WO2015191034A1 - Inhibiteurs de xanthine oxydase à petites molécules et procédés d'utilisation - Google Patents

Inhibiteurs de xanthine oxydase à petites molécules et procédés d'utilisation Download PDF

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WO2015191034A1
WO2015191034A1 PCT/US2014/041590 US2014041590W WO2015191034A1 WO 2015191034 A1 WO2015191034 A1 WO 2015191034A1 US 2014041590 W US2014041590 W US 2014041590W WO 2015191034 A1 WO2015191034 A1 WO 2015191034A1
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dhnb
compounds
subject
gout
allopurinol
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PCT/US2014/041590
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English (en)
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Changyi Chen
Jian-ming LU
Qizhi Yao
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Baylor College Of Medicine
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Publication of WO2015191034A1 publication Critical patent/WO2015191034A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/11—Aldehydes

Definitions

  • Gout is caused by hyperuricemia, namely, abnormally high levels of uric acid in the blood. Gout is usually present as acute inflammatory arthritis, as well as tophi, kidney stones, or urate nephropathy. Gout affects 1-2% of adults in developed countries and represents the most common case of inflammatory arthritis in men. In the United States, gouty arthritis accounts for millions of outpatient visits annually. Furthermore, gout and hyperuricemia are associated with chronic diseases such as hypertension, diabetes mellitus, metabolic syndrome, and renal and cardiovascular disease.
  • Xanthine oxidase is a form of a molybdoflavin protein, xanthine oxidoreductase (XOR). It plays an important role in the catabolism of purines in humans, as it catalyzes the oxidation of hypoxanthine to xanthine and then catalyzes the oxidation of xanthine to uric acid. Meanwhile, reactive oxygen species (ROS), including superoxide and H 2 O 2 , are generated during this process. Uric acid can serve as an antioxidant to prevent macromolecular damage by ROS.
  • ROS reactive oxygen species
  • overproduction of uric acid can cause hyperuricemia and lead to gout and other diseases. Therefore, maintaining uric acid at normal levels represents an important therapeutic goal for the prevention of gout and related disorders. For most patients with primary gout, overproduction of uric acid is the primary cause of hyperuricemia.
  • Allopurinol is the most commonly used therapy for chronic gout and has been used clinically for more than 40 years. Allopurinol lowers uric acid production by inhibiting XO activity, and is used as a first-line urate-lowering phamacotherapy. Allopurinol, a structural isomer of hypoxanthine, is hydroxylated by XO to oxypurinol, which coordinates tightly to the reduced form of the molybdenum center, replacing the Mo-OH group of the native enzyme. Unfortunately, while rare, allopurinol has life-threatening side effects such as a toxicity, for which the mortality rate approaches 20%.
  • a class of xanthine oxidase inhibitors described herein includes compounds of the following structure:
  • R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, hydroxyl, nitro, cyano, fluoro, chloro, bromo, trifluoromethyl, sulfonyl, and aldehyde, wherein R 1 , R 2 , R 3 , R 4 , and R s are not simultaneously hydrogen.
  • suitable xanthine oxidase inhibitors as described herein include the following compounds:
  • a xanthine oxidase inhibitor suitable for the methods described herein also includes the following compound:
  • a method of treating gout or hyperuricemia in a subject includes administering to the subject an effective amount of a xanthine oxidase inhibitor as described herein.
  • the methods for treating gout or hyperuricemia in a subject can further include administering a second therapeutic agent, such as an anti-gout agent (e.g., allopurinol, benzbromarone, colchicine, probenecid, or sulfinpyrazone), an antiinflammatory agent, or an antioxidant, to the subject.
  • a second therapeutic agent such as an anti-gout agent (e.g., allopurinol, benzbromarone, colchicine, probenecid, or sulfinpyrazone), an antiinflammatory agent, or an antioxidant.
  • an anti-gout agent e.g., allopurinol, benzbromarone, colchicine, probenecid, or sulfinpyrazone
  • the methods include administering to the subject an effective amount of a xanthine oxidase inhibitor as described herein. production further comprise selecting a subject having gout or hyperuricemia.
