US20210053931A1 - Ester nitrates derivatives of aromatic aldehydes with multiple pharmalogic properties to treat sickle cell disease - Google Patents
Ester nitrates derivatives of aromatic aldehydes with multiple pharmalogic properties to treat sickle cell disease Download PDFInfo
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- US20210053931A1 US20210053931A1 US17/088,063 US202017088063A US2021053931A1 US 20210053931 A1 US20210053931 A1 US 20210053931A1 US 202017088063 A US202017088063 A US 202017088063A US 2021053931 A1 US2021053931 A1 US 2021053931A1
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- Prior art keywords
- compound
- arh
- compounds
- sickling
- mmol
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- -1 Ester nitrates derivatives Chemical class 0.000 title abstract description 19
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- 238000000034 method Methods 0.000 claims abstract description 28
- 150000003839 salts Chemical class 0.000 claims description 21
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 125000003545 alkoxy group Chemical group 0.000 claims description 7
- 125000003118 aryl group Chemical group 0.000 claims description 7
- 229910052799 carbon Inorganic materials 0.000 claims description 7
- 229910052736 halogen Inorganic materials 0.000 claims description 7
- 150000002367 halogens Chemical class 0.000 claims description 7
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- 230000001965 increasing effect Effects 0.000 abstract description 12
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/58—One oxygen atom, e.g. butenolide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
- C07D213/28—Radicals substituted by singly-bound oxygen or sulphur atoms
- C07D213/30—Oxygen atoms
Definitions
- the invention relates to aromatic aldehydes and their use in the treatment of Sickle Cell Disease.
- Sickle cell disease is the consequence of substitution of Glu by Val in the 6 th position of the ⁇ -globin chain of hemoglobin (Hb) resulting in the formation of sickle Hb (HbS).
- Intracellular polymerization of deoxygenated sickle Hb into long, rigid and insoluble fibres causes the pathophysiology associated with SCD; facilitating a cascade of adverse events that include decreased nitric oxide (NO) bioavailability, endothelial cell activation, compensatory vasoconstriction, increase in neutrophil count, adhesion of red blood cells (RBCs) to tissue endothelium, vaso-occlusion and impaired microvascular blood flow.
- NO nitric oxide
- RBCs red blood cells
- TNF- ⁇ pro-inflammatory cytokines
- TNF- ⁇ also stimulates production of free radicals and other inflammatory mediators, such as IL-1 and PGE2, and induce changes in coagulant and anticoagulant properties.
- Individuals with SCD have also shown decreased NO physiological levels in vascular endothelium that have also been linked to hemolysis, inflammation, vaso-occlusion and multiple organ stress. Produced naturally by the vascular endothelium, NO provides favorable microvasculature effects (i.e., dilation and increased blood flow), and also possesses anti-inflammatory properties.
- HU Hydroxyurea
- SCD gamma globin expression
- HbF fetal Hb
- NO-donor compounds such as S-nitrosocysteine, detaNONOate and thalidomides have also been shown to increase gamma-globin expression.
- HbF is able to prevent RBC sickling by inhibiting HbS polymerization.
- the NO also has a direct positive vasodilation and anti-inflammation effects, as well as inhibition of platelet aggregation; reducing the severity and duration of vaso-occlusive crises. Nonetheless, it has been reported that HU treatment stimulates the production of pro-inflammatory cytokines, such as TNF- ⁇ , IL-1A, IL-1B, etc., counteracting the beneficial effects of the drug.
- the invention relates to aromatic aldehydes and their use in the treatment of Sickle Cell Disease.
- the invention provides new compounds that have greater potency in increasing the affinity of Hb for oxygen, greater potency for preventing sickling, and additional pharmacologic properties that ameliorate other symptoms of SCD.
- the invention further provides methods for treating SCD by providing a subject having SCD with a compound according to the invention.
- the invention provides new anti-sickling aromatic aldehydes that have greater potency for increasing the affinity of hemoglobin (Hb), and especially sickle cell Hb (scHb) and/or reducing the ability of scHb to polymerize, thus reducing the sickling of sickle red blood cells.
- Hb hemoglobin
- scHb sickle cell Hb
- R1 and R4 are the same or different and are H, OH, alkyl, alkoxy, halogen, aryl or O-aryl; and M and Q are different and are O or (CH 2 )q; and R2 and R3 are the same or different and are H or OH; and X or Y or Z are the same or different and are C or N; and n is 0-5; and p is 0-5; and q is 0-5; with the proviso that when M is O, then either n is 1-5 or R1 is not H; and salts thereof.
- Representative compounds selected from the group above include:
- the invention provides new aromatic aldehydes that are metabolized to produce NO 2 and anti-sickling aromatic aldehydes.
- Compounds according to this aspect have a structure selected from:
- R1 and R4 are the same or different and are H, OH, alkyl, alkoxy, halogen, aryl or O-aryl; and M and Q are different and are O or (CH 2 )q; and R2 and R3 are the same and are ONO 2 or different with either as ONO 2 and the other as H or OH; and X or Y or Z are the same or different and are C or N; and n is 0-5; and p is 0-5; and q is 0-5; and salts thereof.
- R1 is H; and M is O; and R2 is H; and R3 is ONO 2 ; and p is 0.
- Representative compounds selected from the group above include:
- the invention provides methods for treating a subject having sickle cell disease (SCD), comprising administering to the subject a therapeutically effective amount of one or more compound selected from the compounds according to the first or second aspect of the invention.
- SCD sickle cell disease
- FIG. 1 shows results of oxygen equilibrium curve studies for 5-HMF and compounds TD-7, TD-7-1, TD-8, TD-8-1, TD-9 and TD-9-1.
- FIG. 2 shows the effect of incubation time on Hb affinity for O 2 with whole blood for 5-HMF and compound TD-7.
- FIG. 3 shows results of a NO release study for compounds TD-1, TD-7-1, TD-8-1 and TD-9-1.
- FIG. 4 shows inhibition of sickling of SS cells by test compounds under 4% O 2 at 37° C. for 5 h.
- FIG. 5A shows binding of TD-7 (green) in deoxyHb.
- FIG. 5B shows a close view of TD-7 (yellow) interaction with the ⁇ F helix of the protein (green)
- the invention relates to aromatic aldehydes and their use in the treatment of Sickle Cell Disease.
- the invention provides new compounds that have greater potency in increasing the affinity of Hb for oxygen, greater potency for preventing sickling, and additional pharmacologic properties that ameliorate other symptoms of SCD.
- the invention further provides methods for treating SCD by providing a subject having SCD with a compound according to the invention.
- the invention provides new anti-sickling aromatic aldehydes that have greater potency for increasing the affinity of hemoglobin (Hb), and especially sickle cell Hb (scHb) and/or reducing the ability of scHb to polymerize, thus reducing the sickling of sickle cell red blood cells.
- Hb hemoglobin
- scHb sickle cell Hb
- Compounds according to the invention have the formula:
- R1 and R4 are the same or different and are H, OH, alkyl, alkoxy, halogen, aryl or O-aryl; and M and Q are different and are O or (CH 2 )q; and R2 and R3 are the same or different and are H or OH; and X or Y or Z are the same or different and are C or N; and n is 0-5; and p is 0-5; and q is 0-5; with the proviso that when M is O, then either n is 1-5 or R1 is not H; and salts thereof.
- Representative compounds selected from the group above include:
- the invention provides new aromatic aldehydes that are metabolized to produce NO 2 and anti-sickling aromatic aldehydes.
- Compounds according to this aspect have a structure selected from:
- R1 and R4 are the same or different and are H, OH, alkyl, alkoxy, halogen, aryl or O-aryl; and M and Q are different and are O or (CH 2 )q; and R2 and R3 are the same and are ONO 2 or different with either as ONO 2 and the other as H or OH; and X or Y or Z are the same or different and are C or N; and n is 0-5; and p is 0-5; and q is 0-5; and salts thereof.
- R1 is H; and M is O; and R2 is H; and R3 is ONO 2 ; and p is 0; and salts thereof.
- Representative compounds selected from the group above include:
- the invention provides methods for treating a subject having sickle cell disease (SCD), comprising administering to the subject a therapeutically effective amount of one or more compound selected from the compounds according to the first or second aspect of the invention.
- SCD sickle cell disease
- the subject is administered from about 20 mg to about 500 mg of the compound. In some embodiments the subject is administered from about 20 mg to about 300 mg of the compound. In some embodiments the subject is administered from about 20 mg to about 200 mg of the compound. In some embodiments the subject is administered from about 20 mg to about 100 mg of the compound. In some embodiments the subject is administered from about 20 mg to about 50 mg of the compound.
- the subject is administered from about 100 mg to about 500 mg of the compound. In some embodiments the subject is administered from about 100 mg to about 300 mg of the compound. In some embodiments the subject is administered from about 100 mg to about 200 mg of the compound.
- the subject is administered from about 200 mg to about 500 mg of the compound. In some embodiments the subject is administered from about 200 mg to about 300 mg of the compound.
