Benzaldehyde compounds with direct polymer destabilizing effects for treating sickle cell disease

Abstract

The present invention relates to compounds and methods for preventing and / or treating one or more symptoms of sickle cell disease (SCD) by administering at least one compound that has been chemically modified to improve bioavailability and activity, for example, to prevent red blood cell (RBC) adhesion and sickling.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 63 / 423,167, filed November 7, 2022, and U.S. Patent Application No. 63 / 442,824, filed February 2, 2023, the entire contents of which are incorporated herein by reference.

[0002] Federally Sponsored Research and Development Statement This invention was made with government support under Contract Nos. H1156158, H1154864, and Md009124 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0003] Technical Field The present invention relates primarily to compounds that exhibit enhanced activity and safety in the treatment of sickle cell disease (SCD), particularly aromatic aldehydes that exhibit highly potent polymer destabilizing activity. [Background technology]

[0004] Related technologies Hemoglobin (Hb) transports oxygen in the blood and releases it to tissues. It functions by balancing two allosteric states: a tense state (T state) with low oxygen affinity and a relaxed state (R state) with high oxygen affinity. Sickle cell disease (SCD) is an inherited blood disorder caused by the substitution of βGlu6 with βVal6 in Hb, resulting in the formation of sickle hemoglobin (HbS). Under conditions of hypoxia or reduced oxygen tension, the concentration of low-affinity deoxygenated Hb (T state) increases, and HbS polymerizes into long, rigid, insoluble fibers, resulting in sickling of red blood cells (RBCs). Polymerization, initiated by a primary interaction with βVal6, is stabilized by multiple secondary contacts between HbS molecules. Hypoxia-induced sickling leads to secondary pathophysiological effects such as erythrocyte adhesion to the endothelium, oxidative stress and damage, erythrocyte hemolysis, inflammation, vascular obstruction, impaired microvascular blood flow, and reduced intravascular bioavailability of nitric oxide, ultimately leading to severe morbidity such as severe pain crises, stroke, renal failure, pulmonary hypertension, and premature death. Until the recent US FDA approval of Voxelotor (trade name: Oxbryta), the only oral medications approved for the treatment of SCD were hydroxyurea (HU) and L-glutamine (Endari). TM ) was the only treatment available. HU induces γ-globin expression, forming fetal hemoglobin (HbF). HbF can directly inhibit HbS polymerization and red blood cell sickling; however, not all patients respond to HU, and its effectiveness is limited. Furthermore, HU can cause bone marrow suppression, a life-threatening side effect. L-glutamine has antioxidant properties that may reduce the frequency of analgesic attacks, but its efficacy is limited and it is not widely used in clinical practice. Given the significant morbidity, mortality, healthcare disparities, and public health burden caused by SCD, the development of more effective oral medications is urgently needed.

[0005] Aromatic aldehydes, such as Voxelotor, bind to the α-cleft of Hb and shift the allosteric equilibrium of HbS toward the high-affinity R state, which prevents polymerization. Allosteric modulators have clinical benefits for patients with SCD. In the phase III HOPE trial, Voxelotor reduced indicators of hemolysis and improved anemia by increasing Hb concentrations by at least 1 g / dL in most patients. However, Voxelotor's disease-modifying clinical effects were limited, and signs of chronic inflammation and hemolysis persisted. For example, there was no significant reduction in vaso-occlusive events. The primary physiological effect of allosteric modulation is HbS dilution. Drug-bound oxygenated HbS tetramers are not incorporated into polymers, thereby reducing the number of polymerizable Hb tetramers. However, the magnitude of clinical benefit from HbS dilution is limited, and patients taking Voxelotor experience significant residual disease sequelae that affect quality and life expectancy. Furthermore, the on-target pharmacodynamic effects of drugs on the allosteric state of Hb are limited by the direct dose, as further increases in oxygen affinity limit oxygen release to tissues and ultimately lead to clinical signs of tissue hypoxia. Drugs that can directly inhibit HbS polymerization may offer more effective disease-modifying effects. For example, individuals with a rare co-inherited condition in which HbF levels remain above 25% until adulthood are known to exhibit little clinical disease. HbF directly destabilizes polymerization by interfering with critical lateral contacts. Similarly, individuals with the rare co-inherited Hb mutation, Hb Stanleyville, which disrupts polymer-stabilizing contacts through lysine mutations on the surface of the αF-helix of HbS, also exhibit a clinically benign phenotype and are virtually disease-free.

[0006] A number of aromatic aldehydes have been discovered that directly inhibit HbS polymerization. These compounds generally contain a benzaldehyde ring with a second ring structure (pyridine or benzene) attached via a methoxy bridge at the ortho position of the aldehyde group. The methoxy bridge ortho to the aldehyde group of the benzaldehyde allows the second ring structure to extend toward the opening of the α-cleft, enabling it to interact with surface residues on the αF-helix of HbS. Compounds that strongly interact with key residues on the αF-helix can destabilize HbS polymers by disrupting secondary contacts that stabilize polymerization. An example of a previously discovered compound is INN-310 (SAJ-310), which has a methoxy group meta to the aldehyde on the benzaldehyde ring and no substituent on the pyridine ring. INN-310 exhibited very weak polymer destabilizing effects in vitro, likely due to limited interactions with αF-helix residues, and also exhibited suboptimal pharmacokinetic properties in vivo. Further examples of potent polymer-destabilizing compounds with improved pharmacokinetic properties include VZHE-039 and PP-14. While INN-310 has a meta-position methoxy group, VZHE-039 and PP-14 possess ortho-position hydroxyl groups on the benzaldehyde, which form intramolecular interactions that stabilize the aldehyde and protect it from rapid oxidative metabolism. Furthermore, VZHE-039 and PP-14 each contain additional chemical substituents on the second ring that can form strong hydrogen bonds and hydrophobic interactions with the αF-helix. As expected, these interactions significantly improved the potency of the polymer destabilizing effect. However, to date, the introduction of chemical substituents on the second ring has presented significant drawbacks that have hindered their development as viable human drug candidates. Substituents on the second ring can be metabolically unstable (carboxyesters or carboxyamides), charged species that may reduce membrane permeability (carboxylic acids), or hepatotoxic (methyl hydroxyls).To date, hundreds of compounds have been investigated that incorporate a wide variety of chemical substituents on the second ring to target the αF-helix.Nevertheless, compounds that have potent polymer destabilizing properties and can safely and reliably reach the therapeutic levels required for chronic oral treatment of SCD have yet to be discovered.

[0007] There is an urgent need to develop novel, non-toxic anti-sickle cell compounds that exhibit more potent and sustained polymer destabilizing effects, have a longer duration of pharmacological action, and have sufficient oral bioavailability to be suitable for oral administration. Summary of the Invention

[0008] Other features and advantages of the present invention will be set forth in the description of the invention that follows, and in part will be obvious from the description, and in part will be learned by the practice of the invention. The present invention will be realized and attained by the compositions and methods particularly pointed out in the specification and claims. It is an object of the present disclosure to provide compounds having the formula: [ka] wherein W, X, Y, and Z may be the same or different and are independently C or N, and at least one of W, X, Y, and Z is N; R1, R2, R3 and R4 may be the same or different and are independently F, Cl or H, as well as pharmaceutically acceptable salts or prodrugs thereof.

[0009] In one embodiment of the invention, the compound [ka] is.

[0010] In other embodiments, the compound is [ka] is.

[0011] In yet another embodiment, the compound is [ka] is.

[0012] In yet another embodiment, at least one of R1, R2, R3, and R4 is independently F or Cl. [ka] is.

[0013] In another embodiment, the compound is [ka] is.