  • Methods of inhibiting xanthine oxidase activity in a cell are also provided herein.
  • the methods include contacting a cell with an effective amount of a xanthine oxidase inhibitor as described herein.
  • the contacting is performed in vivo.
  • the contacting is performed in vitro.
  • Figure 1A is a graph showing the inhibition of xanthine oxidase (XO) activity by test compounds (DH6NB, DHBA, THB-CHO, and DHNB) and a control compound (allopurinol).
  • Figure 1B is a Dixon Plot for DHNB at varying concentrations of xanthine.
  • Figure 1C is a graph showing the effects of pH on the inhibitory effect of DHNB on XO.
  • Figure 2 is a graph comparing the xanthine oxidase inhibitory effects of catechol compounds, each at a concentration of 20 ⁇ . The control represents no inhibitor added.
  • Figure 3 is a graph showing the time course of inhibition of xanthine oxidase activity by DHNB and allopurinol.
  • XO activity was determined under standard conditions and started by adding 20 nM XO (open symbols) or by adding 50 ⁇ xanthine following 4 min pre-incubation of XO and inhibitor (solid symbols). Circles, control - no inhibitor added; squares, with 6.67 ⁇ allopurinol; triangle, with 6.67 ⁇ DHNB.
  • Figure 4 is a graph showing the influence of pre-incubation of 20 ⁇ inhibitors with 20 nM XO on XO activity. Vanillin, DHB-CHO, DH6NB, THB-CHO, allopurinol (at 3.3 ⁇ ), and DHNB (at 6.6 ⁇ and 20 ⁇ ) were the tested compounds.
  • Figure 5 is a graph demonstrating that DHNB inhibition of XO is not reversible by reducing agents.
  • XO 10 mU/ml
  • 20 ⁇ DHNB were pre-incubated for 10 min at 25 °C in phosphate buffer ( 100 mM pH 7.4). Then a high level of GSH (20 mM), 2- mercaptoethanol (2-ME, 20 mM), or dithiothreitol (DTT, 20 mM) was added for 15 min, of the reagent DHNB, GSH, 2-ME, and/or DTT.
  • a "-" signifies that the reagent was not added.
  • Data represent the mean ⁇ S.E. at least three independent determinations.
  • Figure 6A is a graph of the time course of absorption change of DHNB (327 nm, arrow indicates decrease) and the formation of the product (279 nm, arrow indicates increase) following the mixing of 30 nM XO and 30 ⁇ DHNB in 0.1 M phosphate buffer (pH 7.4).
  • Figure 6B is a graph showing the effects of pH on the conversion of DHNB by XO enzyme.
  • Figure 6C contains an HPLC profile of DHNB (control) and a DHNB/XO mixture after incubation for 3 days.
  • Figure 6D is a MS/MS spectrum of DHNB.
  • Figure 6E is a MS/MS spectrum of the DHNB/XO product, DHNB-COOH.
  • Figure 7 contains graphs showing the antioxidant activities of DHNB, DH6NB, DHBA, DHB-CHO, THB-CHO, and allopurinol on the scavenging of free radical DPPH (panel A), hypochlorous acid (HOC1) (panel B), peroxynitrite (ONOO ⁇ ) (panel C), and/or superoxide ion (O2 -. ) (panel D).
  • Vitamin C Vit C
  • GSH glutathione
  • Figure 8 is a graph showing the concentration dependent DPPH scavenging activities of DHNB, DHBA, DHB-CHO, and allopurinol. Vitamin C (Vit C) and Vitamin E (Vit E) were used as controls.
  • Figure 9 is a graph showing the concentration dependent HOCI scavenging activities of DHNB, caffeic acid (CA), DHBA, DHB-CHO, DHB-COOH, and allopurinol.
  • Vitamin C Vit C was used as a control.
  • Figure 10 is a graph showing the concentration dependent peroxynitrite scavenging activity of DHNB, DHBA, DHB-CHO, DH6NB, DMB-CH 2 OH, caffeic acid, THB-CHO, gallic acid, DHB-COOH, and vanillin.
