AVASOPASEM MANGANESE

AVASOPASEM

Average: 518.83
Monoisotopic: 517.134397

Chemical FormulaC₂₁H₃₅Cl₃MnN₅

manganese(2+);(4S,9S,14S,19S)-3,10,13,20,26-pentazatetracyclo[20.3.1.0^4,9.0^14,19]hexacosa-1(26),22,24-triene;dichloride

• Manganese, dichloro((4aS,13aS,17aS,21aS)-1,2,3,4,4a,5,6,12,13,13a,14,15,16,17,17a,18,19,20,21,21a-eicosahydro-7,11-nitrilo-7H-dibenzo(b,H)-5,13,18,21-tetraazacycloheptadecine-kappaN5,kappaN13,kappaN18,kappaN21,kappaN22)-, (pb-7-11-2344’3′)-

CAS 435327-40-5

• A superoxide dismutase mimetic.

• GC 4419
• M-40419
• SC-72325A

• For the Reduction of The Severity and Incidence of Radiation and Chemotherapy-Induced Oral Mucositis

Avasopasem manganese, also known as GC4419, is a highly-selective small molecule mimetic of superoxide dismutase (SOD) being investigated for the reduction of radiation-induced severe oral mucositis.^1,2 This drug has potential application for radiation-induced esophagitis and oral mucositis, in addition to being currently tested against COVID-19.

Avasopasem manganese is a superoxide dismutase mimetic that rapidly and selectively converts superoxide to hydrogen peroxide and oxygen in order to protect normal tissue from radiation therapy-induced damage.¹ This drug is currently being investigated against oral mucositis, esophagitis, and COVID-19.

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2018152353

Transition metal pentaaza 15-membered macrocyclic ring complexes having the macrocyclic ring system corresponding to Formula A have been shown to be effective in a number of animal and cell models of human disease, as well as in treatment of conditions afflicting human patients.

For example, in a rodent model of colitis, one such compound, GC4403, has been reported when administered by intraperitoneal (ip) injection to significantly reduce the injury to the colon of rats subjected to an experimental model of colitis (see Cuzzocrea et al., Europ. J. Pharmacol., 432, 79-89 (2001)).

GC4403 administered ip has also been reported to attenuate the radiation damage arising both in a clinically relevant hamster model of acute, radiation-induced oral mucositis (Murphy et al., Clin. Can. Res., 74(13), 4292 (2008)), and lethal total body irradiation of adult mice (Thompson et al., Free Radical Res., 44(5), 529-40 (2010)).

Similarly, another such compound, GC4419, administered ip has been shown to attenuate VEGFr inhibitor-induced pulmonary disease in a rat model (Tuder, et al., Am. J. Respir. Cell Mol. Biol., 29, 88-97 (2003)), and to increase the anti-tumor activity of anti-metabolite and anti-mitotic agents in mouse cancer models (see, e.g., WO2009/143454). In other studies, GC4419 and GC4403 have been shown to be similarly potent in various animal models of disease. Additionally, another such compound, GC4401, administered ip has been shown to provide protective effects in animal models of septic shock (S. Cuzzocrea, et. al., Crit. Care Med., 32(1 ), 157 (2004)) and pancreatitis (S. Cuzzocrea, et. al., Shock, 22(3), 254-61 (2004)).

[0003] Certain of these compounds have also been shown to possess potent anti-inflammatory activity and prevent oxidative damage in vivo. For example, GC4403 administered ip has been reported to inhibit inflammation in a rat model of inflammation (Salvemini, et.al., Science, 286, 304 (1999)), and prevent joint disease in a rat model of collagen-induced arthritis (Salvemini et al., Arthritis & Rheumatism, 44(12), 2009-2021 (2001)). In addition, these compounds have been reported to possess analgesic activity and to reduce inflammation and edema by systemic administration in the rat-paw carrageenan hyperalgesia model, see, e.g., U.S. Pat. No. 6,180,620.