[0014] Also provided are methods for preventing or treating one or more symptoms or conditions of sickle cell disease (SCD), comprising administering to a subject in need thereof a therapeutically effective amount of at least one compound having the following formula: [ka] wherein W, X, Y, and Z may be the same or different and are independently C or N, and at least one of W, X, Y, and Z is N; and R1, R2, R3, and R4 may be the same or different and are independently F, Cl, or H. In one embodiment of the invention, the one or more symptoms or conditions are selected from the group consisting of red blood cell (RBC) sickling, adhesion of red blood cells to tissue endothelium, oxidative stress and / or damage, hemolysis of red blood cells, inflammation, vascular occlusion, impaired microvascular blood flow, stroke, pain, and death. In yet another embodiment, the step of administering is performed orally. [Brief explanation of the drawings]

[0015] [Figure 1] Time-dependent sickling under anoxic conditions. [Figure 2] Occupancy-dependent p50 shift in human non-sickle blood. [Figure 3AB] Crystal structure of Hb in the R2 conformation in complex with two molecules of compounds 1, 2, 5, and 8 bound to the α-cleft. Hb is shown as a cartoon, compounds 1, 2, 5, and 8 as stick figures, F-helix residues as stick figures, and water molecules as spheres. [Figure 3CD] Same as above [Figure 3EF] Same as above [Figure 3G] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0016] The benzaldehyde compounds disclosed herein unexpectedly exhibit potent and persistent polymer destabilization and allosteric modulation of HbS's affinity for oxygen, despite the lack of chemical substituents in the second ring (pyridine or benzene) that directly interact with αF-helix residues. These compounds lack a carboxyl ester, carboxyl amide, methyl hydroxyl, or similar pharmacophore in the second ring that could form hydrogen bonds with surface residues of the αF-helix. Unexpectedly, however, these compounds form very strong interactions with αF-helix residues through either entirely water-mediated interactions via the pyridine, or alternatively, pyrimidine or triazine nitrogen, or entirely hydrophobic interactions via the benzene ring. Even more surprisingly, the direct polymer destabilization of some compounds in this group is the most potent yet observed, demonstrating sustained inhibition of erythrocyte sickling in vitro for over 150 minutes. This result was unexpected because previously discovered compounds, such as INN-310, lacking additional chemical substituents on the second ring to interact with the αF-helix, had very weak polymer-destabilizing activity, presumably due to the lack of hydrogen-bonding interactions. Therefore, it is a surprising discovery that the compounds of the present invention are not only potent but are the most potent ever discovered. This unexpected result likely arises from the fact that replacing the meta-methoxy group on the benzaldehyde ring of INN-310 with an ortho-hydroxyl group alters the orientation of the compound within the α-cleft of Hb, serendipitously shifting the second ring structure to a position where it can form strong interactions with αF-helix residues without the need for any chemical substituents. This is supported by the fact that replacing the ortho-hydroxyl group with a halogen completely abolished the polymer-destabilizing activity of the compound. This also explains why TD-7, an analog of VZHE-039 that, like INN-310, has a meta-methoxy group on the benzaldehyde ring, exhibited poor in vitro activity.Surface residues of the αF-helix of HbS are important for stabilizing polymers through secondary interactions with neighboring HbS molecules. It is well known and widely accepted that the Stanleyville mutant, an HbS variant with a cognate mutation (αAsn78 ⇔ αLys78) on the surface of the αF-helix, is restricted in polymerization. Without being bound by theory, it is believed that the compounds of the present invention interact with a site located on the surface of the αF-helix of HbS, and that this interaction stereospecifically inhibits polymer formation by HbS. In other words, binding of these ligands to the αF-helix induces a conformational change that disrupts the αF-helix and eliminates interactions between HbS molecules, thereby weakening polymer stability and preventing sickling. While it was previously believed that compounds required hydrogen-bonding functional groups, such as esters, amides, methyl hydroxyls, charged sulfates, or phosphates, to achieve the desired pharmacodynamic effects, the present invention demonstrates that highly potent interactions can be formed entirely through water-mediated or hydrophobic interactions. Furthermore, in addition to their surprising activity, this group of compounds of the present invention lacks the negative side effects observed in previous compounds with additional functional groups in the second ring, such as VZHE-039 and PP-14. They exhibit rapid distribution to erythrocytes, good oral bioavailability, excellent pharmacokinetics, and no evidence of toxicity. In some variants of these compounds, hydrogens on the benzaldehyde or second ring structure are replaced with one or more halogens. While these halogens do not interact with the αF-helix residues, they may modify the metabolic pathway of the compounds by limiting their interaction with metabolic enzymes and eliminating potentially toxic metabolic intermediates.

[0017] The compounds disclosed herein are benzaldehydes that contain a second ring (e.g., pyridine, pyrimidine, etc.) without any hydrogen bond donor / acceptor groups or substituents, such as ester, amide, hydroxyl, sulfate, or phosphate groups, and that interact directly with the αF helix of sickle hemoglobin (HbS). Binding of the compounds to the αF helix of HbS prevents or reduces interactions between HbS molecules, thereby preventing red blood cell (RBC) sickling.

[0018] These compounds have the following general formula I: [ka] wherein W, X, Y, and Z may be the same or different and are independently C or N, and at least one of W, X, Y, and Z is N; and R1, R2, R3, and R4 may be the same or different and are independently halogen or H.

[0019] Exemplary halogens include, but are not limited to, F and Cl.

[0020] Exemplary compounds include, but are not limited to, those having the following formula: [ka] wherein W, X, Y and Z may be the same or different and are independently C or N, and at least one of W, X, Y and Z is N.

[0021] Compounds of this type include, but are not limited to: [ka]

[0022] Additionally, additional representative compounds having the following general formula: [ka] wherein W, X, Y, and Z may be the same or different and independently represent C or N, and at least one of W, X, Y, and Z is N; R1, R2, R3, and R4 may be the same or different and independently represent halogen or H, and at least one of R1, R2, R3, and R4 is halogen. Exemplary halogens include, but are not limited to, F and Cl. Examples of these additional compounds include, but are not limited to: [ka] Pharmaceutically acceptable salts and oral prodrugs of these compounds are also included. Examples of suitable promoieties for oral prodrugs include, but are not limited to, thiazolidins, which replace the aldehyde group to form the prodrug.

[0023] Exemplary Compounds Exemplary compounds of the present invention include, but are not limited to, the following: [ka] JPEG2025537075000015.jpg160170

[0024] These compounds rapidly partition into red blood cells (RBCs) and bind to the N-terminal valine amine in the α-cleft of HbS via a Schiff base interaction between the aldehyde moiety and the N-terminal αVal1 nitrogen of relaxed hemoglobin. Mechanistically, the Schiff base interaction stabilizes the relaxed (R) state, increasing the oxygen affinity of hemoglobin and resulting in anti-sickling activity. Like all aldehydes, the aldehyde moiety of these compounds is susceptible to rapid metabolism, e.g., by aldehyde oxidase, which can convert it to an inactive carboxylate derivative, potentially shortening the duration of the compound's pharmacological action. Consistently, the aromatic aldehyde anti-sickling agent 5-HMF was discontinued in human clinical trials due to its metabolic instability. In one embodiment, the aldehyde group of the compounds presented herein is protected via an intramolecular bond with the adjacent ortho hydroxyl. This ortho hydroxyl also fortuitously positions the second ring for interaction with the F helix.

[0025] pharmaceutically acceptable salts Pharmaceutically acceptable salts of the compounds of the present invention are also encompassed. Examples of such salts include, but are not limited to, salts with inorganic acids (e.g., hydrochloric acid (HCl), hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.) and salts with organic acids (e.g., acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, and polygalacturonic acid). Other salts include pharmaceutically acceptable quaternary salts known to those skilled in the art, particularly those of the formula: --NR + Z ---wherein R represents hydrogen, alkyl, or benzyl, and Z is a counterion including chloride, bromide, iodide, --O-alkyl, toluenesulfonate, methanesulfonate, sulfonate, phosphate, or carboxylate (e.g., benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamate, mandelate, benzilate, and diphenylacetate).

[0026] Oral Prodrugs Oral prodrugs of the present compounds are also included. Oral prodrugs can be used, for example, to improve oral formulation and / or oral bioavailability, or to improve absorption characteristics and gastrointestinal tolerance. Examples of such prodrugs include, but are not limited to, prodrugs formed by replacing the aldehyde group with a promoiety such as thiazolidine, imine, acetal, hemiacetal, ester, or alcohol.

[0027] In some embodiments, the compound is administered as a prodrug, including but not limited to: [ka] wherein W, X, Y, and Z may be the same or different and are each independently C or N; at least one of W, X, Y and Z is N; R1, R2, R3 and R4 may be the same or different and are each independently F, Cl or H; and P is a promoiety, such as a thiazolidine, imine, acetal, hemiacetal, ester, or alcohol.

[0028] In some embodiments, the promoiety (P) is [ka] wherein A and B may be the same or different and each independently represent O, NH, S, OCH, NHCH, or SCH; R and R may be the same or different and each independently represent COOH, COOR, CHOH, COCH, or -(CH) n CH3, where n is 0-5, and R7 is alkyl, aryl, or heterocycle; the bond marked with an * is attached directly to a carbon of the benzene ring.

[0029] In some embodiments, R5 and R6 can form a ring as follows: [ka] Here, the ring structure is a 3- to 8-membered ring, which may or may not be substituted.

[0030] In some embodiments, the compounds may be administered as prodrugs, including, but not limited to: [ka]

[0031] An example synthetic scheme for producing the prodrugs disclosed herein is as follows: [ka] wherein W, X, Y, and Z may be the same or different and independently represent C or N, and at least one of W, X, Y, and Z is N. R1, R2, R3, and R4 may be the same or different and independently represent F, Cl, or H.