  • Vitamin C Vit C was used as a control.
  • Figure 11 is a graph showing the concentration dependent superoxide ion scavenging activity of caffeic acid and DHBA. Glutathione (GSH) was used as a control.
  • Figure 12 is a graph showing the dose dependent hyperuricemic effects of allantoxanamide in mice. DHNB and allopurinol on the allantoxanamide induced hyperuricemic mice.
  • Figure 14A is a photograph of allopurinol treated mice at 2.5 weeks.
  • Figure 14B is a photograph of allopurinol treated mice at 3 weeks.
  • Figure 14C is a photograph of allopurinol treated mice at 4 weeks.
  • Figure 14D is a photograph of allopurinol treated mice at 6 weeks.
  • Figure 14E is a photograph of DHNB treated mice at 4 weeks.
  • Figures 15A and 15B are graphs showing the inhibitory effects of DNSA and NHBA, respectively, on XO activity by measuring the initial rate of uric acid formation.
  • Figure 16 is a graph comparing the xanthine oxidase inhibitory effects of catechol compounds at a concentration of 20 ⁇ . The control represents no inhibitor added.
  • Figure 17 is a graph demonstrating the influence of pre-incubation of DNSA with XO on XO activity.
  • Figure 18 is a graph demonstrating XO activity after pre-incubation with inhibitors DNSA and DHNB for 20 hours.
  • xanthine oxidase inhibitors and methods for their use in treating gout or hyperuricemia in a subject.
  • the xanthine oxidase inhibitors are administered in an effective amount to treat gout or hyperuricemia in a subject.
  • a class of xanthine oxidase inhibitors described herein is re resented b Formula
  • R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from hydrogen, hydroxyl, nitro, cyano, fluoro, chloro, bromo, trifluoromethyl, sulfonyl, and aldehyde (i.e., -CHO).
  • the sulfonyl is methylsulfonyl or sulfonic acid.
  • R 1 , R 2 , R 3 , R 4 , and R 5 are not simultaneously hydrogen.
  • Examples of Formula I include the following compounds:
  • the compounds described herein or derivatives thereof can be provided in a pharmaceutical composition.
  • the forms such as, for example, tablets, suppositories, pills, capsules, powders, liquids, or suspensions, preferably in unit dosage form suitable for single administration of a precise dosage.
  • the compositions will include a therapeutically effective amount of the compound described herein or derivatives thereof in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, or diluents.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, which can be administered to an individual along with the selected compound without causing unacceptable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.
  • the term carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations.
  • a carrier for use in a composition will depend upon the intended route of administration for the composition.
  • the preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21 st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Wil liams & Wilkins, Philadelphia Pa., 2005.
  • physiologically acceptable carriers include buffers, such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN® (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and
  • compositions containing the compounds described herein or derivatives thereof suitable for parenteral injection may comprise physiologically acceptable sterile aqueous reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • compositions may also contain adjuvants, such as preserving, wetting, emulsifying, and dispensing agents.
  • adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • Prevention of the action of microorganisms can be promoted by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Isotonic agents for example, sugars, sodium chloride, and the like may also be included.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Solid dosage forms for oral administration of the compounds described herein or derivatives thereof include capsules, tablets, pills, powders, and granules.
  • the compounds described herein or derivatives thereof is admixed with at least one inert customary excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellu!ose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example, paraffin, (f) ab
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others known in the art. They may contain opacifying agents and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration of the compounds described herein or derivatives thereof include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1 ,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures of these substances, and the like.
  • inert diluents commonly used in
  • composition can also include additional agents, such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.
  • additional agents such as wetting, emulsifying, suspending, sweetening, flavoring, or perfuming agents.
  • Suspensions in addition to the active compounds, may contain additional agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • additional agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • compositions of the compounds described herein or derivatives thereof for rectal administrations are optionally suppositories, which can be prepared by mixing the compounds with suitable non-irritating excipients or carriers, such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but the active component.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but the active component.
  • Dosage forms for topical administration of the compounds described herein or derivatives thereof include ointments, powders, sprays, and inhalants.
