COMPOSITIONS AND METHODS FOR INHIBITING RIBOSOME INACTIVATING PROTEINS

SEQUENCE LISTING

The ASCII text file named “370602-7018US1 Sequence Listing.txt” created on Mar. 8, 2022, comprising 4.6 Kbytes, is hereby incorporated by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

Ribosome inactivating proteins (RIPs) are a class of protein synthesis inhibitors that act at the ribosome. Trichosanthin, a plant toxin derived from Trichosanthes kirilowii, is an example of a type I ribosome inactivating protein (RIP), which comprises an active A chain (RTA). The plant toxin ricin and Shiga toxins (Stxs) are type II ribosome inactivating proteins (RIPs) or AB-toxins, which comprise an active A chain (RTA) or A1 chain (Stx2A1) covalently linked to a cell binding B chain (RTB or Stx2B). Ricin is one of the most lethal substances known, and Stxs produced by Shigella dysenteriae and E. coli O157:H7 (STEC) are responsible for food-borne outbreaks of dysentery and hemolytic-uremic syndrome (HUS). HUS is the most common cause of renal failure in infants and young children in the US. Both Ricin and Stxs depurinate a universally conserved adenine in the highly conserved sarcin-ricin loop (SRL) of the large rRNA and inhibit protein synthesis. Currently, no FDA-approved vaccine or therapeutic exists to protect against ricin or Shiga toxins. In light of the spreading of multi-drug resistant Shigella and E. coli infections in the US, the length of time required for diagnosis (around a month), and vulnerability of children and elderly, quick diagnosis and treatment are national and international research priorities.

Although small-molecule RIP inhibitors have been reported, none of them exhibit potent protection against RIPs. Hence, there is an unmet need in the art to develop potent RIP-inhibitors to treat toxicity caused by RIPs. The present disclosure addresses this unmet need.

BRIEF SUMMARY OF THE DISCLOSURE

The present disclosure relates in part to methods of treating, ameliorating, or preventing toxicity caused by a ribosome inactivating protein (RIP) in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I), or a salt, solvate, stereoisomer, geometric isomer, and/or tautomer thereof:

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wherein A, B, R¹, R², X¹, and X²are defined elsewhere herein. In certain embodiments, the RIP is a type I RIP. On other embodiments, the RIP is a type II RIP. The present disclosure further relates to pharmaceutical compositions comprising at least one pharmaceutically acceptable carrier and at least one compound contemplated herein, or a salt, solvate, stereoisomer, geometric isomer, and/or tautomer thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of certain embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, specific embodiments are shown in the drawings. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 1 shows structure of ricin holotoxin (left PDB ID: 2AAI) and RTA (right PDB ID: 1RTC). Arginine residues at the RTA/RTB interface are labeled and residues in the hydrophobic pocket are also labeled.

FIGS. 2A-2B show cytotoxicity and depurination level of RTA mutants in yeast. FIG. 2A: viability of yeast at 8 hpi (hours post-injection). FIG. 2B: Depurination level was measured at 1 hpi using qRT-PCR and expressed relative to no toxin control.

FIG. 3 provides binding controls for the fragment screen with Biacore. Adenine and P11 peptide (SEQ ID NO: 1) were used as positive controls and myo-inositol and PT peptide (SEQ ID NO: 2) were used as negative controls. The molecular weights of each are indicated. The structure of the last 6 amino acids of P11 (underlined) with RTA have been solved.

FIGS. 4A-4P provide illustrative results of kinetic screening from one plate, including the sensorgrams of fragment inhibitors at 62.5, 125, 250, and 500 μM.

FIGS. 5A-5P provide illustrative results of kinetic screening from one plate, including the kinetic fittings of the sensorgrams of fragment inhibitors at 62.5, 125, 250, and 500 μM.

FIG. 6 is a table showing illustrative dissociation constant (K_D) and percent inhibition of 79 fragments with yeast and rat liver ribosomes at 100 μM fragment concentration.

FIG. 7 shows illustrative inhibition of RTA depurination by fragment inhibitors as determined by qRT-PCR, wherein yeast or rat ribosomes were treated with 1.0 or 0.2 nM RTA, respectively, in the presence of each fragment at 100 μM concentration. Five inhibitors were selected for X-ray crystallography.