[0032] A typical synthesis method involves condensing benzaldehyde 1 with an equimolar amount of L-cysteine ​​ethyl ester 2 in the presence of N-ethyldiisopropylamine hydrochloride (EDA) or triethylamine (NET3) to give the ethyl ester prodrug 3. The ethyl ester protecting group of 3 is hydrolyzed with sodium hydroxide to give the acid prodrug 4.

[0033] Advantages of the compound The compounds described herein exhibit improved pharmacological activity, i.e., greater potency and / or extended (e.g., longer duration) half-life, and / or increased bioavailability under physiological conditions (e.g., 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more potency in the blood circulation compared to other benzaldehyde compounds). For example, the compounds exhibit a half-life in vivo of at least about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, or 12.0 hours or more (e.g., about 12-60 hours, i.e., about 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, or 60 hours). In certain embodiments, the compounds exhibit a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or greater enhancement in efficacy in preventing red blood cell (RBC) sickling and / or HbS polymer formation compared to vanillin or TD-7. In certain embodiments, the compound exhibits 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or greater potency in polymer destabilization or direct inhibition of HbS polymer formation compared to INN-310 and TD-7; Voxelotor does not exhibit direct inhibition of HbS polymer formation. In certain embodiments, the compound can be administered at clinically relevant blood concentrations without toxicity (e.g., hepatocyte hypertrophy or apoptosis) compared to VZHE-039. In certain embodiments, the compound is metabolically unstable and metabolized to a charged species (e.g., a carboxylic acid) and does not contain substituents that reduce the permeability of erythrocyte membranes compared to PP-14. Thus, in certain embodiments, compositions containing the compound can be administered less frequently and / or at lower doses compared to prior art compounds, while still achieving comparable or greater beneficial effects in subjects administered the compound. In certain embodiments, the compound can be administered to clinically effective levels without causing toxicity.

[0034] Examples of treatment methods using this compound The compounds described herein are used to treat or prophylactically treat diseases and / or conditions associated with SCD (Sickle Cell Disease) and other diseases / conditions described below. As used herein, the terms "prophylactically treat" (e.g., "prophylactic treatment," "prophylactically treating") and "prevent" (e.g., "prevention," "preventing") refer to avoiding or inhibiting the onset of at least one symptom of a disease or unwanted condition (e.g., at least one symptom of SCD), which may be accomplished by prophylactically administering to a subject in need thereof a composition comprising at least one compound described herein. Generally, the terms "prophylactic" or "prophylaxis" relate to reducing the likelihood that a subject will develop a disease or its symptoms. Typically, a subject is considered by those skilled in the art to be at risk or susceptible to developing at least one symptom of the disease or undesirable condition, or to have a high likelihood of developing at least one symptom of the disease / condition in the absence of medical intervention. In one embodiment, "prevention" or "prophylactic treatment" is performed when the subject does not yet have symptoms of the disease (condition, disorder, syndrome, etc.; unless otherwise specified, these terms are used interchangeably herein) or when symptoms are not yet known or confirmed to be present. In other words, symptoms may not yet be evident or observable, or may be very "early stage" symptoms. A subject may be considered at risk due to a variety of factors, including, but not limited to, genetic predisposition, the presence of "early" symptoms, test results such as blood tests, etc. In such embodiments, treatment of a subject may prevent adverse or deleterious effects or outcomes of a fully developed disease."Prevention" or "prophylactic treatment" may include preventing the onset of observable symptoms of a disease or condition altogether, or may include reducing or alleviating the degree, severity, or duration of at least one symptom of the disease compared to what would occur in the absence of the medical intervention provided herein.

[0035] The term "treating" (e.g., "treatment," "therapeutic treatment"), as used herein, refers to administering a composition containing at least one compound to a subject already exhibiting at least one symptom of a disease, such as SCD. In other words, at least one indicator known to be associated with the disease has been measured, detected, experienced, or observed in the subject. For example, the symptom may be the primary pathophysiology of red blood cell (RBC) sickling associated with sickle cell disease. Additionally, the compounds disclosed herein ameliorate a range of secondary adverse events in SCD, including adhesion of red blood cells to tissue endothelium, oxidative stress, hemolysis of red blood cells, reduced intravascular nitric oxide (NO) bioavailability, vascular occlusion, impaired microvascular blood flow, elevated blood pressure, stroke, and painful crises resulting from, for example, red blood cell polymerization. For example, such compounds generally do one or more of the following: increase the O2 affinity of HbS; inhibit fibrogenesis; reduce the mechanical fragility of sickle red blood cells; reduce red blood cell hemolysis; attenuate hypoxia-induced cell necrosis and apoptosis; improve microvascular function (e.g., during recovery from the sequelae of SCD or hemorrhagic shock); improve hemodynamics and oxygenation during hypoxic conditions (e.g., maintaining blood pressure and heart rate, preserving microvascular blood flow, reducing hypoxic areas in the heart and brain); reduce the frequency of pain or pain crises; decrease lactate dehydrogenase (LDH) and / or red blood cell hemolysis; lower diastolic blood pressure; increase blood oxygen concentration (SpO2) during hypoxic stress, etc.

[0036] "Treatment" includes reducing or alleviating, and in some cases completely eliminating, at least one symptom of a disease that was present before or at the time of administration of the composition.

[0037] Exemplary Compositions and Methods of Administration Provided herein are compositions containing at least one compound described herein and methods of administering the same to treat, for example, SCD (sickle cell disease), hypoxia, and the like. Practice of these methods generally involves identifying a patient suffering from or at risk of developing a disease or condition described herein (e.g., SCD or hypoxia) and administering a composition described herein by an appropriate route. The exact dose administered may vary depending on the individual patient's age, sex, weight, and general health, the severity of disease symptoms, other treatments the patient is receiving, and the progression or extent of the disease state and its precise etiology being treated. However, generally, for administration to a mammal (e.g., a human), a sufficient amount of the composition is administered to achieve a dose in the range of about 0.1 to 1000 mg or more per kg of body weight per 24 hours. Examples of doses include about 1 to 500 mg, 5 to 100 mg, or 10 to 50 mg per kg of body weight per 24 hours. Generally, a therapeutically effective dose is approximately 20-150 mg per kg of body weight per 24 hours. Dosage will vary depending on the route of administration, bioavailability, the particular formulation being administered, and the nature of the condition being prevented or treated.

[0038] The compositions generally contain suitable excipients, elixirs, binders, etc. (collectively, "pharmaceutically and physiologically acceptable carriers") and are administered in a pharmaceutically acceptable formulation that is compatible with the active ingredient. The prodrug or derivative may be present in the formulation in the form of a pharmaceutically acceptable salt (e.g., alkali metal salts such as sodium, potassium, calcium, lithium, etc., ammonium salts, etc.) or other complexes. It should be understood that pharmaceutically acceptable formulations herein generally include solid, semi-solid, or liquid materials used to prepare dosage forms such as solids, semi-solids, and liquids (e.g., tablets, capsules, liquids, aerosol formulations, various injectable dosage forms (e.g., for intravenous administration), etc.). Suitable pharmaceutical carriers include, but are not limited to, inert solid diluents or fillers, sterile aqueous solutions, various organic solvents, etc. Examples of solid carriers (diluents, excipients) include lactose, starch, conventional disintegrants, coating agents, terra alba (earth alba), sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, lower alkyl ethers of cellulose, etc. Examples of liquid carriers include, but are not limited to, various aqueous or oily media, saline, glucose, water, glycerol, ethanol, isopropanol, phosphate buffer, syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, isopropyl myristate, ethyl cocoate, octyl cocoate, polyoxyethylated hydrogenated castor oil, paraffin, liquid paraffin, propylene glycol, celluloses, parabens, stearyl alcohol, polyethylene glycol, phenoxyethanol, etc., or mixtures thereof. Water may be used as a carrier for the composition, although conventional buffers and agents for adjusting the tonicity of the composition may also be included. Formulations for oral administration may contain various thickeners, flavorings, diluents, emulsifiers, dispersing aids, binders, coating agents, etc. The compositions of the present disclosure may contain any of these additional ingredients to make them suitable for the desired route of administration. Furthermore, the compositions may contain minor amounts of auxiliary ingredients such as wetting agents, emulsifiers, pH buffering agents, etc.Similarly, the carrier or diluent may include sustained-release materials known to those skilled in the art (e.g., glycerol monostearate or glycerol distearate, alone or mixed with wax). Other usable additives and substances, preferably those generally recognized as safe (GRAS), include colorants, flavorings, surfactants (e.g., TWEEN®, oleic acid), solvents, stabilizers, binders, and encapsulating agents (e.g., lactose, liposomes). Preservatives such as methylparaben and benzalkonium chloride may also be used. Depending on the formulation, the active ingredient (e.g., at least one prodrug or derivative) is expected to comprise from about 1% to about 99% of the composition, and the carrier from about 1% to about 99%. The pharmaceutical compositions of the present disclosure may contain suitable pharmaceutically acceptable additives and adjuvants, so long as they do not interfere with or hinder the therapeutic efficacy of the composition. Further examples of other suitable formulations that can be used in the present disclosure are described, for example, in Remington's Pharmaceutical Sciences, 22nd Edition, edited by Allen, Loyd V., Jr (September 2012) and Akers, Michael J., "Sterile Drug Products: Formulation, Packaging, Manufacturing and Quality," Informa Healthcare (2010).