  • the compounds described herein or derivatives thereof are admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellents as may be required.
  • Ophthalmic formulations, ointments, powders, and solutions are also contemplated as being within the scope of the compositions.
  • compositions can include one or more of the compounds described herein and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable salt refers to those salts of the compound described herein or derivatives thereof that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds described herein
  • salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds described herein.
  • salts can be prepared in situ during the isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonate salts, and the like.
  • alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, and the like
  • non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • acceptable salts thereof can be carried out using therapeutically effective amounts of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein for periods of time effective to treat a disorder.
  • the effective amount of the compounds and compositions described herein or pharmaceutically acceptable salts thereof as described herein may be determined by one of ordinary skill in the art and includes exemplary dosage amounts for a mammal of from about 0.5 to about 200mg/kg of body weight of active compound per day, which may be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day.
  • the dosage amount can be from about 0.5 to about 150mg/kg of body weight of active compound per day, about 0.5 to 100mg/kg of body weight of active compound per day, about 0.5 to about 75mg/kg of body weight of active compound per day, about 0.5 to about 50mg/kg of body weight of active compound per day, about 0.5 to about 25mg/kg of body weight of active compound per day, about 1 to about 20mg/kg of body weight of active compound per day, about 1 to about 1 Omg/kg of body weight of active compound per day, about 20mg/kg of body weight of active compound per day, about 1 Omg/kg of body weight of active compound per day, or about 5mg/kg of body weight of active compound per day.
  • the compounds described herein can be prepared in a variety of ways known to one skilled in the art of organic synthesis or variations thereon as appreciated by those skilled in the art.
  • the compounds described herein can be prepared from readily available starting materials. Optimum reaction conditions may vary with the particular art.
  • Variations on Formula I and the compounds described herein include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, the chirality of the molecule can be changed. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.
  • Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, i.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art.
  • product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
  • spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry
  • chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
  • the compounds described herein can be obtained from commercial sources.
  • the compounds can be obtained from, for example, Sigma Chemical Co. (St. Louis, MO), VWR International (Radnor, PA), or Oakwood Products, Inc. (West Columbia, SC).
  • the methods include administering to a subject an effective amount of one or acceptable salt thereof.
  • effective amount when used to describe an amount of compound in a method, refers to the amount of a compound that achieves the desired pharmacological effect or other effect, for example, an amount that results in uric acid production reduction.
  • the compounds and compositions described herein or pharmaceutically acceptable salts thereof are useful for treating gout or hyperuricemia in humans, including, without limitation, pediatric and geriatric populations, and in animals, e.g., veterinary applications.
  • the methods are used to treat conditions associated with elevated uric acid levels, including chronic gouty arthritis, acute inflammatory arthritis, uric acid nephropathy, kidney stones, or tophi.
  • the methods include administering to the subject one or more of the compounds as described herein.
  • the methods can further comprise selecting a subject having gout or hyperuricemia.
  • compositions and methods can include one or more additional agents.
  • the one or more additional agents and the compounds described herein or pharmaceutically acceptable salts thereof can be administered in any order, including concomitant, simultaneous, or sequential administration. Sequential administration can be temporally spaced order of up to several days apart.
  • the methods can also include more than a single administration of the one or more additional agents and/or the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof.
  • the administration of the one or more additional agents and the compounds described herein or pharmaceutically acceptable salts or prodrugs thereof can be by the same or different routes and concurrently or sequentially.
  • Therapeutic agents include, but are not limited to, anti-gout agents.
  • the anti-gout agent can be allopurinol, benzbromarone, colchicine, probenecid, or sulfinpyrazone.
  • Therapeutic agents also include anti-inflammatory agents.
  • suitable anti-inflammatory agents include, for example, steroidal and nonsteroidal antiinflammatory drugs (e.g., ibuprofen and prednisone).
  • the therapeutic agent can also be, tocopherol, beta-carotene, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), caffeic acid, lutein, lycopene, selenium, tert-butylhydroquinone (TBHQ), Vitamin A, Vitamin C, and Vitamin E.