FIG. 8 shows illustrative results from NMR Saturation Transfer Difference (STD) experiments to demonstrate binding of Adenine and 5 Maybridge Fragments to RTA. The protein and ligand concentrations were 20 and 400 μM, respectively. Peaks in these difference spectra arise from magnetization transfer from the RTA protein to the bound ligand.

FIG. 9 depicts chemical structures of eight illustrative fragments.

FIGS. 10A-10B provide illustrative inhibitory fragments co-crystallized with RTA. FIG. 10A provides CC10501 (5-phenylthiophene-2-carboxylic acid; CC70601 (4-(thien-2-ylmethyl) benzoic acid); and BTB13068 (9-oxofluorene-4-carboxamide). FIG. 10B provides inhibitor bound structures (stereoviews), the superposition of RTA structures in complex with CC10501, CC70601, and BTB13068.

FIG. 11 provides illustrative results of a single-dose screening from one plate. Both positive and negative controls were tested repeatedly in between the screening cycles. DMSO corrections were run at the beginning, after 50 cycles and at the end of the screening cycles. Interaction sensorgrams of CC10501 and CC70601 are also provided.

FIG. 12A shows illustrative X-ray crystal structure of CC10501 with RTA. It binds at the ribosome binding site and makes a key interaction with Arg235 imitating the interaction of Arg235 with the last Asp of P6.

FIG. 12B shows illustrative X-ray crystal structure of CC70601 with RTA. It binds at the ribosome-binding site and makes a key interaction with Arg235.

FIGS. 13A-13B show an illustrative table of the data collection and refinement statistics of RTA complexes of RTA+CC10501, RTA+CC70601, and RTA+BTB13068.

FIG. 14 provides an illustrative omit density map (F_o−F_c) of CC10501, CC70601, and BTB13068 inhibitors bound to the RTA. The omit map was calculated after 15 cycle of omit refinement by REFMAC6, leaving out the inhibitors. The contour levels are at 2.5σ.

FIGS. 15A-15B show illustrative superposition of the RTA-inhibitor complexes with the P stalk peptide (stereoview). FIG. 15A shows the electrostatic surface representation of CC10501, CC70601, and C-terminal stalk protein (P2) in the hydrophobic pocket of RTA. FIG. 15B shows a cartoon representation (zoomed in) of CC10501 (stick), CC70601 (stick), and C-terminal stalk protein (P2, stick) in the hydrophobic pocket of RTA.

FIGS. 16A-16C provide illustrative binding interactions of RTA in complex with inhibitors (stereoview). Inhibitor complexes of CC10501 (FIG. 16A), CC70601 (FIG. 16B), and BTB13068 (FIG. 16C) are provided. Amino acids interacting with inhibitors from RTA are highlighted. Selected hydrogen bond interactions are shown with orange dotted lines. The RTA residues interacting with inhibitors within 4 Å are highlighted.

FIG. 17 provides an illustrative electrostatic surface representation (stereoview) of BTB13068 binding in a remote hydrophobic pocket close to helix D4. The binding of BTB13068 is mostly driven by hydrophobic interactions.

FIGS. 18A-18B show an illustrative superposition of the RTA structure in complex with inhibitors with the RTA-RTB complex. FIG. 18A shows superposition of RTA structure in complex with CC10501, CC70601, BTB13068 with the RTA-RTB complex. FIG. 18B shows close views of CC10501 and CC70601 binding in the hydrophobic pocket. The binding position of Phe262 from RTB into the hydrophobic RTA pocket is indicated.

FIGS. 19A-19C show illustrative binding sensorgrams of the three fragments to RTA; CC10501 (FIG. 19A), CC70601 (FIG. 19B), and BTB13068 (FIG. 19C). The fragment concentrations were 12.5, 25, 50, 100, and 200 μM. The binding measurements were repeated five different times using four different chips with three replicates each time.

FIGS. 20A-20C show illustrative kinetic fittings resulting of the binding sensorgrams resulting from binding of the three fragments to RTA: CC10501 (FIG. 20A), CC70601 (FIG. 20B), and BTB13068 (FIG. 20C). The K_Dvalues are shown as the mean±standard deviation. The steady state affinity constant R_maxmodel with global fitting was used to determine the K_Dvalues for CC10501 and CC70601. The surface activity was calculated on the basis of the binding affinity of P6 was 69%. The steady state affinity model with global fitting was used to determine the K_Dvalue for BTB13068 using the Biacore T200 evaluation software 3.0.