[0004] Compounds of the class comprising GC4419 have also been shown to be safe and effective in the prevention and treatment of disease in human subjects. For example, GC4419 administered by intravenous (iV) infusion has been shown to reduce oral mucositis in head-and-neck cancer patients undergoing chemoradiation therapy (Anderson, C, Phase 1 Trial of Superoxide Dismutase (SOD) Mimetic GC4419 to Reduce Chemoradiotherapy (CRT)-lnduced Mucositis (OM) in Patients (pts) with Mouth or Oropharyngeal Carcinoma (OCC), Oral Mucositis Research Workshop,

MASCC/ISOO Annual Meeting on Supportive Care in Cancer, Copenhagen, Denmark (June 25, 2015)).

[0005] However, the administered dose when delivered systemically, for example by a parenteral route, can be limited in animal models and particularly in humans by systemic exposure and resulting toxicity that appears to be similar in nature among the pentaaza 15-membered macrocyclic ring dismutase mimetics of Formula A, particularly GC4403, GC4419, GC4401 and related compounds sharing the dicyclohexyl and pyridine motif in the macrocycle ring (e.g., compounds sharing the dicyclohexyl and pyridine motif generally include compounds according to Formula (I) below herein having W as an unsubstituted pyridine moiety, and wherein U and V are transcyclohexanyl fused rings) . For example, the maximum tolerated dose of GC4403 delivered as a 30-minute iv infusion in humans is 25 mg, or roughly 0.35 mg/kg in a 70-kg subject, and similar limitations exist for animal parenteral dosing. Thus, the efficacy of treatment of conditions such as local inflammatory disease or tissue damage of the alimentary canal may be limited when using systemic delivery of GC4403 and similar compounds.

[0006] In each of these compounds comprising the pentaaza 15-membered macrocyclic ring of Formula A, the five nitrogens contained in the macrocyclic ring each form a coordinate covalent bond with the manganese (or other transition metal coordinated by the macrocycle) at the center of the molecule. Additionally, manganese (or other appropriate transition metal coordinated with the macrocycle) forms coordinate covalent bonds with “axial ligands” in positions perpendicular to the roughly planar macrocycle. Such coordinate covalent bonds are characterized by an available “free” electron pair on a ligand forming a bond to a transition metal via donation and sharing of the electron pair thus forming a two-electron bond between the metal and the donor atom of the ligand (Cotton, F.A. & G. Wilkinson, Advanced Inorganic Chemistry, Chapter 5, “Coordination Compounds”, 2^nd revised edn., Interscience Publishers, p.139 (1966); lUPAC Gold Book, online version http://goldbook.iupac.org/C01329.html). The coordinate covalent nature of the bonds between manganese (or other such appropriate transition metal) and the five macrocyclic ring nitrogens and between manganese (or other such transition metal) and each of the two chloro axial ligands is evidenced, for example, by the “single crystal” X-ray crystal structure of GC4403 (Fig. 11 ) and GC4419 (Fig. 12).

[0007] Coordination compounds contrast with ionic compounds, for example, salts, where in the solid state the forces between anions and cations are strictly coulombic electrostatic forces of attraction between ions of opposite charge. Thus, in salts, discrete cations and anions provide the force to maintain the solid state structure; e.g., such as the chloride ion and the sodium ion in a typical salt such as sodium chloride (Cotton, F.A. & G. Wilkinson, Advanced Inorganic Chemistry, Chapter 5, “The Nature of Ionic Substances”, 2^nd revised edn., Interscience Publishers, pp. 35-36, 45-49 (1966).

[0008] Although pentaaza 15-membered macrocyclic ring complexes have been disclosed in the literature for a number of anti-inflammatory indications, the representative disclosures discussed above illustrate that such compounds are generally administered by intraperitoneal (ip) or intravenous (iv) injection to potentiate systemic bioavailability. Local (e.g. topical) administration has been reported as ineffective in animal models of inflammatory disease, particularly when measured against the efficacy of systemic administration methods (Murphy et al., Clin. Can. Res., 74(13), 4292 (2008); WO 2008/045559). One research group has reported inhibition of colonic tissue injury and neutrophil accumulation by intracolonic administration of a prototype pentaaza macrocycle superoxide dismutase mimetic (MnPAM) (having a different structure from GC4403), though that disclosure neither addresses systemic bioavailability of the compounds described therein, nor explore limitations resulting from systemic bioavailability impacting safety and/or efficacy of that specific compound (Weiss et al., J. Biol. Chem., 271(42): 26149-26156 (1996); Weiss, R. and Riley, D., Drugs Future, 21 (4): 383-389 (1996)).