  • suitable antioxidants include putative antioxidant botanticals, such as, for example, grape seeds, green tea, Scutellaria baicalensis, American ginseng, ginkgo biloba, and the like.
  • any of the aforementioned therapeutic agents can be used in any combination with the compositions described herein.
  • Combinations are administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second).
  • the term combination is used to refer to concomitant, simultaneous, or sequential administration of two or more agents.
  • a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein are administered to a subject prior to onset (e.g., before obvious signs of gout or hyperuricemia), during early onset (e.g., upon initial signs and symptoms of gout or hyperuricemia), or after the development of gout or hyperuricemia.
  • Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of gout or hyperuricemia.
  • Therapeutic treatment involves administering to a subject a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein after gout or hyperuricemia is diagnosed.
  • the methods and compounds described herein are also useful in inhibiting xanthine oxidase activity in a cell.
  • the methods include contacting a cell with an effective amount of a xanthine oxidase inhibitor as described herein.
  • the contacting is performed in vivo.
  • the contacting is performed in vitio.
  • kits for treating or preventing gout or hyperuricemia in a subject can include any of the compounds or compositions described herein. For herein.
  • a kit can further include one or more additional agents, such as anti-gout agents (e.g., allopurinol, benzbromarone, colchicine, probenecid, or sulfinpyrazone), antiinflammatory agents, or antioxidants.
  • a kit can include an oral formulation of any of the compounds or compositions described herein.
  • a kit can additionally include directions for use of the kit (e.g., instructions for treating a subject), a container, a means for administering the compounds or compositions, and/or a carrier.
  • treatment refers to a method of reducing one or more symptoms of a disease or condition.
  • treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the seventy of one or more symptoms of the disease or condition.
  • a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms or signs of the disease in a subject as compared to a control.
  • control refers to the untreated condition.
  • the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
  • prevent, preventing, and prevention of a disease or disorder refer to an action, for example, administration of a composition or therapeutic agent, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or severity of one or more symptoms of the disease or disorder.
  • references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level. Such terms can include, but do not necessarily include, complete elimination.
  • subject means both mammals and non-mammals.
  • Mammals include, for example, humans; non-human primates, e.g., apes and monkeys; cattle;
  • Non-mammals include, for example, fish and birds. of these publications in their entireties are hereby incorporated by reference into this application.
  • PBS phosphate buffered saline
  • KN0 2 potassium nitrite
  • MnCh dioxide manganese
  • DTP A diethylene-triamine-pentaacetic acid
  • EDTA ferrous ammonium sulf
  • XO activity was determined by the method of continuous spectrophotometric rate determination by monitoring the increase of absorption at 295 nm of uric acid in 67 mM phosphate buffer (pH 7.4) containing 20 nM xanthine oxidase with an activity of 5 mU/ml, with or without the compounds as described herein. After pre-incubation for 1 to 5 min at 25 °C, the formation of uric acid in the reaction mixture was initiated by the addition of 50 ⁇ xanthine.
  • the test compounds and positive control are shown in Scheme 1. Allopurinol was used as a positive control. All compounds, including allopurinol, were dissolved in H 2 O or an aqueous solution. H 2 O was used as the negative control.
  • XO inhibition The inhibitory activity of xanthine oxidase by DHNB, DHBA, DH6NB and THB-CHO was determined in vitro by the formation of uric acid, which was measured spectrophotometrically by following the increase in absorbance of uric acid at 295 nm.
  • 20 nM XO was mixed with increasing concentrations of allopurinol, DHNB, DH6NB, DHBA, or THB-CHO (Scheme 1), the initial rate of uric acid formation showed a concentration-dependent decrease compared to the control, reflecting the decrease of XO activity (see Fig. 1 A).
  • DHNB significantly inhibited XO activity with an IC 50 value of 3 ⁇ , which is close to allopurinol 's value of 1.8 ⁇ .
  • the IC 50 values for DHBA and DH6NB were 76 and 96 ⁇ , respectively, indicating weak inhibition of XO activity.
  • the IC 5 o value for THB-CHO was too high to determine.
  • xanthine was added to initiate the reaction. The initial rate of uric acid formation did not change with increasing concentrations of xanthine.