- the subject is administered from about 500 mg to about 10,000 mg of the compound. In some embodiments the subject is administered from about 1,000 mg to about 10,000 mg of the compound. In some embodiments the subject is administered from about 5,000 mg to about 10,000 mg of the compound.
- Administration of the compounds can be by any suitable route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, inhalation, intratracheal, or intrarectal. In some preferred embodiments, administration is orally, by injection, or by inhalation.
- the compounds used in the present invention may be present in any suitable diluent (including water), carrier or excipient.
- salts may form salts which are also within the scope of this invention.
- salt(s) denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases.
- Pharmaceutically acceptable (i.e., non-toxic, exhibiting minimal or no undesired toxicological effects, physiologically acceptable) salts are preferred.
- salts are intended to mean salts that retain the desired biological activity of the compounds and exhibit minimal or no undesired toxicological effects.
- examples of such salts include, but are not limited to, salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, methanesulfonic acid, p-toluenesulfonic acid and polygalacturonic acid.
- inorganic acids for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like
- organic acids such as ace
- salts include pharmaceutically acceptable quaternary salts known by those skilled in the art, which specifically include the quaternary ammonium salt of the formula —NR+Z—, wherein R is hydrogen, alkyl, or benzyl, and Z is a counterion, including chloride, bromide, iodide, —O-alkyl, toluenesulfonate, methylsulfonate, sulfonat, phosphate, or carboxylat (such as benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate, benzyloate, and dip henylacetate).
- R is hydrogen, alkyl, or benzyl
- Z is a counterion, including chloride, bromide, iodide, —O-alkyl, toluenesulfonate, methylsulfonate
- a “therapeutically effective amount” is an amount sufficient to alleviate or eliminate signs or symptoms of the disease.
- TD-7 increase the oxygen affinity of Hb by binding at the ⁇ -cleft and restraining the two ⁇ -subunits from transitioning from the oxyHb to the deoxy Hb conformation as previously described for 5HMF; based on the structure it seems that the improved potency of TD-7 is also in part due to increased perturbation of the ⁇ F-helix that leads to increased destabilization of the ⁇ F-helix mediated polymer contact.
- Trifluoroacetic anhydride (5.5 mL, 2 mmol) was added to a suspension of lithium nitrate (2.7 g, 2 mmol) in anhydrous CH 3 CN (30 mL) at 20° C. The reaction mixture was stirred until clear (30 min) and cooled to 0° C. (ice-bath). Sodium carbonate (4.2 g, 2 mmol) and 5-(hydroxymethyl) furfural (2.5 g, 1 mmol) were added together and the reaction mixture was allowed to stir for 7 h at 0° C. (ice-bath). The reaction mixture was poured into an ice-cold solution of saturated NaHCO 3 and extracted with EtOAc (2 ⁇ 10 mL).
- Compound TD-7-1 was prepared according to literature procedure for a similar compound. (Fotopoulou, T.; Iliodromitis, E. K.; Koufaki, M.; Tsotinis, A.; Zoga, A.; Gizas, V.; Pyriochou, A.; Papapetropoulos, A.; Andreadou, I.; Kremastinos, D. T., Bioorg. Med. Chem. 2008, 16, 4523-4531.)
- Triphenylphosphine 140 mg, 0.6 mmol
- tetrabromomethane 180 mg, 0.6 mmol
- 2-((6-(hydroxymethyl)pyridin-2-yl)methoxy)-5-methoxybenzaldehyde 100 mg, 0.4 mmol
- the reaction mixture was allowed to stir for 2 h at room temperature.
- the reaction mixture was neutralized with saturated NaHCO 3 at 0° C. (ice-bath) and extracted with EtOAc (10 mL). The organic portion was washed with brine (2 ⁇ 10 mL), dried over Na 2 SO 4 and evaporated under reduced pressure to yield a crude product.
- Compound TD-8-1 was prepared according to literature procedure for a similar compound. (Fotopoulou, T.; Iliodromitis, E. K.; Koufaki, M.; Tsotinis, A.; Zoga, A.; Gizas, V.; Pyriochou, A.; Papapetropoulos, A.; Andreadou, I.; Kremastinos, D. T., Bioorg. Med. Chem. 2008, 16, 4523-4531.).
- Triphenylphosphine 140 mg, 0.6 mmol
- tetrabromomethane 180 mg, 0.6 mmol
- the mixture was allowed to stir for 2 h at room temperature.
- the reaction mixture was neutralized with saturated NaHCO 3 at 0° C. (ice-bath) and extracted with EtOAc (10 mL). The organic portion was washed with brine (2 ⁇ 10 mL), dried over Na 2 SO 4 and evaporated under reduced pressure to yield a crude product.
- Compound TD-9-1 was prepared according to literature procedure for a similar compound. (Fotopoulou, T.; Iliodromitis, E. K.; Koufaki, M.; Tsotinis, A.; Zoga, A.; Gizas, V.; Pyriochou, A.; Papapetropoulos, A.; Andreadou, 1.; Kremastinos, D. T., Bioorg. Med. Chem. 2008, 16, 4523-4531.).
- Triphenylphosphine 140 mg, 0.6 mmol
- tetrabromomethane 180 mg, 0.6 mmol
- 2-((6-(hydroxymethyl)pyridin-2-yl)methoxy)-4-methoxybenzaldehyde 100 mg, 0.4 mmol
- the reaction mixture was neutralized with saturated NaHCO 3 at 0° C. (ice-bath) and extracted with EtOAc (10 mL). The organic portion was washed with brine (2 ⁇ 10 mL), dried over Na 2 SO 4 and evaporated under reduced pressure to yield a crude product.
- TD-7, TD-7-1, TD-8, TD-8-1, TD-9 and TD-9-1 were compounds with 100 mM stock solution in DMSO and normal whole blood (AA).
- TD-7, TD-8 and TD-9 are compounds with a hydroxymethyl group on the pyridine ring, while TD-7-1, TD-8-1 and TD-9-1 are the respective nitrate ester derivatives.
- the whole blood samples had hematocrit values ranging from 29-36%, and cell-free purified Hb of 10-12 g/dL in the absence or presence of effectors solubilized in DMSO at 2 mM or in some cases with varying compound concentration.
- the blood samples were incubated in an IL 237 tonometer (Instrumentation Laboratories, Inc., Lexington, Mass.) for about 5-7 min at 37° C. against a gas mixture containing O 2 concentrations of 0.804%, 2.935% and 5.528% and allowed to equilibrate at O 2 tensions of 6, 20 and 40 mmHg. After equilibration, the sample was removed via syringe and aspirated into a ABL 700 series table top automated blood gas analyzer (Radiometer America, Inc., Westlake, Ohio) to determine total hemoglobin (tHb), hematocrit (Hct), pH, pCO 2 , partial pressure of oxygen (pO 2 ), and the Hb oxygen saturation values (sO 2 ).
- tHb total hemoglobin
- Hct hematocrit
- pH pH
- pCO 2 partial pressure of oxygen
- sO 2 partial pressure of oxygen saturation values
- Hemoglobin was prepared from centrifuging human blood at 2500 rpms for 20 min at 4° C. The supernatant solution, debris and plasma were discarded from the centrifuge bottles, leaving behind RBCs. The RBCs were washed thrice with an excess volume of 0.9% NaCl, and once with 1.0% NaCl, each time centrifuging and discarding the supernatant solution. The RBCs were pooled together into a chilled flask and hemolyzed to free Hb by adding 1-2 volume excess of 50 mM Tris buffer, pH 8.6 (containing EDTA). The mixture was allowed to stand in ice for 30 min with occasional stirring. The Hb solution was centrifuged at 10,000 rpm for 2 h at 4° C.
- the supernatant Hb solution was pooled into a chilled flask and NaCl (40-60 mg/mL Hb solution) was slowly added with stirring. Hemoglobin solution was centrifuged at 10,000 rpm for 2 h at 4° C. to remove any cell stroma remnants. The clear supernatant Hb solution was pooled into a chilled flask and the “syrupy” pellet was discarded. Hemoglobin solution was dialyzed against 50 mM Tris buffer, pH 8.6 (containing EDTA) at 4° C. to remove NaCl and/or other low molecular weight impurities.
- NaCl 40-60 mg/mL Hb solution
- This diazonium salt further reacts with N-naphthyl-ethylenediamine (NED) to form a stable azo compound which possesses an intense purple color.
- NED N-naphthyl-ethylenediamine
- the absorbance of the azo compound was measured at 540 urn and was found to be directly proportional to the NOT concentration in the sample.