FIGS. 21A-21C provide illustrative 50% inhibitory concentration (IC₅₀) of fragments against RTA: CC10501 (FIG. 21A), CC70601 (FIG. 21B), and BTB13068 (FIG. 21C). Different colors indicate different measurements, which we repeated 4 to 6 times. The Michaelis-Menten model was used to fit the inhibition curves using the Origin software.

FIG. 22 shows an illustrative time course of depurination under the conditions employed for the 50% inhibitory concentration (IC₅₀) experiments with fragments against RTA. The data are expressed as the mean±standard deviation of three to four technical replicates.

FIG. 23 shows an illustrative summary of the medicinal chemistry efforts towards improvement of CC10501 and CC70601.

FIG. 24 shows an illustrative inhibition of RTA depurination by fragment inhibitors with inhibitor concentrations of 50 μM and 100 μM, wherein yeast ribosomes were treated with 1.0 nM RTA, as determined by qRT-PCR.

FIGS. 25A-25B provide illustrative binding interactions of RTA in complex with CC10501 (FIG. 25A) and RU-NT-70 (FIG. 25B).

FIG. 26 shows an illustrative inhibitory concentration (IC₅₀) of RU-NT-70 against RTA, wherein the measurements were repeated 4 to 6 times. The Michaelis-Menten model was used to fit the inhibition curves using the Origin software.

FIGS. 27A-27B show illustrative 50% inhibitory concentration (IC₅₀) of RU-NT-102 (FIG. 27A) and RU-NT-136 (FIG. 27B) against RTA. Depurination inhibitory activity of the fragments was determined by qRT-PCR and fragment concentrations were varied depending on the inhibitory activity of the fragment. The different measurements which were repeated 4 to 6 times. The Michaelis-Menten model was used to fit the inhibition curves using the Origin software.

FIG. 28 is a table showing a illustrative nalogues of CC10501 having a higher affinity and inhibitory activity against RTA compared to CC10501.

FIGS. 29A-29B show illustrative structures of ricin (FIG. 29A) and shiga toxin (FIG. 29B).

FIG. 30 illustrates that ricin and Stxs depurinate the sarcin/ricin loop (SRL) of the 28S rRNA and inhibits translation.

FIG. 31 show illustrative structures of bacterial, yeast, and human stalk.

FIG. 32 show illustrative models of yeast and human stalk and overlapping conserved amino acid sequences: Hs-P0 (SEQ ID NO: 3), Hs-P1 (SEQ ID NO: 4), Hs-P2 (SEQ ID NO: 5), Sc-P0 (SEQ ID NO: 6), Sc-P1α (SEQ ID NO: 7), Sc-P1β (SEQ ID NO: 8), Sc-P2a (SEQ ID NO: 9), and Sc-P2β (SEQ ID NO: 10).

FIG. 33 illustrates a set-up and working principle of surface plasmon resonance. Thin gold film deposited on glass is excited by plane polarized light to induce an evanescent wave; an increase in mass on the sensor surface causes an increase in the refractive index; measures change in the resonance angle in real time as a change in response units (ΔRU); the sensor gram is a continuous display of RU over time.

FIG. 34 shows that RTA binding to ribosomes is reduced in the ΔP1 and ΔP2 mutants. Biacore 3000 was used to examine the interaction between N-His RTA as the ligand and ribosomes (5 nM) isolated from the ΔP1, ΔP2 and ΔP1 ΔP2 mutants as the analyte.

FIG. 35 shows that RTA interacts with wild type ribosomes via two distinct types of electrostatic interactions.

FIG. 36 Illustrates a model of how RTA depurinates the ribosome. Step 1: RTA molecules are concentrated on the ribosomal surface by electrostatic interactions. Step 2: Interaction with the C-termini of P stalk proteins reorient the active site of RTA towards the SRL. Step 3: P stalk binding stimulates the catalysis of depurination by delivering RTA to the SRL.

FIG. 37 shows that P stalk binding site of RTA is at the RTA/RTB interface.

FIG. 38 shows that R235A mutation causes the greatest reduction in toxicity in yeast.

FIG. 39 illustrates that arginine mutations disrupt the interactions with the P stalk.

FIG. 40 shows an illustrative structure of the RTA-P6 complex with C-terminal sequence (SEQ ID NO: 1).

FIG. 41 shows that L232A mutation reduces toxicity more than the other residues in the hydrophobic pocket.

