded to each of the second, third, fourth and fifth polyvalent atoms, thereby providing a substituent formed by five polyvalent atoms, wherein the open valence on the polyvalent atom is filled by one or more monovalent atoms. The examples of these substituents are well known to those of ordinary skill in the art and are provided herein, such as ethyl ester (CO2CH2CH3), S-ethyl thiocarbonate (C(O)SCH2CH3), N-ethylformamide (C(O)NHCH2CH3) and N, N-dimethylformamide (C(O)N(CH3)2).

[0408] In one embodiment, the substituent on Ar1 contains 6 polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by the first polyvalent atom, which in turn directly or indirectly bonds to each of the second, third, fourth, fifth and sixth polyvalent atoms, thereby providing a substituent formed by six polyvalent atoms, wherein the open valence on the polyvalent atom is filled by one or more monovalent atoms. Examples of these substituents are well known to those of ordinary skill in the art and are provided herein, such as N-cyclopropylcarboxamide (C(O)NH-cyclopropyl), N-propylcarboxamide (C(O)NHCH2CH2CH3), N-(2-hydroxyethyl)carboxamide (C(O)NHCH2CH2OH) and N-carboxamidinecarboxamide (C(O)NHC(=NH)NH2).

[0409] In one embodiment, the substituent on Ar1 contains 7 polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by the first polyvalent atom, which in turn directly or indirectly bonds to each of the second, third, fourth, fifth, sixth and seventh polyvalent atoms, thereby providing a substituent formed by seven polyvalent atoms, wherein the open valence on the polyvalent atom is filled by one or more monovalent atoms. Examples of these substituents are well known to those of ordinary skill in the art and are provided herein, such as N-(n-butyl)formamide (C(O)NHCH2CH2CH2CH3), N-(tert-butyl)formamide (C(O)NHC(CH3)3), N,N-diethylformamide (C(O)N(CH2CH3)2) and N-cyclobutylformamide (C(O)NH(cyclobutyl)).

[0410] In one embodiment, the substituent on Ar1 contains 8 polyvalent atoms.In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by the first polyvalent atom, and the first polyvalent atom is directly or indirectly bonded to each of the second, third, fourth, fifth, sixth, seventh and eighth polyvalent atoms, so a substituent formed by eight polyvalent atoms is provided, wherein the open valence on the polyvalent atom is filled by one or more monovalent atoms.The examples of these substituents are well-known to those of ordinary skill in the art and are provided in this article, such as N-cyclopentylformamide (C (O) NH (cyclopentyl)), (piperidin-1-yl) ketone (C (O)-piperidin-1-yl) and (morpholine-4-yl) ketone (C (O)-morpholine-4-yl).

[0411] In one embodiment, the substituent on Ar1 contains 9 polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by a first polyvalent atom, which in turn is directly or indirectly bonded to each of the second, third, fourth, fifth, sixth, seventh, eighth, and ninth polyvalent atoms, thereby providing a substituent formed by nine polyvalent atoms, wherein the open valences on the polyvalent atoms are filled by one or more monovalent atoms. Examples of such substituents are well known to those of ordinary skill in the art and are provided herein, such as di-(isopropyl)formaldehyde (C(O)O(CH(CH3)2)2, di-(n-propyl)formaldehyde (C(O)O(CH2CH2CH3)2), N-cyclohexylcarboxamide (C(O)NH(cyclohexyl)), (4-methylpiperazin-1-yl)methanone (C(O)(4-methylpiperazin-1-yl), 2-(acetylamino)ethylcarboxamide (C(O)NHCH2CH2NHC(O)CH3) and N-phenylcarboxamide (C(O)NH(phenyl)).

[0412] In one embodiment, the substituent on Ar contains 10 polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar are replaced by the first polyvalent atom, which in turn directly or indirectly bonds to each of the second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth polyvalent atoms, thereby providing a substituent formed by ten polyvalent atoms, wherein the open valence on the polyvalent atom is filled by one or more monovalent atoms. Examples of these substituents are well known to those of ordinary skill in the art and are provided herein, such as N-benzylformamide (C (O) NHCH2 (phenyl)).

[0413] In one embodiment, Ar1 is a substituted aryl group, wherein at least one substituent on Ar1 is selected from the group consisting of halogen, hydroxyl, thiol, nitro, and nitrile.

[0414] In one embodiment, Ar1 is a substituted aryl group, wherein at least one substituent on Ar1 is selected from: halogen, -OH, -CN, -NO2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C1-C6 cycloalkyl, -OR 0 、-CONH2、-C(O)NR 1 R 1′ 、-NR 1 R 1′ 、-NR 1 C(O)R 3 、-C(O)SR 3 、-COR 3 、-OC(O)R 3 、-C(O)OR 3 , thiol, -R 4 -H, -SOR 1 、-S(O)2R 1 、-S(O)2NR 1 R 1′ and -NS(O)2R 3 ; and wherein (a) R 0 is independently selected at each occurrence from C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; (b) R 1 and R 1′is independently selected at each occurrence from H, hydroxy, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl and substituted or unsubstituted heteroaryl, -C(=NH)NH2, -CH2CO2R 0 , -CH2C(O)NHCH2CO2H, -CH2CH2OH, -CH2CH2NHC(O)R 3 、-CH2C(O)NHCH2CO2H、 (c) where R 1 and R 1′ can form a heterocyclic ring, including but not limited to azetidine, pyrrolidine, piperidine, piperazine, morpholine, (d) R 2 is independently selected at each occurrence from C2-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy; (e) R 3 is independently selected at each occurrence from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine; (f) R 4 is independently selected at each occurrence from alkylene interrupted by one or more heteroatoms wherein the heteroatoms are O, S, NH or combinations thereof.

[0415] In one embodiment, Ar1 is substituted aryl, wherein at least one substituent on Ar1 is selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted haloalkyl, and substituted or unsubstituted haloalkoxy.

[0416] The Ar1 group will include an aromatic moiety as explained herein, wherein the aromatic moiety may be optionally substituted as described herein, except for (triazole-Ar2) mIn one embodiment, exemplary substituents of Ar1 are halides (e.g., fluorides, chlorides, and bromides), alkyl groups having 1 to 6 carbon atoms (e.g., methyl and ethyl), haloalkyl groups having 1 to 6 carbon atoms (e.g., trifluoromethyl), cyano, formyl, and formamide. In another embodiment, exemplary substituents of Ar1 are nitro (—NO2), cyano (—CN), formic acid (—COOH or its salt), formamide (—C(O)NH2), C1-C6 alkoxy (including methoxy), C1-C6 alkyl (including methyl), C1-C6 haloalkyl (e.g., trifluoromethyl), C1-C6 heteroalkyl (including amide (e.g., —NHC(O)(C1-C6 alkyl), —NHC(O)(C1-C6 heteroalkyl), —C(O)NH(C1-C6 alkyl), —C(O)NH(C1-C6 alkyl), C6 heteroalkyl), -C(O)N(C1-C6 alkyl)(C1-C6 alkyl), -C(O)N(C1-C6 alkyl)(C1-C6 heteroalkyl), and -C(O)N(C1-C6 heteroalkyl)(C1-C6 heteroalkyl), including -NHC(O)CH3, C(O)NHCH3, -C(O)N(CH3)2, -NHC(O)CH2CH3, C(O)NHCH2CH3, -C(O)N(CH3)CH2CH3, -C(O)N(CH2CH3), -C(O)NH(C 1-C6 cycloalkyl) and -NHC(O)(C1-C6 cycloalkyl) (e.g., C(O)NH(cyclopropyl), -NHC(O)-cyclopropyl, C(O)NH(cyclohexyl), NHC(O)-cyclohexyl), C(O)NHCH2CH2CH2CH3, -C(O)NH(C(CH3)3), -C(O)NH(CH2CH2OH)), ketones (e.g., -C(O)(C1-C6 alkyl) (including -C(O)CH3), -C(O)(cycloalkyl) (including -C(O)-cyclohexyl and C(O)-(heterocycloalkyl), wherein the heterocycloalkyl can be, for example, morpholinyl, piperidinyl, piperazinyl, N-methylpiperazinyl)), esters (for example, -CO2-(C1-C6 alkyl), including -CO2CH3, -CO2CH2CH3, -CO2CH2CH2CH3, -CO2CH2(CH3)2) and thioesters (for example, C(O)-S-(C1-C6 alkyl), including -C(O)-S-CH3 and -C(O)-S-CH2CH3).

[0417] In one embodiment, Ar1 is a substituted aryl group, wherein at least one substituent on Ar1 is selected from the group consisting of: -O-(C 1-6 Alkyl), C 1-6 Alkyl, C 1-6 Halogenated alkyl, -CO2-C 1-6 Alkyl, -CONH-C1-6 Alkyl, -CONH2, CN; and -NO2.

[0418] When n is 1 or 2, the compound of formula (I) will include a linker L. In one embodiment, the linker L can be a direct bond. In another embodiment, the linker is not a direct bond, but one or more atoms, particularly selected from carbon, nitrogen, oxygen, and sulfur atoms. In another embodiment, the linker can be an alkylene group (e.g., C1-C6 alkylene) or a substituted alkylene. The linker can be a heteroalkylene linker, which refers to a substituted or unsubstituted alkylene, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur, and the heteroatom is at and / or placed at one or more terminal positions of the parent chain. In one embodiment, L is a heteroalkylene with a length of 2 to 10 carbon atoms, wherein one or more carbon atoms are replaced by at least one heteroatom selected from oxygen, nitrogen, and sulfur. In one embodiment, L can be a heteroalkylene linker with at least one N, O, or S heteroatom, wherein the heteroalkylene can be linear or cyclized and optionally substituted, wherein exemplary substituents include oxo, --OH, C 1-4 Alkyl and C 1-4 Examples of heteroalkylene linking groups include heteroalkylene groups containing amides, such as -C(O)NH-alkylene- and -C(O)NH-alkylene-NHC(O)-, wherein the alkylene is optionally C1-C6 alkylene. Other examples of heteroalkylene groups include heteroalkylene groups containing esters, such as -C(O)O-alkylene- and -C(O)O-alkylene-OC(O)-, wherein in one embodiment, the alkylene is an unsubstituted C1-C6 alkylene, and in another embodiment, the alkylene is a substituted C1-C6 alkylene. 1- C6 alkylene. In one embodiment, the linker is hydrolytically stable so it does not decompose or degrade or otherwise break down when the PEM is placed in water.

[0419] The linking group L does not usually need to be too long; in one embodiment, it contains 1 to about 25 atoms (atomic counts exclude hydrogen and halogens), wherein in addition to hydrogen and halogens, the linking group can be optionally composed of atoms selected from carbon, nitrogen, oxygen and sulfur. In various other embodiments, the linking group has less than 25 atoms (excluding hydrogen and halogens), for example, it contains 1 to about 20 atoms or 1 to about 15 atoms or 1 to about 10 atoms or 1 to about 5 atoms, in each case, hydrogen and halogens are excluded from the atomic count, wherein the counted atoms can be optionally selected from carbon, oxygen, nitrogen and sulfur.

[0420] In one embodiment, the triazole ring in the compound of formula (I) may be substituted in addition to being directly bonded to Ar1 and Ar2. In general, the compounds of the present disclosure may optionally be described as comprising the formula Wherein Ar1 and Ar2 are defined elsewhere herein, and M can be hydrogen (in this case the triazole ring is only replaced by Ar1 and Ar2), C1-C4 alkyl, or M can be a halide substituent, such as fluoride, chloride, bromide or iodide. In one embodiment, the compounds of the present disclosure have a triazole ring substituted only by Ar1 and Ar2, that is, M is hydrogen. In another embodiment, the compounds of the present disclosure have a triazole ring substituted by Ar1, Ar2 and C1-C4 alkyl. In another embodiment, the compounds of the present disclosure have a triazole ring substituted by Ar1, Ar2 and a halide. In another embodiment, the compounds of the present disclosure include a triazole ring substituted by an iodide, that is, M is an iodide. In another embodiment, the compounds of the present disclosure have an M-substituent on the triazole ring, wherein M is selected from hydrogen and iodide.

[0421] Therefore, in one embodiment, the present disclosure provides the formula Compound, wherein Ar1 and Ar2 are defined elsewhere herein, and M is selected from hydrogen, C1-C4 alkyl and halide. Optionally, as described above, M can be hydrogen, or in another selection, M can be, for example, a halide, such as an iodide, as shown in compound 4,4′-((pyridine-2,6-diyl)bis(5-iodo-1H-1,2,3-triazole-4,1-diyl)-)bis(2-hydroxybenzoic acid). When, as in the above structure, the compound of the present disclosure has more than one M-substituted triazole ring, M is independently selected at each occurrence. However, in one embodiment, M is the same atom at each occurrence in the compound of the present disclosure. For example, the present disclosure provides a compound wherein M is hydrogen at each occurrence of M. In another example, the present disclosure provides a compound wherein M is an iodide at each occurrence of M.

[0422] The compound of formula (I) includes at least one triazole-Ar2 moiety. In one embodiment, the disclosed compound includes two or more triazole-Ar2 moieties, such as Compound. When the compound of the present disclosure includes two or more than two triazole-Ar2 parts, the Ar2 part can optionally have the same chemical structure at each occurrence. However, when the PEM compound of the present disclosure contains multiple triazole-Ar2 parts, in one embodiment, these Ar2 parts are not necessarily the same as each other, and in fact they may not be the same. The Ar2 part can be different from each other in terms of Ar2 ring atoms and / or substitution on Ar2 ring atoms. For example, if one Ar2 group is phenyl and another Ar2 group is pyridyl, the two Ar2 groups are different in terms of the ring atoms that constitute the Ar2 group. As another example, if both Ar2 groups are phenyl, but one phenyl is substituted by carboxyl and the other phenyl is substituted by methoxy, for example, 4-(4-(3-(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)benzoic acid, then it is considered that the compound has two different Ar2 groups. Another example, two Ar2 groups can be positional isomers of each other, such as when both Ar2 groups are phenyl, and both benzene rings are substituted with hydroxyl and carboxyl, but the positions of hydroxyl and / or carboxyl groups on the two benzene rings are different, for example, if on one benzene ring, triazole is located at the 3 position (meta position) relative to the carboxyl group, and on the other benzene ring, triazole is located at the 4 position (para position) relative to the carboxyl group, then the two Ar2 groups are considered to be positional isomers and are not identical. In one embodiment, the Ar2 ring is identical in all aspects at each occurrence in the compounds of the present disclosure. In one embodiment, the Ar2 ring atoms are identical at each occurrence of Ar2, but the substitution on the Ar2 ring is not identical at each occurrence of Ar2. In another embodiment, the Ar2 ring atoms are not identical at each occurrence of Ar2, and the substitution on the Ar2 ring can be identical or different.

[0423] Formula (I) compound includes at least one Ar2 part, wherein Ar2 is a monocyclic aromatic ring selected from phenyl and pyridyl in one embodiment, which can be optionally substituted. In one embodiment, Ar2 is a monocyclic 6-membered aromatic ring, wherein examples are such as phenyl, pyridyl and pyrazinyl, wherein the Ar2 group optionally includes a substituent on the ring atom again. In another embodiment, Ar2 is a 5-membered monocyclic aromatic ring, which can be optionally substituted. In another embodiment, Ar2 is a 5-membered or 6-membered aromatic ring, which can be optionally substituted. In another embodiment, Ar2 is a 9-membered or 10-membered fused bicyclic ring comprising two 5-membered and / or 6-membered monocyclic rings fused together, wherein at least one of the two monocyclic rings is an aromatic ring. In another embodiment, Ar2 is a 9-membered or 10-membered fused bicyclic ring comprising two 5-membered and / or 6-membered monocyclic rings fused together, wherein both monocyclic rings are aromatic rings. In one embodiment, Ar2 can be any of these options, that is, Ar2 is selected from (a) a 5-membered monocyclic aromatic ring, (b) a 6-membered monocyclic aromatic ring, (c) a 9-membered fused bicyclic ring comprising a 5-membered and a 6-membered monocyclic ring fused together, wherein at least one of the two monocyclic rings and optionally both monocyclic rings are aromatic rings, and (d) a 10-membered fused bicyclic ring comprising two 6-membered monocyclic rings fused together, wherein at least one of the two monocyclic rings and optionally both monocyclic rings are aromatic rings,

[0424] In the compounds of formula (I), optionally, Ar2 is a 5-membered monocyclic aromatic ring selected from the group consisting of thiophene, 1,2-thiazole, 1,3-thiazole, furan, 1,2-oxazole, 1,3-oxazole, 1H-pyrrole, 1H-pyrazole, oxadiazole, thiadiazole, 1,2,4-triazole, 1,2,3-triazole and 1H-imidazole.

[0425] In the compounds of formula (I), optionally, Ar2 is a 6-membered monocyclic aromatic ring selected from the group consisting of benzene, pyridine, pyridazine, pyrimidine and pyrazine.

[0426] In the compounds of formula (I), optionally, Ar2 is a 9-membered fused bicyclic aromatic ring system selected from the group consisting of benzofuran, 1,3-benzoxazole, furano[3,2-b]pyridine, furano[3,2-c]pyridine, furano[2,3-c]pyridine, furano[2,3-b]pyridine, indole, 1H-benzimidazole, 1H-pyrrolo[3,2-b]pyridine, 1 H-pyrrolo[3,2-c]pyridine, 1H-pyrrolo[2,3-c]pyridine, 1H-pyrrolo[2,3-b]pyridine, benzothiophene, 1,3-benzothiazole, thieno[3,2-b]pyridine, thieno[3,2-c]pyridine, thieno[2,3-c]pyridine, benzoxadiazole, benzothiadiazole, benzisoxazole, benzotriazole and thieno[2,3-b]pyridine.