- Sodium nitrite (NaNO 2 ) was used as a standard for the Griess assay. NaNO 2 samples in deionized water were prepared of concentrations within the linear range of the assay (1-100 ⁇ M). The samples were incubated for 30 min at 37° C., after which Griess reagent was added and absorbance was measured at 540 nm. The measured absorbances were used to obtain a standard curve ( FIG. 3 ). For the measurement of NO 2 ⁇ release for all the test compounds (TD-1, TD-7-1, TD-8-1 and TD-9-1), the absorbances were extrapolated from the
- test compound 1 mL of a 2 mM solution in 0.1 M phosphate buffer (pH 7.4)
- a freshly prepared solution of L-cysteine 1 mL of a 36 mM solution in 0.1 M phosphate buffer, pH 7.4
- the NO released (quantitated as NO 2 ⁇ release in ⁇ M) was calculated from the standard nitrite curve by extrapolation of the absorbance. Presence of NO 2 ⁇ shows formation of a pink color, the intensity of which is directly proportional to the amount of NO 2 ⁇ present in the mixture.
- Table 4 represents NO 2 ⁇ release for compounds TD-1, TD-7-1, TD-8-1 and TD-9-1.
- Table 5 represents the concentrations of the effectors along with the % inhibition of sickling cells.
- the hemolysates that had been prepared from SS cells after incubation with test compounds were also subjected to cation-exchange HPLC as previously reported (Abdulmalik et al., 2005).
- Table 5 shows the effect of the various concentrations of the test compounds on the amount of modified Hb S which reflects Schiff-base adduct formation between the aromatic aldehyde and the protein.
- TD-7 either the free base or HCl salt
- TD-7 showed a very potent anti-sickling effect, as much as 95% inhibition at 0.5 mM concentration.
- a plateau effect was observed as the concentration of TD-7 was increased.
- the water soluble HCl salt of TD-7 interestingly barely showed any antisickling effect at 0.5 mM, but remarkably inhibited 100% sickling at 1.0 mM showing a plateau with increasing drug concentration.
- INN-312 the second-generation positive control, also decreased the percent of sickled cells in a dose-dependent manner but at 0.5 mM it only inhibited sickling to 15% compared to the 95% by TD-7.
- TD-7-1 the ester nitrate of TD-7 although preventing RBC sickling, hemolyzed the RBC at higher drug concentrations. Also, it seems to swell/hydrate the RBC due to possible intracellular uptake of water which could be beneficial to counteract the rigidity of sickle cells.
- TD-8 and TD-9 were not as potent as the TD-7 analog, only inhibiting 38 and 57% RBC sickling, respectively at 2 mM. In comparison, vanillin which is the parent scaffold for the INN and TD compounds only inhibited 12% RBC sickling at 5 mM.
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Abstract
Description
- The invention relates to aromatic aldehydes and their use in the treatment of Sickle Cell Disease.
- Sickle cell disease (SCD) is the consequence of substitution of Glu by Val in the 6th position of the β-globin chain of hemoglobin (Hb) resulting in the formation of sickle Hb (HbS). Intracellular polymerization of deoxygenated sickle Hb into long, rigid and insoluble fibres causes the pathophysiology associated with SCD; facilitating a cascade of adverse events that include decreased nitric oxide (NO) bioavailability, endothelial cell activation, compensatory vasoconstriction, increase in neutrophil count, adhesion of red blood cells (RBCs) to tissue endothelium, vaso-occlusion and impaired microvascular blood flow. The clinical condition is characterized by chronic hemolytic anemia, frequent and severe painful crises, and multi-system pathology that impact nearly every organ.
- Individuals with SCD have shown significant levels of pro-inflammatory cytokines, such as TNF-α which can trigger the painful vaso-occlusive crises and lead to the emergence of infectious and inflammatory episodes. TNF-α also stimulates production of free radicals and other inflammatory mediators, such as IL-1 and PGE2, and induce changes in coagulant and anticoagulant properties. Individuals with SCD have also shown decreased NO physiological levels in vascular endothelium that have also been linked to hemolysis, inflammation, vaso-occlusion and multiple organ stress. Produced naturally by the vascular endothelium, NO provides favorable microvasculature effects (i.e., dilation and increased blood flow), and also possesses anti-inflammatory properties.
- Hydroxyurea (HU) is the only drug approved for treating SCD. When metabolized in vivo it releases NO which is responsible for inducing gamma globin expression and the formation of fetal Hb (HbF). Several NO-donor compounds, such as S-nitrosocysteine, detaNONOate and thalidomides have also been shown to increase gamma-globin expression. HbF is able to prevent RBC sickling by inhibiting HbS polymerization. The NO also has a direct positive vasodilation and anti-inflammation effects, as well as inhibition of platelet aggregation; reducing the severity and duration of vaso-occlusive crises. Nonetheless, it has been reported that HU treatment stimulates the production of pro-inflammatory cytokines, such as TNF-α, IL-1A, IL-1B, etc., counteracting the beneficial effects of the drug.
- Delivery of oxygen to tissues is partly influenced by the affinity of Hb for oxygen. In normal circulation, oxygen is increasingly released in the following order—in larger vessels, at the arterioles, and at the capillaries. However, in patients with SCD, the existing compensatory low oxygen affinity (P50=32±2 mm Hg; normal P50=26±2 mm Hg), paradoxically causes more oxygen to be released at the arteries and arterioles. Vasoconstriction, which occurs at the arterioles to counteract this process, exacerbates the untoward cell sickling, vaso-occlusion, and inefficient tissue oxygenation at the capillaries. Several aromatic aldehydes, such as vitamin B6 (pyridoxal), vanillin, pyridyl derivatives of vanillin (INN compounds), 12C79 and 5-HMF have been studied that show promise in preventing tissue hypoxia by increasing the oxygen affinity of sickle Hb and ensuring availability of more oxygen at the capillaries. Some of these compounds also stercospecifically inhibit polymer formation. 5HMF, a non-toxic by-product of sugar metabolism is currently being studied by NIH/Baxter for treating SCD.
- There is a need for new compounds that have greater potency in increasing the affinity of Hb for oxygen, greater potency for preventing sickling, and additional pharmacologic properties that ameliorate other symptoms of SCD.
- The invention relates to aromatic aldehydes and their use in the treatment of Sickle Cell Disease. The invention provides new compounds that have greater potency in increasing the affinity of Hb for oxygen, greater potency for preventing sickling, and additional pharmacologic properties that ameliorate other symptoms of SCD. The invention further provides methods for treating SCD by providing a subject having SCD with a compound according to the invention.
- In a first aspect, the invention provides new anti-sickling aromatic aldehydes that have greater potency for increasing the affinity of hemoglobin (Hb), and especially sickle cell Hb (scHb) and/or reducing the ability of scHb to polymerize, thus reducing the sickling of sickle red blood cells.
- Compounds according to the invention have the formula:
- wherein R1 and R4 are the same or different and are H, OH, alkyl, alkoxy, halogen, aryl or O-aryl; and M and Q are different and are O or (CH2)q; and R2 and R3 are the same or different and are H or OH; and X or Y or Z are the same or different and are C or N; and n is 0-5; and p is 0-5; and q is 0-5; with the proviso that when M is O, then either n is 1-5 or R1 is not H; and salts thereof.
- Representative compounds selected from the group above include:
- In a second aspect, the invention provides new aromatic aldehydes that are metabolized to produce NO2 and anti-sickling aromatic aldehydes. Compounds according to this aspect have a structure selected from:
- wherein R1 and R4 are the same or different and are H, OH, alkyl, alkoxy, halogen, aryl or O-aryl; and M and Q are different and are O or (CH2)q; and R2 and R3 are the same and are ONO2 or different with either as ONO2 and the other as H or OH; and X or Y or Z are the same or different and are C or N; and n is 0-5; and p is 0-5; and q is 0-5; and salts thereof. In some embodiments, R1 is H; and M is O; and R2 is H; and R3 is ONO2; and p is 0.
- Representative compounds selected from the group above include:
- In a third aspect, the invention provides methods for treating a subject having sickle cell disease (SCD), comprising administering to the subject a therapeutically effective amount of one or more compound selected from the compounds according to the first or second aspect of the invention.
-
FIG. 1 shows results of oxygen equilibrium curve studies for 5-HMF and compounds TD-7, TD-7-1, TD-8, TD-8-1, TD-9 and TD-9-1. -
FIG. 2 shows the effect of incubation time on Hb affinity for O2 with whole blood for 5-HMF and compound TD-7. -
FIG. 3 shows results of a NO release study for compounds TD-1, TD-7-1, TD-8-1 and TD-9-1. -
FIG. 4 shows inhibition of sickling of SS cells by test compounds under 4% O2 at 37° C. for 5 h. -
FIG. 5A shows binding of TD-7 (green) in deoxyHb.FIG. 5B shows a close view of TD-7 (yellow) interaction with the αF helix of the protein (green) - The invention relates to aromatic aldehydes and their use in the treatment of Sickle Cell Disease. The invention provides new compounds that have greater potency in increasing the affinity of Hb for oxygen, greater potency for preventing sickling, and additional pharmacologic properties that ameliorate other symptoms of SCD. The invention further provides methods for treating SCD by providing a subject having SCD with a compound according to the invention.