FIG. 42 shows that the conserved LF motif of P6 is inserted in a pocket lined by Tyr183 and Phe240. C-terminal sequence (SEQ ID NO: 1) FIG. 43 demonstrates that double, triple, and quadruple mutants containing L232A show further reduction in toxicity and activity in yeast.

FIG. 44 illustrates that combining hydrophobic mutations with R235A eliminates activity of RTA in Vero cells.

FIG. 45 is a table showing that illustrative peptide mimics of the P stalk C-termini inhibit the activity of RTA by preventing ribosome binding. Peptide mimics P11 (SEQ ID NO: 1), P10 (SEQ ID NO: 11), P9 (SEQ ID NO: 12), P8 (SEQ ID NO: 13), P7 (SEQ ID NO: 14), P6 (SEQ ID NO: 15), P5 (SEQ ID NO: 16), P4 (SEQ ID NO: 17), and P3 (SEQ ID NO: 18).

FIGS. 46A-46D shows illustrative X-ray crystal structure of RTA with C-termini of ribosomal P protein (PDB ID: 5GU4, FIG. 46A) with three different fragments bound to RTA (FIG. 46B; FIG. 46C; and FIG. 46D).

FIGS. 47A-47C show the hydrophobic pocket of ricin toxin A subunit: with P protein peptide (FIG. 47A); with an inhibitor (FIG. 47B); and covered by the B subunit (FIG. 47C).

FIG. 48 shows an illustrative interaction of RTA with the P stalk to access SRL.

FIG. 49A: Structures of Stx2A1 with citrate; FIG. 49B: shows citrate together with P11; FIG. 49C: shows Stx2A1 with BTB13086; FIG. 49D: shows BTB13086 together with P11.

FIG. 50 shows a comparison of RU-NT-93 with Retro2 in Vero cells. Vero cells (0.5 mL/well) were plated at 10⁵cells/mL and grown for 24 h, then media with serum was removed and replaced by serum free media, vortexed with additions of compounds from 100% DMSO stocks to a final DMSO concentration of 0.5%. After 1 h, ricin was add (200 pM). Wells were harvested 2 h after ricin addition, with addition of 350 μL lysis buffer, as described for Qiagen Plus RNA Mammalian cell kid. Total RNA was extracted from Vero cells using the RNeasy Plus Mini Kit (Qiagen) either immediately or after storage at −80° C. The high capacity cDNA reverse transcription kit (Thermo Fisher Scientific) was used for cDNA conversion of approximately 375 ng of total RNA in a 20 μL reaction. Percent depurination was measured by qRT-bars showing the standard error. A Dunnet test was used to compare each compound to control (−cpd) ***P<0.001.

FIG. 51 shows protection in A549 cells against ricin administration with administration of compounds of the present disclosure. Human epithelial lung cells (A549) were grown and treated with holotoxin as described elsewhere herein. Cells were exposed to ricin (200 pm) for 2 h, within the linear range for dose and time.

FIG. 52 shows protection in Vero cells against ricin administration with administration of RU-NT-47 and RU-NT-57. Vero cell protection from ricin holotoxin was performed as described elsewhere herein, except that the compound (i.e., RU-NT-47 or RU-NT-57) and toxin were administered simultaneously.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure is related to the discovery of RIP inhibitors that exhibit potent (nanomolar) protection against RIPs. The paucity of promising RIP inhibitor candidates is primarily due to the large active site pocket and the strong electrostatic interactions at the interface between RIP and ribosome that make some drug-like molecules ineffective in competing with the ribosome for binding to RIPs. This technical barrier to developing small-molecule inhibitors of protein/ribosome complex has stymied the progress of RIP inhibitor development.

To address this barrier at the root level, P-proteins of the ribosomal stalk were identified as the host target of ricin and Stxs, then it was shown that the ribosome binding surface of RTA is on the opposite face of the active site. Further, it was demonstrated that blocking the interaction of RTA with the ribosome reduces its depurination activity and cytotoxicity. These studies established toxin/ribosome interactions as a new target for drug discovery and arginine 235 as a key interacting residue at the ribosome-binding site of RTA as an initial step in inhibitor discovery.