[0427] In the compounds of formula (I), optionally, Ar2 is a 10-membered fused bicyclic aromatic ring system selected from the group consisting of naphthylene, quinoline, quinazoline, quinoxaline, 1,5-naphthyridine, 1,6-naphthyridine, 1,7-naphthyridine, 1,8-naphthyridine, isoquinoline, phthalazine, 2,6-naphthyridine and 2,7-naphthyridine.

[0428] As mentioned above, the compounds of the present disclosure include at least one Ar2 group, wherein the Ar2 group includes at least one aromatic ring and optionally includes one or more substituents on the aromatic ring. In one embodiment, Ar2 includes at least one, i.e. one or more substituents, such as 1-5 or 1-4 or 1-3 or 1-2 substituents on the aromatic ring. Optionally, Ar2 includes just one substituent on the aromatic ring. In another selection, Ar2 includes just two substituents on the aromatic ring. In another selection, Ar2 includes just three substituents on the aromatic ring. In a further selection, Ar2 includes just four substituents on the aromatic ring. In an optional embodiment, Ar2 includes two or more substituents on the aromatic ring.

[0429] In one embodiment, one or more substituents on the ring atoms of Ar2 are selected from substituents optionally named "G", wherein the substituents are selected from halogen, C1-C6 alkyl, C1-C6 haloalkyl, -E-CO2H, -E-CHO, -EC(O)R 3 、-EC(O)NH(OH)、-EC(O)NHR 1 、-E-CONR 1 R 1′ 、-E-NR 1 R 1′ AND-E-OR 2 wherein (a) E is selected from a direct bond and C1-C6 alkylene; (b) R0 is independently selected at each occurrence from C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; (c) R 1 and R 1′ is independently selected at each occurrence from H, hydroxy, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl and substituted or unsubstituted heteroaryl, -C(=NH)NH2, -CH2CO2R 0 , -CH2C(O)NHCH2CO2H, -CH2CH2OH, -CH2CH2NHC(O)R3 、-CH2C(O)NHCH2CO2H、 (d) where R 1 and R 1′ can form a heterocyclic ring, including but not limited to azetidine, pyrrolidine, piperidine, piperazine, morpholine, (e)R 2 is independently selected at each occurrence from C2-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy; (f) R 3 is independently selected at each occurrence from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine.

[0430] In one embodiment, the substitution on Ar2 includes amino (-NH2). In one embodiment, the substitution on Ar2 includes alkoxy, such as C1-C6 alkoxy. For example, in one embodiment, the substitution on Ar2 includes methoxy. In one embodiment, the substitution on Ar2 includes formic acid or alkylene-formic acid. For example, in one embodiment, the substitution on Ar2 of the PEM compound of formula (I) includes formic acid. In one embodiment, the substitution on Ar2 includes formate, or alkylene-formate. For example, in one embodiment, the substitution on Ar2 of the PEM compound of formula (I) includes -CH2-CO2-CH3. In one embodiment, the substitution on Ar2 includes a haloalkyl group, such as a C1-C6 haloalkyl group. For example, in one embodiment, the substitution on Ar2 of the PEM compound of formula (I) includes trifluoromethyl. In one embodiment, the substitution on Ar2 includes hydroxyl or hydroxyl-substituted alkyl, such as hydroxyl-substituted C1-C6 alkyl. For example, in one embodiment, the substitution on Ar2 of the compound of formula (I) includes hydroxyl (-OH).

[0431] In one embodiment, the substitution on Ar2 includes a group selected from formic acid and alkylene-formic acid (e.g., C1-C6 alkylene-formic acid) and another group selected from hydroxyl and hydroxyl-substituted alkyl (e.g., C1-C6 alkyl substituted with a hydroxyl). For example, in one embodiment, the substitution on Ar2 is or includes a formic acid or a hydroxyl.

[0432] In one embodiment, the substitution on Ar2 includes a group selected from formic acid and alkylene-formic acid (e.g., C1-C6 alkylene-formic acid) and a group selected from haloalkyl (e.g., C1-C6 haloalkyl). For example, in one embodiment, the substitution on Ar2 is or includes a formic acid group and a trifluoromethyl group.

[0433] In one embodiment, the substitution on Ar2 includes a group selected from hydroxyl and hydroxy-substituted alkyl (e.g., C1-C6 alkyl substituted by hydroxyl) and another group selected from haloalkyl (e.g., C1-C6 haloalkyl). For example, in one embodiment, the substitution on Ar2 is or includes a hydroxyl group and a trifluoromethyl group.

[0434] In one embodiment, the substitution on the Ar2 ring of formula (I) includes at least one of the following: a) formic acid and alkylene-formic acid, such as C1-C6 alkylene-formic acid; b) hydroxyl and hydroxyl-substituted alkyl, such as C1-C6 alkyl substituted with one hydroxyl; and c) haloalkyl, such as C1-C6 haloalkyl. For example, at least one of formic acid, hydroxyl and trifluoromethyl.

[0435] In one embodiment, the substitution on the Ar2 ring of formula (I) includes at least two of the following: a) formic acid and alkylene-formic acid, such as C1-C6 alkylene-formic acid; b) hydroxyl and hydroxyl-substituted alkyl, such as C1-C6 alkyl substituted with one hydroxyl; and c) haloalkyl, such as C1-C6 haloalkyl. For example, at least two of formic acid, hydroxyl and trifluoromethyl.

[0436] In one embodiment, the substitution on the Ar2 ring of formula (I) includes all of the following three: a) formic acid and alkylene-formic acid, such as C1-C6 alkylene-formic acid; b) hydroxyl and hydroxyl-substituted alkyl, such as C1-C6 alkyl substituted with one hydroxyl; and c) haloalkyl, such as C1-C6 haloalkyl. That is, Ar2 can be substituted with formic acid, hydroxyl and trifluoromethyl.

[0437] For example, in one embodiment, the Ar2 group is a substituted phenyl group selected from:

[0438]

[0439] In one embodiment, the Ar2 group is a substituted phenyl group, wherein the substituent of the phenyl group is further replaced by G 2 , G 3 , G 4 and G 5 substituted aryl, and in more specific embodiments, the phenyl group is further substituted with G 2 , G 3 , G4 and G 5 Substituted phenyl, for example further with G 2 Or by G 2 and G 3 Substituted phenyl.

[0440] As described above, in one embodiment, the PEM compound of the present disclosure may have a hydroxyl group and a formic acid substitution on Ar2. These two groups may be located at different positions on the Ar2 ring. For example, in one embodiment, the present disclosure provides a PEM compound of formula (I) described by the following formula:

[0441]

[0442] In yet another embodiment, the PEM compound of formula (I) of the present disclosure has hydroxyl and formic acid substitution on Ar2, as provided by the following formula:

[0443]

[0444] In yet another embodiment, the PEM compound of formula (I) disclosed herein has hydroxyl and formic acid substitution on Ar2, as shown in the following formula:

[0445]

[0446] In one embodiment, the PEM compound of formula (I) disclosed herein has at least hydroxyl and carboxyl substitutions on Ar2, and may have other substitutions on Ar2. For example, Ar2 may be substituted with hydroxyl, formic acid, and alkyl, such as C1-C6 alkyl, to provide, for example, a compound of the following formula:

[0447]

[0448] As mentioned above, in one embodiment, the PEM compound of formula (I) of the present disclosure may have a haloalkyl group and a formic acid substitution on Ar2 instead of a hydroxyl group and a formic acid as shown in the above structure. As an example, the PEM compound of the present disclosure may be described by the following formula:

[0449]

[0450] The PEM compounds of formula (I) include solvates, including hydrates, chelates and salt forms thereof. In some cases, the PEM compound may be amorphous, while in other cases, the PEM compound may be crystalline. In addition, some crystalline forms of the compound may exist in the form of polymorphs, which are also contemplated herein. In addition, some compounds may also form solvates with water or other organic solvents. Such solvates are similarly included within the scope of the compounds described herein.

[0451] The PEM compound of formula (I) may be in the form of a chelate, such as a copper chelate. The copper chelate may be formed by combining the PEM compound of the present disclosure with copper sulfate. Depending on the substituents on the Ar1 and Ar2 groups, the PEM compound of formula (I) may be in the form of a salt (acid addition salt or base addition salt).

[0452] The PEM structure includes all its stable stereoisomeric forms. Therefore, the PEM compounds described herein can have one or more chiral (or asymmetric) centers, so enantiomers, diastereomers and other stereoisomeric forms that can be defined as (R)- or (S)- according to absolute stereochemistry can be produced. When the compounds described herein contain olefinic double bonds or other geometric asymmetric centers, unless otherwise stated, it is intended to represent that these compounds contain both E and Z geometric isomers (for example, cis or trans) simultaneously. Similarly, unless otherwise stated, all possible isomers, and their racemic and optically pure forms, and are also intended to include all tautomeric forms. Therefore, it is expected that various stereoisomers and mixtures thereof include "enantiomers", which refer to two stereoisomers whose molecules are non-superimposable mirror images. Therefore, compounds can exist in any isomeric form, including racemates, racemic mixtures and as separate enantiomers or diastereomers.

[0453] The PEM compounds of the present disclosure are generally water soluble. One measure of water solubility is the logP value of the compound. The logP value can be calculated based on the chemical structure of the compound using commercial software. For example, CHEMDRAW chemical drawing software (Cambridgesoft Limited, a subsidiary of PerkinElmer Holdings) can calculate the logP value of a drawn chemical structure. In one embodiment, the PEM compounds of the present disclosure have a logP of at least 4.9.

[0454] The compounds of the present disclosure, such as the PEM compounds of formula (I) as described above, can generally be synthesized by reacting a diacetylene compound of formula Arl(C≡CH)2 with an azide compound of formula Ar2-N3 in the presence of a Cu(I) catalyst. See also Crowley JD, McMorran DA (2012) "Click-Triazole" Coordination Chemistry: Exploiting 1,4-Disubstituted-1,2,3-Triazoles as Ligands. In: J. (ed.) Click Triazoles. Topics in Heterocyclic Chemistry, Volume 28. Springer, Berlin, Heidelberg doi.org / 10.1007 / 7081_201167.

[0455] Specific and similar reactants can also be identified by indexes of known chemicals compiled by the Chemical Abstracts service of the American Chemical Society, which are available in most public and university libraries, as well as through online databases (the American Chemical Society in Washington, D.C. can be contacted for more details). Chemicals that are known but not commercially available in the catalog can be prepared by custom chemical synthesis companies, many of which standard chemical supply companies (e.g., those listed above) provide custom synthesis services. A reference for the preparation and selection of pharmaceutical salts of the present disclosure is P. H. Stahl & C. G. Wermuth "Handbook of Pharmaceutical Salts," Verlag Helvetica Chimica Acta, Zurich, 2002.

[0456] Compounds of formula Ar1(C≡CH) are commercially available, for example, from TCI America (Portland, OR, USA), which sells, for example, 1,3-diethynylbenzene, 1,4-diethynylbenzene, 2,6-diethynylpyridine, and 3,6-diethynylcarbazole.

[0457] Typically, ethynyl aromatic compounds can be prepared from aromatic aldehydes via a Seyferth-Gilbert homologation reaction using dimethyl (diazomethyl)phosphonate available from MilliporeSigma Corp. (St. Louis, MO, USA). Alternatively, dimethyl (diazomethyl)phosphonate can be generated in situ from dimethyl 1-diazo-2-oxopropylphosphonate (Ohira-Bestmann reagent). See, for example, Seyforth et al., J. Org. Chem. 36(10): 1379-1386 (1971). doi: 10.1021 / jo00809a014 and Bestman et al., Synlett. 1996(06): 521-522 (1996). doi: 10.1055 / s-1996-5474.

[0458] Another approach to prepare ethynyl aromatic compounds is the Sonogashira coupling of halogenated aromatic compounds with (tert-butyldimethylsilyl)acetylene in the presence of a palladium catalyst. The ethynyl aromatic is formed upon subsequent deprotection of the silyl group. See, e.g., Sonogashira, Organomet. Chem., 653: 46-49 (2002). doi: 10.1016 / s0022-328x(02)01158-0.

[0459] The following reactions (I), (II) and (III) illustrate exemplary preparations of diethynyl aromatic compounds. In reaction (I), 2,6-dibromopyridine-4-amine is converted to the corresponding 2,6-diethynylpyridine-4-amine compound. In reaction (II), 2,6-diiodo-4-nitroaniline is converted to the corresponding 2,6-diethynyl-4-nitroaniline. In reaction (III), 2-hydroxy-3,5-diiodobenzoic acid is converted to the corresponding 3,5-diethynyl-2-hydroxybenzoic acid. In each case, the conversion is carried out through an intermediate ditrimethylsilyl (TMS) compound, as shown.

[0460]

[0461] Each of these reaction products, namely 2,6-diethynylpyridin-4-amine and 2,6-diethynyl-4-nitroaniline and 3,5-diethynyl-2-hydroxybenzoic acid, can be used as a precursor for preparing Arl in the PEM of the present disclosure. Therefore, each of them represents an Arl(C≡CH)2 compound, which can react with an azide compound of formula Ar2-N3 in the presence of a Cu(I) catalyst to provide a PEM. Reactions (I), (II) and (III) illustrate the preparation of precursors of the substituted Arl moieties of the present disclosure.

[0462] Compounds of the formula Ar2-N3 are also commercially available, for example, from TCI America (Portland, Oregon, USA), Synthonix (Wake Forest, North Carolina, USA), SigmaAldrich (St. Louis, Missouri, USA), Toronto Research Chemicals (Toronto, Canada), and AnaSpec (Fremont, California, USA). In general, azides of the formula Ar2-N3 can be prepared by nucleophilic displacement of an electrophilic compound such as an alkyl, benzyl or allylic iodide or bromide with sodium azide.

[0463] In general, the compounds used in the reactions described herein can be prepared according to organic synthesis techniques known to those skilled in the art starting from commercially available chemicals and / or from compounds described in the chemical literature. "Commercially available chemicals" can be obtained from standard commercial sources such as Across Organics (Pittsburgh, Pa.), Aldrich Chemical (Milwaukee, Wis., including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Manor, U.K.), Avocado Research (Lancashire, U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (Westchester, Pa.), Crescent Chemical Co. (Hawpoague, N.Y.), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, N.Y.), Fisher Scientific Co. (Pittsburgh, Pa.), Fisons Chemicals (Leicestershire, U.K.), Frontier Scientific (Logan, Utah), ICN Biomedicals, Inc. (Costa Mesa, Calif.), Key Organics (Cornwall, U.K.), Lancaster Synthesis (Wyndham, N.H.), Maybridge Chemical Co. Ltd. (Cornwall, U.K.), Parish Chemical Co. (Orem, Utah), Pfaltz & Bauer, Inc. (Waterbury, Conn.), Polyorganix (Houston, Texas), Pierce Chemical Co. (Rockford, Ill.), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Products, Inc. (New Brunswick, N.J.), TCI America (Portland, Oregon), TransWorld Chemicals, Inc. (Rockville, Md.), and Wako Chemicals USA, Inc. (Richmond, Va.).

[0464] In one embodiment, the PEM compounds of the present disclosure, such as the PEM compounds of formula (I), are present in a composition. For example, the PEM compounds of the present disclosure can be present in a composition that also comprises an aqueous buffer. In one embodiment, the PEM compounds of the present disclosure are present in a composition comprising a biomolecule (e.g., a polypeptide and / or a polynucleotide). The polypeptide can be an enzyme, such as a DNA polymerase. The following definitions may be helpful in understanding these compositions and certain uses thereof.

[0465] As used herein, "nucleic acid", also known as polynucleotide, is a series of covalently linked nucleotides, in which the 3' position of the pentose of one nucleotide is connected to the 5' position of the next through a phosphodiester group. Nucleic acid molecules can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a combination of the two. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are biological polynucleotides in which the nucleotide residues are connected in a specific sequence through phosphodiester bonds. As used herein, the terms "nucleic acid", "polynucleotide" or "oligonucleotide" include any polymer compound having a linear backbone of nucleotides. Oligonucleotides, also known as oligomers, are typically shorter chain polynucleotides. If targeted for sequencing, nucleic acid is typically referred to as a "target nucleic acid" or "target sequence".

[0466] As used herein, the term "template-dependent manner" is intended to refer to a process involving template-dependent extension of a primer molecule (e.g., synthesis of DNA by a DNA polymerase). The term "template-dependent manner" refers to polynucleotide synthesis of RNA or DNA, wherein the sequence of the newly synthesized polynucleotide chain is determined by the well-known complementary base pairing rules (see, e.g., Watson, JD et al., In: Molecular Biology of the Gene, 4th Ed., WA Benjamin, Inc., Menlo Park, Calif. (1987)).