- In a first aspect, the invention provides new anti-sickling aromatic aldehydes that have greater potency for increasing the affinity of hemoglobin (Hb), and especially sickle cell Hb (scHb) and/or reducing the ability of scHb to polymerize, thus reducing the sickling of sickle cell red blood cells. Compounds according to the invention have the formula:
- wherein R1 and R4 are the same or different and are H, OH, alkyl, alkoxy, halogen, aryl or O-aryl; and M and Q are different and are O or (CH2)q; and R2 and R3 are the same or different and are H or OH; and X or Y or Z are the same or different and are C or N; and n is 0-5; and p is 0-5; and q is 0-5; with the proviso that when M is O, then either n is 1-5 or R1 is not H; and salts thereof.
- Representative compounds selected from the group above include:
- In a second aspect, the invention provides new aromatic aldehydes that are metabolized to produce NO2 and anti-sickling aromatic aldehydes. Compounds according to this aspect have a structure selected from:
- wherein R1 and R4 are the same or different and are H, OH, alkyl, alkoxy, halogen, aryl or O-aryl; and M and Q are different and are O or (CH2)q; and R2 and R3 are the same and are ONO2 or different with either as ONO2 and the other as H or OH; and X or Y or Z are the same or different and are C or N; and n is 0-5; and p is 0-5; and q is 0-5; and salts thereof. In some embodiments, R1 is H; and M is O; and R2 is H; and R3 is ONO2; and p is 0; and salts thereof.
- Representative compounds selected from the group above include:
- In a third aspect, the invention provides methods for treating a subject having sickle cell disease (SCD), comprising administering to the subject a therapeutically effective amount of one or more compound selected from the compounds according to the first or second aspect of the invention.
- In some embodiments the subject is administered from about 20 mg to about 500 mg of the compound. In some embodiments the subject is administered from about 20 mg to about 300 mg of the compound. In some embodiments the subject is administered from about 20 mg to about 200 mg of the compound. In some embodiments the subject is administered from about 20 mg to about 100 mg of the compound. In some embodiments the subject is administered from about 20 mg to about 50 mg of the compound.
- In some embodiments the subject is administered from about 100 mg to about 500 mg of the compound. In some embodiments the subject is administered from about 100 mg to about 300 mg of the compound. In some embodiments the subject is administered from about 100 mg to about 200 mg of the compound.
- In some embodiments the subject is administered from about 200 mg to about 500 mg of the compound. In some embodiments the subject is administered from about 200 mg to about 300 mg of the compound.
- In some embodiments the subject is administered from about 500 mg to about 10,000 mg of the compound. In some embodiments the subject is administered from about 1,000 mg to about 10,000 mg of the compound. In some embodiments the subject is administered from about 5,000 mg to about 10,000 mg of the compound.
- Administration of the compounds can be by any suitable route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, inhalation, intratracheal, or intrarectal. In some preferred embodiments, administration is orally, by injection, or by inhalation.
- The compounds used in the present invention may be present in any suitable diluent (including water), carrier or excipient.
- The compounds used in the method of the present invention may form salts which are also within the scope of this invention. The term “salt(s)”, as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases. Pharmaceutically acceptable (i.e., non-toxic, exhibiting minimal or no undesired toxicological effects, physiologically acceptable) salts are preferred.
- As used herein, the term “pharmaceutically acceptable salts” is intended to mean salts that retain the desired biological activity of the compounds and exhibit minimal or no undesired toxicological effects. Examples of such salts include, but are not limited to, salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmoic 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 by those skilled in the art, which specifically include the quaternary ammonium salt of the formula —NR+Z—, wherein R is hydrogen, alkyl, or benzyl, and Z is a counterion, including chloride, bromide, iodide, —O-alkyl, toluenesulfonate, methylsulfonate, sulfonat, phosphate, or carboxylat (such as benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate, benzyloate, and dip henylacetate).
- A “therapeutically effective amount” is an amount sufficient to alleviate or eliminate signs or symptoms of the disease.
- Without wishing to be bound by theory, we have determined the binding of TD-7 in deoxyHb to help elucidate its antisickling activity. Two molecules of TD-7 bind in a symmetry-related fashion at the α-cleft of the Hb, the aldehydes forming Schiff-base interaction with the two α-subunits N-terminal Val1 nitrogens (
FIG. 5A ). Formation of the Schiff-base disposes the methoxy-pyridine-methoxyl substituent group toward the mouth of the central water cavity, where the hydroxyl of the methoxyl makes hydrogen-bond interactions with the amide nitrogen and oxygen atoms of αAsp75 and αAsp74, respectively (FIG. 5B ); both residues located on the αF-helix. These interactions appear to perturb the αF-helix, as well as result in a significant conformational change in the side-chain of αAsn78. The αF-helix residue, Asn78 has been implicated in stabilizing the Hb S fiber, and the Hb variant, Stanclyvillc (αAsn78 aLsy78) is known to inhibit Hb S polymerization and RBS sickling.20 Thus, not only does TD-7 increase the oxygen affinity of Hb by binding at the α-cleft and restraining the two α-subunits from transitioning from the oxyHb to the deoxy Hb conformation as previously described for 5HMF; based on the structure it seems that the improved potency of TD-7 is also in part due to increased perturbation of the αF-helix that leads to increased destabilization of the αF-helix mediated polymer contact. - The following examples are provided to further illustrate certain preferred embodiments of the invention and are not intended to be construed as limiting the scope of the invention.
-
- Compound TD-1 was prepared according to literature procedure for a similar compound. (Gavrila, A.; Andersen, L.; Skrydstrup, T., Tetrahedron Lett. 2005, 46, 6205-6207.)
- Trifluoroacetic anhydride (5.5 mL, 2 mmol) was added to a suspension of lithium nitrate (2.7 g, 2 mmol) in anhydrous CH3CN (30 mL) at 20° C. The reaction mixture was stirred until clear (30 min) and cooled to 0° C. (ice-bath). Sodium carbonate (4.2 g, 2 mmol) and 5-(hydroxymethyl) furfural (2.5 g, 1 mmol) were added together and the reaction mixture was allowed to stir for 7 h at 0° C. (ice-bath). The reaction mixture was poured into an ice-cold solution of saturated NaHCO3 and extracted with EtOAc (2×10 mL). The combined organic portion was washed with brine (10 mL), dried over Na2SO4 and evaporated under reduced pressure to yield a crude product. The crude residue was purified by column chromatography (petroleum ether/EtOAc; 2:1) to afford 2.9 g (85%) of TD-1 as a colorless viscous oil: IR (KBr, cm−1): 1678 (C═O), 1631, 1276 (NO2), 845 (ON); 1H-NMR (CDCl3): δ 5.48 (s, 2H, CH2), 6.73 (d, J=3.5 Hz, 1H, ArH), 7.25 (d, J=3.6, 1H, ArH), 9.68 (s, 1H, CHO); 13C-NMR (CDCl3): δ 177.9, 153.4, 151.5, 120.9, 114.5, 65.4. Anal. Calcd for (C6H5NO5) C, 42.12; H, 2.95; N, 8.19. Found: C, 42.02; H, 2.86; N, 8.26. The purity of the compound was checked by HPLC and was found to be 98.8% pure.
-
- Compound TD-7 was prepared according to literature procedure for a similar compound. (Nnamani, I. N.; Joshi, G. S.; Danso-Danquah, R.; Abdulmalik, O.; Asakura, T.; Abraham, D. J.; Safo, M. K., Chem. Biodivers. 2008, 5, 1762-1769.)
- A mixture of 2-hydroxy-5-methoxybenzaldehyde (0.20 mL, 1.5 mmol), 6-(bromomethyl)-2-pyridinemethanol (0.30 g, 1.5 mmol), and K2CO3 (0.25 g, 1.8 mmol) in anhydrous DMF (20 mL) was allowed to stir at room temperature for 12 h. The reaction mixture was diluted with EtOAc (20 mL), washed with H2O (2×20 mL), brine (2×10 mL), dried over Na2SO4 and evaporated under reduced pressure to yield a crude product. The residue was purified by column chromatography (petroleum ether/EtOAc; 1:1) to yield 0.32 g (78%) of TD-7 as a yellow-colored solid: mp 84-85° C.; IR (KBr, cm−1): 3396 (O—H), 1678 (C═O); 1H-NMR (CDCl3): δ 3.51 (s, 1H, OH), 3.81 (s, 3H, OCH3), 4.79 (s, 2H, CH2), 5.29 (s, 2H, CH2), 6.99 (d, J=9.0 Hz, 1H, ArH), 7.09-7.13 (dd, J=9.0, 3.2 Hz, 1H, ArH), 7.21 (d, J=7.7 Hz, 1H, ArH), 7.37 (d, J=3.2 Hz, 1H, ArH), 7.43 (d, J=7.6 Hz, 1H, ArH), 7.74 (t, 1H, ArH), 10.58 (s, 1H, CHO); 13C-NMR (CDCl3): δ 189.24, 158.80, 155.61, 154.17, 154.17, 137.66, 125.56, 123.40, 119.84, 119.64, 114.92, 110.917, 71.65, 64.02, 55.86. MS (ESI) m/z found 274.15 (M+H)+, 296.14 (M+Na)+. The purity of the compound was checked by HPLC and was found to be 97.8% pure.