[0039] The compositions (formulations) of the present disclosure are formulated to be administered by many suitable means commonly known to those skilled in the art, including, but not limited to, oral, injection, rectal, inhalation, intravaginal, intranasal, transdermal, ophthalmic, spray, transdermal, sublingual, rectal and buccal administration, inhalation of aerosols, administration by microneedle, etc. In one embodiment, the route of administration is oral, injection, or intravenous, preferably by tablet administered orally.

[0040] The administration of the compounds of the present disclosure may be intermittent, or may be gradual, continuous, constant, or controlled (for example, by sustained-release formulations that further extend the duration of bioavailability). Furthermore, the time of administration of the pharmaceutical formulation and the number of times administered per day may vary and are best determined by a skilled medical professional, such as a physician. Generally, the compounds are administered at least once a day, and may be administered, for example, twice, three times, four times, or more times a day. In critical situations, the administration frequency may be higher.

[0041] Administration of the compound by either means may be performed as a sole therapy or in combination with other therapies or treatments, such as antibiotics, analgesics, hydroxyurea, vaccinations, blood transfusions, oxygen supplements, gene therapy, nitric oxide, drugs that promote fetal hemoglobin production, statins, etc. Furthermore, if other coexisting conditions (e.g., cardiac disease) are included as sequelae of the disease, additional treatments for cardiac disease, including surgery, may be administered. Other treatment options include various nutraceuticals, dietary modifications, exercise modifications, etc. The term "in conjunction with" refers both to the administration of one or more additional components as a separate formulation and to the inclusion of one or more additional components in a composition of the present disclosure.

[0042] The subject to which the compositions are administered is generally a mammal, often, but not always, a human. Veterinary applications of the technology are also contemplated, including companion animals (e.g., cats, dogs, etc.), domestic and farm animals, horses, and even "wild" animals under veterinary care that have special value or require special care (e.g., animals in sanctuaries or zoos, injured animals undergoing rehabilitation, etc.).

[0043] Diseases and conditions to be treated In one embodiment, the disease or condition to be prevented or treated as described herein is sickle cell disease (SCD) and pathophysiology associated with SCD, such as sickling of red blood cells (RBC).Furthermore, the compound improves a series of secondary adverse events, such as adhesion of red blood cells to tissue endothelium, oxidative stress, hemolysis of red blood cells, reduced intravascular NO bioavailability, vascular occlusion, impaired microvascular blood flow, elevated blood pressure, stroke and pain crisis.In addition, the compound also has the effects of increasing the oxygen affinity of HbS, inhibiting fibrogenesis, reducing the mechanical fragility of sickle red blood cells, and inhibiting hemolysis of red blood cells.

[0044] In another embodiment, the compounds may be used to treat or prevent symptoms of hypoxia, whether associated with SCD or not. As used herein, "hypoxia" (also known as hypoxia) refers to a lack of adequate oxygen supply throughout the body or at the tissue level in a particular area of ​​the body. Hypoxia can be classified as either "systemic" or "local," with the former affecting the entire body and the latter affecting a specific area of ​​the body. There are four types of hypoxia: (1) hypoxemia, in which the partial pressure of oxygen in the blood flowing to tissues is low and insufficient to saturate hemoglobin; (2) anemia, in which the amount of functional hemoglobin is too low, reducing the blood's oxygen-carrying capacity; (3) stasis, in which blood is normal but blood flow to tissues is reduced or unevenly distributed; and (4) histotoxicity, in which tissues or cells are damaged by toxins and cannot properly utilize oxygen. Diseases of the blood, heart and circulatory system, and lungs can all cause hypoxia in some form.

[0045] Systemic hypoxia can occur, for example, when healthy individuals ascend to high altitudes, causing altitude sickness and potentially fatal complications such as high-altitude pulmonary edema (HAPE) and high-altitude cerebral edema (HACE). Hypoxia can also occur in healthy individuals when inhaling gas mixtures with low oxygen concentrations, such as during underwater diving or space travel. This is particularly true when using closed-circuit rebreather systems, which regulate the amount of oxygen in the air being delivered. Hypoxia can also occur in premature infants due to immature lung development. Hypoxia resulting from ischemia (insufficient blood flow to organs and tissues) is called ischemic hypoxia and can be induced, for example, by embolic events, myocardial infarctions that reduce systemic blood flow, tissue trauma, or intentionally by medical procedures such as stent placement or tourniquet application. Conditions such as peripheral vascular disease can also cause localized hypoxia. Other causes include altered respiratory drive (e.g., respiratory alkalosis), physiological or pathological shunting of blood, diseases interfering with pulmonary function (causing a mismatch between ventilation and perfusion), and pulmonary embolism. Alterations in the environment or oxygen tension in the alveoli can also contribute. A lack of hemoglobin can lead to anemia, which can reduce tissue perfusion and cause "anemic hypoxia." Carbon monoxide poisoning can also cause hypoxia, for example, acute poisoning from smoke inhalation or chronic exposure to smoking or smog. Formation of methemoglobin, resulting from odorless asphyxiating gases such as nitrogen and methane, cyanide poisoning, or ingestion of sodium nitrite or other drugs or chemicals, can also cause hypoxia. The compounds described herein are used to prevent or treat one or more symptoms of these hypoxia-related conditions. In addition, the compounds inhibit hypoxia-induced cell necrosis and apoptosis, improve microvascular function during resuscitation from hemorrhagic shock, and have beneficial effects on hemodynamics and oxygenation in hypoxic conditions (e.g., maintenance of blood pressure and heart rate, preservation of microvascular blood flow, reduction of hypoxic areas in the heart and brain, etc.).In addition, several clinical symptoms improve, including: reduced pain, decreased lactate dehydrogenase (LDH) and / or decreased red blood cell hemolysis, decreased diastolic blood pressure, and increased blood oxygen saturation (SpO2) during hypoxia testing.

[0046] Compound manufacturing method A general synthetic scheme for preparing the compounds disclosed herein is shown in Scheme I below. Briefly, substituted hydroxybenzaldehyde was reacted with substituted chloromethylpyridine or substituted bromomethylpyridine under basic conditions at room temperature for 2 hours, followed by heating at 60°C for 4 to 6 hours to obtain compounds 1 to 13 in yields of 9 to 47%. [ka]

[0047] It is to be understood that the present invention is not limited to the particular embodiments described above and below, which may, of course, vary, and that the terminology used herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0048] Unless the context or description clearly indicates otherwise, when a range of numerical values ​​is given, it is understood that all intermediate values ​​between the upper and lower limits of that range (to the nearest tenth of the unit of the lower limit) are included in the range and encompassed by the present invention. Furthermore, it is understood that narrower ranges between any two values ​​within that range are also encompassed by the present invention, unless the context or description clearly indicates otherwise.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art. Representative and exemplary methods and materials are described herein, although methods and materials similar or equivalent can also be used in the practice or testing of the present invention.

[0050] All publications and patents cited herein are incorporated by reference as if each publication or patent was individually expressly incorporated by reference, and are incorporated herein for the purpose of disclosing and describing the methods and / or materials for which they are cited. The citation of any publication is for the sole purpose of indicating its publication prior to the filing date of the present application and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, any publication dates provided may be different from the actual public availability dates, which must be independently confirmed.

[0051] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" are to be construed as including plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any element. Accordingly, this description is intended to support the use of exclusive terms such as "solely," "only," or negative limitations in the claims, such as "absent [specific feature or element]," "excluding [specific feature or element]," or "not including (not present, not containing, etc.) [specific feature or element]."