[0467] As used herein, "nucleic acid polymerase" is an enzyme that is generally used to connect 3'-OH 5'-triphosphate nucleotides, oligomers and their analogs. Polymerases include, but are not limited to, DNA-dependent DNA polymerase, DNA-dependent RNA polymerase, RNA-dependent DNA polymerase, RNA-dependent RNA polymerase, T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, DNA polymerase 1, Klenow fragment, Thermomyces DNA polymerase, Tth DNA polymerase, DNA polymerase (New England Biolabs), Deep DNA polymerase (New England Biolabs), Bst DNA polymerase large fragment, Stoeffel fragment, 9 0 N DNA polymerase, 9 0 N DNA polymerase, Pfu DNA polymerase, Tfl DNA polymerase, Tth DNA polymerase, RepliPHIPhi29 polymerase, Tli DNA polymerase, eukaryotic DNA polymerase β, telomerase, Therminator TM Polymerase (New England Biolabs), KOD HiFi TM DNA polymerase (Novagen), KOD1 DNA polymerase, Q-beta replicase, terminal transferase, AMV reverse transcriptase, M-MLV reverse transcriptase, Phi6 reverse transcriptase, HIV-1 reverse transcriptase. The polymerase according to the present invention may be a variant, mutant or chimeric polymerase.

[0468] As used herein, "DPO4-type DNA polymerase" is a DNA polymerase naturally expressed by archaea, Sulfolobus, or related Y-family DNA polymerases, which generally function in the replication of damaged DNA by a process called translesion synthesis (TLS). Y-family DNA polymerases are homologous to DPO4 polymerases; examples include prokaryotic enzymes PolII, PolIV, PolV, archaeal enzyme Dbh, and eukaryotic enzymes Rev3p, Rev1p, Polη, REV3, REV1, and Polκ DNA polymerases, and chimeras thereof. The modified recombinant DPO4-type DNA polymerase comprises one or more mutations relative to a naturally occurring wild-type DPO4-type DNA polymerase, for example, one or more mutations that increase the ability to utilize large nucleotide analogs as substrates or another polymerase property, and may include additional changes or modifications to the wild-type DPO4-type DNA polymerase, such as one or more deletions, insertions, and / or fusions of additional peptide or protein sequences (e.g., for immobilizing the polymerase on a surface or otherwise labeling the polymerase). Examples of variant polymerases according to the present invention are variants of Sulfolobus DPO4 described in published PCT patent applications WO2017 / 087281 A1 and PCT patent application numbers PCTUS201 8 / 030972 and PCTUS201 8 / 64794, which are incorporated herein by reference in their entirety.

[0469] As used herein, "nucleic acid polymerase reaction" refers to an in vitro method for preparing a new nucleic acid chain or extending an existing nucleic acid (e.g., DNA or RNA) in a template-dependent manner. According to the present invention, the nucleic acid polymerase reaction includes a primer extension reaction, which results in the incorporation of nucleotides or nucleotide analogs into the 3' end of the primer, such that the incorporated nucleotides or nucleotide analogs are complementary to the corresponding nucleotides of the target polynucleotide. The primer extension products of the nucleic acid polymerase reaction can be further used for single molecule sequencing or as a template to synthesize additional nucleic acid molecules.

[0470] Primer extension reaction reagents generally include (i) polymerase; (ii) buffer; and (iii) one or more extendable nucleotides or nucleotide analogs. Primer extension reactions can be used to measure the length of the resulting nucleic acid product under specific experimental conditions, and to determine the effects of various polymerase reaction additives (e.g., PEM) on polymerase activity by comparing the length of the extended primer product (e.g., by gel electrophoresis).

[0471] As used herein, "enhancing a nucleic acid polymerase reaction" refers to the ability of an additive (eg, a PEM) to enable a nucleic acid polymerase to synthesize a primer extension product that is at least one subunit longer in length than in the absence of the PEM.

[0472] As used herein, the rate of a nucleic acid polymerase reaction refers to the average speed at which a nucleic acid polymerase extends a polymer chain. As used herein, the terms "rate" and "elongation rate" are used interchangeably. The nucleotide incorporation assay of Hogrefe et al. (Methods in Enzymol. Vol. 334, pp. 91-116 (2001)) can be used to measure polymerization rate. In short, polymerase activity can be measured by 32 p-dCTP is measured by incorporation into activated salmon sperm DNA (purchased from Pharmacia; activation protocol see, Procedures in Nucl. Acid Res. (Cantoni and Davies eds.), p. 263-276 (1966) at p. 264). The reaction buffer can be, for example, 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM dithiothreitol (DTT), 50 μg / ml bovine serum albumin (BSA) and 4% (v / v) glycerol. Nucleotide substrates and DNA are used in large amounts, usually at least 10 times the Km of the polymerase being tested, for example, 200 μM each of dATP, dTTP and dGTP, 195 μM dCTP plus 5 μM labeled dCTP, and 250 μg / ml activated DNA. The reaction is quenched on ice and an aliquot of the reaction mixture is spotted on an ion exchange filter (e.g., Whatman DE81). Unincorporated nucleotides are washed away and then scintillation counting is performed to measure incorporated radioactivity.

[0473] As used herein, "increased rate" means an increase of 5%-10%, 10%-50%, or 50%-100% or more compared to a polymerization reaction lacking a PEM having an increased rate as defined herein.

[0474] As used herein, "processivity" refers to the degree to which a nucleic acid polymerase polymerizes during a single contact between the polymerase and its template, i.e., its property of continuing to act on a substrate rather than dissociating from the substrate. The degree of polymerization refers to the number of nucleotides or nucleotide analogs added by the polymerase during a single contact between the polymerase and its template. Processivity depends on the properties of the polymerase, the sequence of the template, the structure of the nucleotide or nucleotide analog substrate, and the reaction conditions, such as salt concentration, temperature, or the presence of specific additives.

[0475] As used herein, "increased processivity" refers to an increase of 5%-10%, 10%-50%, or 50%-100% or more compared to a polymerization reaction lacking a PEM that increases processivity as defined herein. Methods for measuring processivity of nucleic acid polymerases are well known in the art, for example, as described in Sambrook et al. 1989, In ​​Molecular Cloning, 2nd Edition, CSH Press, 7.79-7.83 and 13.8, as described in U.S. Published Patent Application No. 2002 / 0119467, Published PCT Application No. WO01 / 92501, and U.S. Pat. No. 5,972,603, the entire contents of which are incorporated herein by reference.

[0476] As used herein, the term "fidelity" refers to the accuracy of nucleic acid polymerization by a template-dependent nucleic acid polymerase. The fidelity of a DNA polymerase is measured by the error rate (the frequency of incorporating inaccurate nucleotides, i.e., nucleotides that are not incorporated in a template-dependent manner). The fidelity or error rate of a DNA polymerase can be measured using assays known in the art (see, e.g., Lundburg et al., 1991 Gene, 108: 1-6). As used herein, "increasing fidelity" refers to an increase of 5%-10%, 10%-50%, or 50%-100% or more compared to a polymerization reaction lacking an additive that increases fidelity as defined herein.

[0477] As used herein, the term "plurality" means "at least two".

[0478] "XNTPs" are extendable, 5' triphosphate-modified nucleotide substrates that are compatible with template-dependent enzymatic polymerization. XNTPs have two distinct functional components; namely, a nucleobase 5'-triphosphate aminoester and a tether attached to each nucleoside triphosphate aminoester, whose position allows controlled extension by intranucleotide cleavage of the phosphoramidate bond. As used herein, XNTPs are exemplary "non-natural, highly substituted nucleotide analog substrates". Exemplary XNTPs and methods for making them are described, for example, in PCT Application No. WO2016 / 081871 published by the applicant, which is incorporated herein by reference in its entirety.

[0479] "Xpandomer intermediates" are intermediates assembled from XNTPs (also referred to herein as "daughter strands") and are formed by polymerase-mediated template-directed assembly of XNTPs using a target nucleic acid template. The newly synthesized Xpandomer intermediate is a constrained Xpandomer. At the process step where the phosphoramidate bond provided by the XNTPs is cleaved, the constrained Xpandomer is no longer constrained and is an Xpandomer product that is extended as the tether is extended.

[0480] "Xpandomer" or "Xpandomer product" is a synthetic molecular construct produced by constrained Xpandomer amplification, which itself is synthesized by template-directed assembly of XNTP substrates. Xpandomer is elongated relative to the target template from which it is generated. It is composed of a series of subunits, each of which is a motif, each motif is a member of a library, including sequence information, tethers, and optionally part or all of the substrate, all of which are derived from the formative substrate construct. Xpandomer is designed to extend longer than the target template, thereby reducing the linear density of the sequence information of the target template along its length. In addition, Xpandomer optionally provides a platform for increasing the size and abundance of reporters, thereby improving the signal-to-noise ratio of detection. Lower linear information density and stronger signals improve resolution and reduce the sensitivity requirements for detecting and decoding the sequence of the template strand.

[0481] "Tether" or "tether member" refers to a polymer or molecular construct having a generally linear dimension and having a terminal portion at each of two opposite ends. The tether is connected to a nucleoside triphosphate by a bond at the terminal portion to form an XNTP. The bond is used to constrain the tether in a "constrained configuration". The tether has a "constrained configuration" and an "extended configuration". The constrained configuration is found in XNTPs and daughter chains or Xpandomer intermediates. The constrained configuration of the tether is a precursor to the extended configuration, as seen in the Xpandomer product. The transition from the constrained configuration to the extended configuration results in the cleavage of the selectively cleavable phosphoramidate bond. The tether contains one or more reporters or reporter constructs along its length that can encode the sequence information of the substrate. The tether provides a method to extend the length of the Xpandomer, thereby reducing the linear density of the sequence information.

[0482] A "tether element" or "tether segment" is a polymer of generally linear dimensions having two ends, wherein the ends form terminal bonds for connecting the tether element. A tether element is a segment of a tether. Such polymers may include, but are not limited to, polyethylene glycol, polyglycol, polypyridine, polyisocyanide, polyisocyanate, poly(triarylmethyl)methacrylate, polyaldehyde, polypyrrolidone, polyurea, polyethylene glycol phosphodiester, polyacrylate, polymethacrylate, polyacrylamide, polyvinyl ester, polystyrene, polyamide, polyurethane, polycarbonate, polybutyrate, polybutadiene, polybutyrolactone, polypyrrolidone, polyvinyl phosphonate, polyacetamide, polysaccharide, polyhyaluronate, polyamide, polyimide, polyester, polyethylene, polypropylene, polystyrene, polycarbonate, polyterephthalate, polysilane, polyurethane, polyether, polyamino acid, polyglycine, polyproline, N-substituted polylysine, polypeptide, side chain N-substituted peptide, poly-N-substituted glycine , peptoids, peptides with side chain carboxyl substituted, homotypic peptides, oligonucleotides, ribonucleic acid oligonucleotides, deoxynucleic acid oligonucleotides, oligonucleotides modified to prevent Watson-Crick base pairing, oligonucleotide analogs, polycytidylic acid, polyadenylic acid, polyuridylic acid, polythymidine, polyphosphates, polynucleotides, polyribonucleotides, polyethylene glycol-phosphodiester, peptide polynucleotide analogs, threonine-polynucleotide analogs, ethylene glycol-polynucleotide analogs, morpholino-polynucleotide analogs, locked nucleotide oligomer analogs, polypeptide analogs, branched polymers, comb polymers, star polymers, dendrimers, random polymers, gradient and block copolymers, anionic polymers, cationic polymers, stem-loop forming polymers, rigid segments and flexible segments.

[0483] A "reporter" consists of one or more reporter elements. The reporter is used to analyze the genetic information of a target nucleic acid.

[0484] A "reporter construct" comprises one or more reporters that can generate a detectable signal, wherein the detectable signal generally comprises sequence information. This signal information is referred to as a "reporter code" and is subsequently decoded into genetic sequence data. The reporter construct may also comprise a tether segment or other structural components, including a polymer, a graft copolymer, a block copolymer, an affinity ligand, an oligomer, a hapten, an aptamer, a dendrimer, a linking group, or an affinity binding group (e.g., biotin).

[0485] The "reporter code" is the genetic information of the measured signal from the reporter construct. The reporter code is decoded to provide sequence-specific genetic information data.

[0486] Thus, in one embodiment, the present disclosure provides a composition comprising a PEM as disclosed herein and a buffer. In another embodiment, the present disclosure provides a composition comprising a PEM as disclosed herein and a plurality of nucleotides and / or nucleotide analogs. In another embodiment, the present disclosure provides a composition comprising a PEM as disclosed herein and a polynucleotide. In another embodiment, the present disclosure provides a composition comprising a PEM as disclosed herein and a protein, wherein optionally the protein is a polymerase, including any of the above-mentioned polymerases.

[0487] In one embodiment, the present disclosure provides a composition comprising a PEM compound of the present disclosure (e.g., a PEM compound of formula (I)) and a molecular crowding agent. In general, molecular crowding agents include a series of large neutral polymers. Examples of useful molecular crowding agents include, but are not limited to, polyethylene glycol (PEG), polysucrose, dextran, or polyvinyl alcohol. Exemplary molecular crowding agents and formulations are described in U.S. Patent No. 7,399,590, which is incorporated herein by reference. In one embodiment, the molecular crowding agent is a polyalkylene glycol, optionally having a number average molecular weight of 4,000-10,000. In one embodiment, the molecular crowding agent is a derivative of polyalkylene glycol, for example, one or both of the terminal hydroxyl groups of the polyalkylene glycol are in the form of an ester group or an ether group. In one embodiment, the molecular crowding agent is an inert water-soluble polymer.

[0488] In one embodiment, the present disclosure provides a composition comprising a PEM compound of the present disclosure and an aqueous buffer. In one embodiment, the PEM compound has formula (I). In one selection, the composition has a pH of about 6 to 8.5, and the buffer helps stabilize the pH of the composition. An exemplary buffer is Tris HCl. Other suitable buffers include those known in the art, such as phosphate buffers, citric acid buffers, sodium acetate buffers, sodium carbonate buffers, etc.

[0489] In one embodiment, the present disclosure provides a composition comprising a PEM compound of the present disclosure (e.g., a PEM compound of formula (I)) and a polynucleotide. In one selection, the polynucleotide is single-stranded, such as a single-stranded DNA or a single-stranded RNA. When the polynucleotide is intended to be used as a primer, the polynucleotide is a single-stranded DNA molecule. When intended to be used as a primer, the polynucleotide may have a length of about 10-60mer oligonucleotides, such as 20-30 oligonucleotides. The polynucleotide may alternatively be used as a template, in which case it may be a single-stranded DNA or a single-stranded RNA, and may have a length from 30 bases to kilobases and above, such as 10k bases and above.

[0490] In one embodiment, the present disclosure provides a composition comprising a PEM compound of the present disclosure (e.g., a PEM compound of formula (I)) and a protein. For example, the protein can be an enzyme, a nucleic acid polymerase, a DNA polymerase. An example of a suitable DNA polymerase is a variant of the DPO4 polymerase as discussed herein.

[0491] In one embodiment, the present disclosure provides a composition comprising at least one PEM compound of the present disclosure, such as a PEM compound of formula (I), and a mixture of nucleotides or nucleotide analogs, wherein relative to the same polymerization reaction in the absence of at least one compound, the at least one compound increases the number and accuracy of nucleotide analogs incorporated into the daughter chain during the template-dependent polymerization reaction. Optionally, the mixture of nucleotide analogs includes nucleoside triphosphate amino esters, wherein each nucleoside triphosphate amino ester has a core base and a polymer tethering portion selected from adenine, guanine, thymine and cytosine, wherein the first end of the polymer tethering portion is connected to the core base, and the second end of the polymer tethering portion is connected to the alpha phosphate of the nucleoside triphosphate amino ester to provide an extension of the nucleotide analog by cutting of the aminophosphoroester bond. Optionally, the composition further includes a buffer comprising one or more of the following: Tris OAc, NH4OAc, PEG, a water-miscible organic solvent (e.g., dimethylformamide (DMF), N-methylpyrrolidone (NMP) or acetone), polyphosphate 60, NMS and MnCl2. Optionally, the composition further comprises a single-stranded binding protein. Optionally, the composition comprises urea. Optionally, the mixture of nucleotide analogs comprises nucleotide analogs comprising a detectable label, wherein the detectable label is optionally one of luminescent, chemiluminescent, fluorescent, fluorescent, chromogenic or chromogenic. In one embodiment, the composition comprises two or more of these options, for example, all of these options.

[0492] In one aspect of the present disclosure, the PEM disclosed herein and its composition can be used to enhance nucleic acid polymerization or improve the properties of the resulting nucleic acid, such as the length or accuracy of the reaction product. Polymerization includes, for example, primer extension reaction, PCR, mutagenesis, isothermal amplification, DNA sequencing and probe labeling. Such methods are well known in the art. Enhancement can be provided by stimulating nucleotide incorporation through mechanisms such as increasing the continuous synthesis ability of the polymerase (i.e., reducing the dissociation of the polymerase from the template), increasing the rate of substrate binding or enzyme catalysis, and increasing the accuracy or fidelity of nucleotide incorporation. In addition, enhancement can be provided by reducing obstacles in the nucleic acid template (e.g., secondary structure and duplex DNA). Overcoming or improving these obstacles by adding PEM can make the polymerization reaction occur more accurately or efficiently, or allow the use of lower denaturation / extension temperatures or isothermal temperatures.