-
- Compound TD-9 was prepared according to literature procedure for a similar compound. (Nnamani, I. N.; Joshi, G. S.; Danso-Danquah, R.; Abdulmalik, O.; Asakura, T.; Abraham, D. J.; Safo, M. K., Chem. Biodivers. 2008, 5, 1762-1769.)
- A mixture of 2-hydroxy-4-methoxybenzaldehyde (113 mg, 0.74 mmol), 6-(bromomethyl)-2-pyridinemethanol (150 mg, 0.74 mmol), and K2CO3 (123 mg, 0.90 mmol) in anhydrous DMF (20 mL) was stirred at room temperature for 12 h. The reaction mixture was diluted with EtOAc (20 mL), washed with H2O (2×20 mL), brine (2×10 mL), dried over Na2SO4 and evaporated under reduced pressure to yield a crude product. The crude was purified by column chromatography (petroleum ether/EtOAc; 1:1) to yield 190 mg (94%) of TD-9 as a yellow-colored solid: mp 104-105° C.; IR (KBr, cm−1): 3153 (O—H), 1599 (C═O); 1H-NMR (CDCl3): δ 3.58 (s, 1H, OH), 3.85 (s, 3H, OCH3), 4.78 (d, 2H, CH2), 5.30 (s, 2H, CH2), 6.53 (d, J=2.2 Hz, 1H, ArH), 6.60 (d, J=2.16 Hz, 1H, ArH), 7.22 (d, J=7.68 Hz, 2H, ArH), 7.47 (d, J=7.76 Hz, 2H, ArH), 7.76 (t, 1H, ArH), 7.85 (d, J=8.64 Hz, 1H, ArH), 10.43 (s, 1H, CHO); 13C-NMR (CDCl3): δ 166.16, 162.27, 158.67, 155.51, 137.74, 131.04, 119.85, 119.69, 118.1, 106.68, 99.16, 70.85, 63.87, 55.66. MS (ESI) m/z found 274.11 (M+H)+, 296.09 (M+Na)+. The purity of the compound was checked by HPLC and was found to be 96.9% pure.
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- Compound TD-8 was prepared according to literature procedure for a similar compound. (Nnamani, I. N.; Joshi, G. S.; Danso-Danquah, R.; Abdulmalik, O.; Asakura, T.; Abraham, D. J.; Safo, M. K., Chem. Biodivers. 2008, 5, 1762-1769.)
- A mixture of 3-hydroxy-4-methoxybenzaldehyde (200 mg, 1.0 mmol), 6-(bromomethyl)-2-pyridinemethanol (152 mg, 1.0 mmol), and potassium carbonate (165 mg, 1.2 mmol) in anhydrous DMF (20 mL) was stirred at room temperature for 12 h. The reaction mixture was diluted with EtOAc (20 mL), washed with H2O (2×20 mL), brine (2×10 mL), dried over Na2SO4 and evaporated under reduced pressure to yield a crude product. The crude product was purified by column chromatography (petroleum ether/EtOAc; 1:1) to yield 220 mg (80%) of TD-8 as a white-colored solid: mp 120-121° C.; IR (KBr, cm−1): 3176 (O—H), 1681 (C═O); 1H-NMR (CDCl3): δ 3.65 (s, 1H, OH), 4.00 (s, 3H, OCH3), 4.78 (d, 2H, CH2), 5.32 (d, 2H, CH2), 7.03 (d, J=8.72 Hz, 1H, ArH), 7.17 (d, J=7.68 Hz, 1H, ArH), 7.44-7.51 (m, 3H, ArH), 7.71 (t, 1H, ArH); 13C-NMR (CDCl3): δ 190.66, 155.53, 154.95, 137.54, 130.11, 126.88, 119.99, 119.60, 111.92, 110.98, 158.79, 71.40, 63.90, 56.22. MS (ESI) m/z found 274.11 (M+H)+, 296.09 (M+Na)+. The purity of the compound was checked by HPLC and was found to be 99.5% pure.
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- Compound TD-7-1 was prepared according to literature procedure for a similar compound. (Fotopoulou, T.; Iliodromitis, E. K.; Koufaki, M.; Tsotinis, A.; Zoga, A.; Gizas, V.; Pyriochou, A.; Papapetropoulos, A.; Andreadou, I.; Kremastinos, D. T., Bioorg. Med. Chem. 2008, 16, 4523-4531.)
- Silver nitrate (76 mg, 0.45 mmol) was added to a solution of 2-((6-(bromomethyl)pyridin-2-yl)methoxy)-5-methoxybenzaldehyde (100 mg, 0.3 mmol) in anhydrous CH3CN (5 mL). The reaction mixture was allowed to stir at 80° C. for 2 h. The reaction mixture was filtered through celite and washed with CH2Cl2 (15 mL), and evaporated under reduced pressure to yield a crude product. The crude residue was purified by column chromatography (petroleum ether/EtOAc; 5:1) to yield 80 mg (83.78%) of TD-7-1 as a white-colored solid: mp 92-93° C.; IR (KBr, cm−1): 1664 (C═O), 1632 (NO2), 1274 (NO2), 872 (ON); 1H-NMR (CDCl3): δ 3.81 (s, 3H, OCH3), 5.28 (d, 2H, CH2), 5.56 (s, 2H, CH2), 6.98 (d, J=9.08 Hz, 1H, ArH), 7.10-7.13 (dd, J=9.08, 3.28 Hz, 1H, ArH), 7.33-7.37 (dd, J=8.4, 3.24 Hz, 2H, ArH), 7.53 (d, J=7.84 Hz, 1H, ArH), 7.80 (t, 1H, ArH), 10.57 (s, 1H, CHO); 13C-NMR (CDCl3): δ 189.18, 157.08, 155.20, 154.20, 152.50, 138.05, 125.52, 123.38, 121.27, 121.19, 114.85, 111.00, 74.15, 71.51, 55.85. MS (ESI) m/z found 319.09 (M+H)′, 341.07 (M+Na)′. The purity of the compound was checked by HPLC and was found to be 94.5% pure.
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- 2-((6-(Bromomethyl)pyridin-2-yl)methoxy)-5-methoxybenzaldehyde, a precursor for the synthesis of TD-7-1 was prepared according to literature procedure for a similar procedure. (Fotopoulou, T.; Iliodromitis, E. K.; Koufaki, M.; Tsotinis, A.; Zoga, A.; Gizas, V.; Pyriochou, A.; Papapetropoulos, A.; Andreadou, 1.; Kremastinos, D. T., Bioorg. Med. Chem. 2008, 16, 4523-4531.). Triphenylphosphine (140 mg, 0.6 mmol) and tetrabromomethane (180 mg, 0.6 mmol) was added to a solution of 2-((6-(hydroxymethyl)pyridin-2-yl)methoxy)-5-methoxybenzaldehyde (100 mg, 0.4 mmol) in anhydrous CH2Cl2 (6 mL). The reaction mixture was allowed to stir for 2 h at room temperature. The reaction mixture was neutralized with saturated NaHCO3 at 0° C. (ice-bath) and extracted with EtOAc (10 mL). The organic portion was washed with brine (2×10 mL), dried over Na2SO4 and evaporated under reduced pressure to yield a crude product. The crude was purified by column chromatography (petroleum ether/EtOAc; 5:1) to yield 100 mg (74.6%) as a yellow-colored solid: 1H-NMR (CDCl3): δ 3.81 (s, 3H, OCH3), 4.56 (s, 2H, CH2), 5.28 (d, 2H, CH2), 6.98 (d, J=9.08 Hz, 1H, ArH), 7.10-7.13 (dd, J=9.08, 3.28 Hz, 1H, ArH), 7.33-7.37 (dd, J=8.4, 3.24 Hz, 2H, ArH), 7.53 (d, J=7.84 Hz, 1H, ArH), 7.80 (t, 1H, ArH), 10.58 (s, 1H, CHO). The following compound was used for the synthesis of TD-7-1.