[0009] Aspects of the present disclosure provide for formulations of pentaaza macrocyclic ring complexes of the class comprising GC4419, GC4403, and GC4401 that exhibit limited systemic bioavailability when administered orally (e.g. less than 20%, less than 15%, and even less than 10% bioavailability when dosed in appropriate oil-based formulations; see Table 1 and when combined with other formulations even less than 5%, and even less than 1%; see Example 28). In general, drug absorption from the gastrointestinal tract occurs via passive uptake so that absorption is favored when the drug is in a non-ionized (neutral) and lipophilic form. See, e.g., Goodman & Gilman’s: The Pharmacological Basis of Therapeutics, Ninth Edition, p. 5-9 (1996). Without wishing to be limited to any particular theory, this is also believed to be the case for this class of compounds, as exemplified by GC4403, where the axial ligands are both chloro moieties forming a coordinate covalent bond to the manganese and a neutral complex results:

The Mn(ll) pentaaza macrocyclic ring dichloro complexes, such as GC4419, GC4401, GC4444, and GC4403 (structures shown below) were synthesized using literature procedures. For GC4403 the chiral R,R-diaminocyclohexane is utilized as starting material,² whereas for GC4419, the mirror-image enantiomer of GC4403, the chiral S,S-diaminocyclohexane is utilized instead.^3,4 The remainder of the synthesis of GC4419 can be identical in all respects to the method published for GC4403.² The synthesis of the GC4401 complex was reported previously in reference 5.

[00213] The synthesis of GC4444 which contains the additional 11-R-Methyl substituent generating a fifth chiral center on carbon (and is also derived from R,R-diaminocyclohexane) is made from the corresponding chiral tetraamine whose synthesis was published in reference 6 as Example 5C.

Syntheses of Axial Ligand Derivatives

[00214] The same Mn(II) pentaaza macrocyclic ring dichloro complexes (GC4419, GC4403, GC4444 and GC4401 ) were also used as the starting material precursors for the syntheses of other axial ligand bound derivatives using a generic synthesis scheme in which a large excess of a salt of an anion is used to displace the chloro ligand thereby generating the new compound.

EXAMPLE 2

[00215] Synthesis of Manganese(ll)bis-acetato[(4aS,13aS,17aS,21aS)-1,2,3,4,48,5,6,12,13,13a,14,15,16,17, 17a,18,19,20,21,21a- Eicosahydro-11,7-nitrilo-7H-dibenzo[b,h][1,4,7,10] tetraazacycloheptadecine-KN5, κΝ13, κΝ18, κΝ21, κΝ22]-, [bis-Acetato (GC4419)]. GC4701

[00216] Using a 500-mL Erlenmeyer, 100 mL of deionized (“DI”) water was added to 5.3 g of GC4419; the mixture was stirred vigorously for 15-20 min, then sonicated for 5 min. The resulting light brownish suspension was filtered through a 10-20 μ fritted funnel (ca. 0.3 g of solid material remained in the funnel). The resulting clear solution was added into a sodium acetate solution (ca. 429 mmol, 21 equiv in 100 mL DI water) as a stream in one portion. No solid separated and the yellowish solution was stirred for 5 additional min. The solution was transferred to a separatory funnel and extracted (3 X 50 mL) with dichloromethane. The organic layers were separated, combined, and transferred back into a separatory funnel. The dichloromethane solution was back-extracted (2 X 50 mL) with aqueous sodium acetate (32 g/100 mL). The dichloromethane layer was dried over MgSO₄ (ca. 10 g) for 30 min (w/stirring), filtered using a 10-20 μ fritted funnel, and the solution taken to dryness using a rotavap. To the yellow oily solid resulting from taking the solution to dryness was added methanol (50 mL). This solution was then again taken to dryness on the rotovap to yield a light yellow foam/glass. This material was dried in vacuo at room temperature for two days.