[0493] In some embodiments, PEM can be used in combination with another type of additive to enhance polymerase reaction. An exemplary class of additives is minor groove binding protein (MGB). In one embodiment, MGB is selected from the group consisting of: distamycin A and its synthetic analogs, sphaerocin, (+)-CC-1065, duocarmycin, pyrrolobenzodiazepines, trabectin and its analogs, Hoechst dyes and derivatives thereof, lexitropsin, thiazotropsin A, diamidines and polyamides. In certain embodiments, at least one minor groove binding portion is a Hoechst dye. For more information about using MGB to enhance polymerase reaction, see the application entitled ENHANCEMENT OF NUCLEIC ACID POLYMERIZATION BY MGBS jointly submitted by the applicant.

[0494] One exemplary polymerase reaction that can be enhanced with PEMs is the polymerization of non-natural nucleotide analogs called "XNTPs," which forms the basis of the "Expansion Sequencing" (SBX) protocol developed by Stratos Genomics (see, e.g., Kokoris et al., U.S. Pat. No. 7,939,259, "High Throughput Nucleic Acid Sequencing by Expansion"). In general, SBX uses this biochemical polymerization to transcribe the sequence of a DNA template onto a measurable polymer called an "Xpandomer." The transcribed sequence is encoded in high signal-to-noise ratio reporters along the Xpandomer backbone, which are spaced approximately 10 nm apart and designed for high signal-to-noise ratio, well-differentiated reactions. These differences provide significant performance enhancements in the efficiency and accuracy of sequence reading from Xpandomers relative to natural DNA. An overview of the SBX process is provided in Figure 1A , Figure 1B , Figure 1C and Figure 1D Described in.

[0495] XNTPs are expandable, 5' triphosphate-modified nucleotide substrates that are compatible with template-dependent enzymatic polymerization. Figure 1A , which emphasizes the unique features of these nucleotide analogs: XNTP 100 has two different functional regions; namely: a selectively cleavable phosphoramidite bond 110, connecting 5'α-phosphate 115 to a nucleobase 105; and a tether 120 connected at certain positions within the nucleoside triphosphate aminoester, which allows for controlled extension by intranucleotide cleavage of the phosphoramidite bond. The tether of the XNTP comprises a linker arm portion 125A and 125B separated by a selectively cleavable phosphoramidite bond. Each linker is connected to one end of a reporter 130 by a linking group (LG), as disclosed in U.S. Pat. No. 8,324,360 to Kokoris et al., which is incorporated herein by reference in its entirety. XNTP 100 is illustrated in a "constrained configuration", which is a characteristic of XNTP substrates and daughter chains after polymerization. The constrained configuration of polymerized XNTPs is a precursor to the extended configuration, as seen in Xpandomer products. The transition from the constrained to the extended conformation occurs upon cleavage of the PN bond of the phosphoramidate within the main backbone of the daughter chain.

[0496] The synthesis of Xpandomers is summarized in Figure 1B and Figure 1C During assembly, monomeric XNTP substrates 145 (XATP, XCTP, XGTP, and XTTP) are polymerized at the extendable end of the nascent daughter chain 150 by a template-directed polymerization process using the single-stranded template 140 as a guide. Typically, the process starts with a primer and proceeds in the 5' to 3' direction. Typically, a DNA polymerase or other polymerase is used to form the daughter chain, and conditions are selected so as to obtain a complementary copy of the template strand. After the daughter chain is synthesized, the coupled tether comprises a constrained Xpandomer that further comprises the daughter chain. The tether in the daughter chain has a "constrained configuration" of the XNTP substrate. The constrained configuration of the tether is a precursor to the extended configuration, as seen in the Xpandomer product.

[0497] like Figure 1CAs shown, the transition from the constrained configuration 160 to the expanded configuration 165 is caused by the cleavage of a selectively cleavable phosphoramidate bond (represented by an unshaded ellipse for simplicity) within the primary backbone of the daughter strand. In this embodiment, the tether comprises one or more reporters or reporter constructs, 130A, 130C, 130G, or 130T, that are specific to the nucleobases to which they are attached, thereby encoding the sequence information of the template. In this way, the tether provides a means to extend the length of the Xpandomer and reduce the linear density of the sequence information of the parent strand.

[0498] Figure 1D Xpandomer 165 is illustrated as translocating from cis reservoir 175 to trans reservoir 185 through nanopore 180. After passing through the nanopore, each reporter of the linearized Xpandomer (labeled "G," "C," and "T" in this illustration) generates a unique and reproducible electronic signal (represented by overlay trace 190) specific for the nucleobase to which it is attached.

[0499] Figure 2 The general structure of XNTP is described in more detail. XNTP 200 comprises a nucleobase triphosphate aminoester 210 having a tether portion 220A and 220B, which are separated by a selectively cleavable phosphoramidate bond 230. The tether is connected to the nucleoside triphosphate at the linking groups 250A and 250B, wherein the first tether end is connected to a heterocycle 260 (represented here by cytosine, although the heterocycle can be any of the four standard nucleobases A, C, G, or T), and the second tether end is connected to the alpha phosphate 270 of the nucleobase backbone. It will be appreciated by those skilled in the art that many suitable coupling chemistries known in the art can be used to form the final XNTP substrate product, for example, the tether coupling can be completed by a triazole bond to conjugate the tether.

[0500] In this embodiment, tether 275 comprises several functional elements, including enhancers 280A and 280B, reporter codes 285A and 285B, and translation control elements (TCE) 290A and 290B. Each of these features performs a unique function during the translocation of Xpandomer through the nanopore and the generation of a unique and repeatable electronic signal. Tether 275 is designed to be translocated by hybridization (TCH). As shown, TCE provides a hybridization region that can form a duplex with a complementary oligomer (CO) and is located near the reporter code. The size of different reporter codes can prevent ions from flowing through the nanopore at different measurable levels. Specific reporter codes can be effectively synthesized using phosphoramidite chemistry commonly used for oligonucleotide synthesis. Reporter can be designed by selecting the sequence of a specific phosphoramidite from a commercially available library. Such libraries include, but are not limited to, polyethylene glycols of 1 to 12 or more ethylene glycol units in length, aliphatics of 1 to 12 or more carbon units in length, deoxyadenosine (A), deoxycytosine (C), deoxyguanosine (G), deoxythymine (T), abasic (Q). The duplex TCE associated with the reporter code also contributes to ion flow obstruction, so the combination of the reporter code and TCE can be referred to as a "reporter". The reporter code is followed by an enhancer, which in one embodiment includes a spermine polymer.

[0501] Figure 3 An example of an Xpandomer cleaved during translocation of an α-hemolysin nanopore is shown. The biological nanopore is embedded in a lipid bilayer membrane that separates and electrically isolates two electrolyte reservoirs. A typical electrolyte is 1 molar KCl buffered to a pH of 7.0. When a small voltage (typically 100 mV) is applied across the bilayer, the nanopore restricts the flow of ionic current and is the main resistor in the circuit. Xpandomer reporters are designed to provide specific levels of ionic current blockade, and the sequence information can be read by measuring the sequence of ionic current levels as the reporter sequence translocates the nanopore.

[0502] The α-hemolysin nanopore is usually oriented so that translocation occurs by entering the vestibule side and exiting the stalk side. Figure 3As shown, the nanopore is oriented to capture the Xpandomer from the stem side first. This orientation is advantageous when using the TCH method because it reduces blocking artifacts that occur when entering the vestibule first. Unless otherwise stated, the stem side is assumed to be the translocation direction. As the Xpandomer translocates, the reporter enters the stem until its duplex TCE stops at the entrance of the stem. The diameter of the duplex is about 2.4nm, and the entrance of the stem is about 2.2nm, so the reporter is held in the stem until the complementary strand 395 of the duplex dissociates (releases) and then translocation proceeds to the next reporter. Free complementary strands are extremely unfavorable for entering the nanopore because the Xpandomer is still translocating and diffusing away from the hole.

[0503] In one embodiment, each member of the reporter code (after the duplex) is formed by an ordered selection of phosphoramidites that can be selected from many commercial libraries. The phosphoramidites of each component contribute to the net ionic resistance according to their position in the nanopore (after the duplex termination), their displacement, their charge, their interaction with the nanopore, their chemical and thermal environment, and other factors. The charge on each phosphoramidite is partially attributed to the phosphate ions that have a nominal charge of -1 but are effectively reduced by counterion shielding. The force pulling the duplex is due to the effect of the local electric field on these effective charges along the reporter. Since each reporter molecule can have different charge distributions, it can apply different forces to the duplex for a given applied voltage. The force transmitted along the reporter backbone is also used to lengthen the reporter to provide a repeatable blocking response.

[0504] The expanded sequencing (SBX) method developed by the present inventors provides significant performance enhancements in terms of sequence reading efficiency and accuracy of Xpandomers relative to natural DNA. However, the initial transcription of natural DNA template sequences into measurable Xpandomers relies on the ability of DNA polymerases to utilize XNTPs as substrates (the general structure of XNTPs is referred to herein). Figure 1A and Figure 2 Discussion). The inventors have found that most DNA polymerases cannot efficiently polymerize XNTPs. However, the inclusion of suitable additives, such as the PEMs disclosed herein, improves the efficiency and accuracy of polymerization of XNTPs into Xpandomers. Therefore, the PEMs disclosed herein can be used in the context of the SBX method to enhance DNA polymerase primer extension reactions using XNTPs as substrates.

[0505] A representative primer extension reaction may include the following reagents: 2 pmol primer, 2.2 pmol 45mer oligonucleotide template, 50 pmol of each XNTP (XATP, XCTP, XGTP and XTTP), 50 mM Tris HCl, pH 6.79, 200 mM NaCl, 20% PEG, 5% NMS, 0.5 nmol polyphosphate 60.19, 0.3 mM MnCl2 and 0.6 μg purified recombinant DNA polymerase protein. PEM is usually added to the mixture at a concentration in the micromolar to millimolar range. The reaction also includes additional additives, such as single-stranded binding protein (SSB), urea and NMS. The reaction is run at 23°C for 1 hour. The reaction product (i.e., constrained Xpandomer) is treated to cleave the aminophosphoryl ester bond to produce a linearized Xpandomer. The reaction products are analyzed using gel electrophoresis on a 4%-12% acrylamide gel to resolve and display Xpandomer products of different lengths.

[0506] Therefore, in one embodiment, the present disclosure provides an aqueous (water-containing) composition comprising a PEM and a buffer, particularly a buffer suitable for DNA polymerization, wherein Tris HCl is an exemplary buffer of this type. In one embodiment, the present disclosure provides a composition comprising a PEM and a DNA polymerase protein. In one embodiment, the present disclosure provides a composition comprising a PEM and a polynucleotide, such as an oligonucleotide of 20-90mer, 20-60mer, 30-90mer or 30-60mer. In one embodiment, the present disclosure provides a composition comprising each of these components, i.e., an aqueous composition comprising a PEM, a buffer, a DNA polymerase protein and a polynucleotide.

[0507] To study the accuracy of enhancing XNTP polymerization, the primer extension products can be sequenced using the SBX protocol. In short, the Xpandomer product of the constrained XNTP polymerization is cut to produce a linearized Xpandomer. This is achieved by first quenching the extension reaction with a solution containing 100mM EDTA, 2mMTHPTA, and 2% Tween-20. The sample is then amine-modified with a DMF solution of 1M NaHCO3 and 1 M succinic anhydride. The cleavage of the aminophosphoryl ester bond is performed with 37% HCl, and the linearized Xpandomer is purified with a QIAquick column (QIAGEN, Inc.).

[0508] For sequencing, protein nanopores were prepared by inserting α-hemolysin into DPhPE / hexadecane bilayer members in buffer B1, which contained 2M NH4Cl and 100mM HEPES, pH 7.4. The cis-pore was perfused with buffer B2, which contained 0.4M NH4Cl, 0.6M GuCl, and 100mM HEPES, pH 7.4. The Xpandomer sample was heated to 70°C for 2 minutes, cooled completely, and then 2μL of the sample was added to the cis-pore. A voltage pulse of 90mV / 390mV / 10μs was then applied and data was acquired by Labview acquisition software.

[0509] The sequence data is analyzed by a histogram display of a population of sequence reads from a single SBX reaction. The analysis software aligns each sequence read to the template sequence and trims the sequence range of the ends of the reads that do not align to the correct template sequence.

[0510] In one embodiment, the present disclosure provides a method for improving the accuracy of enhancing XNTP polymerization, wherein the method comprises adding a PEM as disclosed herein to a DNA polymerization reaction as described above.

[0511] In one embodiment, the present disclosure provides a kit, wherein the kit can be used in the methods described herein. The kit will include at least one compound of the present disclosure, and one or more of the following: a) a molecular crowding agent, b) an aqueous buffer, c) a protein such as a polymerase, d) a polynucleotide that can be used as, for example, a primer and / or a polynucleotide that can be used as, for example, a template.

[0512] For example, in one embodiment, the present disclosure provides a kit for sequencing a nucleic acid template. The kit includes a mixture of at least one compound of the present disclosure and nucleotide analogs. Relative to the same polymerization reaction in the absence of at least one compound of the present disclosure, the compound of the present disclosure can be used to increase the quantity and accuracy of the nucleotide analogs incorporated into the daughter chain during the polymerization reaction process dependent on the template. Optionally, the mixture of nucleotide analogs includes nucleoside triphosphate amino esters, wherein each nucleoside triphosphate amino ester includes a core base and a polymer tethering part selected from the group consisting of adenine, guanine, thymine and cytosine, wherein the first end of the polymer tethering part is connected to the core base, and the second end of the polymer tethering part is connected to the alpha phosphate of the nucleoside triphosphate amino ester to provide the expansion of the nucleotide analogs by the cutting of the phosphoramidate bond. Optionally, the mixture of nucleotide analogs includes a nucleotide analog comprising a detectable label, wherein the detectable label is an optically detectable label selected from the group consisting of luminescence, chemiluminescence, fluorescence, fluorescence, coloration or chromogenic labels. Optionally, the kit includes an aqueous buffer comprising Tris OAc, NH4OAc, PEG, a water-miscible organic solvent (e.g., dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), acetone, etc.), polyphosphate 60, NMS, and MnCl2. Optionally, the kit includes a single-stranded binding protein. Optionally, the kit includes urea. Optionally, the kit includes two or more of these components, e.g., 3 or 4, or all of the specified components.

[0513] Compounds can be prepared by methods known to those of ordinary skill in the art, wherein such methods can be determined by various reference books and databases. Detailed description of the synthesis of reactants that can be used to prepare the disclosed compounds or suitable reference books and papers that provide reference to articles describing the preparation include, for example, "Synthetic Organic Chemistry, " John Wiley & Sons, Inc., New York; SR Sandler et al., "Organic Functional Group Preparations, " 2nd edition, Academic Press, New York, 1983; HO House, "Modem Synthetic Reactions, " 2nd edition, W.A. Benjamin, Inc. Menlo Park, Calif 1972; T.L. Gilchrist, "Heterocyclic Chemistry, " 2nd edition, John Wiley & Sons, New York, 1992; J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure, " 4th edition, Wiley-Interscience, New York, 1992. Other suitable reference books and papers detailing the synthesis of reactants that can be used to prepare the compounds of the present disclosure, or providing references to articles describing the preparations, include, for example, Fuhrhop, J. and Penzlin G. "Organic Synthesis: Concepts, Methods, Starting Materials", 2nd Edition, Revised Supplement (1994) John Wiley & Sons ISBN: 3-527-29074-5; Hoffman, RV "Organic Chemistry, An Intermediate Text" (1996) Oxford University Press, ISBN 0-19-509618-5; Larock, RC "Comprehensive Organic Transformations: A Guide to Functional Group Preparations" 2nd Edition (1999) Wiley-VCH, ISBN: 0-471-19031-4; March, J."Advanced Organic Chemistry: Reactions, Mechanisms, and Structure" 4th edition (1992) John Wiley & Sons, ISBN: 0-471-60180-2; Otera, J. (editor) "Modern Carbonyl Chemistry" (2000) Wiley-VCH, ISBN: 3-527-29871-1; Patai, S. "Patai's 1992Guide to the Chemistry of Functional Groups" (1992) Interscience ISBN: 0-471-93022-9; Quin, LD et al. "A Guide to Organophosphorus Chemistry" (2000) Wiley-Interscience, ISBN: 0-471-31824-8; Solomons, TWG "OrganicChemistry" 7th Edition (2000) John Wiley & Sons, ISBN: 0-471-19095-0; Stowell, JC, "Intermediate Organic Chemistry" 2nd edition (1993) Wiley-Interscience, ISBN: 0-471-57456-2; "Industrial Organic Chemicals: Starting Materials and Intermediates: An Ullmann's Encyclopedia" (1999) John Wiley & Sons, ISBN: 3-527-29645-X, in 8 volumes; "Organic Reactions" (1942-2000) John Wiley & Sons, in over 55 volumes; and "Chemistry of Functional Groups" John Wiley & Sons, in 73 volumes. .

[0514] Examples

[0515] The compounds shown in Table 1 were prepared according to the general examples disclosed herein.