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- Compound TD-8-1 was prepared according to literature procedure for a similar compound. (Fotopoulou, T.; Iliodromitis, E. K.; Koufaki, M.; Tsotinis, A.; Zoga, A.; Gizas, V.; Pyriochou, A.; Papapetropoulos, A.; Andreadou, I.; Kremastinos, D. T., Bioorg. Med. Chem. 2008, 16, 4523-4531.). Silver nitrate (76 mg, 0.45 mmol) was added to a solution of 3-((6-(bromomethyl)pyridine-2-yl)methoxy)-4-methoxybenzaldehyde (100 mg, 0.3 mmol) in anhydrous CH3CN (5 mL). The reaction mixture was allowed to stir at 80° C. for 2 h. The reaction mixture was filtered through celite and washed with CH2Cl2 (15 mL) and evaporated under reduced pressure to yield a crude product. The crude was purified by column chromatography (petroleum ether/EtOAc; 5:1) to yield 94 mg (98%) of TD-8-1 as a yellow-colored solid: mp 43-44° C.; IR (KBr, cm−1): 1683 (C═O), 1632 (NO2), 850 (ON); 1H-NMR (CDCl3): δ 3.99 (s, 3H, OCH3), 5.31 (s, 2H, CH2), 5.57 (s, 2H, CH2), 7.02 (d, J=8.24 Hz, 1H, ArH), 7.31 (d, J=7.68 Hz, 1H, ArH), 7.46 (d, J=2.16 Hz, 1H, ArH), 7.49-7.52 (dd, J=8.2, 1.76 Hz, 1H, ArH), 7.55 (d, J=7.76 Hz, 1H, ArH), 9.82 (s, 1H, CHO); 13C-NMR (CDCl3): δ 190.56, 157.157, 155.01, 152.01, 152.47, 148.42, 137.91, 130.20, 126.78, 121.34, 121.00, 112.24, 111.08, 74.27, 71.43, 56.24. MS (ESI) m/z found 341.08 (M+Na)′. The purity of the compound was checked by HPLC and was found to be 96.9% pure.
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- 3-((6-(Bromomethyl)pyridine-2-yl)methoxy)-4-methoxybenzaldehyde, a precursor for the synthesis of TD-8-1 was prepared according to literature procedure for a similar procedure. (Fotopoulou, T.; Iliodromitis, E. K.; Koufaki, M.; Tsotinis, A.; Zoga, A.; Gizas, V.; Pyriochou, A.; Papapetropoulos, A.; Andreadou, I.; Kremastinos, D. T., Bioorg. Med. Chem. 2008, 16, 4523-4531.). Triphenylphosphine (140 mg, 0.6 mmol) and tetrabromomethane (180 mg, 0.6 mmol) was added to a solution of 3-((6-(hydroxymethyl)pyridine-2-yl)methoxy)-4-methoxybenzaldehyde (100 mg, 0.4 mmol in anhydrous CH2Cl2 (6 mL). The mixture was allowed to stir for 2 h at room temperature. The reaction mixture was neutralized with saturated NaHCO3 at 0° C. (ice-bath) and extracted with EtOAc (10 mL). The organic portion was washed with brine (2×10 mL), dried over Na2SO4 and evaporated under reduced pressure to yield a crude product. The crude was purified by column chromatography (petroleum ether/EtOAc; 5:1) to yield 76 mg (56.5%) as a yellow-colored solid: 1H-NMR (CDCl3-d1): δ 3.99 (s, 3H, OCH3), 4.56 (s, 2H, CH2), 5.31 (s, 2H, CH2), 7.02 (d, J=8.2 Hz, 1H, ArH), 7.38 (d, J=7.8 Hz, 1H, ArH), 7.46 (d, J=7.8 Hz, 1H, ArH), 7.49 (d, J=1.84 Hz, 1H, ArH), 7.52 (d, J=1.76 Hz, 1H, ArH), 7.72 (t, 1H, ArH), 9.82 (s, 1H, CHO). The following compound was used for the synthesis of TD-8-1.
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- Compound TD-9-1 was prepared according to literature procedure for a similar compound. (Fotopoulou, T.; Iliodromitis, E. K.; Koufaki, M.; Tsotinis, A.; Zoga, A.; Gizas, V.; Pyriochou, A.; Papapetropoulos, A.; Andreadou, 1.; Kremastinos, D. T., Bioorg. Med. Chem. 2008, 16, 4523-4531.).
- Silver nitrate (76 mg, 0.5 mmol) was added to a solution of 2-((6-(bromomethyl)pyridin-2-yl)methoxy)-4-methoxybenzaldehyde (100 mg, 0.3 mmol) in anhydrous CH3CN (5 mL). The reaction mixture was allowed to stir at 80° C. for 2 h. The reaction mixture was filtered through celite and washed with CH2Cl2 (15 mL) and evaporated under reduced pressure to yield a crude product. The crude product was purified by column chromatography (petroleum ether/EtOAc; 5:1) to yield 53 mg (56%) of TD-9-1 as a white-colored solid: mp 70-71° C.; IR (KBr, cm−1): 1677 (C═O), 1285 (NO2), 871 (ON); 1H-NMR (CDCl3-d1): δ 3.85 (s, 3H, OCH3), 5.29 (s, 2H, CH2), 5.56 (s, 2H, CH2), 6.52 (d, J=2.16 Hz, 1H, ArH), 6.58-6.61 (m, 1H, ArH), 7.34 (d, J=7.68 Hz, 1H, ArH), 7.58 (d, J=7.88 Hz, 1H, ArH), 7.79-7.85 (m, 2H, ArH), 10.42 (s, 1H, CHO); 13C-NMR (CDCl3): δ 166.19, 163.05, 158.71, 155.53, 138.15, 131.18, 121.31, 121.28, 118.15, 106.95, 99.07, 74.14, 70.80, 55.68. MS (ESI) m/z found 341.08 (M+Na)+. The purity of the compound was checked by HPLC and was found to be 99.8% pure.
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- 2-((6-(Bromomethyl)pyridin-2-yl)methoxy)-4-methoxybenzaldehyde, a precursor for the synthesis of TD-9-1 was prepared according to literature procedure for a similar procedure. (Fotopoulou, T.; Iliodromitis, E. K.; Koufaki, M.; Tsotinis, A.; Zoga, A.; Gizas, V.; Pyriochou, A.; Papapetropoulos, A.; Andreadou, I.; Kremastinos, D. T., Bioorg. Med. Chem. 2008, 16, 4523-4531.)
- Triphenylphosphine (140 mg, 0.6 mmol) and tetrabromomethane (180 mg, 0.6 mmol) was added to a solution of 2-((6-(hydroxymethyl)pyridin-2-yl)methoxy)-4-methoxybenzaldehyde (100 mg, 0.4 mmol) in anhydrous CH2Cl2 (6 mL). The mixture was allowed to stir for 2 h at room temperature. The reaction mixture was neutralized with saturated NaHCO3 at 0° C. (ice-bath) and extracted with EtOAc (10 mL). The organic portion was washed with brine (2×10 mL), dried over Na2SO4 and evaporated under reduced pressure to yield a crude product. The product was purified by column chromatography (petroleum ether/EtOAc; 5:1) to yield 100 mg (75%) as a yellow-colored solid: 1H-NMR (CDCl3): δ 3.85 (s, 3H, OCH3), 4.56 (s, 2H, CH2), 5.30 (s, 2H, CH2), 6.53 (d, J=2.16 Hz, 1H, ArH), 6.57-6.59 (m, 1H, ArH), 7.40 (d, J=7.72 Hz, 1H, ArH), 7.50 (d, J=7.8 Hz, 1H, ArH), 7.76 (t, 1H, ArH), 7.84 (t, J=8.68 Hz, 1H, ArH), 10.43 (s, 1H, CHO). The resulting compound was used for the synthesis of TD-9-1.
- A solution of 1.0 M HCl (5 mL) in diethyl ether was added in a drop-wise fashion to a stirring solution of TD-7 (200 mg) in absolute ethanol (3 mL), resulting in precipitation of the hydrochloride salt of TD-7. Ethyl ether (10 mL) was added to the mixture to completely precipitate all the HCl salt. The compound was filtered by vacuum filtration, washed with more ether and further dried under vacuum. The yield obtained was 88%.