[0052] It will be apparent to those skilled in the art after reading this specification that each individual embodiment described and illustrated herein has individual components and features which may be readily separated or combined with the features of other embodiments without departing from the scope or spirit of the invention, and that methods described may be performed in the order described, or in any other order which is logically possible. [Example]

[0053] Recently, we serendipitously discovered a group of benzaldehyde compounds that exhibited very strong polymer destabilizing effects. However, these compounds lacked any chemical substituents on the second ring (pyridine or benzene) that would be expected to strongly interact with the αF helix residues, except when hydrogen atoms were replaced by halogens such as F or Cl. Initially, these compounds were synthesized and evaluated as chemical scaffolds for discovering novel polymer-destabilizing benzaldehydes by screening new chemical substituents on the second ring. However, due to the lack of substituents that strongly interact with the αF helix, as in VZHE-039 and PP-14, their polymer destabilizing effects were presumed to be very weak, similar to INN-310. However, contrary to expectations, these compounds were found to be able to exhibit very strong interactions with the αF helix residues through a completely water-mediated pathway via the pyridine, or alternatively, pyrimidine or triazine nitrogens, or through a completely hydrophobic pathway via the benzene ring. In some embodiments, halogens replace hydrogen atoms in the ring structure, but these halogens do not interact with the αF helix residues; rather, they may have been introduced to limit or modify interactions with metabolic enzymes or to remove potentially toxic metabolic intermediates. This unexpected result may be due to the fact that the replacement of the meta-methoxy group in INN-310 with the ortho-hydroxy group on the benzaldehyde ring slightly altered the orientation of the compound within the α-cleft of Hb, fortuitously positioning the second ring structure in a position that allows for strong interactions with the αF helix residues without the need for any hydrogen bond donor / acceptor substituents. In contrast, compounds 9 and 13, in which the ortho-hydroxy group on the benzaldehyde ring was replaced with a fluorine atom, completely abolished the polymer destabilizing effect, while compound 12, in which a meta-fluorine atom was introduced next to the ortho-hydroxy group on the benzaldehyde ring, exhibited significantly reduced inhibitory potency.

[0054] Thus, a series of novel analog compounds with unexpected polymer destabilizing effects and surprising activity were prepared. Indeed, compounds 1 and 2 (Table 1) are the most potent polymer destabilizing agents discovered to date, consistently demonstrating rapid uptake into red blood cells (RBCs) under completely anaerobic conditions and potent and sustained inhibition of RBC sickling, demonstrating greater activity than VZHE-039 and PP-14. Compounds 1, 2, and 5 also exhibit acceptable absorption, distribution, metabolism, and excretion (ADME) properties without metabolic instability, consistent with their utility as orally administered small molecule drugs suitable for the treatment of sickle cell disease. In contrast, reference compound A, which forms entirely hydrophobic interactions with the αF helix via its benzene ring and exhibits potent polymer destabilizing effects, is not bioavailable. Compounds 1, 2, and 5 have each been confirmed safe at levels above expected therapeutic drug concentrations in nonclinical toxicity studies, with no evidence of hepatotoxicity, indicating an acceptable therapeutic window for human clinical trials. These are the first polymer-destabilized benzaldehydes with properties suitable for progressing to human clinical trials. However, while hundreds of compounds with different chemical substituents on the second ring have been tested, many have not become viable drug candidates due to various issues stemming from the second ring substituents.

[0055] Results and Discussion synthesis / chemistry Compounds 1–13, listed in Table 1, were synthesized as shown in Scheme 4. Briefly, substituted hydroxybenzaldehydes were reacted with substituted chloromethylpyridines or bromomethylpyridines under basic conditions at room temperature for 2 hours, followed by heating at 60°C for 4–6 hours, to give compounds 1–13 in 9–47% yields. Detailed descriptions of the synthesis of each compound are provided in the Experimental Section. The synthetic methods for the comparative compounds listed in Table 2, such as TD-7, INN-310, PP-14, and Voxelotor, have been reported in the literature (Deshpande et al., 2018; Oksenberg et al., 2016; Pagare et al., 2018, 2020). [ka]

[0056] Table 1. Structures of example target compounds [Table 1] JPEG2025537075000024.jpg240170JPEG2025537075000025.jpg143170

[0057] Table 2. Structures of comparative compounds [Table 2]

[0058] In vitro functional and biological evaluation Compounds 1–13 (Table 1) were investigated along with the comparative compounds INN-310, TD-7, VZHE-039, PP-14, and Voxelotor (Table 2). The syntheses of the comparative compounds have been reported in the literature (Deshpande et al., 2018; Oksenberg et al., 2016; Pagare et al., 2018, 2020). All comparative compounds are aromatic aldehydes known to increase the oxygen affinity of hemoglobin (Hb) and, accordingly, inhibit hypoxia-induced red blood cell (RBC) sickling (Deshpande et al., 2018; Oksenberg et al., 2016; Pagare et al., 2018, 2020). PP-14 and VZHE-039 exhibit potent anti-sickling activity via direct polymer destabilization, while Voxelotor, INN-310, and TD-7 exhibit limited or no such activity ( Deshpande et al., 2018 ; Oksenberg et al., 2016 ; Pagare et al., 2018 , 2020 ).

[0059] Compounds 1–13 formed adducts with Hb and exhibited potent anti-sickling effects after incubation under hypoxic conditions. Furthermore, contrary to expectations, the majority of compounds in this class exhibited potent polymer destabilizing effects accompanied by anti-sickling effects even under anoxic conditions (Table 3). In comparison, Voxelotor showed no anti-sickling effects under anoxic conditions. In time-course experiments under anoxic conditions (Figure 1), compounds 1 and 2 exhibited the most potent and sustained polymer destabilizing effects observed to date, generally exhibiting activity comparable to or superior to many previously tested compounds, such as VZHE-039 and PP-14, which possess chemical substituents that directly interact with αF helix surface residues. In contrast, INN-310 exhibited weak activity under anoxic conditions. Despite their potent antisickling effects, compounds 1, 2, and 5 showed relatively weaker changes in Hb p50 in human blood compared with Voxelotor under conditions of equal drug-Hb occupancy (Figure 2), indicating the relative importance of the direct polymer destabilization effect in this class of compounds in drug efficacy.

[0060] In the partitioning studies, all compounds showed high partition ratios of over 90% (Table 4), indicating that these compounds rapidly permeate the erythrocyte membrane. Therefore, the differences in drug-Hb adduct formation primarily reflect differences in the Hb-binding affinity of each compound. The most potent compounds, Compounds 1, 2, and 5, showed over 90% Hb adduct formation after incubation at 2 mM in AA blood.

[0061] Table 3. Hemoglobin modification and anti-sickling studies using blood suspensions from homozygous SCD patients [Table 3] Biological effect demonstrated by compound at 2 mM concentration, 4 biological replicates

[0062] Table 4. Hemoglobin modification studies using non-sickling AA blood [Table 4] Biological effect demonstrated by compound at 2 millimolar concentration, 6 biological replicates

[0063] Atomic-level X-ray crystallographic analysis of the destabilizing effect of Hb polymers Compounds 1–13 were cocrystallized with coordinated hemoglobin (carboxymonoxyhemoglobin, COHb) under low-salt conditions. Only compounds 1, 2, 5, and 8 formed Hb crystals of suitable quality for X-ray diffraction, allowing the structures to be determined at 1.9–2.1 Å resolution. As expected, the resulting crystal structures of the tetrameric Hb-bound compound complexes were in a relaxed R2 state conformation, similar to the R2 state tetrameric Hb structures previously determined in complex with the reference compounds INN-310, VZHE-039, PP-14, and Voxelotor (Deshpande et al., 2018; Oksenberg et al., 2016; Pagare et al., 2018, 2020). The overall structures of all complexes were indistinguishably similar when compared with each other, the native R2-state structure (PDB code 1BBB), or Hb complexes with reference compounds, with RMSDs ranging from 0.3 to 0.5 Å. As expected, similar to INN-310, VZHE-039, and PP-14 (but unlike Voxelotor), two molecules of each compound bound symmetrically to the α-cleft of the Hb tetramer, forming Schiff bases with the two αVal1 residues (the N-terminal amines of the α1 and α2 subunits) and also forming interactions with several other proteins. The bulkiness of Voxelotor precludes two-molecule binding, resulting in only one molecule binding to the α-cleft and forming only one Schiff base. The formation of Schiff bases by these compounds is consistent with the modification state of Hb, as confirmed by their distinct peaks on cation-exchange HPLC, distinct from those of unmodified Hb. Specific interactions with proteins include intra- and intersubunit hydrophobic interactions and / or water-mediated or direct hydrogen bonds between αAla130, αSer131, αThr134, αThr134, and / or αLeu2 via the benzaldehyde and / or pyridine rings. In bimolecularly bound compounds (except Voxelotor), the two pyridine rings formed extensive face-to-face π-π stacking interactions (Figure 3).These interactions with Hb are consistent with the results in Table 3, which show that benzaldehydes stabilize the relaxed Hb state and increase the oxygen affinity of Hb, thereby exerting an oxygen-dependent anti-sickling effect, as previously reported for other benzaldehydes (Deshpande et al., 2018; Oksenberg et al., 2016; Pagare et al., 2018, 2020).