[0516] Materials and Methods. 4-Azidosalicylic acid and 2,6-dibromo-4-pyridinecarboxylic acid were purchased from Toronto Research Chemicals, Inc. (Toronto, Ontario, Canada). 4-Azido-2-(trifluoromethyl)benzoic acid, 3-amino-5-hydroxybenzoic acid, and 4-aminoisophthalic acid were purchased from Matrix Scientific (Columbia, South Carolina, USA). 3-Amino-6-(trifluoromethyl)benzoic acid hydrochloride, 1-(4-aminophenyl)-2,2,2-trifluoroethan-1-one, glycylglycine methyl ester hydrochloride, 3,3,3-trifluoropropan-1-amine, and diethyl 3-aminopropan-1-ylphosphonate were purchased from Enamine LLC (Monmouth Junction, New Jersey, USA). Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluoro-phosphate (HATU), 1,3-diethynylbenzene, 2,6-diethynylpyridine, 3,5-diethynylpyridine, 3,6-diethynylcarbazole, 4-azidobenzoic acid, cyclopropylamine, 6-amino-2-naphthoic acid, 4-amino- Phthalic acid, 4-amino-3-hydroxybenzoic acid, 4-amino-2-methylbenzoic acid, 4-amino-2,3,5,6-tetrafluorobenzoic acid, 4-amino-2-nitrobenzoic acid, n-(2-aminoethyl)acetamide, 4-(aminophenyl)phosphonic acid, 2,6-dichloro-9H-purine, dimethyl aspartate hydrochloride, 4-amino-2-(trifluoromethyl)benzonitrile, 3,5-diaminobenzoic acid, and 2,5-dibromofuran were purchased from TCI America (Portland, OR, USA). 4-Methoxy-2,6-dibromopyridine, 4-nitro-2,6-dibromopyridine, and 2,6-dibromo-4-pyridinecarboxylic acid, glycine methyl ester hydrochloride were purchased from Chem-Impex International, Inc. (Wood Dale, IL).Tetrakis(triphenylphosphine)palladium(0), trimethylsilyl acetylene, DMSO, DMF, MeOH, EtOAc, sodium ascorbate, copper sulfate, diisopropylamine, EDTA, morpholine, diethylamine, ammonium hydroxide, ethylamine, ethanol, azetidine, n-ethyl-n-ethylamine, methyl 2,6-dichloropyridine-4-carboxylate, ethyl 2,6-dibromopyridine-4-carboxylate, 4-methyl-2,6-dichloropyridine, 2-chloro-4-cyanopyridine, 4-amino-2-(trifluoromethyl)benzoic acid, 2-bromo-4-cyanopyridine Pyridine, methyl azidoacetate, 4-azidoaniline hydrochloride, 4-methoxyphenylazide, 4-amino-2-fluorobenzoic acid, n-butylamine, 3-amino-5-(trifluoromethyl)benzoic acid, 3-(4-aminophenyl)propionic acid, 4-(4-aminophenyl)-butyric acid, 4-amino-2-methoxybenzoic acid, 2-amino-3-(trifluoromethyl)benzoic acid, aminoethanol, 1,4-diaminobutane, and 2,5-dibromothiophene were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). TLC and flash chromatography solvents were purchased from Sigma-Aldrich or Thermo Fisher Scientific Inc. (Waltham, MA, USA).

[0517] Flash chromatography was carried out on a Reveleris preparative purification system purchased from Buchi Corp. (New Castle, Delaware, USA). The system was equipped with a hand-packed column (2.3cm in diameter x 8cm in height) filled with C18 spherical silica gel (Cat.No.76646-01) from Sorbent Technologies, Inc (Norcross, Georgia) and sealed with a polypropylene frit. 1 to 1.5mL of sample was directly loaded onto the top of the column. The mobile phase was water (A) and acetonitrile (B). The gradient was from 0 to 2% B in 2 minutes, then from 2% to 100% B in 20 minutes, and the flow rate was 28ml / min. UV was monitored at 220nm, 260nm and 280nm. Fractions were collected at a UV threshold of 0.1AU. Thin layer chromatography was performed using aluminum backed TLC Silica Gel 60F254 (Cat. No. 1.05534.0001) from EMD Millipore Corp. (Billerica, MA, USA). ESI mass spectra were performed by Numega Resonance Lab (San Diego, CA, USA) using a Perkin Elmer PE-SCIEX API-150 mass spectrometer in positive and negative modes.

[0518] Table 1

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[0545]

[0546]

[0547]

[0548]

[0549] Example 1

[0550] Synthesis of 4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0551]

[0552] Compound 1 was prepared by mixing 4-azidosalicylic acid B (1.79 mg, 10 μmol) and 2,6-diethynylpyridine A (0.67 mg, 5 μmol) in DMSO (150 μL). The solution was mixed with a solution of TBTA (5.1 mg, 0.96 μmol) and sodium ascorbate (6.4 mg, 32 μmol) in DMSO (95 μL). The click reaction was initiated by adding 20 Mm copper sulfate (5 μL) under stirring. The extent of the reaction was analyzed by TLC (94:5:1 ethyl acetate:methanol:acetic acid), and the reaction was completed within 5 minutes based on the consumption of azide and alkyne. The reaction mixture volume was brought to 1 M1 with DMSO and 0.5 M EDTA (100 μL). The solid was isolated and dissolved in additional DMSO. The DMSO solution was combined and purified by flash chromatography as described in the materials and methods above. The product formed a glassy solid upon rotary evaporation in 50% to 75% yield. 1 H NMR (300MHz, DMSO-d6) δppm3.29 (2H, br.S., ( Q (18) H and Q (33)H)), 7.17-7.29(4H,m,( C (7) H, C (11)H, C (28)H, C (32)H))7.85(2H, d, J=8.11( C (10) H and C (31)H)), 8.06 (3H, s, ( C (15)H, C (16)H, C (17) H)) 9.36 (2H, s, ( C (5) H and C (25)H)).

[0553] Example 2

[0554] Synthesis of 4,4′-(pyridine-3,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0555]

[0556] According to the method of Example 1, compound 2 was prepared using 4-azidosalicylic acid B and 3,5-diethynylpyridine C. 1 HNMR (300MHz, DMSO-d6) δppm 3.29 (2H, br.S., ( Q (18) H and O (33) H)) 7.17-7.29 (4H, m, ( C (7) H, C (11)H, C (28)H, C (31)H))7.85(2H, d, J=8.11Hz, ( C (10) H and C (31)H))8.06(3H,s,,( C (13)H, C (15)H, C (17) H)) 9.36 (2H, s, ( C (5) H and C (25)H)).

[0557] Example 3

[0558] Synthesis of 4,4′-(1,3-phenylenebis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0559]

[0560] According to the method of Example 1, compound 3 was prepared using 4-azidosalicylic acid B and 1,3-diethynylbenzene D. 1 HNMR (300MHz, DMSO-d6) δppm 3.29 (2H, br.S., ( O (18) H and O (33) H))7.16-7.27 (4H, m, ( C (7) H, C (11)H, C (28)H, C (32) H))7.57-7.67(1H,m,( C (16) H)) 7.80-7.88 (2H, m, ( C (10) H andC (31)H))7.94(2H, d, J=7.63Hz, ( C (15) H and C (17) H) 8.57 (1H, s, ( C (13) H)) 9.35 (2H, s, ( C (5) H and C (25)H).

[0561] Example 4

[0562] Synthesis of 4,4′-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0563]

[0564] According to the method of Example 1, compound 4 was prepared using 4-azidosalicylic acid B and 3,6-diethynylcarbazole E. 1 HNMR (300MHz, DMSO-d6) δppm 3.29 (2H, br.S., ( O (25)H and O (40)H))7.16-7.27(4H,m,( C (7) H, C (11)H, C (35)H, C (39)H))7.61(2H, d, J=8.34Hz, ( C (10) H and C (38)H))7.84(2H, d, J=7.87Hz, ( C (22)H and C (24)H))8.02(2H, d, J=8.34Hz, ( C (21)H and C (23)H))8.79(2H,s,( C (13) H and C (19)H))9.28(2H,s,( C (5) H and C (32)H))11.53(1H,s,( N (16)H).

[0565] Example 5

[0566] Synthesis of 4,4′-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))diphenylamine

[0567]

[0568] According to the method of Example 1, compound 5 was prepared using 4-azidoaniline hydrochloride F and 3,6-diethynylcarbazole E.

[0569] Example 6

[0570] Synthesis of 4,4′-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))dibenzoic acid

[0571]

[0572] According to the method of Example 1, compound 6 was prepared using 4-azidobenzoic acid G and 3,6-diethynylcarbazole E.

[0573] Example 7

[0574] Synthesis of 3,6-bis(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)-9H-carbazole

[0575]

[0576] According to the method of Example 1, compound 7 was prepared using 4-azidoanisole H and 3,6-diethynylcarbazole E.

[0577] Example 8

[0578] Synthesis of dimethyl 2,2′-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))diacetate

[0579]

[0580] According to the method of Example 1, compound 8 was prepared using methyl azidoacetate I and 3,6-diethynylcarbazole E.

[0581] Example 9

[0582] Synthesis of 4,4′-((4-methoxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0583]

[0584] The preparation of compound 9 was started by synthesizing 4-methoxy-2,6-diethynylpyridine J from 4-methoxy-2,6-dibromopyridine and trimethylsilyl acetylene using the conditions described by Sonogashira (Organomet. Chem., 653: 46-49 (2002). doi: 10.1016 / s0022-328x(02)01158-0). According to the method of Example 1, the synthesis of compound 9 was completed by clicking 4-azidosalicylic acid B and compound J.

[0585] Example 10

[0586] Synthesis of 4,4′-((4-carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0587]

[0588] The preparation of compound 10 was started by synthesizing 4-cyano-2,6-diethynylpyridine K from 4-cyano-2,6-dibromopyridine and trimethylsilyl acetylene using the conditions described by Sonogashira (Organomet. Chem., 653: 46-49 (2002). doi: 10.1016 / s0022-328x(02)01158-0). According to the method of Example 1, the synthesis of compound 10 was completed by clicking 4-azidosalicylic acid B and compound K.

[0589] Example 11

[0590] Synthesis of 4,4′-((4-nitropyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0591]

[0592] The preparation of compound 11 was started by synthesizing 4-nitro-2,6-diethynylpyridine L from 4-nitro-2,6-dibromopyridine and trimethylsilyl acetylene using the conditions described by Sonogashira (Organomet. Chem., 653: 46-49 (2002). doi: 10.1016 / s0022-328x(02)01158-0). According to the method of Example 1, the synthesis of compound 11 was completed by clicking 4-azidosalicylic acid B and compound L.

[0593] Example 12

[0594] Synthesis of 5,5′-((4-cyanopyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0595]

[0596] According to the method of Example 10, compound 12 was prepared using 5-azidosalicylic acid M and 4-cyano-2,6-diethynylpyridine K.

[0597] Example 13

[0598] Synthesis of 4,4′-((4-methylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0599]

[0600] Compound 13 was synthesized from 4-methyl-2,6-dichloropyridine and trimethylsilyl acetylene using the conditions described by Sonogashira (Organomet. Chem., 653: 46-49 (2002). doi: 10.1016 / s0022-328x(02)01158-0). According to the method of Example 1, the synthesis of compound 11 was completed by clicking 4-azidosalicylic acid B and 4-methyl-2,6-diethynylpyridine N.

[0601] Example 14

[0602] Synthesis of 4,4′-((4-(ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0603]

[0604]

[0605] Compound 14 was prepared by synthesizing ethyl 2,6-diethynylpyridine-4-carboxylate O from ethyl 2,6-dibromopyridine-4-carboxylate and trimethylsilyl acetylene using the conditions described by Sonogashira (Organomet. Chem., 653: 46-49 (2002). doi: 10.1016 / s0022-328x(02)01158-0). According to the method of Example 1, the synthesis of compound 14 was completed by clicking 4-azidosalicylic acid B and ethyl 2,6-diethynylpyridine-4-carboxylate O.

[0606] Example 15

[0607] Synthesis of 5,5′-((4-(ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0608]

[0609] According to the method of Example 14, compound 15 was prepared by click reaction using 5-azidosalicylic acid M and ethyl 2,6-diethynylpyridine-4-carboxylate O.

[0610] Example 16

[0611] Synthesis of 4,4′-((4-(methoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0612]

[0613]

[0614] Compound 16 was synthesized from 2,6-dichloropyridine-4-carboxylic acid methyl ester P (Organomet. Chem., 653: 46-49 (2002). doi: 10.1016 / s0022-328x(02)01158-0) from 2,6-dichloropyridine-4-carboxylic acid methyl ester and trimethylsilyl acetylene using the conditions described by Sonogashira. According to the method of Example 1, the synthesis of compound 16 was completed by clicking 4-azidosalicylic acid B and 2,6-diethynylpyridine-4-carboxylic acid methyl ester P.

[0615] Example 17

[0616] Synthesis of 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0617]

[0618] The synthesis of compound 17 was started by mixing 2,6-dibromo-4-pyridinecarboxylic acid (0.2 g, 0.71 mmol), DIPEA (0.18 g, 1.42 mmol) and HATU (0.27 g, 0.71 mmol) in DMF (900 ul). Ethylamine (0.154 ml, 1.78 mmol-) was added immediately and mixed for 1 hour. The reaction was detected to be complete by TLC and purified by silica gel flash chromatography using a gradient of ethyl acetate / hexane. N-ethyl-2,6-dibromo-4-formamide was separated as a yellow solid with a yield of 69%. N-ethyl-2,6-diethynyl-4-formamide Q was prepared using the Sonogashira method described in Example 16 using ethyltrimethylsilane. The synthesis of compound 17 was completed by clicking 4-azido salicylic acid B and N-ethyl-2,6-diethynyl-4-formamide Q according to the method of Example 1.

[0619] Example 18

[0620] Synthesis of 4,4′-((4-(methylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0621]

[0622] Compound 18 was prepared according to the method of Example 17 using methylamine to form the amide.

[0623] Example 19

[0624] Synthesis of 4,4′-((4-carbamoylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0625]

[0626] Compound 19 was prepared according to the method of Example 17 using ammonia to form the amide.

[0627] Example 20

[0628] Synthesis of 4,4′-(pyrazine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0629]

[0630]

[0631] Compound 20 was synthesized from 2,6-dichloropyrazine and trimethylsilyl acetylene using the Sonogashira method described by Bhowmick, S. et al. (App. Organomet. Chem. 31(12): e3824(2017)). The synthesis of compound 20 was completed by clicking 4-azidosalicylic acid B and 2,6-diethynylpyrazine T according to the method of Example 1.

[0632] Example 21

[0633] Synthesis of 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0634]

[0635] The synthesis of compound 73 began with the diazotization of 4-amino-2-(trifluoromethyl)benzoic acid with sodium nitrite and sulfuric acid, followed by nucleophilic displacement with azide (Org. Synthesis 1942, 22, 96) to form 4-azido-2-(trifluoromethyl)benzoic acid ( U ), which was purified by flash chromatography. According to the method of Example 1, 4-azido-2-(trifluoromethyl)benzoic acid was added by clicking U and N-ethyl-2,6-diethynyl-4-carboxamide Q to complete the synthesis of compound 73.

[0636] Example 22

[0637] Synthesis of 7,7′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxy-1,8-naphthyridine-4-carboxylic acid)

[0638]

[0639] The synthesis of compound 74 began with the diazotization of 7-amino-2-hydroxy-1,8-naphthyridine-4-carboxylic acid with sodium nitrite and sulfuric acid, followed by nucleophilic displacement with azide (Org. Synthesis 1942, 22, 96) to form 7-azido-2-hydroxy-1,8-naphthyridine-4-carboxylic acid ( V ), which was purified by flash chromatography. According to the method of Example 1, 7-azido-2-hydroxy-1,8-naphthyridine-4-carboxylic acid was added by clicking V The synthesis of compound 74 was completed by using N-ethyl-2,6-diethynyl-4-carboxamide Q.

[0640] Example 23

[0641] Synthesis of 4-(4-(3-(1-(4-carboxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxybenzoic acid

[0642]

[0643] The synthesis of compound 28 was completed in two steps. First, 2,6-diethynylbenzene was D 4-Azidosalicylic acid B was cleaved with half the amount of pyridinium according to the method of Example 3 to prepare 4-(4-(3-ethynylphenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxybenzoic acid. In the second step, 4-azidobenzoic acid G was cleaved according to the method of Example 1 to give 4-(4-(3-(1-(4-carboxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxybenzoic acid 28.

[0644] Example 24

[0645] Synthesis of 4-(4-(4-cyanopyridin-2-yl)-1H-1,2,3-triazol-1-yl)-2-hydroxybenzoic acid

[0646]

[0647] The preparation of compound 43 was started by synthesizing 4-cyano-2-ethynylpyridine W from 4-cyano-2-chloropyridine and trimethylsilyl acetylene using the conditions described by Sonogashira (Organomet. Chem., 653: 46-49 (2002). doi: 10.1016 / s0022-328x(02)01158-0). According to the method of Example 1, the synthesis of compound 43 was completed by clicking 4-azidosalicylic acid B and compound W.

[0648] Example 25

[0649] Synthesis of 4,4′-((pyridine-2,6-diyl)bis(5-iodo-1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0650]

[0651] The preparation of compound 31 begins with the synthesis of 2,6-bis(iodoethynyl)pyridine from 2,6-diethynylpyridine A according to the method of Tepper et al. (Org. Lett., 2015, 17(23), pp 5740-574), which involves treatment with N-iodosuccinimide and silver nitrate and separation by flash chromatography. According to the method of Example 1, the synthesis of compound 31 is completed by clicking 4-azidosalicylic acid B and compound X.