[00217] The isolated yellowish brittle (4.11 g, 75% yield based on GC4419) was analyzed by HPLC and showed a purity of 99.7% and elemental analysis showed 0.98 wt. % residual chlorine. The elemental analysis is consistent with the expected bis-(acetato) structure C₂₅H₄₁MnN₅O₄●2H₂O. Anal Cal’d: C, 53.00% ; H, 8.01 %; N, 12.36%, and Mn, 9.70%. Anal Found: C, 53.10% ; H, 8.34% ; Mn, 9.86%, N, 12.56%, and CI (as total halogen content), 0.98 wt. %.

Patent

WO 2002071054

https://patents.google.com/patent/WO2002071054A1/en

Examples 1 (traditional mobile phase) and 2 (mobile phase containing excess of salt of a coordinating anion).

+X^“

Scheme 2 Any metal complex possessing a metal that is capable of coordinating a monodentate ligand can be used in the present invention. Examples of such metal complexes include, but are not limited to, complexes of the metals Mn and Fe. The metal complexes of the invention preferably have therapeutic and diagnostic utilities. These therapeutic and diagnostic utilities include, but are not limited to, use as superoxide dismutase mimetic compounds, MRI imaging enhancement agents, peroxynitrite decomposition catalysts, and catalase mimics. The preferred metal complexes for use in the invention are superoxide dismutase mimetic compounds. Examples of such superoxide dismutase mimetic compounds include, but are not limited to, the following complexes of the metals Mn and Fe. Iron based superoxide dismutase mimetics include, but are not limited to, Fe^ra(salen) complexes, Fe^ra(l,4,7,10,13-pentaazacyclopentadecane) derivatives and Fe^ffl(porphyrinato) complexes. Manganese based superoxide dismutase mimetic compounds include, but are not limited to, metal complexes containing manganese(π) or manganese(m). Examples of manganese based superoxide dismutase mimetic compounds include Mn^m(porphyrinato) complexes, Mn^ffl(salen) complexes, and Mn^π(l ,4,7, 10, 13-pentaazacyclopentadecane) derivatives. Mn^π(l ,4,7, 10,13- pentaazacyclopentadecane) derivatives are more preferred for use in the invention. Examples of Mn^π(l,4,7,10,13-pentaazacyclopentadecane) derivatives preferred for use in the invention include, but are not limited to, M40403 and M40401, as shown in Scheme 3 below.

Furthermore, stereoisomers of all of the above metal complexes can be used in the process of the present invention. Diastereomers of the same metal complexes can also be detected and separated by the method of the present invention. As it is often desirable to separate stereoisomers as a means of evaluating the chemical and optical purity of production samples, the metal complexes can also comprise products of a reaction stream. Enantiomers of any of the metal complexes referenced above can be used in the chiral HPLC method of the invention for the separation of enantiomers of a metal complex.

M40403 M40401

M40484

Scheme 3

The ligand is a coordinating anion that binds to the metal cation of the metal complex. The coordinating anion can serve as an axial ligand for a superoxide dismutase mimetic compound. Examples of such anions include, but are not limited to, chloride anions, thiocyanate anions, stearate anions, acetate anions, trifluoroacetate anions, carboxylate anions, formate anions, or azide anions. Preferred anions include chloride anions, thiocyanate anions, and formate anions. More preferred anions are chloride anions. The more preferred anions in the chiral HPLC embodiment of the invention are thiocyanate anions. When present in an excess, the thiocyanate anions bind to the coordinating metal of the complexes preferentially to the chloride anions. An excess of thiocyanate anions will produce the bis(thiocyanato) complexes of M40403 and M40419 as shown in Scheme 4.

M40403 M40403-(SCN)₂

M40419 M40419-(SCN)₂

Scheme 4

An example of the use of the acetate anion as the coordinating anion with M40403 is shown in Scheme 5 below. Scheme 6 illustrates the use of the formate anion as the coordinating anion with M40403.