Fragment-based lead discovery (FBLD), using surface plasmon resonance (SPR) together with nuclear magnetic resonance (NMR), has emerged as an important tool for discovery of inhibitors of protein function. FBLD has not previously been used to identify inhibitors of ricin. FBLD involves screening small molecules (100-300 Da) with expected affinities in 0.1-10 mM range, which may become fragments for constructing drug-like compounds. Its advantages over conventional high throughput screening (HTS) are that: 1) a tremendous chemical diversity can be represented in fragment collections of as few as 1000 molecules; 2) observed hit rates for fragment screens are 10-1000× higher than conventional HTS due to greater exploration of chemical space; 3) low molecular weight compounds tend to be more soluble; and 4) the method of detection, SPR is simpler, more robust and is more reliable than HTS because it is less prone to artifacts.

Low molecular mass fragments tend to bind with low affinity. Thus, compounds require screening at high concentrations to be detected, which can lead to high false positive hit rates in biochemical assays. Therefore, to detect low affinity interactions, SPR was employed. The primary advantage of SPR versus complementary biophysical screening methods, such as NMR, X-ray crystallography, and calorimetry, is its low reagent requirements. Typically, 1-5 μg of target protein is immobilized on the SPR chip surface, and each fragment is used at 100-500 μM.

FBLD is used in combination with SPR and NMR to identify ricin-specific inhibitors that target ribosome binding and/or depurination activity. Eight fragments that show dose-dependent binding to RTA and inhibit its activity in a cell-free depurination assay are identified herein. The selectivity of fragments to RTA was verified and these ligands validated using NMR.

Toxin-specific inhibitors of ricin that target the ribosome-binding site have not been previously identified. These inhibitors reveal previously undescribed ribosome and SRL binding mechanisms and are useful in providing important insights into the mechanism of toxicity. These inhibitors can be used alone or in synergetic combination with immunotherapeutics or vaccines as pre-therapeutics against ricin. These inhibitors can also be used as immunotoxin rescue therapeutics since they can inhibit off-target effects of RTA containing immunotoxins.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods and materials are described. As used herein, each of the following terms has the meaning associated with it in this section.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

A disease or disorder is “alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, pulmonary and topical administration.

A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

The terms “patient,” “subject,” or “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.

As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the disclosure within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the disclosure. Other additional ingredients that can be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, Pa.), which is incorporated herein by reference.

As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids or bases, organic acids or bases, solvates, hydrates, or clathrates thereof. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present disclosure, such as for example utility in process of synthesis, purification or formulation of compounds useful within the methods of the disclosure. Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include sulfate, hydrogen sulfate, hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic, galactaric, galacturonic acid, glycerophosphonic acids and saccharin (e.g., saccharinate, saccharate). Suitable pharmaceutically acceptable base addition salts of compounds of the disclosure include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N′-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

As used herein, the terms “pharmaceutically effective amount” and “effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

As used herein, the term “treatment” or “treating” is defined as the application or administration of a therapeutic agent, i.e., a compound of the disclosure (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a condition contemplated herein, a symptom of a condition contemplated herein or the potential to develop a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein, the symptoms of a condition contemplated herein or the potential to develop a condition contemplated herein. Such treatments can be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term “substantially free of” as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt % to about 5 wt % of the material, or about 0 wt % to about 1 wt %, or about 5 wt % or less, or less than, equal to, or greater than about 4.5 wt %, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt % or less. The term “substantially free of” can mean having a trivial amount of, such that a composition is about 0 wt % to about 5 wt % of the material, or about 0 wt % to about 1 wt %, or about 5 wt % or less, or less than, equal to, or greater than about 4.5 wt %, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt % or less, or about 0 wt %.

The term “organic group” as used herein refers to any carbon-containing functional group. Examples can include an oxygen-containing group such as an alkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group; a carboxyl group including a carboxylic acid, carboxylate, and a carboxylate ester; a sulfur-containing group such as an alkyl and aryl sulfide group; and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, OC(O)N(R)₂, CN, CF₃, OCF₃, R, C(O), methylenedioxy, ethylenedioxy, N(R)₂, SR, SOR, SO₂R, SO₂N(R)₂, SO₃R, C(O)R, C(O)C(O)R, C(O)CH₂C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)₂, OC(O)N(R)₂, C(S)N(R)₂, (CH₂)_0-2N(R)C(O)R, (CH₂)_0-2N(R)N(R)₂, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)₂, N(R)SO₂R, N(R)SO₂N(R)₂, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)₂, N(R)C(S)N(R)₂, N(COR)COR, N(OR)R, C(═NH)N(R)₂, C(O)N(OR)R, C(═NOR)R, and substituted or unsubstituted (C₁-C₁₀₀)hydrocarbyl, wherein R can be hydrogen (in examples that include other carbon atoms) or a carbon-based moiety, and wherein the carbon-based moiety can be substituted or unsubstituted.