[0652] Example 26

[0653] Synthesis of 4,4′-((3,5-dimethylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0654]

[0655] Compound 30 was synthesized from 2,6-dibromo-3,5-dimethylpyridine and trimethylsilyl acetylene using the Sonogashira method described by Bhowmick, S. et al. (App. Organomet. Chem. 31(12): e3824(2017)). The synthesis of compound 30 was completed by clicking 4-azidosalicylic acid B and 2,6-diethynyl-3,5-dimethylpyridine Y according to the method of Example 1.

[0656] Example 27

[0657] Synthesis of 4,4′-((9-acetyl-9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0658]

[0659] Compound 33 was synthesized from 9-acetyl-3,6-diiodocarbazole and trimethylsilyl acetylene using the Sonogashira method described by Bhowmick, S. et al. (App. Organomet. Chem. 31(12): e3824(2017)). The synthesis of compound 33 was completed by clicking 4-azido salicylic acid B and 9-acetyl-3,6-diethynylcarbazole Z according to the method of Example 1.

[0660] Example 28

[0661] Synthesis of 4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(N,2-dihydroxybenzamide)

[0662]

[0663] The synthesis of compound 34 began with a DMF (72ul) solution of mixed N-hydroxysuccinimide 4-azido salicylate (40mg, 0.145mmol), to which a water (72ul-) solution of hydroxylamine hydrochloride (30mg, 0.43mmol-) was added and mixed overnight. The product was detected by TLC, and the reaction was purified by silica gel flash chromatography using a gradient of dichloromethane and methylenepyridine-MeOH. 4-azido-N, 2-dihydroxybenzamide AA was separated with a yield of 57%. According to the method of Example 1, the synthesis of compound 34 was completed by clicking 4-azido-N, 2-dihydroxybenzamide AA and 2,6-diethynylpyridine A.

[0664] Example 29

[0665] Synthesis of 5-(4-(6-(4-(3-carboxy-4-hydroxy-5-methylphenyl)-1H-1,2,3-triazol-1-yl)-4-(methoxycarbonyl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)-2-hydroxy-3-methylbenzoic acid

[0666]

[0667] The synthesis of compound 44 begins with the diazotization of 5-amino-2-hydroxy-3-methylbenzoic acid with sodium nitrite and sulfuric acid, followed by nucleophilic displacement with azide (Org. Synthesis 1942, 22, 96) to form 5-azido-2-hydroxy-3-methylbenzoic acid ( BB ), which was purified by flash chromatography. According to the method of Example 1, 5-azido-2-hydroxy-3-methylbenzoic acid was purified by clicking BB The synthesis of compound 44 was completed with 2,6-diethynylpyridine-4-carboxylate P.

[0668] Example 30

[0669] Synthesis of 4,4′-((4-(but-3-yn-1-ylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0670]

[0671] The synthesis of compound 47 was started by mixing 2,6-dibromo-4-pyridinecarboxylic acid (0.2 g, 0.71 mmol), DIPEA (0.18 g, 1.42 mmol) and HATU (0.27 g, 0.71 mmol) in DMF (900 ul). Butynylamine (0.154 ml, 1.78 mmol) was added immediately and mixed for 1 hour. The reaction was detected to be complete by TLC and purified by silica gel flash chromatography using a gradient of ethyl acetate / hexane. N-(but-3-yn-1-yl)-2,6-dibromoisonicotinamide was isolated as a solid. N-(but-3-yn-1-yl)-2,6-diethynylisonicotinamide CC was prepared using the Sonogashira method described in Example 16 using ethyltrimethylsilane. According to the method of Example 1, the synthesis of compound 47 was completed by clicking 4-azidosalicylic acid B and N-(but-3-yn-1-yl)-2,6-diethynylisonicotinamide CC.

[0672] Example 31

[0673] Synthesis of 4,4′-(naphthalene-2,7-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0674]

[0675] Compound 63 was synthesized from 2,7-dibromonaphthalene and trimethylsilyl acetylene using the Sonogashira method described by Bhowmick, S. et al. (App. Organomet. Chem. 31(12): e3824(2017)). The synthesis of compound 63 was completed by clicking 4-azidosalicylic acid B and 2,7-diethynylnaphthalene DD according to the method of Example 1.

[0676] Example 32

[0677] Synthesis of 4,4′-(naphthalene-2,3-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0678]

[0679] Compound 64 was initiated by synthesizing 2,3-diethynylnaphthalene EE from 2,3-dibromonaphthalene and trimethylsilyl acetylene using the Sonogashira method described by Bhowmick, S. et al. (App. Organomet. Chem. 31(12): e3824 (2017). The synthesis of compound 64 was completed by clicking 4-azidosalicylic acid B and 2,3-diethynylnaphthalene EE according to the method of Example 1.

[0680] Example 33

[0681] Synthesis of 4,4′,4″,4″′-((((butane-1,4-diylbis(azanediyl))bis(carbonyl))bis(pyridine-4,2,6-triyl))tetrakis(1H-1,2,3-triazole-4,1-diyl))tetrakis(2-hydroxybenzoic acid)

[0682]

[0683] Compound 71 was synthesized from 2,6-dibromo-4-pyridinecarboxylic acid and trimethylsilyl acetylene using the Sonogashira method described by Bhowmick, S. et al. (App. Organomet. Chem. 31(12): e3824 (2017)). 2,6-Diethynyl-4-pyridinecarboxylic acid was treated with HATU, DIPEA and 1,4-diaminobutane to give N,N'-(butane-1,4-diyl)bis(2,6-diethynylisonicotinamide) (FF) after separation by flash chromatography. The synthesis of compound 71 was completed by clicking 4-azidosalicylic acid B and N,N'-(butane-1,4-diyl)bis(2,6-diethynylisonicotinamide) (FF) according to the method of Example 1.

[0684] Example 34

[0685] Synthesis of 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3,5,6-trichloropicolinic acid)

[0686]

[0687] The synthesis of compound 72 begins with the diazotization of 4-amino-3,5,6-trichloropyridine-2-carboxylic acid with sodium nitrite and sulfuric acid, followed by nucleophilic displacement with azide (Org. Synthesis 1942, 22, 96) to form 4-azido-3,5,6-trichloropyridine-2-carboxylic acid (GG), which is purified by flash chromatography. According to the method of Example 1, the synthesis of compound 72 is completed by clicking 4-azido-3,5,6-trichloropyridine-2-carboxylic acid GG with N-ethyl-2,6-diethynyl-4-carboxamide Q.

[0688] Example 35

[0689] Synthesis of 4,4′-((4-(methylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0690]

[0691] According to Example 17, N-methyl-2,6-diethynylpyridine-4-carboxamide HH was prepared using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and methylamine. Compound 79 was prepared using copper click method using diethynyl HH and azide U in Example 1. Mass spectrum of compound 79 (ESI negative ion mode): C 27 H 16 F6N8O5 calculated value: 646.47; found value: 645[MH + ].

[0692] Example 36

[0693] Synthesis of 4,4′-((4-(morpholine-4-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0694]

[0695] According to Example 17, N-morpholino-2,6-diethynylpyridine-4-carboxamide II was prepared by HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and morpholine. Compound 80 was prepared using the copper click method using diethynyl II and azide U in Example 1. Mass spectrum of compound 80 (ESI negative ion mode): C30H20F6N8O6 calculated value: 702.53; found value: 701.1 [M-H+]

[0696] Example 37

[0697] Synthesis of 4,4′-((4-(diethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0698]

[0699] According to Example 17, N,N-diethylamino-2,6-diethynylpyridine-4-carboxamide JJ was prepared by HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and N,N-diethylamine. Compound 81 was prepared using the copper click method using diethynyl JJ and azide U in Example 1. Mass spectrum of compound 81 (ESI negative ion mode): C 30 H 22 F6N8O5 calculated value: 688.55; found value: 687[MH + ]

[0700] Example 38

[0701] Synthesis of 4,4′-((4-carbamoylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0702]

[0703] According to Example 17, 2,6-diethynylpyridine-4-carboxamide KK was prepared using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and ammonia. Compound 82 was prepared using copper click method using diethynyl KK and azide U in Example 1. Mass spectrum of compound 82 (ESI negative ion mode): C 26 H 14 F6N8O5 calculated value: 632.44; measured value: 631.1 [MH + ]

[0704] Example 39

[0705] Synthesis of 4,4′-((4-(ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0706]

[0707] Compound 83 was prepared using the copper click method using ethyl 2,6-diethynylpyridine-4-carboxylate O and azide U in Example 1. Mass spectrum of compound 83 (ESI negative ion mode): C 28 H 17 F6N7O6 calculated value: 661.48; measured value: 659.9 [MH + ].

[0708] Example 40

[0709] Synthesis of 4,4′-((4-(azetidine-1-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0710]

[0711] According to Example 17, N-azetidinyl-2,6-diethynylpyridine-4-carboxamide LL was prepared using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and azetidine. Compound 84 was prepared using copper click method using diethynyl LL and azide B in Example 1. Mass spectrum of compound 84 (ESI negative ion mode): C 27 H 20 N8O7 calculated value: 568.51; measured value: 567[MH +].

[0712] Example 41

[0713] Synthesis of 4,4′-((4-(ethyl(methyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0714]

[0715] According to Example 17, N-methyl-N-ethyl-2,6-diethynylpyridine-4-carboxamide MM was prepared using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and N-methyl-N-ethylamine. Compound 85 was prepared using copper click method using diethynyl MM and azide B in Example 1. Mass spectrum of compound 85 (ESI negative ion mode): C 27 H 22 N8O7 calculated value: 570.52; measured value: 569.1 [MH + ].

[0716] Example 42

[0717] Synthesis of N-ethyl-2,6-bis(1-(4-(2,2,2-trifluoroacetyl)phenyl)-1H-1,2,3-triazol-4-yl)isonicotinamide

[0718]

[0719] 1-(4-aminophenyl)-2,2,2-trifluoroethan-1-one was diazotized with sodium nitrite and sulfuric acid, and then displaced with azide (Org. Synthesis 1942, 22, 96 DOI: 10.15227 / orgsyn.022.0096) to form 1-(4-azidophenyl)-2,2,2-trifluoroethan-1-one NN, which was purified by flash chromatography. Compound 86 was prepared using a copper click method using azide NN and diacetylene Q in Example 1. Compound 86 mass spectrum (ESI negative ion mode): C 28 H 18 F6N8O3 calculated value: 628.5; found value: 627[MH + ].

[0720] Example 43

[0721] Synthesis of 4,4′-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0722]

[0723] Compound 87 was prepared using azide U and 2,6-diethynylpyridine A using the copper click method in Example 1. Mass spectrum of compound 87 (ESI negative ion mode): C 25 H 13 F6N7O4 calculated value: 589.41; measured value: 588.1 [MH + ].

[0724] Example 44

[0725] 4,4′-((4-(cyclopropylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-

[0726] Synthesis of (trifluoromethyl)benzoic acid

[0727]

[0728] According to Example 17, N-cyclopropylamino-2,6-diethynylpyridine-4-carboxamide OO was prepared using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and cyclopropylamine. Compound 88 was prepared using copper click method using diethynyl OO and azide U in Example 1. Mass spectrum of compound 88 (ESI negative ion mode): C 29 H 18 F6N8O5 calculated value: 672.50; found value: 671.1 [MH + ].

[0729] Example 45

[0730] Synthesis of 4,4′-((4-(butylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid-)

[0731]

[0732]

[0733] According to Example 17, N-butylamino-2,6-diethynylpyridine-4-carboxamide PP was prepared by HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and 1-aminobutane. Compound 89 was prepared using copper click method using diethynyl PP and azide U in Example 1. Mass spectrum of compound 89 (ESI negative ion mode): C 30 H 22 F6N8O5 calculated value: 688.55; measured value: 687.1 [MH + ].

[0734] Example 46

[0735] Synthesis of 5,5′-((4-(diethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0736]

[0737] Diazotization of 5-amino-2-(trifluoromethyl-)benzoic acid was performed according to Example 22 to prepare 5-azido-2-(trifluoromethyl)benzoic acid QQ. Compound 90 was prepared using the copper click method using azide QQ and diacetylene JJ in Example 1. Mass spectrum of compound 90 (ESI negative ion mode): C 30 H 22 F6N8O5 calculated value: 688.55; measured value: 687.1 [MH + ].

[0738] Example 47

[0739] Synthesis of 5,5′-((4-(morpholine-4-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0740]

[0741] Compound 91 was prepared using azide QQ and diacetylene II using the copper click method in Example 1. Mass spectrum of compound 91 (ESI negative ion mode): C 30 H 20 F6N8O6 calculated value: 702.53; found value: 701[MH + ].

[0742] Example 48

[0743] Synthesis of 4,4′-(pyridazine-3,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

[0744]

[0745] 3,6-Dibromopyridazine was used to prepare 3,6-diethynylpyridazine according to the Sonogashira method described in Example 9. Compound 92 was prepared using the copper click method using azide B and 3,6-diethynylpyridazine in Example 1. Mass spectrum of compound 92 (ESI negative ion mode): C 22 Calculated value for H14N8O6: 486.1; Found: 485[MH + ].

[0746] Example 49

[0747] Synthesis of 5,5′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-(trifluoromethyl)benzoic acid)

[0748]

[0749]

[0750] Diazotization of 3-amino-5-(trifluoromethyl)benzoic acid was performed according to Example 22 to prepare 3-azido-5-(trifluoromethyl)benzoic acid RR. Compound 93 was prepared using the copper click method using azide RR and diacetylene Q in Example 1. Mass spectrum of compound 93 (ESI negative ion mode): C 28 H 18 F6N8O5 calculated value: 660.49; found value: 659 [MH + ].

[0751] Example 50

[0752] Synthesis of 4,4′-((4-carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0753]

[0754] Compound 94 was prepared using azide U and 2,6-diethynylpyridine-4-carboxylic acid using the copper click method in Example 1. Mass spectrum of compound 94 (ESI negative ion mode): C 26 H 13 F6N7O6 calculated value: 633.42; measured value: 632[MH + ].

[0755] Example 51

[0756] Synthesis of 3,3′-(((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(4,1-phenylene))dipropionic acid

[0757]

[0758] Diazotization of 3-(4-aminophenyl)propionic acid was performed according to Example 22 to prepare 3-(4-azidophenyl)propionic acid SS. Compound 95 was prepared using the copper click method using azide SS and diacetylene Q in Example 1. Mass spectrum of compound 95 (ESI negative ion mode): C 30 H 28N8O5 calculated value: 580.61; measured value: 579[MH + ].

[0759] Example 52

[0760] 4,4′-(((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(4,1-

[0761] Synthesis of phenylene) dibutyric acid

[0762]

[0763]

[0764] Diazotization of 4-(4-aminophenyl)butyric acid was performed according to Example 22 to prepare 4-(4-azidophenyl)butyric acid TT. Compound 96 was prepared using the copper click method using azide TT and diacetylene Q in Example 1. Mass spectrum of compound 96 (ESI negative ion mode): C 32 H 32 N8O5 calculated value: 608.66; measured value: 607[MH + ].

[0765] Example 53

[0766] Synthesis of 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))diphthalic acid

[0767]

[0768] Diazotization of 4-aminophthalic acid was performed according to Example 22 to prepare 4-azidophthalic acid UU. Compound 97 was prepared using the copper click method using azide UU and diacetylene Q in Example 1. Mass spectrum of compound 97 (ESI negative ion mode): C 28 H 20 N8O9 calculated value: 612.52; measured value: 611.1 [MH + ].

[0769] Example 54

[0770] Synthesis of 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-methoxybenzoic acid)

[0771]

[0772] Diazotization of 4-amino-2-methoxybenzoic acid was completed according to Example 22 to prepare 4-azido-2-methoxybenzoic acid VV. Compound 98 was prepared using the copper click method using azide VV and diacetylene Q in Example 1. Mass spectrum of compound 98 (ESI negative ion mode): C 28 Calculated value for H24N8O7: 584.55; Found: 583[MH + ].

[0773] Example 55

[0774] Synthesis of 5,5′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))diisophthalic acid

[0775]

[0776] Diazotization of 4-aminoisophthalic acid was performed according to Example 22 to prepare 4-azidoisophthalic acid WW. Compound 99 was prepared using the copper click method using azide WW and diacetylene Q in Example 1. Mass spectrum of compound 99 (ESI negative ion mode): C 28 Calculated value for H20N8O9: 612.52; Found: 611[MH + ].

[0777] Example 56

[0778] Synthesis of 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-hydroxybenzoic acid)

[0779]

[0780] Diazotization of 4-amino-3-hydroxybenzoic acid was performed according to Example 22 to prepare 4-azido-3-hydroxybenzoic acid XX. Compound 100 was prepared using the copper click method using azide XX and diacetylene Q in Example 1. Mass spectrum of compound 100 (ESI negative ion mode): C 26 Calculated value for H20N8O7: 556.50; Found: 555.1 [MH + ].

[0781] Example 57

[0782] Synthesis of diethyl (3-(4-(6-(1-(3-(diethoxyphosphoryl)propyl)-1H-1,2,3-triazol-4-yl)-4-(ethylcarbamoyl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)propyl)phosphonate

[0783]

[0784]

[0785] Diazotization of O,O-diethyl (3-aminopropyl) phosphonate was performed according to Example 22 to prepare O,O-diethyl (3-azidopropyl) phosphonate YY. Compound 101 was prepared using the copper click method using azide YY and diacetylene Q in Example 1. Mass spectrum of compound 101 (ESI negative ion mode): C 26 H42N8O7P2 calculated value: 640.62; found value: 639.3 [MH + ].