M40403 M40403-(OAc)₂

Scheme 5

M40403 M40403-(Formate)₂

Scheme 6

The coordinating anion is supplied by a salt of the coordinating anion. Salts of the chloride anion include, but are not limited to, sodium chloride, lithium chloride, potassium chloride, ammonium chloride, or tetraalkylammonium chloride. Preferred salts of the chloride anion include sodium chloride, lithium chloride and tetrabutylammonium chloride. Salts of the thiocyanate anion include, but are not limited to, sodium thiocyanate, potassium thiocyanate, ammonium thiocyanate, or lithium thiocyanate. Preferred salts of the thiocyanate anion include sodium thiocyanate and potassium thiocyanate. Salts of the acetate anion include, but are not limited to, potassium acetate, sodium acetate, ammonium acetate, ammonium trifluoroacetate and lithium acetate. Preferred salts of the acetate anion include ammonium acetate. Salts of the formate anion include, but are not limited to, potassium formate, sodium formate, ammonium formate and lithium formate. Preferred salts of the formate anion include ammonium formate. Salts of the cyanate anion include but are not limited to, sodium cyanate, potassium cyanate, or ammonium cyanate. Salts of the carboxylate anion include, but are not limited to, potassium carboxylate, ammonium carboxylate and sodium carboxylate. Salts of the stearate anion include, but are not limited to, lithium stearate and sodium stearate. Salts of the azide anion include, but are not limited to, sodium azide, potassium azide, and lithium azide. The salt added to the mobile phase can also be a mixture of any of these salts. Examples include a mixture of tetrabutylammonium chloride and lithium chloride.

EXAMPLES

Experimental For Examples 1-8 Chemicals, Solvents and Materials

All solvents used in the study were HPLC grade or equivalent. All chemicals were ACS reagent grade or equivalent.

HPLC System and Data Analysis

The HPLC chromatography was performed using a Gilson system (Model 306 pump, Model 155 UN-V detector, Model 215 liquid handler, Unipoint Software,

Win98), a Narian system (Model 310 pump, Model 340 UN-N detector, Model 410 autosampler Star Workstation, Win98) or SSI system (Acuflow Series IN pump, Acutect 500 UV-N detector, Alcott Model 718 autosampler, HP Model 3395 integrator).

Example 1

HPLC Analysis of M40403 using Method 1

M40403 Method 1: Analytical Column: Waters YMC ODS-AQ S5 120A (4.6 x 50 mm); System A: 0.1% trifluoroacetic acid in H₂O; System B: 0.08% trifluoroacetic acid in acetonitrile; Gradient: 10-50% system B over 10 min; Flow rate: 3ml/min; Detector wavelength: 265. Injected 20 μl of stock solution of M40403 prepared by dissolving 1 mg in 1 ml of water and diluting with 1 ml of system A. The HPLC chromatogram of M40403 using method 1 is shown in Figure 1. Example 2 HPLC Analysis of M40403 using Method 2

Method 2: Analytical Column: Waters YMC 9DS-AQ S5 12θΛ (4.6 x 50 MM); System A: 0.5 N aqueous NaCl; System B: 1 :4 water/CH₃CN; Gradient: 10-50% system B over 9 min; Flow rate: 3mL/min; Detector wavelength: 265 nm. Injected 20 μl of stock solution of M40403 prepared by dissolving 1 mg in 1 ml of system A. The HPLC chromatogram of M40403 using method 2 is shown in Figure 2.

Example 3 HPLC Analysis of M40403 using Method 3

Method 3: Analytical Column: Waters Symmetry Shield RP18, 5 μm, 250 x 4.6 mm;

Mobile Phase: Acetonitrile: 0.125 M Tetrabutylammonium Chloride in water (pH 6.5), 5%: 95% H₂0(v/v); Flow rate: 1 mL/min; Detection wavelength: 265nm. Injected 20 μl of stock solution of M40403 prepared by dissolving 1 mg in 1 ml of mobile phase. The HPLC chromatogram of M40403 using method 3 is shown in Figure 3.

The HPLC chromatogram of M40403 and related compounds using method 3 is shown in Figure 3a. Method 3 allows a separation of M40402 (bisimine of M40403), M40414 (monoimine of M40403) and M40475 (free ligand of M40403) (see chromatogram in Figure 3a).

Example 4

HPLC Analysis of M40403 using Method 4

Method 4: Analytical Column: Waters Symmetry Shield RP18, 5 μm, 250 x 4.6 mm; Mobile Phase: Acetonitrile: 0.125 M Tetrabutylammonium Chloride and 0.5 M LiCl in water (pH 6.5), 5%: 95% H₂0 (v/v); Flow rate: lmL/min; Detection wavelength: 265 nm. Injected 20 μl of stock solution of M40403 prepared by dissolving 1 mg in 1 ml of system A. The HPLC chromatogram of M40403 using method 4 is shown in Figure 4.