  • a Dixon plot of a steady-state kinetic study of DHNB xanthine concentrations when the concentration of DHNB was fixed see Fig. IB).
  • the pH dependence of DHNB inhibition indicated that neutral or slightly acidic solutions benefit the inhibition (see Fig. 1C).
  • XO inhibition of DHNB is irreversible - DHNB displayed time-dependent inhibition of XO activity, similar to that of allopurinol.
  • XO (20 nM) was added to the mixture of xanthine (50 ⁇ ) and the inhibitor (6.67 ⁇ ) to start the reaction, both DHNB and allopurinol showed time-dependent inhibition (see Fig. 3).
  • An excess of 6.67 ⁇ DHNB or 6.67 ⁇ allopurinol reduced the rate gradually and finally reached a steady state level of catalytic activity. No complete inactivation was observed at the tested condition.
  • XO was treated with 20 ⁇ DHNB to induce inhibition; the reaction mixture was then treated with high levels of glutathione (GSH; 20 mM), dithiothreitol (DTT; 20 mM) or 2-mercaptoethanol (2-ME; 20 mM), which did not abolish the inhibition (see Fig. 5).
  • GSH glutathione
  • DTT dithiothreitol
  • 2-ME 2-mercaptoethanol
  • the reaction kinetics of DHNB with XO at different pH values were measured using a spectrophotometer by monitoring the decay of DHNB at 327 nm in a system of 30 nM XO with 30 ⁇ DHNB in phosphate buffer with pH 6.5 to 8.5.
  • the extinction coefficient of DHNB at 327 nm was measured as 15,600 M '1 cm "1 .
  • the sample for product analysis by mass spectroscopy and HPLC was prepared by mixing 0.3 U XO with 4 mg DHNB in 1 mL phosphate buffer (pH 7.4) for 3 days.
  • DHNB/XO samples were analyzed by HPLC (Bio-Rad BioLogic DuoFlow; Hercules, CA) equipped with a 250 x 4.6 mm, 5 micron Phenomenex C-18 (2) Luna column, with a mobile phase of 40% acetonitrile/water. DHNB and its product were monitored by the optical absorption at 279 nm and 327 nm.
  • Negative electrospray ionization-mass spectrometry (ESI-MS) and tandem (MS- MS) were applied to detect and confirm the reaction products of DHNB with XO. All mass spectrometric experiments were performed on an API 3200-Qtrap triple quadrupole mass spectrometer (Applied Biosystem/MDS SCIEX; Foster City, CA) equipped with a follows: ion-spray voltage, -4.5 kV; ion source temperature, 6001; gas 1 , 40 psi; gas 2, 40 psi; curtain gas, 20 psi; collision gas, high.
  • the absorption of DHNB at 327 nm decreased with time and a new peak appeared at 270 nm (see Fig. 6A).
  • the decay rate was in the range of 10 " '° mol/L/s and was pH dependent, i.e., the higher the pH value, the faster DHNB decayed (see Fig. 6B).
  • DHNB itself was very stable. At room temperature, DHNB was converted by XO enzyme to a product which has no inhibitory effect on XO and thus recovered the XO activity as the concentration of DHNB decreased. The UV-VIS spectrum of the product was different from that of DHNB. HPLC analysis of DHNB/XO showed a new peak which was more polar than DHNB (see Fig 6C). Mass spectrometric analysis of the product gave a molecular ion ([M-H] " ) peak at m/z 198 in the EI mass spectrum, while DHNB showed [M-H] " peak at m/z 182 (see Fig 6D, E).
  • the MS/MS of m/z 182 of DHNB gave several typical fragments such as m/z 165 ([M-H-OH] " ), 152 ([M-H-CHO-H] " ) and 135 (m/z 152-OH).
  • the MS/MS of molecular ion at m/z 198 of the product gave a first main fragment at m/z 154, a mass difference of 44 indicating a loss of CO2, which further loses a -OH to give a fragment at m/z 137.
  • the product is 3,4-dihydroxy-5-nitrobenzoic acid, implying that DHNB is oxidized to the acid by the enzyme.