[0786] Example 58

[0787] Synthesis of 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-methylbenzoic acid)

[0788]

[0789] Diazotization of 4-amino-2-methylbenzoic acid was performed according to Example 22 to prepare 4-amino-2-methylbenzoic acid ZZ. Compound 102 was prepared using the copper click method using azide ZZ and diacetylene Q in Example 1. Mass spectrum of compound 102 (ESI negative ion mode): C 28 H 24 N8O5 calculated value: 552.55; measured value: 551.1 [MH + ].

[0790] Example 59

[0791] Synthesis of 4,4′-((5-carboxy-1,3-phenylene)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0792]

[0793] Compound 103 was prepared using azide Q and 3,5-diethynylbenzoic acid using the copper click method in Example 1. Mass spectrum of compound 103 (ESI negative ion mode): C 27 H 14 F6N6O6 calculated value: 632.44; found value: 631[MH + ].

[0794] Example 60

[0795] Synthesis of 2,2′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-(trifluoromethyl)benzoic acid)

[0796]

[0797] Diazotization of 2-amino-3-(trifluoromethyl)benzoic acid was completed according to Example 22 to prepare 2-azido-3-(trifluoromethyl)benzoic acid AAA. Compound 104 was prepared using the copper click method using azide AAA and diacetylene Q in Example 1. Mass spectrum of compound 104 (ESI negative ion mode): C 28 H 18 F6N8O5 calculated value: 660.49; found value: 659 [MH + ].

[0798] Example 614, Synthesis of 4′-((4-((2-hydroxyethyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0799]

[0800] According to Example 17, N-(2-hydroxyethyl)amino-2,6-diethynylpyridine-4-carboxamide BBB was prepared by HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and aminoethanol. Compound 105 was prepared using the copper click method using diethynyl BBB and azide U in Example 1. Mass spectrum of compound 105 (ESI negative ion mode): C 28 H 18 F6N8O6 calculated value: 676.49; found value: 675[MH + ].

[0801] Example 62

[0802] Synthesis of 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-nitrobenzoic acid)

[0803]

[0804] Diazotization of 4-amino-2-nitrobenzoic acid was completed according to Example 22 to prepare 4-azido-2-nitrobenzoic acid CCC. Compound 106 was prepared using the copper click method using azide CCC and diacetylene Q in Example 1. Mass spectrum of compound 106 (ESI negative ion mode): C 26 H 18 N10 O9 calculated value: 614.49; found value: 613 [MH + ].

[0805] Example 63

[0806] Synthesis of 4,4′-((4-((3,3,3-trifluoropropyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0807]

[0808] According to Example 17, N-(3,3,3-trifluoropropyl)-2,6-diethynylpyridine-4-carboxamide DDD was prepared by HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and 3,3,3-trifluoropropan-1-amine. Compound 107 was prepared using the copper click method using diethynyl DDD and azide U in Example 1. Mass spectrum of compound 107 (ESI negative ion mode): C 29 H 17 F9N8O5 calculated value: 728.49; found value: 727[MH + ].

[0809] Example 64

[0810] Synthesis of 4,4′,4″,4″′-((((butane-1,4-diylbis(azanediyl))bis(carbonyl))bis(pyridine-4,2,6-triyl))tetrakis(1H-1,2,3-triazole-4,1-diyl))tetrakis(2-(trifluoromethyl)benzoic acid)

[0811]

[0812] Compound 108 was prepared using the copper click method using diacetyl FF and azide U in Example 1. Mass spectrum of compound 108 (ESI negative ion mode): C 56 H 34 F 12 N 16 O 10 Calculated value: 1318.97; Measured value: 1316.9[MH + ].

[0813] Example 65

[0814] Synthesis of (4-(4-(4-(ethylcarbamoyl)-6-(1-(4-phosphonophenyl)-1H-1,2,3-triazol-4-yl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)phenyl)phosphonic acid

[0815]

[0816] Diazotization of (4-aminophenyl)phosphonic acid was performed according to Example 22 to prepare (4-azidophenyl)phosphonic acid EEE. Compound 109 was prepared using the copper click method using azide EEE and diacetylene Q in Example 1. Mass spectrum of compound 109 (ESI negative ion mode): C 24 H 22 N8O7P2 calculated value: 596.44; found value: 595[MH + ].

[0817] Example 66

[0818] Synthesis of 2,2′-((4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))diacetic acid

[0819]

[0820] Methyl (4-azido-2-(trifluoromethyl)benzoyl)glycine FFF was prepared by coupling azide U with glycine methyl ester according to the HATU condensation method described in Example 17. Methyl ester FFF was saponified with 1N NaOH and then neutralized to give (4-azido-2-(trifluoromethyl)benzoyl)glycine GGG. Compound 110 was prepared using the copper click method using diacetylene Q and azide GGG in Example 1. Mass spectrum of compound 110 (ESI negative ion mode): C 32 H 24 F6N 10 O7 calculated value: 774.60; found value: 773 [MH + ].

[0821] Example 67

[0822] Synthesis of dimethyl 2,2′-((4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))diacetate

[0823]

[0824] Compound 111 was prepared using azide FFF and diacetylene Q using the copper click method in Example 1. Mass spectrum of compound 111 (ESI negative ion mode): C 34 H 28 F6N 10 O7 calculated value: 802.65; measured value: 801.1 [MH+ ].

[0825] Example 68

[0826] Synthesis of (2S,2'S)-2,2'-((4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))disuccinic acid

[0827]

[0828] (4-azido-2-(trifluoromethyl)benzoyl)aspartate HHH was prepared according to the HATU condensation method described in Example 17, and azide U was coupled with dimethyl aspartate to give (4-azido-2-(trifluoromethyl)benzoyl)aspartate dimethyl ester. The dimethyl ester was saponified with 1N NaOH and then neutralized to give HHH. Compound 112 was prepared using the copper click method using diacetylene Q and azide HHH in Example 1. Mass spectrum of compound 112 (ESI negative ion mode): C 36 H 28 F6N 10 O 11 Calculated value: 890.67; Measured value: 889[MH + ].

[0829] Example 69

[0830] Synthesis of 2,2′-((2,2′-((4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))bis(acetyl))bis(azanediyl))diacetic acid

[0831]

[0832] (4-azido-2-(trifluoromethyl)benzoyl)glycylglycine III was prepared by coupling azide U with glycylglycine methyl ester according to the HATU condensation method described in Example 17. The methyl ester was saponified with 1 N NaOH and then neutralized to give (4-azido-2-(trifluoromethyl)benzoyl)-glycylglycine III. Compound 113 was prepared using the copper click method using diacetylene Q and azide III in Example 1. Compound 113 mass spectrum (ESI negative ion mode): C 36 H 30 F6N 12 O9 calculated value: 888.70; found value: 887 [MH + ].

[0833] Example 70

[0834] Synthesis of 2,6-bis(1-(4-cyano-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-N-ethylisonicotinamide

[0835]

[0836] Diazotization of 4-amino-2-(trifluoromethyl)benzonitrile was performed according to Example 22 to prepare 4-azido-2-(trifluoromethyl)benzonitrile JJJ. Compound 114 was prepared using the copper click method using azide JJJ and diacetylene Q in Example 1. Mass spectrum of compound 114 (ESI negative ion mode): C 28 H 16 F6N 10 O calculated value: 622.50; found value: 621 [MH + ].

[0837] Example 714, Synthesis of 4′-((5-carboxy-1,3-phenylene)bis(1H-1,2,3-triazole-1,4-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0838]

[0839] Diazotization of 3,5-diaminobenzoic acid was performed according to Example 22 to prepare 3,5-diazidebenzoic acid KKK. 4-Ethynyl-2-(trifluoromethyl)benzoic acid LLL was prepared using 4-bromo-2-(trifluoromethyl)benzoic acid according to the Sonogashira method described in Example 9. Compound 115 was prepared using the copper click method using bisazide KKK and monoethynyl LLL in Example 1. Compound 115 mass spectrum (ESI negative ion mode): C 27 H 14 F6N6O6 calculated value: 632.44; found value: 631[MH + ].

[0840] Example 72

[0841] Synthesis of 4,4′-((5-(ethylcarbamoyl)-1,3-phenylene)bis(1H-1,2,3-triazole-1,4-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0842]

[0843] 3,5-Diazido-N-ethylbenzamide MMM was prepared by coupling bisazide acid KKK with ethylamine according to the HATU condensation method described in Example 17. Compound 116 was prepared using the copper click method using monoethynyl LLL and bisazide MMM in Example 1. Mass spectrum of compound 116 (ESI negative ion mode): C 29 H 19 F6N7O5 calculated value: 659.51; measured value: 658.1 [MH + ].

[0844] Example 73

[0845] Synthesis of 4,4′-(thiophene-2,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0846]

[0847] 2,5-Dibromothiophene was used to prepare 2,5-diethynylthiophene NNN according to the Sonogashira method described in Example 9. Compound 117 was prepared using azide U and diethynyl NNN using the copper click method in Example 1. Mass spectrum of compound 117 (ESI negative ion mode): C 24 H 12 F6N6O4 calculated value: 594.45; found value: 593[MH + ].

[0848] Example 74

[0849] Synthesis of 4,4′-(furan-2,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

[0850]

[0851] 2,5-Dibromofuran was used to prepare 2,5-diethynylfuran OOO according to the Sonogashira method described in Example 9. Compound 118 was prepared using azide U and diethynyl OOO using the copper click method in Example 1. Mass spectrum of compound 118 (ESI negative ion mode): C 24 H 12 F6N6O5 calculated value: 578.39; found value: 577[MH + ].

[0852] Example 75

[0853] Synthesis of 3′-(4-(4-(ethylcarbamoyl)-6-(1-(3′-(trifluoromethyl)-[1,1′-biphenyl]-3-yl)-1H-1,2,3-triazol-4-yl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)-3-(trifluoromethyl)-[1,1′-biphenyl]-4-carboxylic acid--Carbon dioxide (1 / 1-)

[0854]

[0855] The synthesis of 3'-amino-3-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylic acid was started by mixing methyl 4-bromo-2-(trifluoromethyl)benzoate (142 mg, 0.5 mmol), 3-aminophenylboronic acid (137 mg, 1 mmol), Pd(PPh3)2Cl2 (35 mg, 0.1 mmol) and K2CO3 (138 mg, 2 mmol) and adding methanol (2 ml). The solution was heated at 45°C for 1.5 hours. The methanol was removed by evaporation. The residual solid was dissolved in dimethylformamide (1 ml) and purified by silica gel chromatography using Reveleris Prep, eluting with EtOAc / hexane mobile phase. The appropriate fractions were collected based on UV and evaporated to give methyl 3'-amino-3-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylate (101 mg, 68% yield).

[0856] Diazotization of 3′-amino-3-(trifluoromethyl)-[1,1′-biphenyl]-4-carboxylic acid methyl ester was completed according to Example 22 to prepare 3′-azido-3-(trifluoromethyl)-[1,1′-biphenyl]-4-carboxylic acid methyl ester. The methyl ester was saponified with KOH, neutralized with 0.25 HCl and recovered with EtOAc to give 3′-azido-3-(trifluoromethyl)-[1,1′-biphenyl]-4-carboxylic acid PPP. Compound 119 was prepared using the copper click method using azide PPP and diacetyl Q in Example 1. Compound 119 mass spectrum (ESI negative ion mode): C 40 H 26 Calculated for N8O5: 812.69.

[0857] Example 76

[0858] Synthesis of 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-cyanobenzoic acid)

[0859]

[0860] Diazotization of 4-amino-2-cyanobenzoic acid was completed according to Example 22 to prepare 4-azido-2-cyanobenzoic acid QQQ. Compound 120 was prepared using the copper click method using azide QQQ and diacetyl Q in Example 1. Mass spectrum of compound 120 (ESI negative ion mode): C 28 H 18 N 10 O5 calculated value: 574.50.

[0861] Example 77

[0862] Synthesis of 4,4′-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-chlorobenzoic acid)

[0863]

[0864] Diazotization of 4-amino-2-chlorobenzoic acid was performed according to Example 22 to prepare 4-azido-2-chlorobenzoic acid RRR. Compound 121 was prepared using the copper click method using azide RRR and diacetylene Q in Example 1. Mass spectrum of compound 121 (ESI negative ion mode): C 26 H 18 Calculated for C12N8O5: 593.38.

[0865] Example 78

[0866] Screening of PEMs for enhancing XNTP polymerization

[0867] The expanded sequencing (SBX) method developed by the present inventors provides significant performance enhancements in terms of sequence reading efficiency and accuracy of Xpandomers relative to natural DNA. However, the initial transcription of natural DNA template sequences into measurable Xpandomers relies on the ability of DNA polymerases to utilize XNTPs as substrates (the general structure of XNTPs is referred to herein). Figure 1A and Figure 2 Discussion). The inventors have found that most DNA polymerases cannot efficiently polymerize XNTPs. In order to improve the efficiency and accuracy of XNTP polymerization into Xpandomers, several PEMs were screened to enhance the ability of DNA polymerase primer extension reactions using XNTPs as substrates.

[0868] A representative primer extension reaction may include the following reagents: 2 pmol primer, 2.2 pmol 45mer oligonucleotide template, 50 pmol of each XNTP (XATP, XCTP, XGTP and XTTP), 50 Mm Tris HCl, Ph 6.79, 200 Mm NaCl, 20% PEG, 5% NMS, 0.5 nmol polyphosphate 60.19, 0.3 Mm MnCl2 and 0.6 μg purified recombinant DNA polymerase protein. The reaction can be run at 23 ° C for 1 hour. The reaction product (i.e., constrained Xpandomer) is treated to cleave the aminophosphoryl ester bond to produce a linearized Xpandomer. The reaction products can be analyzed using gel electrophoresis on a 4%-1 2% acrylamide gel to resolve and display Xpandomer products of different lengths. For the above-mentioned PEM screening, PEM is typically tested in the micromolar to millimolar range.

[0869] Surprisingly, several PEMs were observed to significantly and reproducibly enhance DNA polymerase-mediated primer extension using XNTPs. An exemplary gel demonstrating this enhancement is shown in Figure 4 and Figure 5 . refer to Figure 4 As shown, under these conditions, the DNA polymerase extended the template-bound primer to only about 14 XNTPs, as seen in lane 1 (no PEM additive). However, the addition of certain PEMs to the primer extension reaction enabled the polymerase to synthesize longer extension products, as can be seen, for example, in lanes 3 (compound 4), 7 (compound 3), and 9 (compound 1). In contrast, several different aromatic compounds had little or no effect on XNTP polymerization (see, e.g., lanes 2, 4-6, and 8), indicating that the PEM activity is specific for compounds 1, 3, and 4.

[0870] Similarly, refer to Figure 5 As shown, in the absence of PEM additives, DNA polymerase showed modest primer extension activity towards XNTP (lane 1, no PEM additive), whereas addition of different concentrations of compound 2 (lanes 8-10) significantly enhanced the primer extension activity. Again, this PEM activity was specific to compound 2, as other unrelated aromatic compounds had no effect (lanes 2-7).

[0871] Example 79

[0872] PEM sequencing enhancement and extension (SBX)

[0873] To investigate the accuracy of the PEM-dependent enhancement of XNTP polymerization, the primer extension products were sequenced using the SBX protocol. In short, the Xpandomer product of the constrained XNTP polymerization was cut to produce a linearized Xpandomer. This was achieved by first quenching the extension reaction with a solution containing 100 MmEDTA, 2 Mm THPTA, and 2% Tween-20. The sample was then amine-modified with a DMF solution of 1 M NaHCO3 and 1 M succinic anhydride. The cleavage of the phosphoramidate bond was performed with 37% HCl, and the linearized Xpandomer was purified with a QIAquick column (QIAGEN, Inc.).

[0874] For sequencing, protein nanopores were prepared by inserting α-hemolysin into a DphPE / hexadecane bilayer member in buffer B1, which contained 2M NH4Cl and 100 Mm HEPES, Ph 7.4. The cis-pore was perfused with buffer B2, which contained 0.4M NH4Cl, 0.6M GuCl, and 100Mm HEPES, Ph 7.4. The Xpandomer sample was heated to 70°C for 2 minutes, cooled completely, and then 2 μL of the sample was added to the cis-pore. A voltage pulse of 90Mv / 390Mv / 10μs was then applied and data was acquired by Labview acquisition software.

[0875] The sequence data is analyzed by a histogram display of a population of sequence reads from a single SBX reaction. The analysis software aligns each sequence read to the template sequence and trims the sequence range at the ends of the reads that do not align to the correct template sequence. An exemplary histogram of SBX sequencing of a 45mer template is shown in Fig. 6A Medium (no additive control) and Figure 6B (SBX in the presence of PEM Compound 1). As can be seen, in the absence of Compound 1, the sequence reads were inaccurate around base 18 of the template. Notably, the addition of Compound 1 to the SBX reaction improved the accuracy of the sequence reads across the entire length of the 45mer template.