The HPLC chromatogram of M40403 and related compounds using method 4 is shown in Figure 4a. Method 4 allows a separation of M40402 (bisimine of M40403), M40414 (monoimine of M40403) and M40475 (free ligand of M40403) and all diastereomers of M40403 (see chromatogram in Figure 4a).

Example 5 HPLC Analysis of M40401 using Method 1

M40401 Method 1: Analytical Column: Waters YMC ODS-AQ S5 120A (4.6 x 50 mm); System A: 0.1 % trifluoroacetic acid in H₂O; System B: 0.08% trifluoroacetic acid in acetonitrile; Gradient: 10-50% system B over 10 min; Flow rate: 3ml/min; Detector wavelength: 265. Injected 20 μl of stock solution of M40401 prepared by dissolving 1 mg in 1 ml of water and diluting with 1 ml of system A. The HPLC chromatogram of M40401 using method 1 is shown in Figure 5.

Example 6 HPLC with various NaCl concentrations:

An HPLC was taken of M40401 with various concentrations of NaCl.

Analytical Column: Waters YMC 9DS-AQ S5 120 A (4.6 x 50 mm);

System A: (A) H₂O (no NaCl) ; (B) 0.01 M NaCl in water; (C) 0.5 M NaCl in water;

System B: acetonitrile; Gradient: 0-100% system B over 10 min; Flow: 3 ml/min;

Detector wavelength: 265 nm. Injected 20 μl of stock solution of M40401 prepared by dissolving 1 mg in 1 ml of system A. The HPLC chromatogram of M40401 using various NaCl concentrations is shown in Figure 6. Example 7 HPLC Analysis of M40401 using Method 2

Method 2: Analytical Column: Waters YMC ODS-AQ S5 12θΛ (4.6 x 50 MM); System A: 0.5 N aqueous NaCl; System B: 1 :4 water/CH₃CN; Gradient 1 : 10-50% system B over 9 min; Flow rate: 3 mL/min; Detector wavelength: 265 nm. Injected 20 μl of stock solution of M40403 prepared by dissolving 1 mg in 1 ml of system A.

The HPLC chromatogram of M40401 using method 2 is shown in Figure 7. Method 2 allows a separation of M40472 (bisimine of M40401), M40473 (monoimine of M40401), free ligand of M40403 and two isomers of M40401 (M40406, M40404).

Example 8

HPLC Analysis of M40401 using Method 3

Method 3: Analytical Column: Waters Symmetry Shield RP18, 5 m, 250 4.6 mm; Mobile Phase: Acetonitrile: 0.125 M Tetrabutylammom^‘um Chloride in H₂0 (pH 6.5), 5: 95%) H₂0 (v/v); Flow rate: lmL/min; Detection wavelength: 265 nm. The HPLC chromatogram of M40401 using method 3 is shown in Figure 8.

Method 3 allows a separation of M40472 (bisimine of M40401), M40473 (monoimine of M40401), free ligand of M40403 and two isomers of M40401 (M40406, M40404).

Example 9 HPLC Analysis of M40401 using Method 4

Method 4: Analytical Column: Waters Symmetry Shield RP18, 5 μm, 250 x 4.6 mm;

Mobile Phase: Acetonitrile: 0.125 M Tetrabutylammonium Chloride and 0.5 M LiCl in water (pH 6.5), 5: 95%> H₂O (v/v); Flow rate: 1 mL/min; Detection wavelength: 265 nm; Injected 20 μl of stock solution of M40401 prepared by dissolving 1 mg in 1 ml of a mobile phase. The HPLC chromatogram of M40401 using method 4 is shown in Figure 9.

The HPLC chromatogram of M40401 and related compounds using method 4 is shown in Figure 9a. Method 4 allows a separation of M40472 (bisimine of M40401), M40473 (monoimine of M40401), free ligand of M40403 and two isomers of M40401 (M40406, M40404). Example 10

HPLC of M40403-(HCOO^“)₂ Using Formate Anion

An HPLC of M40403 employing the formate anion was taken. Analytical Column: Waters YMC 9DS-AQ S5 120 A (4.6 x 50 mm); System A: 0.025 M ammonium formate in water; System B: 1 : 4 = 0.125 M ammonium formate in water/ acetonitrile; Gradient: 0-100% system B over 10 min; Flow: 3 ml/min;

Detector wavelength: 265 nm. Injected 20 μl of stock solution of M40403-(Formate)₂ prepared by dissolving 1 mg in 1 ml of system A. The HPLC chromatogram of M40403-(HCOO^“)₂ is shown in Figure 10.