- Studies were performed on compounds TD-7, TD-7-1, TD-8, TD-8-1, TD-9 and TD-9-1 using 100 mM stock solution in DMSO and normal whole blood (AA). TD-7, TD-8 and TD-9 are compounds with a hydroxymethyl group on the pyridine ring, while TD-7-1, TD-8-1 and TD-9-1 are the respective nitrate ester derivatives. The whole blood samples had hematocrit values ranging from 29-36%, and cell-free purified Hb of 10-12 g/dL in the absence or presence of effectors solubilized in DMSO at 2 mM or in some cases with varying compound concentration. The blood samples were incubated in an IL 237 tonometer (Instrumentation Laboratories, Inc., Lexington, Mass.) for about 5-7 min at 37° C. against a gas mixture containing O2 concentrations of 0.804%, 2.935% and 5.528% and allowed to equilibrate at O2 tensions of 6, 20 and 40 mmHg. After equilibration, the sample was removed via syringe and aspirated into a ABL 700 series table top automated blood gas analyzer (Radiometer America, Inc., Westlake, Ohio) to determine total hemoglobin (tHb), hematocrit (Hct), pH, pCO2, partial pressure of oxygen (pO2), and the Hb oxygen saturation values (sO2). Similar procedures were followed for concentration and time dependence studies. The measured values of pO2 and sO2 at each oxygen saturation level were then subjected to a non-linear regression analysis using the program Scientist (Micromath, Salt Lake City, Utah) with the following equation:
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- This equation was used to calculate P50 and Hill coefficient (N) values. Corresponding control experiments without the test compound (P50control) but containing DMSO were also performed. 5HMF is known to exhibit optimal left-shifting activity between 1 to 2 h when incubated with whole blood necessitating the use of 1.5 h for the incubation time for our studies. The results are shown in Table 1 and
FIG. 1 . The most potent left-shifter was TD-7 with a ΔP50 shift of −17.0 mmHg, more potent than the positive control, 5HMF (ΔP50 of −11.1 mmHg). -
TABLE 1 Effects of left-shifting compounds on Hb affinity for O2 with normal whole blood. Concentration P50 ΔP50 b Compound (mM) (mmHg) Na (mmHg) 5HMF 2 20.2 ± 2.0c† 1.8 ± 0.1c† −11.1 TD-7 0.1 30.1d* 2.5d* −0.5 0.5 25.9d* 2.1d* −4.7 1 23.2d* 1.7d* −7.4 2 14.3 ± 4.3c† 1.3 ± 0.3c† -17.0 TD-8 2 25.9 ± 2.5c† 1.9 ± 0.1c† −5.4 TD-9 2 21.1 ± 1.2c† 1.6 ± 0.3c† −10.2 TD-1 2 27.4 ± 2.3c† 1.9 ± 0.1c† −3.9 TD-7-1 2 23.4 ± 1.6c† 1.7 ± 0.1c† −7.9 TD-8-1 2 308d† 2.1d† TD-9-1 2 28.6d† 1.9d† −2.7 aHill coefficient; bΔP50 is (P50 (control)-P50 (sample)) expressed in mmHg; cmeasurements were conducted in n ≥ 3; dmeasurements were conducted in n < 3 - Hemoglobin was prepared from centrifuging human blood at 2500 rpms for 20 min at 4° C. The supernatant solution, debris and plasma were discarded from the centrifuge bottles, leaving behind RBCs. The RBCs were washed thrice with an excess volume of 0.9% NaCl, and once with 1.0% NaCl, each time centrifuging and discarding the supernatant solution. The RBCs were pooled together into a chilled flask and hemolyzed to free Hb by adding 1-2 volume excess of 50 mM Tris buffer, pH 8.6 (containing EDTA). The mixture was allowed to stand in ice for 30 min with occasional stirring. The Hb solution was centrifuged at 10,000 rpm for 2 h at 4° C. The supernatant Hb solution was pooled into a chilled flask and NaCl (40-60 mg/mL Hb solution) was slowly added with stirring. Hemoglobin solution was centrifuged at 10,000 rpm for 2 h at 4° C. to remove any cell stroma remnants. The clear supernatant Hb solution was pooled into a chilled flask and the “syrupy” pellet was discarded. Hemoglobin solution was dialyzed against 50 mM Tris buffer, pH 8.6 (containing EDTA) at 4° C. to remove NaCl and/or other low molecular weight impurities. Strips of standard cellulose dialysis tubing that had been washed 3-4 times and boiled for 10 min in deionized water were used for dialysis. The dialyzed Hb was further purified by ion-exchange chromatography using DEAE sephacel to exclude 2,3-DPG. The resin was equilibrated with 50 mM Tris buffer, pH 8.6, and the Hb solution was run through the column with 50 mM Tris buffer, pH 8.6 (containing EDTA). The pooled fractions were concentrated with an Amicon stirred cell (Amicon, Model 402), to a final HbA concentration of about 80-120 mg/mL. Concentrated HbA was stored at −80° C.
- As a result of the low left-shifting activities of TD-8 and TD-9, as well as the nitrate ester derivatives, we further evaluated the activity of the compounds TD-1, TD-7, TD-7-1, TD-8, and TD-9 using cell-free Hb (devoid of any proteins, enzymes, plasma and cell membranes) to determine whether plasma protein or RBC membrane could have any effect on compound biological activity. Previous studies have shown limited effect of plasma protein and RBC membrane on 5HMF activity. The same tonometry procedure used on whole blood was used with the cell-free hemoglobin using 2 mM compound and 12 mg/mL of Hb. The results are shown in Table 2.
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TABLE 2 Effects of left-shifting compounds on Hb affinity for O2 with cell-free Hb. P50 ΔP50 b Compound (mmHg) Na (mmHg) Control 12.5 2.6 — 5HMF 8.3 2.0 −4.2 TD-7 7.6 1.8 −4.9 TD-8 7.6 1.9 −4.9 TD-9 6.8 1.7 −5.7 TD-1 8.4 2.0 −4.1 TD-7-1 7.7 2.1 −3.9 aHill coefficient; bΔP50 is (P50 (control)-P50 (sample)) expressed in mmHg; measurements were conducted in n = 1. - Unlike the results obtained from the whole blood study (Table 1), the results depicted in Table 2 show that all compounds exhibit significant left-shift in the OEC with cell-free Hb. TD-8 which barely shifted the OEC with whole blood now shows comparable shift as TD-7 with cell-free Hb. Also, TD-9 appears to be an even more potent left-shifter in cell-free Hb than TD-7, which was opposite to what was observed with whole blood. Although the nitrate esters still show less left-shift compared to the parent compounds, there is still significant improvement in their left-shifting potency with cell-free Hb. This observation suggests that the functional activities of both TD-8 and TD-9 are significantly affected by RBC membrane permeability issues and/or plasma protein binding resulting in their reduced allosteric activities.
- This study shows time-dependent left-shifting effect of TD-7 compared to 5HMF. All samples were pre-incubated with whole blood (Hct ˜34%) with the specified compounds 0.5 mM concentrations for the specified time. The control experiment without test compound (P50 (control)) contains the same concentration of DMSO used to solubilize the test solution (P50 (sample)). The results are shown in Table 3 and
FIG. 2 . -
TABLE 3 Effect of incubation time on Hb affinity for O2 with whole blood for left-shifting compounds. Incubation Time P50 ΔP50 b Compound (Hours) (mmHg) Na (mmHg) Control 1 32.7 ± 0.2 2.5 ± 0.1 — 2 33.0 ± 0.4 2.5 ± 0.1 — 4 33.1 ± 0.6 2.5 ± 0.1 — 6 34.2 ± 0.3 2.5 ± 0.1 — 10 35.1 ± 0.3 2.5 ± 0.1 — 12 36.1 ± 0.4 2.4 ± 0.1 — 5HMF 1 29.8 ± 0.2 2.2 ± 0.1 −2.9 2 29.2 ± 0.3 2.2 ± 0.1 −3.8 4 29.2 ± 0.1 2.1 ± 0.1 −3.9 6 30.0 ± 0.1 2.1 ± 0.1 −4.2 10 31.8 ± 0.1 2.1 ± 0.1 −3.3 12 32.8 ± 0.1 2.0 ± 0.1 −3.3 Incubation Time P50 ΔP50 Compound (Hours) (mmHg) N (mmHg) TD-7 1 28.2 ± 0.9 2.2 ± 0.2 −4.5 2 28.2 ± 0.8 2.1 ± 0.1 −4.8 4 28.8 ± 0.6 2.1 ± 0.1 −4.3 6 29.1 ± 0.6 2.0 ± 0.2 −5.1 10 30.6 ± 1.0 2.0 ± 0.1 −4.5 12 31.1 ± 0.7 2.0 ± 0.2 −5.0 aHill coefficient; bΔP50 is (P50 (control)-P50 (sample)) expressed in mmHg; cmeasurements were conducted in ≥ 3;. The P50 values of control were found to increase consistently, with incubation. While the P50 values for 5HMF seem to return to baseline after 6 h, there was no recovery for TD-7 even after 12 h suggesting TD-7 will have a longer sustained effect (FIG. 2). - Griess assay was performed on the left-shifting nitrate ester compounds (TD-1, TD-74, TD-8-1 and TD-9-1) to measure the release of NO. Griess assay is a colorimetric assay, used for detection of NO in the form of nitrite ions (NO2 −). NO released in solution is highly unstable, with a half-life of 1 to 30 s. It gets rapidly oxidized to NO2 − which is detected by the Griess reagent. In this method, NO2 −, first reacts with the sulfanilamide present in the reagent to form a transient diazonium salt. This diazonium salt further reacts with N-naphthyl-ethylenediamine (NED) to form a stable azo compound which possesses an intense purple color. The absorbance of the azo compound was measured at 540 urn and was found to be directly proportional to the NOT concentration in the sample. Sodium nitrite (NaNO2) was used as a standard for the Griess assay. NaNO2 samples in deionized water were prepared of concentrations within the linear range of the assay (1-100 μM). The samples were incubated for 30 min at 37° C., after which Griess reagent was added and absorbance was measured at 540 nm. The measured absorbances were used to obtain a standard curve (
FIG. 3 ). For the measurement of NO2 − release for all the test compounds (TD-1, TD-7-1, TD-8-1 and TD-9-1), the absorbances were extrapolated from the standard NaNO2 curve. - In this assay, a solution of the test compound (1 mL of a 2 mM solution in 0.1 M phosphate buffer (pH 7.4)) was mixed thoroughly with a freshly prepared solution of L-cysteine (1 mL of a 36 mM solution in 0.1 M phosphate buffer, pH 7.4), and incubated at 37° C., for 1 and 16 h under anaerobic conditions. After exposure to air for 10 min at 25° C., an aliquot of the Griess reagent (1 mL) [freshly prepared by mixing equal volumes of 1.0% sulfanilamide (prepared and stored in aqueous 5% phosphoric acid) and 0.1% N-naphthylethylenediamine dihydrochloride in water] was added to an equal volume (1 mL) of each incubation solution after mixing. After 10 min, absorbance was measured at 540 nm using a Hewlett Packard (Agilent) 8453 UV-visible Spectroscopy. NaNO2 solutions of 1-100 μM concentrations were used to prepare a standard nitrite curve of absorbance (nm) versus concentration (μM). The NO released (quantitated as NO2 − release in μM) was calculated from the standard nitrite curve by extrapolation of the absorbance. Presence of NO2 − shows formation of a pink color, the intensity of which is directly proportional to the amount of NO2 − present in the mixture. Table 4 represents NO2 − release for compounds TD-1, TD-7-1, TD-8-1 and TD-9-1.