As used herein, the term “alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e. C1-6 means one to six carbon atoms) and including straight, branched chain, or cyclic substituent groups. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, and cyclopropylmethyl. A non-limiting example is (C₁-C₆)alkyl, particularly ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term “alkenylene”, employed alone or in combination with other terms, means, unless otherwise stated, a stable mono-unsaturated or di-unsaturated straight chain or branched chain hydrocarbon group having the stated number of carbon atoms wherein the group has two open valencies. Heteroalkenylene substituents can a group consisting of the stated number of carbon atoms and one or more heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be placed at any position of the heteroalkenyl group, including between the rest of the heteroalkenyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkenyl group.

As used herein, the term “alkynylene”, employed alone or in combination with other terms, means, unless otherwise stated, a stable straight chain or branched chain hydrocarbon group with a triple carbon-carbon bond, having the stated number of carbon atoms wherein the group has two open valencies. Heteroalkynylene substituents can a group consisting of the stated number of carbon atoms and one or more heteroatoms selected from the group consisting of O, N, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be placed at any position of the heteroalkynyl group, including between the rest of the heteroalkynyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkynyl group.

As used herein, the term “substituted alkyl” means alkyl as defined above, substituted by one, two or three substituents selected from halogen, —OH, alkoxy, —NH₂, —N(CH₃)₂, —C(═O)OH, trifluoromethyl, —C(═O)O(C₁-C₄)alkyl, —C(═O)NH₂, —SO₂NH₂, —C(═NH)NH₂, and —NO₂, preferably containing one or two substituents selected from halogen, —OH, alkoxy, —NH₂, trifluoromethyl, —N(CH₃)₂, and —C(═O)OH, more preferably selected from halogen, alkoxy and —OH. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

The term “substituted” as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term “functional group” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R)₂, CN, NO, NO₂, ONO₂, azido, CF₃, OCF₃, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)₂, SR, SOR, SO₂R, SO₂N(R)₂, SO₃R, C(O)R, C(O)C(O)R, C(O)CH₂C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)₂, OC(O)N(R)₂, C(S)N(R)₂, (CH₂)_0-2N(R)C(O)R, (CH₂)_0-2N(R)N(R)₂, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)₂, N(R)SO₂R, N(R)SO₂N(R)₂, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)₂, N(R)C(S)N(R)₂, N(COR)COR, N(OR)R, C(═NH)N(R)₂, C(O)N(OR)R, and C(═NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (C₁-C₁₀₀)hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl.

The term “alkenyl” as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, —CH═CH(CH₃), —CH═C(CH₃)₂, —C(CH₃)═CH₂, —C(CH₃)═CH(CH₃), —C(CH₂CH₃)═CH₂, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.

The term “alkynyl” as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to —C≡CH, —C≡C(CH₃), —C≡C(CH₂CH₃), —CH₂C≡CH, —CH₂C≡C(CH₃), and —CH₂C≡C(CH₂CH₃) among others.

The term “acyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is bonded to a hydrogen forming a “formyl” group or is bonded to another carbon atom, which can be part of an alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. An acyl group can include 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. An acyl group can include double or triple bonds within the meaning herein. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning herein. A nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a “haloacyl” group. An example is a trifluoroacetyl group.

The term “cycloalkyl” as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4-2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbornyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term “cycloalkenyl” alone or in combination denotes a cyclic alkenyl group.

The term “aryl” as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof.

The term “aralkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.

The term “heterocyclyl” as used herein refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. A heterocyclyl group designated as a C₂-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C₄-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase “heterocyclyl group” includes fused ring species including those that include fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted as discussed herein. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with groups such as those listed herein.

The term “heteroaryl” as used herein refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C₂-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C₄-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein.

Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N-hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3-anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl), indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl 1,2,3-triazol-4-yl, 1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7-benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3-dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2-benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6-benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3-dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro-benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro-benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1-benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H-dibenz[b,f]azepin-1-yl, 5H-dibenz[b,f]azepine-2-yl, 5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,11-dihydro-5H-dibenz[b,f]azepine (10,11-dihydro-5H-dibenz[b,f]azepine-1-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-3-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-5-yl), and the like.