Example 11 HPLC of M40403-(OAc)₂ Using Acetate Anion

An HPLC of M40403 employing the acetate anion was taken.

Analytical Column: Waters YMC 9DS-AQ S5 120 A (4.6 x 50 mm); System A: 0.025 M ammonium acetate in water; System B: 1: 4 = 0.125 M ammonium acetate in water/ acetonitrile; Gradient: 0-100% system B over 10 min; Flow: 3 ml/min;

Detector wavelength: 265 nm. Injected 20 μl of stock solution of M40403-(OAc)₂ prepared by dissolving 1 mg in 1 ml of system A. The HPLC chromatogram of M40403 -(OAc)₂ is shown in Figure 11.

Example 12

An HPLC method to separate the diastereomers of superoxide dismutase mimetic compound M40403. Four stereoisomer mixtures were prepared (Part A) as shown in Schemes 5-9 and then separated (Part B) via reversed-phase high performance liquid chromatography. Part A: Synthesis of Stereoisomers Of M40403

M40403 is synthesized from its single-isomer, tetra-amine precursor M40400 in the reaction shown in Scheme 7.

M40400 M40402

M40403

Scheme 7

The various stereoisomers of M40403 are synthesized from the various isomers of 1,2-diaminocyclohexane which provides the chiral carbon centers in M40403. The 1,2-diaminocyclohexane isomers used to prepare the R,R+R,S) M40403 stereoisomer mixture of Set 1 are shown in Scheme 6. Similarly, the 1,2-diaminocyclohexane isomers used to prepare the (R,R+S,S) M40403 stereoisomer mixture of Set 2 are shown in Scheme 7. The 1,2-diaminocyclohexane isomers used to prepare the (R,S+R,S) M40403 stereoisomer mixture of Set 3 are shown in Scheme 8. The 1,2- diaminocyclohexane isomers used to prepare the (S,S+R,S) M40403 stereoisomer mixture of Set 4 are shown in Scheme 9. As shown in Schemes 6-9 the M40403 diastereomers are prepared by template cyclization, followed by reduction with sodium borohydride.

Scheme 8

(S.S.S.S)

Scheme 9

(S.R.R.S)

Scheme 10

Scheme 11

Table 1

Part B: Separation of Stereoisomer Mixtures

Chemicals, Materials, and Methods

Tetrabutylammonium chloride hydrate (98%, 34,585-7) was purchased from Aldrich Chemical Company. Sodium chloride (99.6%, S-9888) was purchased from Sigma Chemical Company. All other solvents (HPLC-grade unless otherwise indicated) and reagents were purchased from Fisher Scientific and were of the finest grade available. The SymmetryShield® RP₁₈ column (4.6 mm x 250 mm, 5 μm particle size) and its corresponding guard column were purchased from Waters Corporation. Reversed-Phase HPLC Experiments

Preparation of Standard Solutions

HPLC Mobile phased was an aqueous solution consisting of 0.125 M tetrabutylammonium chloride (TBAC) and 0.5 M LiCl, prepared by adding tetrabutylammonium chloride hydrate (36.99 g) and solid LiCl (21.2 g) to a 1 L volumetric flask, diluting to volume with Millipore water, and inverting the flask several times to obtain a homogeneous solution. The resulting solution was filtered through a 0.45 μm nylon filter prior to use. Mobile phase B was HPLC-grade acetonitrile. Samples of each diastereoisomer set for HPLC-UN analysis were prepared at concentrations of ~ 3.0 mg/mL in a 50:50 mixture of 0.5 M LiCl in MeOH:

PATENT

WO/2021/163397

SOLID STATE FORMS OF AVASOPASEM MANGANESE AND PROCESS FOR PREPARATION THEREOF

Avasopasem manganese (GC4419), has the following chemical structure:

[0003] Avasopasem manganese is a highly selective small molecule superoxide dismutase (SOD) mimetic which is being developed for the reduction of radiation-induced severe oral mucositis (SOM). The compound is described in U.S. Patent No. 8,263,568.