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TABLE 4 Absorbance and NO2 − release values for Compounds TD-1, TD-7-1, TD-8-1 and TD-9-1. Incubation Time NO2 − releasea (μM) NO2 − releasea (μM) (Hours) after 1 hour after 16 hours TD-1 4.02 11.93 TD-7-1 7.61 59.25 TD-8-1 21.94 102.54 TD-9-1 20.45 78.66 aThe NO2 − release (μM) was calculated from the standard NaNO2 curve by extrapolating the absorbance (nm) of the test compounds against the concentration (μM) of NaNO2. - All four compounds (TD-1, TD-7-1, TD-8-1 and TD-9-1) exhibited significant nitric oxide release (4.0-21.9 μM range at 1 h and 59-102.5 μM range at 16 h) upon incubation in the presence of L-cysteine. The NO release after 16 h incubation was found to be 3-10 fold higher than 1 h incubation. From Table 4, it can be interpreted that for all the compounds, the NO release in the form of NO2 − is found to increase with time, with TD-1 showing the least release, while TD-8-1 showed the most release.
- In vitro RBC morphological (anti-sickling) analysis was conducted at the Children's Hospital of Philadelphia (by Drs. Toshio Asakura and Osheiza Abdulmalik). The anti-sickling effects of 5HMF, TD-7 HCl, TD-7, TD-1, and TD-7-1 were studied by incubating suspensions of SS cells in the absence or presence of three different concentrations of the effectors in the presence of air for 1 h at 37° C. followed by incubation under 4% oxygen at 37° C. for 5 h. All samples were pre-incubated for 1 h with various compounds and then incubated under 4% O2 for 5 h. Hb had a hematocrit of 30%. Table 5 represents the concentrations of the effectors along with the % inhibition of sickling cells. The hemolysates that had been prepared from SS cells after incubation with test compounds were also subjected to cation-exchange HPLC as previously reported (Abdulmalik et al., 2005). Table 5 shows the effect of the various concentrations of the test compounds on the amount of modified Hb S which reflects Schiff-base adduct formation between the aromatic aldehyde and the protein.
-
TABLE 5 Effects of various concentrations of compounds on inhibition of SS cell sickling. Concentration Inhibition of Compound (mM) sickled cells (%) 5HMF 0.5 4 1.0 17 2.0 35 5.0 75 TD-7 HCl salt 1.0 100 2.0 100 5.0 100 TD-7 0.5 95 1.0 95 2.0 100 5.0 100 TD-8 0.5 14 1.0 20 2.0 38 TD-9 0.5 26 1.0 35 2.0 57 TD-7-1 0.5 72 1.0 100 2.0 100 - The anti-sickling effects of 5HMF, TD-7, TD-7 HCl salt, TD-1, and TD-7-1 were studied and the results shown in Table 5 and
FIG. 4 (% inhibition). In the absence of test compounds, about 95% of the Hb underwent sickling in 5 h. In the presence of the positive control, 5HMF, the percentage of the sickled cells decreased in a dose-dependent manner with 4, 17, 35 and 75% inhibition at 0.5 mM, 1 mM, 2 mM and 5 mM concentration, respectively. In comparison, TD-1, the nitrate ester analog of 5HMF showed only a modest decrease in the anti-sickling effect (Not shown in Table). This observation may be partly related to poor RBC membrane passage. Remarkably TD-7, either the free base or HCl salt, showed a very potent anti-sickling effect, as much as 95% inhibition at 0.5 mM concentration. A plateau effect was observed as the concentration of TD-7 was increased. The water soluble HCl salt of TD-7 interestingly barely showed any antisickling effect at 0.5 mM, but remarkably inhibited 100% sickling at 1.0 mM showing a plateau with increasing drug concentration. INN-312, the second-generation positive control, also decreased the percent of sickled cells in a dose-dependent manner but at 0.5 mM it only inhibited sickling to 15% compared to the 95% by TD-7. TD-7-1, the ester nitrate of TD-7 although preventing RBC sickling, hemolyzed the RBC at higher drug concentrations. Also, it seems to swell/hydrate the RBC due to possible intracellular uptake of water which could be beneficial to counteract the rigidity of sickle cells. TD-8 and TD-9 were not as potent as the TD-7 analog, only inhibiting 38 and 57% RBC sickling, respectively at 2 mM. In comparison, vanillin which is the parent scaffold for the INN and TD compounds only inhibited 12% RBC sickling at 5 mM. -
- 1 Wishner, B. C.; Ward, K. B.; Lattman, E. E.; Love, W. E. Crystal structure of sickle-cell deoxyhemoglobin at 5 Å resolution. J. Mol. Biol. 1975, 98, 179-194.
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- 4 Nnamani, I. N.; Joshi, G. S.; Danso-Danquah, R.; Abdulmalik, O.; Asakura, T.; Abraham, D. J.; Safo, M. K. Pyridyl derivatives of benzaldehyde as potential antisickling agents. Chem. Biodivers. 2008, 5, 1762-1769.
- 5 Abdulmalik, O.; Safo, M. K.; Chen, Q.; Yang, J.; Brugnara, C.; Ohene-Frempong, K.; Abraham, D. J.; Asakura, T. 5-Hydroxymethyl-2-furfural modifies intracellular sickle haemoglobin and inhibits sickling of red blood cells. Br. J. Haematol. 2005, 128, 552-561.
- 6 Abdulmalik, O.; Ghatge, M. S.; Musayev, F. N.; Parikh, A.; Chen, Q.; Yang, J.; Nnamani, I.; Danso-Danquah, R.; Eseonu, D. N.; Asakura, T.; Abraham, D. J.; Venitz, J.; Safo, M. K. Crystallographic analysis of human hemoglobin elucidates the structural basis of the potent and dual antisickling activity of pyridyl derivatives of vanillin. Acta Crystallogr. D: Biol. Crystallogr. 2011, 67, 920-928.
- 7 Parikh, A.; Ghatge, M.; Safo, M; Venitz, J. Comparative pharmacodynamic (PD) profiling of the in-vitro time- and concentration-dependent effects of known, synthetic allosteric effectors of hemoglobin (AEH). Presented at the 2011 American Association of Pharmaceutical Scientists (AAPS) Annual Meeting and Exposition, Washington, D.C., October 2011.
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Nitric Oxide 2002, 6, 178-185. - 11 Mack, A. K.; Kato, G. J. Sickle cell disease and nitric oxide: A paradigm shift? Int. J. Biochem. Cell Biol. 2006, 38, 1237-1243.
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- 13 Hunter, C. J.; Dejam, A.; Blood, A. B.; Shields, H.; Kim-Shapiro, D. B.; Machado, R. F.; Tarekegn, S.; Mulla, N.; Hopper, A. O.; Schechter, A. N.; Power, G. G.; Gladwin, M. T. Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator. Nat. Med. 2004, 10, 1122-1127.
- 14 Weiner D. L.; Hibberd, P. L.; Betit, P.; Cooper, A. B.; Botelho, C. A.; Brugngra, C. Preliminary assessment of inhaled nitric oxide for acute vaso-occlusive crisis in pediatric patients with sickle cell disease. JAMA 2003, 289, 1136-1142.
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- 17 Kahn, M. J.; Maley, J. H.; Lasker, G. F.; Kadowitz, P. J. Updated role of nitric oxide in disorders of erythrocyte function. Cardiovasc. Hematol. Disord. Drug Targets 2013, 13, 83-87.
- 18 Perutz, M. F. Structure and mechanism of haemoglobin. Br. Med. Bull. 1976, 32, 195-208.
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