The term “heterocyclylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein. Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl.

The term “heteroarylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.

The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.

The term “amine” as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)₃wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R—NH₂, for example, alkylamines, arylamines, alkylarylamines; R₂NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R₃N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term “amine” also includes ammonium ions as used herein.

The term “amino group” as used herein refers to a substituent of the form —NH₂, —NHR, —NR₂, —NR₃⁺, wherein each R is independently selected, and protonated forms of each, except for —NR₃⁺, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An “alkylamino” group includes a monoalkylamino, dialkylamino, and trialkylamino group.

The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

The term “haloalkyl” group, as used herein, includes mono-halo alkyl groups, poly-halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, and the like.

As used herein, the term “optionally substituted” means that the referenced group can be substituted or unsubstituted. In certain embodiments, the referenced group is optionally substituted with zero substituents, i.e., the referenced group is unsubstituted. In other embodiments, the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from groups described herein.

In certain embodiments, the substituents are independently selected from the group consisting of halogen, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂, alkyl (including straight chain, branched and/or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoro alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, —S-alkyl, —C(═O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], —C(═O)N[H or alkyl]₂, —OC(═O)N[substituted or unsubstituted alkyl]₂, —NHC(═O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], —NHC(═O)alkyl, —N[substituted or unsubstituted alkyl]C(═O)[substituted or unsubstituted alkyl], —NHC(═O)[substituted or unsubstituted alkyl], —C(OH)[substituted or unsubstituted alkyl]₂, and —C(NH₂)[substituted or unsubstituted alkyl]₂. In other embodiments, by way of example, an optional substituent is selected from oxo, fluorine, chlorine, bromine, iodine, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂, —CH₃, —CH₂CH₃, —CH(CH₃)₂, —CF₃, —CH₂CF₃, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —OCF₃, —OCH₂CF₃, —S(═O)₂—CH₃, —C(═O)NH₂, —C(═O)—NHCH₃, —NHC(═O)NHCH₃, —C(═O)CH₃, and —C(═O)OH. In yet one embodiment, the substituents are independently selected from the group consisting of C₁-6 alkyl, —OH, C₁-6 alkoxy, halo, amino, acetamido, oxo and nitro. In yet other embodiments, the substituents are independently selected from the group consisting of C₁-6 alkyl, C₁-6 alkoxy, halo, acetamido, and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain can be branched, straight or cyclic.

Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

As used herein, the term “type I ribosome inactivating protein” refers to a single polypeptide chain, comprising an A (active) domain capable of inhibiting protein translation.

As used herein, the term “type II ribosome inactivating protein” refers to a heterodimeric species consisting of an A chain, functionally equivalent to that of a type I ribosome-inactivating protein, linked to a B subunit by a disulfide bond, endowed with lectin-binding properties, and capable of inhibiting protein translation.

The following abbreviations are used herein: RIP=ribosome inactivating protein; RTA=active A; Stxs=Shiga toxins; SRL=sarcin-ricin loop; SPR=surface plasmon resonance; and FBLD=Fragment-based lead discovery.

Compounds and Compositions

In certain embodiments, the compound has the structure of Formula (I), or a salt, solvate, stereoisomer, geometric isomer, and/or tautomer thereof:

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wherein:

A is a bond or an optionally substituted C₁-C₂linker selected from the group consisting of optionally substituted C₁-C₂alkylene, —CH═CH—, —C≡C—,

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- wherein, if present, the C₁-C₂alkylene is optionally substituted with at least one substituent selected from the group consisting of hydroxyl, C₁-C₆alkoxy, N(R^a)(R^a), and halogen,
- wherein * indicates the bond from A to the 5-membered ring;

B is selected from the group consisting of a bond,

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- wherein ** indicates the bond from B to R²;

R¹is selected from the group consisting of H, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted benzyl, optionally substituted C₁-C₆alkyl, optionally substituted C₃-C₆cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted C₂-C₆alkenyl, optionally substituted C₂-C₆alkynyl, C(O)—C₁-C₆alkyl, C(O)-aryl, C(O)NR^a₂, cyano, and halogen,