[0004] Polymorphism, the occurrence of different crystalline forms, is a property of some molecules and molecular complexes. A single molecule may give rise to a variety of polymorphs having distinct crystal structures and physical properties like melting point, thermal behaviors (e.g., measured by thermogravimetric analysis (“TGA”), or differential scanning calorimetry (“DSC”)), X-ray diffraction (XRD) pattern, infrared absorption fingerprint, and solid state (¹³C) NMR spectrum. One or more of these techniques may be used to distinguish different polymorphic forms of a compound.

[0005] Different salts and solid state forms (including solvated forms) of an active pharmaceutical ingredient may possess different properties. Such variations in the properties of different salts and solid state forms and solvates may provide a basis for improving formulation, for example, by facilitating better processing or handling characteristics, changing the

dissolution profile in a favorable direction, or improving stability (polymorph as well as chemical stability) and shelf-life. These variations in the properties of different salts and solid state forms may also offer improvements to the final dosage form, for instance, if they serve to improve bioavailability. Different salts and solid state forms and solvates of an active pharmaceutical ingredient may also give rise to a variety of polymorphs or crystalline forms, which may in turn provide additional opportunities to assess variations in the properties and characteristics of a solid active pharmaceutical ingredient.

[0006] Discovering new solid state forms and solvates of a pharmaceutical product may yield materials having desirable processing properties, such as ease of handling, ease of processing, storage stability, and ease of purification or as desirable intermediate crystal forms that facilitate conversion to other polymorphic forms. New solid state forms of a pharmaceutically useful compound can also provide an opportunity to improve the performance characteristics of a pharmaceutical product. It enlarges the repertoire of materials that a formulation scientist has available for formulation optimization, for example by providing a product with different properties, including a different crystal habit, higher crystallinity, or polymorphic stability, which may offer better processing or handling characteristics, improved dissolution profile, or improved shelf-life (chemical/physical stability). For at least these reasons, there is a need for additional solid state forms (including solvated forms) of Avasopasem manganese.

EXAMPLES

Preparation of starting materials

[00119] Avasopasem manganese can be prepared according to methods known from the literature, for example U.S. Patent No. 8,263,568. Alternatively, Avasopasem manganese can be prepared by the template method reported for the enantiomeric analogue GC4403, which has the formula:

GC4403 is disclosed in International Appl. No. WO 98/58636 (as compound SC-72325) and Riley, D.P, and Schall, O.F., Advances in Inorganic Chemistry (2007), 59, 233-263. Thus, GC4403 can be synthesized via the template route described in the literature using the chiral R,R-l,2-diamminocyclohexane [Salvemini, D., et ah, Science (1999), 286, 304-6 , and Aston, K, et al., Inorg. Chem. (2001), 40(8), 1779-89] Avasopasem manganese (GC4419) can be prepared by the same method except that the chiral R,R-l,2-diamminocyclohexane is replaced with S,S-1 ,2-diamminocyclohexane.

Example 1: Preparation of Avasopasem manganese Form AMI

[00120] Avasopasem manganese (0.1 grams) was dissolved in dichloromethane (0.5 ml) at 25-30°C in a test tube. The solution was filtered through 0.45 micron filter and the clear solution was subjected to slow solvent evaporation at 25°C by covering the tube with paraffin film with a pin hole. After, 2 days, the obtained solid was analyzed by XRD- Form AMI; as shown in Figure 1

1. GlobeNewswire: Galera Therapeutics Announces Avasopasem Manganese Improved Markers of Chronic Kidney Disease in Patients Receiving Cisplatin [Link]
2. Galera Therapeutics: AVASOPASEM (GC4419) [Link]

///////////AVASOPASEM, Avasopasem manganese, GC-4419,  GC4419, GC 4419, M 40419, M40419; M-40419, SC 72325A, SC-72325A, SC72325A,

[Cl-].[Cl-].[Mn++].C1CC[C@@H]2NCC3=CC=CC(CN[C@H]4CCCC[C@@H]4NCCN[C@H]2C1)=N3