  • the compounds described herein can serve as antioxidants. This was determined by testing the ability of the compounds to scavenge three times and the data are presented as mean ⁇ SD.
  • DPPH scavenging assay - The abilities of the polyphenols described herein to scavenge the DPPH radical were measured optically by monitoring the decreases of their absorptions at 429 nm.
  • DPPH was used at a concentration of 100 ⁇ .
  • Their scavenging activities were compared with that of vitamin C. As shown in Fig. 7A, DHNB, DH6NB, DHBA, DHB- CHO, and THB-CHO have as strong of a DPPH scavenging effect as vitamin C;
  • HOCI scavenging assay - HOCI was prepared immediately before use by adjusting the pH of a 1% (v/v) solution of NaOCl to pH 6.2 with 0.6 M sulfuric acid. The concentration was further determined spectrophotometrically at 235 nm using the molar extinction coefficient of 100 Nf'cm " '.
  • 5-Thio-2-nitrobenzoic acid (TNB) was prepared by reducing 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) with sodium borohydride in phosphate buffer.
  • the final concentrations of reagents used in the assay are as follows: 25 ⁇ HOCI, 70 ⁇ TNB, 0 to 200 ⁇ antioxidants, phosphate buffer, 50 mM, pH 6.6.
  • the HOCI scavenging assay was based on the inhibition of TNB oxidation to DTNB induced by HOCI.
  • THB-CHO had a stronger HOCI scavenging effect than that of vitamin C.
  • DHNB had a moderate scavenging effect
  • other compounds including DHBA, DHB-CHO, and DH6NB
  • DH6NB had a weak scavenging effect on HOCI (Fig. 7B).
  • the concentration dependent effect of these compounds, including DHNB, caffeic acid, DHBA, DHB-CHO, DHB-COOH, and allopurinol, on HOC! scavenging activity were also studied and compared with that of Vitamin C (Fig. 9).
  • Peroxynitrite scavenging assay - Peroxynitrite (ONOO-) was generated by mixing 5 mL acidic solution (0.6 M HCI) of H 2 O 2 (0.7 M) and 5 mL of 0.6 M KN0 2 in an ice bath for 1 second and the reaction was quenched with 5 mL of ice-cold 1.2 M and the reaction mixture was then left overnight at -20°C. Concentrations of ONOO- were determined before each experiment at 302 nm using a molar extinction coefficient of 1 ,670 M -1 cm " '.
  • the final concentrations of reagents used in the assay are as follows: 25 uM ONOO " , 10 ⁇ DTPA, 5 ⁇ DHR 123, 0.1 M phosphate buffer, pH 7.4.
  • the ONOO " scavenging assay was performed by monitoring the oxidation of
  • DHNB, DHBA, DHB-CHO, DH6NB, caffeic acid, THB-CHO, gallic acid, vanillin, and DMB-CH 2 OH were compared with that of vitamin C.
  • DHNB, DHBA, DHB-CHO, DH6NB, caffeic acid, THB-CHO and gallic acid had a strong scavenging effect on ONOO " (Fig. 7C).
  • Vitamin C was used as a positive control.
  • concentration dependent effects of these compounds on ONOO " scavenging were also studied and compared with that of vitamins C and E (Fig. 10).
  • Superoxide scavenging assay - Superoxide ( O 2 -. ) scavenging activity was assayed in the xanthine-xanthine oxidase system and determined by the inhibition of the reduction of nitro blue tetrazolium (NBT) to form blue formazan which has an absorption at 560 nm.
  • the final concentrations of reagents used in the assay are as follows: 16.8 mU xanthine oxidase, 25 ⁇ xanthine, 50 ⁇ NBT, and 0.1 M phosphate buffer (pH 8.5). O 2 -.
  • DHBA and THB-CHO at the concentration of 20 ⁇ , had strong scavenging effects on superoxide (Fig. 7D).
  • the concentration dependent effect of DHBA and caffeic acid on superoxide scavenging was also studied and compared with that of glutathione (Fig. 11).