[0876] These results inspired additional experiments to test the ability of PEM compound 1 to enhance SBX of even longer templates. Fig. 7A and 7B Histograms of SBX sequencing of 60mer and 80mer templates are shown, respectively. Surprisingly, compound 1 was able to perform accurate sequence reads, intact to the end of each longer template. These results demonstrate that the novel PEM can robustly and accurately enhance XNTP polymerization activity, which strongly improves the ability of SBX to provide nanopore-based nucleic acid sequence information.

[0877] Example 80

[0878] PEM enables the synthesis of long Xpandomer products

[0879] After successful accurate replication of templates up to 80 nucleotides long into the Xpandomer, XNTP polymerization reactions were performed using four longer templates consisting of 88, 127, 227, and 277 nucleotides in length. A variant of DPO4 DNA polymerase, referred to as C4552 (SEQ ID NO: 1), was used in these polymerization reactions, and the reaction conditions were optimized for C4552 activity in the presence of PEM compound 1. Other suitable DPO4 polymerase variants include, but are not limited to, those of SEQ ID NOs: 2-5. In addition to 1 Mm compound 1, the reaction additives also included 1 Mm urea and 2.75 μg single-strand binding protein (Eco SSB). Extension reactions were performed using 0.85 pmol template, 0.5 pmol oligonucleotide primer, and 1 nmol of each XNTP in a final volume of 10 μL. Reactions were performed in a buffer consisting of 50 Mm TrisCl, Ph8.84, 200 Mm NH4Oac, and 20% PEG8K, supplemented with 5% NMS, 3 or 4 nmol of polyphosphate PP-60.20, and 2 Mm MnCl2. 1.2 μg of purified recombinant DNA polymerase protein was used in each extension reaction, and the reactions were run at 23°C for 1-2 hours. The results of an exemplary extension reaction using a longer template are shown in Figure 8 Remarkably, in the presence of compound 1, the polymerase was able to polymerize XNTPs to generate a complete Xpandomer copy of each longer template, ranging in length from 88 (lanes 1 and 6) to 277 (lanes 5 and 10) nucleotides. Lanes 1-5 and 6-10 represent identical extension reactions, except for the amount of PP-60.20 additive, which was 3 nmol in lanes 1-5 and 4 nmol in lanes 6-10. These results highlight the surprising advantage conferred by compound 1 in reactions requiring polymerization of unnatural, highly substituted nucleotide analogs by DNA polymerases, and suggest that this compound, as well as other PEMs, can greatly expand the potential of SBX sequencing protocols.

[0880] Example 81

[0881] Next-generation PEMs enhance polymerization of XNTPs to generate long Xpandomer products

[0882] Based on the favorable properties observed for PEM Compound 1, next generation PEM compounds were designed with the goal of improving certain properties, including but not limited to the water solubility of the molecule. Exemplary next generation PEM structures are described in Examples 9-34 and Table 1.

[0883] The PEM activity of compounds 9-11 was tested in a primer extension assay using three 100mer templates derived from HIV1, 2 and 3 genomes. The primer extension reaction included the following reagents: 75MmTrisCl, Ph 8.44, 175Mm NH4Oac, 20% PEG8K, 5% NMS, 0.8nmol PP-60.20, 0.6Mm MnCl2, 2.3μg Tth single-strand binding protein (SSB), 0.5M or 1M urea, 200pmol of each XNTP, 1.1pmol template, 1pmol oligonucleotide primer, 1.2μg purified recombinant C4552 DNA polymerase and 0.5Mm PEM. The 10 μL primer extension reaction was run at 23°C for 30 minutes, and the reaction products were analyzed by gel electrophoresis. An exemplary gel showing primer extension products is shown in Fig. 9 As shown in lanes 1 (HIV1 template), 2 (HIV2 template), and 4 (HIV3 template, no SSB and 1M urea), compound 1 was able to polymerize XNTPs into full-length Xpandomer copies of three different 100mer templates (arrows indicate the gel migration position of the 100mer). Similarly, each of compounds 9 (lanes 13-16), 10 (lanes 9-12), and 11 (lanes 5-8) was able to polymerize XNTPs at least as efficiently as compound 1 on each of the three different 100mer templates. These results suggest that PEM activity can be optimized by increasing various physicochemical properties of the compounds, such as water solubility.

[0884] Compound 12 was tested for PEM activity in a primer extension assay using HIV2 100mer template. The primer extension reaction included the following reagents: 50 Mm TrisCl, 200 Mm NH4Oac, 20% PEG8K, 5% NMS, 0.6 nmol PP-60.20, 0.6 Mm MnCl2, 2.75 μg / μl Eco single-strand binding protein (SSB), 1 M urea, 50 pmol of each XNTP, 1.1 pmol template, 1 pmol oligonucleotide primer, 1.2 μg / μl purified recombinant C4760 DNA polymerase (SEQ ID NO: 2), and 0.5, 1 or 1.5 Mm PEM. The 10 μL primer extension reaction was run at 23°C for 30 minutes, and the reaction products were analyzed by gel electrophoresis. An exemplary gel showing primer extension products is shown in Fig.10As shown in lanes 5 (0.5 Mm PEM), 6 (1 Mm PEM), and 7 (1.5 Mm PEM), compound 12 is able to polymerize XNTPs into full-length Xpandomer copies of a 100-mer template in a manner comparable to compound 10 (lane 1) (the gel migration position of the 100-mer is indicated by an arrow). Lanes 2-4 show primer extension products from reactions with structurally related additives that lack robust PEM activity. These results suggest that PEM activity may be determined by very specific chemical structures.

[0885] Compounds 13 and 14 were tested for PEM activity in a primer extension assay using HIV2 100mer template. The primer extension reaction included the following reagents: 50 Mm TrisCl, 200 Mm NH4Oac, 20% PEG8K, 5% NMS, 0.6 nmol PP-60.20, 0.6 Mm MnCl2, 2.75 μg / μl Eco single-strand binding protein (SSB), 1 M urea, 50 pmol of each XNTP, 1.1 pmol template, 1 pmol oligonucleotide primer, 1.2 μg / μl purified recombinant C4760 DNA polymerase (variant of DPO4, see SEQ ID NO: 2), and 0.5, 1, 1.52 or 2.5 Mm PEM. 10 μL primer extension reactions were run at 23°C for 30 minutes, and the reaction products were analyzed by gel electrophoresis. An exemplary gel showing primer extension products is shown in Fig.11 (The position of the full-length HIV2 100mer is indicated by an arrow). As shown in lanes 5-7 (various concentrations of compound 14) and 8-10 (various concentrations of compound 13), each of these next-generation PEMs was able to polymerize XNTPs into full-length Xpandomer copies of the 100mer template in a manner comparable to compound 10 (lanes 1-4).

[0886] Compound 15 was tested for PEM activity in a primer extension assay using a 411mer amplicon template. The primer extension reaction included the following reagents: 50 Mm TrisCl, 200 Mm NH4Oac, 20% PEG8K, 5% NMS, 3 nmol PP-60.20, 2 Mm MnCl2, 2 μg Kod single-strand binding protein (SSB), 1 M urea, 250 pmol of each XNTP, 1 pmol template, 1 pmol oligonucleotide primer, 1.2 μg purified recombinant C4760 DNA polymerase (variant of DPO4, see SEQ ID NO: 2), and 2 (lane 2) or 3 (lane 3) Mm PEM. The 10 μL primer extension reaction was run at 37°C for 20 minutes, and the reaction products were analyzed by gel electrophoresis. An exemplary gel showing primer extension products is shown in Fig.12(the position of the 277mer is indicated by an arrow). As shown in lanes 2 and 3 (two different concentrations of compound 15), this next generation PEM is able to polymerize XNTPs into long Xpandomer copies of the 411mer template in a manner comparable to compound 14 (lane 1). Notably, the polymerase is completely dependent on the addition of the PEM to the reaction in order to be able to synthesize these long Xpandomer products. Even longer extension products can be obtained by optimizing various reaction parameters (e.g., extension time and / or concentrations of various additives).

[0887] Compounds 16, 17 and 18 and combinations thereof were tested for PEM activity in a primer extension assay using a 100mer template derived from HIV2. The primer extension reaction included the following reagents: 50 Mm TrisCl, 200 Mm NH4Oac, 20% or 25% PEG8K, 5% NMS, 0.6 nmol PP-60.20, 0.6 Mm MnCl2, 2 μg Kod single-strand binding protein (SSB), 1 M urea, 50 pmol of each XNTP, 1 pmol template, 1 pmol oligonucleotide primer, 1.2 μg purified recombinant C4760 DNA polymerase (variant of DPO4, see SEQ ID NO: 2) and 0.5-2 Mm PEM. The 10 μL primer extension reaction was run at 37°C for 30 minutes, and the reaction products were analyzed by gel electrophoresis. An exemplary gel showing primer extension products is shown in Fig.13 (the position of the 100mer product is indicated by an arrow). As shown in lanes 2 (2 Mm compound 16) and 3-6 (0.5, 1, 2 and 3 Mm compound 17), these next-generation PEMs enable XNTPs to be polymerized into long Xpandomer copies of the 100mer template in a manner comparable to compound 14 (lane 1). In addition, the combination of 2 Mm compound 14 and 0.1 Mm (lane 7) or 0.3 Mm (lanes 8 and 9) compound 17 also polymerizes XNTPs into long Xpandomer copies of the 100mer template, indicating that the combination of PEMs may allow the use of lower doses of each individual PEM. Similarly, the combination of 2 Mm compound 16 and 0.1 Mm (lane 10) or 0.3 Mm (lanes 11 and 12) compound 18 also appears to allow the use of lower doses of each individual PEM to polymerize XNTPS into full-length copies of the 100mer template.

[0888] Compound 19 was tested for PEM activity in a primer extension assay using a 411mer amplicon template. The primer extension reaction included the following reagents: 50 Mm TrisCl, 200 Mm NH4Oac, 20% PEG8K, 5% NMS, 3 nmol PP-60.20, 2 Mm MnCl2, 2 μg Kod single-strand binding protein (SSB), 1 M urea, 250 pmol of each XNTP, 0.5 pmol template, 0.5 pmol oligonucleotide primer, 1.2 μg purified recombinant C4760 DNA polymerase (variant of DPO4, see SEQ ID NO: 2), and 0.5 (lane 2), 1 (lane 3) or 1.5 Mm (lane 4) PEM. The 10 μL primer extension reaction was run at 37°C for 30 minutes, and the reaction products were analyzed by gel electrophoresis. An exemplary gel showing primer extension products is shown in Fig.14 (the position of the 277mer product is indicated by an arrow). As shown in lanes 2-4, this next-generation PEM is able to polymerize XNTPs into long Xpandomer copies of the 411mer template, although its efficiency is lower than that of compound 14 (lane 1). These results suggest that PEM activity may be specific to the structure of the PEM and / or the length of the template.

[0889] Example 82

[0890] Third Generation PEM Compounds

[0891] To further explore the relationship between PEM structure and polymerase enhancing activity, as well as to optimize various physicochemical properties, "third generation" PEM compounds were synthesized, as described herein, e.g., Table 1 (Compounds 79-118) and Examples 35-74. These PEMs were tested in primer extension reactions using DNA templates of the following lengths: A) 45mer; B) 100mer; C) 150mer; and D) 222mer. The primer extension reactions were carried out under conditions comparable to those described in Examples 77 and 78, and the extension products were similarly analyzed by gel electrophoresis. Preliminary functional characterization of third generation compounds showing PEM activity is summarized in Table 2 below.

[0892] Table 2

[0893] Summary of PEM Activity

[0894]

[0895]

[0896] Example 83

[0897] PEM enables synthesis of Xpandomer copies of RNA templates

[0898] To begin investigating the potential of PEMs to enhance multiple polymerase reactions, the ability of PEMS to enable DNA polymerases to utilize RNA as a template was tested. Several compounds listed in Table I were screened using a 45mer RNA template derived from the HIV2 genome sequence. The primer extension reaction included the following reagents: 50 Mm TrisCl, 200 Mm NH4Oac, 20% PEG8K, 5% NMS, 0.6 nmol PP-60.20, 0.6 Mm MnCl2, 2 μg Kod single-strand binding protein (SSB), 1 M urea, 50 pmol of each XNTP, 0.5 pmol template, 0.5 pmol oligonucleotide primer, 1.2 μg purified recombinant C4760 DNA polymerase (variant of DPO4, see SEQ ID NO: 2) and 2 Mm PEM. 10 μL primer extension reactions were run at 37°C for 30 minutes, and the reaction products were analyzed by gel electrophoresis. Surprisingly, several compounds were found to show PEM activity on RNA templates; these RNA PEMs include the following compounds: compounds 51, 73, 49, 17, 75, 76, 93, 105, 106, 110, 116, and 118. These results suggest that some PEMs may play a role in sequencing by extending RNA templates.

[0899] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patents, and non-patent disclosures mentioned in this specification and / or listed in the application data sheet, including but not limited to U.S. Provisional Patent Application No. 62 / 867,049 filed on June 26, 2019, are incorporated herein by reference in their entirety. For the purpose of describing and disclosing materials and methods such as those described in publications, these documents may be incorporated by reference, which may be used in conjunction with the invention currently described. The publications discussed above and throughout the text are provided only for their disclosure prior to the filing date of this application. Nothing herein should be construed as admitting that the inventor has no right to rely on prior inventions prior to any cited publications.

Claims

1. A compound of formula (I) or a solvate, hydrate, tautomer, chelate or salt thereof, wherein: m is 1, 2 or 3; m′ is 1, 2 or 3; n is 0, 1 or 2; p is 0, 1, or 2; When X is C, W is N, or when X is N, W is C; is a single bond or a double bond, wherein the double bond starts at any one of W or X that is carbon; L is a linking group; M is independently selected at each occurrence from hydrogen, halogen and C1-C4 alkyl; Ar1 at each occurrence is independently selected from optionally substituted pyridine, pyrazine, pyridazine, furan, thiophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine and carbazole, wherein: The substituents of Ar1 are independently selected at each occurrence from halogen, -OH, -CN, -NO2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C1-C6 cycloalkyl, -OR 0 、-CONH2、-C(O)NR 1 R 1′ 、-NR 1 R 1′ 、-NR 1 C(O)R 3 、-C(O)SR 3 、-COR 3 、-OC(O)R 3 、-C(O)OR 3 , thiol, -R 4 -H, -SOR 1 、-S(O)2R 1 、-S(O)2NR 1 R 1′ and -NS(O)2R 3 ; and among them R 0 is independently selected at each occurrence from C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; R 1 and R 1′ is independently selected at each occurrence from H, hydroxy, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, and substituted or unsubstituted heteroaryl, -C(=NH)NH2, -CH2CO2R 0 , -CH2C(O)NHCH2CO2H, -CH2CH2OH, -CH2CH2NHC(O)R 3 、-CH2C(O)NHCH2CO2H、 and Where R 1 and R 1′ can form a heterocyclic ring, including but not limited to azetidine, pyrrolidine, piperidine, piperazine, morpholine, R 2 is independently selected at each occurrence from C2-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy; R 3 is independently selected at each occurrence from H, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine; R 4 is independently selected at each occurrence from alkylene interrupted by one or more heteroatoms, wherein the heteroatoms are O, S, NH or a combination thereof; Y is independently selected at each occurrence from Ar2, -(CH2)3PO(OEt)2 or -CH2CO2Me; Ar2 is independently selected at each occurrence from substituted 5-membered and 6-membered monocyclic aromatic rings and 9-membered and 10-membered fused bicyclic rings comprising two monocyclic rings taken together, wherein at least one of the two monocyclic rings is aromatic, wherein: Ar2 by G 1 , G 2 , G 3 , G 4 and G 5 Replace, where: When Ar2 is singly substituted, G 1 is independently selected at each occurrence from oxo, -NH2, -COR 3 、-E-CO2H、-C(O)NR 1 R 1′ 、-E-PO(OR 1 )2. And the G 2 , G 3 , G 4 and G 5 substituted aryl; G 2 , G 3 , G 4 and G 5 is independently selected at each occurrence from being absent or from a group comprising: halogen, -CN, C1-C6 alkyl, C1-C6 haloalkyl, -E-CO2H, -E-CHO, -EC(O)R 3 、-EC(O)NH(OH)、-EC(O)NHR 1 、-E-CONR 1 R 1′ 、-E-NR 1 R 1′ AND-E-OR 2 ,in: E at each occurrence is independently selected from a direct bond and C1-C6 alkylene.

2. The compound according to claim 1, wherein Ar1 is a monocyclic heteroaryl group.

3. The compound according to claim 2, wherein Ar1 is selected from: The triazole ring is located at position k on Ar1. The compound according to claim 1 , wherein Ar 1 is a bicyclic aromatic group.

5. The compound according to claim 4, wherein Ar1 is a bicyclic carbocyclic aromatic group selected from: The triazole ring is located at position k on Ar1.

6. The compound according to claim 4, wherein Ar1 is a bicyclic heterocyclic aryl group selected from: The triazole ring is located at position k on Ar1. The compound according to claim 1 , wherein Ar 1 is a tricyclic aryl group.

8. The compound according to claim 7, wherein Ar1 is a tricyclic carbocyclic aromatic group selected from:

9. The compound according to claim 7, wherein Ar1 is a tricyclic heteroaryl group selected from: The triazole ring is located at position k on Ar1.

10. The compound according to claim 7, wherein Ar1 is a tricyclic heteroaryl group selected from The triazole ring is located at position k on Ar1.

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