[0064] Most importantly, consistent with the compounds' oxygen-independent anti-sickling effects under fully anoxic conditions, compounds 1, 2, 5, and 8, like VZHE-039 and PP-14 but unlike INN-310 and Voxelotor, form hydrogen-bonding interactions with surface-located αF-helix residues (Figure 3). As noted above, the αF-helix plays a critical role in stabilizing the Hb polymer, and disruption of this helix by interaction with the second ring of the benzaldehyde leads to direct destabilization of the polymer. The differences in each compound's unique interactions with the αF-helix are consistent with the strength of their polymer-destabilizing effects, as determined by their inhibition of sickling under anoxic conditions. VZHE-039 and PP-14 contain a methyl hydroxyl group and a carboxyl ester group on the pyridine ring, respectively, which form hydrogen-bonding interactions with residues of the αF-helix. On the other hand, compounds 1, 2, 5, and 8 unexpectedly form water-mediated interactions with the αF-helix residues through the pyridine nitrogen, despite the absence of any heteroatom-containing substituents on the pyridine ring. Compound 1 forms very strong water-mediated hydrogen-bonding interactions with the αF-helix residues through one or two water molecules. Compounds 2, 5, and 8 each interact with the αF-helix through at least three water molecules. Therefore, compound 1 appears to form the strongest hydrogen-bonding interactions with the αF-helix residues compared with VZHE-039 and PP-14, explaining the high affinity of compound 1 (dissociation constant KD of 0.06 mM, VZHE-039 0.1 mM, PP-14 0.15 mM). INN-310 forms only weak to moderate hydrophobic interactions with the αF-helix, but no hydrogen-bonding interactions, resulting in very weak polymer destabilization and limited anti-sickling effect under anoxic conditions. This is why it was previously expected that the compounds of the present invention would not exhibit strong polymer destabilizing effects. In contrast, the bulky substituents of Voxelotor orient the pyridine moiety away from the αF-helix, explaining the complete lack of polymer destabilizing effect.

[0065] In vivo pharmacokinetic evaluation Single-dose PK study Single-dose pharmacokinetics (PK) of each compound was evaluated using male Sprague Dawley (SD) rats (n = 4), and the maximum or peak concentrations in whole blood and the area under the curve (AUC) are shown in Table 5. Compounds 1, 2, and 5 demonstrated the highest oral exposure levels observed to date for polymer-destabilized benzaldehydes, particularly on a molar equivalent basis. Although not shown, PP-14 is rapidly metabolized to a carboxylic acid metabolite after oral administration.

[0066] Table 5. Summary of single-dose pharmacokinetic properties in rats [Table 5] Single oral dose is 100 mg / kg

[0067] 14-day repeated dose PK study Male and female SD rats (n = 3) were orally administered each compound for 14 days. The steady-state peak drug concentrations and area under the curve for each compound are shown in Table 6. Compounds 2 and 5 demonstrated the highest oral exposure levels observed to date, particularly on a molar equivalent basis. The oral exposure level of compound 1 was comparable to that of VZHE-039 and higher than that of Voxelotor, particularly on a molar equivalent basis. The steady-state exposure at the final dosing interval on day 14 was similar to the total compound exposure obtained from single-dose PK. No clinical or histopathological changes were observed after 14 days of exposure to compounds 1, 2, and 5. Both clinical and histopathological changes in the liver, including hepatocellular necrosis, were observed in animals treated with VZHE-039 for 14 days.

[0068] Table 6. Summary of repeated-dose pharmacokinetic properties in rats [Table 6] Repeated oral administration of 100mg / kg for 14 days

[0069] Experimental Section Detailed synthesis method Synthesis of 2-hydroxy-6-(pyridin-3-ylmethoxy)benzaldehyde (compound 1): To a stirred solution of 2,6-dihydroxybenzaldehyde (36.2 mmol) and potassium carbonate (36.2 mmol) in 3-pentanone was added 3-bromomethylpyridine HBr (36.2 mmol). The reaction was refluxed for 3 hours, and upon completion, the solid precipitate was filtered and washed with 3-pentanone. The crude solid was purified by flash chromatography (1:1 hexane / ethyl acetate) to yield 1.62 g (19.5%) of a pale yellow granular solid. Mp 99-103. 1 H NMR (80 MHz, CDCl3) δ 11.93 (br s, 1H), 10.31 (s, 1H), 8.64 (dt, 2H, J=1.7, 4.4 Hz), 7.7-7.9 (m, 1H), 7.2-7.5 (m, 2H), 6.49 (dd, 2H, J=5.2, 8.4 Hz), 5.13 (s, 2H). 13 C NMR (20 MHz, CDCl3) δ 193.9, 163.6, 161.1, 149.8, 148.9, 138.4, 135.3, 131.5, 123.7, 110.9, 110.5, 102.1, 68.1.

[0070] Synthesis of 2-hydroxy-6-(pyridin-2-ylmethoxy)benzaldehyde (compound 2): 2-Bromomethylpyridine hydrobromide (60.966 mmol), 2,6-dihydroxybenzaldehyde (1 equiv.), K2CO3 (3 equiv.), and dry acetone were added to a dry conical flask equipped with a magnetic stirrer and flushed with N2 gas. After sealing with a rubber septum, N2 was bubbled through the reaction mixture using a long needle. The mixture was stirred at room temperature for 1 h and then at 60 °C for an additional 1 h using a fine-gauge needle. After TLC confirmed the reaction was complete, the mixture was vacuum filtered while hot through a bed of silica and washed with hot acetone. After evaporation, the crude material was purified by column chromatography using a 2:1 Hex / EtOAc system to yield 6.58 g (47%) of a pale yellow solid. Purity: 99%. Mp = 127.5-129.2 °C. 1 H NMR (80 MHz, CDCl3) δ 12.03 (s, 1H), 10.54 (s, 1H), 8.67 (d, J = 4.9 Hz, 1H), 7.81 (t, J = 7.5 Hz, 1H), 7.44 (dd, J = 10.2, 6.6 Hz, 3H), 6.55 (t, J = 8.5 Hz, 2H), 5.34 (s, 2H).

[0071] Synthesis of 2-hydroxy-6-(pyridin-4-ylmethoxy)benzaldehyde (compound 3): A mixture of 2,6-dihydroxybenzaldehyde (1 eq), K2CO3 (3 eq), KI (0.1 eq), EtOAc (2 mL), and DMF (2 mL) in 50 mL of 3-pentanone was sonicated for 5–10 min. 4-(Chloromethyl)pyridine hydrochloride was then added and sonicated at 60 °C for 90 min. The reaction mixture was transferred to a hot plate and stirred at 75 °C until complete by TLC. The mixture was vacuum filtered while hot and washed with hot acetone. The filtrate was evaporated and purified by flash chromatography (Biotage Selekt, Safar HC 25 g) to give 0.252 g (11%) of a pale yellow solid: purity: 97%; melting point: 140.8 °C. 1H NMR (80 MHz, ) δ 12.00 (s, 1H), 10.51 (s, 1H), 8.70 (s, 2H), 7.59-7.25 (m, 4H), 6.62 (d, J = 8.5 Hz, 1H), 6.40 (d, J = 7.4 Hz, 1H), 5.22 (s, 2H).

[0072] General procedure for the synthesis of compounds 4-8: To a solution of the substituted pyridine in dry acetone was added 2,6-dihydroxybenzaldehyde (1 equiv.), K2CO3 (2 or 3 equiv.), and KI (0.1 equiv.). After bubbling N2 through the solution, the reaction mixture was stirred at room temperature for 1 h and then at 60 °C with a fine-gauge needle attached. After TLC showed the reaction was complete, the mixture was vacuum filtered while hot through a bed of silica and washed with hot acetone. After evaporation, the crude product was purified by column chromatography.

[0073] Synthesis of 2-((5-fluoropyridin-3-yl)methoxy)-6-hydroxybenzaldehyde (compound 4) 3-(Chloromethyl)-5-fluoropyridine hydrochloride (10 mmol) was reacted with 2,6-dihydroxybenzaldehyde according to the general procedure described above. Purification of the crude material afforded 0.942 g (38.1%) of a pale yellow solid with the following characteristic profile: purity: 97%; melting point: 128-129 °C; 1 H NMR (80 MHz, CDCl3) δ 11.92 (s, 1H), 10.35 (s, 1H), 8.46 (d, J = 2.6 Hz, 2H), 7.57-7.18 (m, 3H), 6.47 (dd, J = 13.6, 8.3 Hz, 2H), 5.15 (s, 2H).