  • mice A hyperuricemia mouse model was used. Allantoxanamide, a potent uricase inhibitor, was used to induce hyperuricemia in mice in this study. Briefly, adutt C57BL/6 mice (15-25 g, 6-8 weeks old, 6 per group) were administrated DHNB at a concentration of 100 mg/kg in 1.0% polyethylene glycol 400 (PEG400 in a volume of 0.1 ml/10 g mouse body weight) via oral gavage. The mice were subsequently intraperitoneal ly injected with allantoxanamide at 200 mg/kg in 0.5% CMC-Na in a volume of 0.1 ml/10 g mouse body weight just after the tested drug oral administration to increase the serum uric acid level.
  • PEG400 polyethylene glycol 400
  • Positive control mice were administered allopurinol at the same concentration as DHNB followed by i.p. allantoxanamide.
  • the negative control mice were administered PEG400 only followed by i.p. allantoxanamide.
  • the normal group mice were administered PEG400 only followed by i.p. CMC-Na only. Food and water were withheld overnight prior to the study.
  • Whole blood samples were collected from mice through orbital vein bleeding at the end of the study. The mice were anaesthetized with diethyl ether inside a chamber. The blood was allowed to clot for 1 h at room temperature and then centrifuged at 2350 x g for 4 min to obtain the serum. The serum was kept on ice and assayed immediately. Serum uric acid was determined with the phosphotungstate method, as known to those of skill in the art.
  • mice C57BL/6 mice were randomized into 3 groups (12/group). Groups 1 to 3 received an oral vehicle solution (PEG400), DHNB (500 mg/kg), and allopurinol (500 mg/kg), respectively. Each mouse was monitored for general health conditions on a daily basis for 28 days, including examination of mortality, body weights, and behavior of the mice.
  • PEG400 oral vehicle solution
  • DHNB 500 mg/kg
  • allopurinol 500 mg/kg
  • DHNB or allopurinol at 500 mg/kg was administrated to 12 mice, respectively, via oral gavage. Control mice received the vehicle solution. The animals were observed daily up to 28 days. DHNB- treated mice did not show any symptoms of general toxicity. There was no difference in body weight and behavior between DHNB-treated mice and control mice. Histology analysis for the liver, kidney, and heart did not show any difference between DHNB- treated mice and control mice. In the allopurinol treated mice, however, 5 mice died within 3 days (mortality 42 %).
  • mice (mixed male and female) gave birth to total 19 pups, but eight died in two days.
  • the survived pups of allopurinol treated mice started to lose hair after two weeks (Fig. 14A) and lost most of the back hair at 3 weeks (Fig. 14B) to 4 weeks (Fig. 14C).
  • the pups After separated from the adult mice, the pups started to grow hair again and returned to normal hair at the age of 6 to 7 weeks (Fig. 14D).
  • this hair loss phenomenon was not observed on DHNB treated mice (see Fig. 14E for DHNB treated mice at 4 weeks).
  • Table 1 A summary of the in vivo toxicities of DHNB and allopurinol in mice is shown in Table 1.
  • XO inhibition effects of the compounds shown in Scheme 2 were compared at a concentration of 20 ⁇ .

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Abstract

L'invention concerne des inhibiteurs de xanthine oxydase à petites molécules, ainsi que des procédés d'utilisation de ceux-ci dans le traitement de la goutte ou de l'hyperuricémie.
PCT/US2014/041590 2014-06-09 2014-06-09 Inhibiteurs de xanthine oxydase à petites molécules et procédés d'utilisation WO2015191034A1 (fr)

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CN114209679A (zh) * 2022-02-21 2022-03-22 中国科学院南海海洋研究所 3，5-二羟基-4-甲氧基苄醇的合成方法及其应用

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Publication number Priority date Publication date Assignee Title
CN114209679A (zh) * 2022-02-21 2022-03-22 中国科学院南海海洋研究所 3，5-二羟基-4-甲氧基苄醇的合成方法及其应用
CN114209679B (zh) * 2022-02-21 2022-06-10 中国科学院南海海洋研究所 3，5-二羟基-4-甲氧基苄醇的合成方法及其应用

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