- wherein each optional substituent comprises at least one substituent selected from the group consisting of C₁-C₆alkyl, C₂-C₆alkenyl, C₁-C₆cycloalkyl, C₁-C₆alkoxy, hydroxyl, NR^a₂, C(O)—C₁-C₆alkyl, CN, CF₃, NO₂, and C(O)-aryl,
  - wherein two adjacent optional C₁-C₆alkyl, C₁-C₆cycloalkyl, C₁-C₆alkoxy, and C(O)—C₁-C₆alkyl substituents may optionally combine to form a 5 or 6-membered fused ring,
  - wherein each optionally substituted aryl, optionally substituted heteroaryl, optionally substituted benzyl, optionally substituted C₁-C₆alkyl, optionally substituted C₂-C₆alkenyl, or optional substituent thereof, may optionally combine with X²to form a 5, 6, or 7-membered fused ring;

R²is selected from the group consisting of

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each occurrence of R^ais independently selected from the group consisting of H, C₁-C₆alkyl, C₁-C₆alkoxy, benzyl, and aryl;

R^bis selected from the group consisting of H, C₁-C₆alkyl, C₁-C₆alkoxy, benzyl, aryl, hydroxyl, and C₁-C₆hydroxyalkyl;

X¹is selected from the group consisting of S, O, and NR^a;

X²is CH or N;

each occurrence of X³is independently O or S;

each occurrence of Y is independently CH or N, wherein 0-3 Y are N in a given ring.

In certain embodiments, the compound of Formula (I) is the compound of Formula (II):

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wherein:

R³is selected from the group consisting of C₁-C₆alkyl, C₂-C₆alkenyl, C₁-C₆cycloalkyl, C₁-C₆alkoxy, hydroxyl, NR^a₂, C(O)—C₁-C₆alkyl, CN, CF₃, NO₂, and C(O)-aryl;

- L¹and L²are each independently a bond or optionally substituted C₁-C₂alkyl.

In certain embodiments, the compound of Formula (I) is not 4-(thiophen-2-ylmethyl)benzoic acid. In certain embodiments, the compound of Formula (I) is 4-(thiophen-2-ylmethyl)benzoic acid.

In certain embodiments, A is a bond. In certain embodiments, A is optionally substituted C₁-C₂alkylene. In certain embodiments, A is optionally substituted —CH═CH—. In certain embodiments, A is —CH═CH—. In certain embodiments, A is —C≡C—. In certain embodiments, A is

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In certain embodiments, A is

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In certain embodiments, A is

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In certain embodiments, A is

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In certain embodiments, A is

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In certain embodiments, B is a bond. In certain embodiments, B is

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In certain embodiments, B is

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In certain embodiments, B is

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In certain embodiments, R²is

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In certain embodiments, R²is

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In certain embodiments, R²is

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In certain embodiments, R²is

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In certain embodiments, R²is

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In certain embodiments, R¹is CN. In certain embodiments, R¹is Br. In certain embodiments, R¹is Me. In certain embodiments, R¹is Et. In certain embodiments, R¹is Ph. In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R¹is

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In certain embodiments, R is

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In certain embodiments, R¹is

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In certain embodiments R²is

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In certain embodiments, R²is

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In certain embodiments, R²is

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In certain embodiments, R²is

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In certain embodiments, R²

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In certain embodiments, R²is

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In certain embodiments, R²is

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In certain embodiments, R²is

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In certain embodiments, A is a bond.

In certain embodiments, A is

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In certain embodiments A is

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In certain embodiments, A is

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In certain embodiments, A is

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In certain embodiments, A is

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In certain embodiments, A is

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In certain embodiments, A is

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In certain embodiments, A is

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In certain embodiments, A is

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In certain embodiments, A is

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In certain embodiments, A is

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In certain embodiments, B is a bond. In certain embodiments, B is

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In certain embodiments, B is

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In certain embodiments, R³is F. In certain embodiments, R³is Br. In certain embodiments, R³is Me. In certain embodiments, R³is NMe₂. In certain embodiments, R³is OMe.

In certain embodiments, L¹is a bond. In certain embodiments, L¹is —CH₂—.

In certain embodiments, L²is —CH₂—. In certain embodiments, L²is —CH₂CH₂—.

In certain embodiments, the compound is selected from the group consisting of:

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In certain embodiments, the compound is selected from the group consisting of:

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In certain embodiments, the compound is selected from the group consisting of:

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