[0074] Synthesis of 2-((3-fluoropyridin-2-yl)methoxy)-6-hydroxybenzaldehyde (compound 5) 2-(Chloromethyl)-3-fluoropyridine hydrochloride (17.141 mmol) was reacted with 2,6-dihydroxybenzaldehyde according to the general procedure described above. Purification and crystallization from TBME gave 1.8 g (42.5%) of a pale yellow solid with the following characteristic profile: purity: 99%; melting point: 132-136 °C; 1 H NMR (80 MHz, CDCl3) δ 12.09 (s, 0H), 10.48 (s, 1H), 8.57 (s, 1H), 7.54 (t, J = 8.7 Hz, 3H), 6.93-6.29 (m, 2H), 5.46 (s, 2H).

[0075] Synthesis of 2-((2-fluoropyridin-3-yl)methoxy)-6-hydroxybenzaldehyde (Compound 6) 3-(Chloromethyl)-2-fluoropyridine (10 mmol) was reacted with 2,6-dihydroxybenzaldehyde according to the general procedure described above. The crude material was purified to give 0.924 g (37.4%) of a pale yellow solid with the following characteristic profile: purity: 97%; melting point: 156-160 °C; 1 H NMR (80 MHz, CDCl3) δ 11.97 (s, 1H), 10.42 (s, 1H), 8.41-7.65 (m, 2H), 7.64-7.04 (m, 3H), 6.76-6.33 (m, 2H), 5.23 (s, 2H).

[0076] Synthesis of 2-((2-chloropyridin-3-yl)methoxy)-6-hydroxybenzaldehyde (compound 7) 2-Chloro-3-(chloromethyl)pyridine (10 mmol) was reacted with 2,6-dihydroxybenzaldehyde according to the general procedure described above. Purification of the crude material afforded 0.447 g (17%) of a pale beige solid with the following characteristic profile: purity: 97%; melting point: 158-159°C; 1 H NMR (80 MHz, CDCl3) δ 11.98 (s, 1H), 10.38 (s, 1H), 8.52 (s, 1H), 7.69-7.16 (m, 3H), 6.58 (d, J = 8.6 Hz, 2H), 5.36 (s, 2H).

[0077] Synthesis of 2-((3-chloropyridin-2-yl)methoxy)-6-hydroxybenzaldehyde (compound 8) 3-Chloro-2-(chloromethyl)pyridine (10 mmol) was reacted with 2,6-dihydroxybenzaldehyde according to the general procedure described above. The crude material was purified to give 0.63 g (23.6%) of a white solid with the following characteristic profile: purity: 99%; melting point: 123-124°C; 1 H NMR (80 MHz, CDCl3) δ 11.84 (s, 1H), 10.26 (s, 1H), 8.39 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.43-6.93 (m, 3H), 6.42 (d, J = 8.2 Hz, 2H), 5.26 (s, 2H).

[0078] Synthesis of 2,4-difluoro-6-(pyridin-2-ylmethoxy)benzaldehyde (compound 9): 2-Bromomethylpyridine hydrobromide (12.7 mmol) was reacted with 2,4-difluoro-6-hydroxybenzaldehyde according to the procedure for compound 2. The crude material was purified with a 1:3 Hex / CHCl system and then crystallized from TBME to give a pale yellow solid material. Purity: 99%. 1 H NMR (80 MHz, CDCl3) δ 10.49 (s, 1H), 8.62 (s, 1H), 7.77 (d, J = 11.1 Hz, 2H), 7.30 (d, J = 4.8 Hz, 1H), 6.64 (d, J = 8.9 Hz, 2H), 5.34 (s, 2H). 19 F NMR (76 MHz, CDCl3) δ -94.41 (dd, J = 23.4, 12.4 Hz), -108.93 (d, J = 12.0 Hz).

[0079] General procedure for the synthesis of compounds 10-11: To a solution of the substituted pyrimidine in dry acetone was added 2,6-dihydroxybenzaldehyde (1 equiv.), K2CO3 (3 equiv.), and KI (0.1 equiv.). N2 was bubbled through the solution, and the reaction mixture was stirred at 60 °C with a fine-gauge needle attached. After TLC showed the reaction was complete, the mixture was vacuum filtered while hot and washed with hot acetone. After evaporation, the crude material was purified by column chromatography.

[0080] Synthesis of 2-hydroxy-6-(pyrimidin-5-ylmethoxy)benzaldehyde (compound 10) 5-(Chloromethyl)pyrimidine hydrochloride (6.06 mmol) was reacted with 2,6-dihydroxybenzaldehyde for 18 hours according to the general procedure described above. The crude material was purified in a 1:2:1 Hex / CHCl3 / EtOAc system and then crystallized from TBME to give 0.206 g (14.8%) of a yellow solid with the following characteristic profile: purity: 99%; H NMR (80 MHz, CDCl3) δ 11.91 (s, 1H), 10.31 (s, 1H), 9.21 (s, 1H), 8.81 (s, 2H), 7.61–7.07 (m, 1H), 6.49 (dd, J = 11.3, 8.4 Hz, 2H), 5.13 (s, 2H).

[0081] Synthesis of 2-hydroxy-6-(pyrimidin-2-ylmethoxy)benzaldehyde (compound 11) 2-(Chloromethyl)pyrimidine hydrochloride (6.06 mmol) was reacted with 2,6-dihydroxybenzaldehyde for 2 h according to the general procedure described above. The crude material was purified with a 1:3 Hex / CHCl system and then crystallized from TBME to give 0.423 g (15.7%) of a yellow solid with the following characteristic profile: purity: 99%; 1 H NMR (80 MHz, CDCl3) δ 12.02 (s, 1H), 10.55 (s, 1H), 8.82 (d, J = 4.9 Hz, 2H), 7.53-7.09 (m, 2H), 6.50 (dd, J = 11.4, 8.4 Hz, 2H), 5.42 (s, 2H).

[0082] Synthesis of 3-fluoro-2-hydroxy-6-(pyridin-3-ylmethoxy)benzaldehyde (compound 12): 3-Bromomethylpyrimidine hydrobromide (12.811 mmol) was reacted with 3-fluoro-2,6-dihydroxybenzaldehyde according to the procedure described above for compound 2. The crude material was purified with a 1:3 Hex / CHCl system, followed by crystallization from TBME to give the product as a pale yellow solid. Purity: 97%. 1 H NMR (80 MHz, CDCl3) δ 11.40 (s, 1H), 10.16 (s, 1H), 8.59 (d, J = 3.2 Hz, 2H), 7.73 (d, J = 7.9 Hz, 1H), 7.43-6.99 (m, 2H), 6.75-6.38 (m, 1H), 5.30 (s, 2H).

[0083] Synthesis of 2,4-difluoro-6-((3-fluoropyridin-2-yl)methoxy)benzaldehyde (compound 13): A solution of 2,4-difluoro-6-hydroxybenzaldehyde (1 equiv.), K2CO3 (2 or 3 equiv.), and KI (0.1 equiv.) in dry acetone was heated at 40 °C for 30 min. 2-(Chloromethyl)-3-fluoropyridine hydrochloride (2.747 mmol) was warmed in dry acetone and added dropwise to the aldehyde solution. The mixture was heated to 65 °C for 2 h. After TLC showed the reaction was complete, the mixture was vacuum filtered while hot and washed with hot acetone. The filtrate was evaporated, and the crude product was purified by flash chromatography (Biotage Selekt, Safar HC 10 g column, 1:3 Hex / CHCl3 isocratic system) to give 64 mg (8.7%) of an off-white solid: purity: 99%; 1 H NMR (80 MHz, CDCl3) δ 10.39 (s, 1H), 8.52 (s, 1H), 7.75-7.15 (m, 3H), 7.00-6.27 (m, 2H), 5.43 (s, 2H).

[0084] Biochemical assays Sickle cell inhibition, Hb adducts, and Hb oxygen equilibrium in SS blood suspensions All compounds (compounds 1–13) and comparator molecules (VZHE-039, PP-14, INN-310, and Voxelotor) were evaluated according to established procedures (Abdulmalik et al., 2005, 2020; Pagare et al., 2020). Blood samples from donors with homozygous SCD (who provided informed consent) were diluted with Hemox buffer supplemented with glucose (10 mM) and BSA (0.2%) to adjust the hematocrit of the blood suspension to 20%. This standardized procedure normalizes the red blood cell-to-drug ratio for assay consistency and reproducibility. Briefly, blood suspensions were incubated at room temperature in the presence of various concentrations of test compounds (0–2 mM) in a 96-well round-bottom plate at 37°C for 1 h. The suspension was then incubated with a hypoxic gas mixture (2.5% oxygen / balance nitrogen) at 37°C for up to 2 hours, and then fixed in a 2% glutaraldehyde solution without air exposure. Fixed blood aliquots were evaluated by microscopic morphological analysis (Image J software) to identify the proportion of sickle and non-sickle red blood cells, and the sickle cell inhibition rate was calculated based on the percentage change from the negative control (DMSO).

[0085] To establish oxygen-independent sickling inhibition, the incubation chamber was opened and exposed to room temperature air for 15 minutes to allow for full reoxygenation, during which time the sickle cells transformed into normal round cells. This transformation was monitored microscopically. The incubation chamber was then closed and incubated in 100% nitrogen gas for 1 hour (for screening) or up to 2.5 hours (for time-course studies). Blood aliquots were withdrawn and fixed at designated intervals, and the assay was repeated. Aliquots were again subjected to microscopic morphological analysis using bright-field imaging to identify the proportion of sickle and non-sickle cells, and the sickle cell inhibition rate was calculated.

[0086] The remaining samples were then washed with phosphate-buffered saline and hemolyzed in hypotonic hemolysis buffer with or without sodium borohydride (50 mM). Aliquots from the lysates, with or without sodium borohydride (to reduce Schiff base adducts), were also evaluated for the level of Hb modification (drug adduct formation) by cation-exchange HPLC (Hitachi D-7000 Series, Hitachi Instruments, Inc., San Jose, CA) using a weak cation-exchange column (Poly CAT A: 50 mm x 4.6 mm, Poly LC, Inc., Columbia, MD). The hemoglobin isotype peaks were eluted with a linear gradient of 0% to 80% B at 410 nm (Mobile Phase A: 40 mM Bis-Tris, 2 mM KCN, pH 6.9; Phase B: 40 mM Bis-Tris, 2 mM KCN, 0.2 M NaCl, pH 6.55). A clarified lysate of normal human adult Hb (Hb A) and a commercial standard (Helena Laboratories, Beaumont, TX) containing approximately equal amounts of Hb F, A, S, and C were used as reference isotypes. Reduced samples were used to assess "fixed adduct" formation (dependent on erythrocyte membrane permeability), whereas nonreduced samples were used to assess drug adduct formation independent of drug permeability.

[0087] Drug Distribution Test Erythrocyte partitioning studies were performed using both SS red blood cells after the sickling test described above and in separate, independent studies using non-sickling blood. Briefly, aliquots of blood samples incubated with test molecules were lysed either in deionized water (unfixed) or in water containing 50 mM sodium borohydride (fixed). Samples were subjected to cation-exchange HPLC analysis as described above. Modified Hb (Hb adducts) was determined as a percentage of total Hb in each sample, and the partition ratio was determined as the relative percentage of Hb modification in fixed samples compared to the corresponding unfixed samples. The partition ratio is the relative percentage of Hb modified intracellularly by molecules that permeated the red blood cell membrane compared to unfixed adducts, which reflect Hb binding that does not permeate the red blood cell membrane.

[0088] Dose-dependent P50 shift in human blood Test compounds were added to a 96-well round-bottom plate at concentrations of 0.25–2 mM and incubated at 37°C for 1 h. After incubation, red blood cells were washed once with PBS and lysed in deionized water. Approximately 100 μl aliquots of each lysate were added to 3 mL of 0.1 M potassium phosphate buffer (pH 7.0) in a cuvette and analyzed using Hemox as previously described (Abdulmalik et al., 2020; Pagare et al., 2020). TM Hemoximetry analysis was performed using an analyzer (TCS Scientific Corp.). The change in p50 value (Δp50) was calculated by subtracting the p50 value of the treated sample from the p50 value of the control sample and expressing the difference as a percentage of the control p50 value.

[0089] In vivo pharmacokinetic and safety studies A single dose of each compound was administered orally at 100 mg / kg as a 0.5% carboxymethylcellulose suspension. Whole blood samples were collected for at least 72 hours after administration. Bioanalysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS).

[0090] Each compound was administered orally at a dose of 100 mg / kg once daily for 14 days as an oral suspension in 0.5% carboxymethylcellulose. During the final dosing interval on day 14, whole blood was collected at seven time points over a maximum of 24 hours. Clinical findings, body weight, and food consumption were recorded throughout the 14-day study period. Clinical chemistry and hematology parameters were assessed, and histopathological analysis of organs was performed after 14 days of study drug exposure.

[0091] X-ray crystal structure analysis Cocrystals of Hb coordinated with compounds 1, 2, 5, and 8 were obtained according to previously published methods (Abdulmalik et al., 2020; Deshpande et al., 2018; Pagare et al., 2018). Briefly, 40–50 mg / mL Hb in a round-bottom flask was placed under vacuum for approximately 1 h to remove oxygen and prepare deoxygenated Hb. Subsequently, CO-coordinated Hb (COHb) was formed by bubbling carbon monoxide through the deoxygenated Hb. COHb was prepared by incubating compounds 1, 2, 5, or 8 in a 10–20 molar excess for approximately 1 h to form the COHb-compound complex. Next, a 10 molar excess of sodium cyanoborate (NaCNBH4) was added to reduce the reversible Schiff base adducts to the corresponding irreversible alkylamine covalent bonds. The COHb-compound complex solution was crystallized by the batch method using 10-20% PEG 6000 and 100 mM HEPES buffer (pH 7.4). X-ray diffraction data were obtained using a Rigaku MicroMax TMCrystals were collected at 100 K using a 007HF X-ray generator, an Eiger R 4M detector, and an Oxford Cobra cryosystem (The Woodlands, TX). Crystals were cryoprotected in 80 μL of mother liquor mixed with 62 μL of 50% PEG 6000. Diffraction data were processed using CrysAlisPro software (Rigaku) ​​and the CCP4 program suite (Winn et al., 2011). The crystal structure of the complex was solved by molecular replacement using the Phenix program (Adams et al., 2011; Echols et al., 2012) with the native R2 crystal structure (PDB ID 1BBB) as the search model. Structural refinement was performed using Phenix and COOT (Adams et al., 2011; Brunger et al., 1998; Echols et al., 2012).

[0092] While the present invention has been described using certain exemplary embodiments, those skilled in the art will recognize that the present invention can be practiced with modification within the concept and scope of the appended claims. Therefore, the present invention should not be limited to the above-described embodiments, but further encompasses all modifications and equivalents that are within the concept and scope of the subject matter described herein.

Claims

1. A compound having the formula: 【Chemistry 1】 provided that W, X, Y, and Z may be the same or different and are independently C or N, and at least one of W, X, Y, and Z is N; R 1 , R 2 , R 3 and R 4 may be the same or different and are independently F, Cl, or H; and pharmaceutically acceptable salts or oral prodrugs thereof.

2. 10. The compound of claim 1, 【Chemistry 2】 A compound which is

3. 2. The compound of claim 1 , 【Transformation 3】 A compound which is

4. 2. The compound of claim 1 , 【Chemistry 4】 A compound which is

5. 2. The compound of claim 1 , R 1 , R 2 , R 3 and R 4 may be the same or different, and at least one is independently F or Cl.

6. 6. The compound of claim 5, 【Transformation 5】 A compound which is

7. 2. The compound of claim 1 , 【Transformation 6】 A compound which is

8. 1. A method of preventing or treating one or more symptoms or conditions of sickle cell disease (SCD) in a subject in need thereof, comprising:

10. A method comprising administering to the subject a therapeutically effective amount of at least one compound of claim 1.

9. 9. The method of claim 8, The method, wherein the one or more symptoms or conditions are selected from the group consisting of red blood cell (RBC) sickling, adhesion of red blood cells to tissue endothelium, oxidative stress and / or damage, red blood cell hemolysis, inflammation, vascular occlusion, impaired microvascular blood flow, stroke, pain, and death.

10. 9. The method of claim 8, The method wherein said administering step is performed orally.

11. 1. A method of preventing or treating one or more symptoms or conditions of sickle cell disease (SCD) in a subject in need thereof, comprising:

1. A method comprising administering to the subject a therapeutically effective amount of a compound having the formula: or a salt or prodrug that is metabolized or converted to said compound in the subject: 【Transformation 7】 provided that W, X, Y, and Z may be the same or different and are independently C or N, and at least one of W, X, Y, and Z is N; R 1 , R 2 , R 3 and R 4 may be the same or different and are independently F, Cl or H.

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