Hydrazine based compounds and methods of use thereof for treatment of disease or neurological injury

Abstract

The invention generally relates to hydrazine based compounds and methods of use thereof for treatment of disease or neurological injury.

Description

[0001] Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0002] PATENT APPLICATION HYDRAZINE BASED COMPOUNDS AND METHODS OF USE THEREOF FOR TREATMENT OF DISEASE OR NEUROLOGICAL INJURY

[0003] Related Application

[0004] The present application claims the benefit of and priority to U. S. provisional patent application serial number 63 / 778,875, filed March 27, 2025, the content of which is incorporated by reference herein in its entirety.

[0005] Field of the Invention

[0006] The invention generally relates to hydrazine based compounds and methods of use thereof for treatment of disease or neurological injury.

[0007] Background

[0008] Oxidative stress has been found to be associated with numerous different diseases.

[0009] Acrolein, a reactive alpha / beta-unsaturated aldehyde, has been reported to be a product of oxidative stress and lipid peroxidation. Furthermore, acrolein has been reported to remain active in the body for several days while more commonly studied oxidative species decay within seconds. Therefore, and without being bound by theory, it is believed herein that acrolein may be a key factor in perpetuating oxidative stress and may cause numerous different diseases associated with oxidative stress.

[0010] Prior work has shown that hydrazine based compounds, such as hydralazine and phenelzine, are able to scavenge acrolein, and therefore my provide treatments for disease that involve a build-up of acrolein in the body.

[0011] However, each of these compounds have side effects within the body that make their use undesirable. For example, phenelzine is a monoamine oxidase inhibitor (MAOI) compound that has previously been used as an antidepressant. There are numerous detrimental side-effects associated with use of phenelzine, all of which stem from the MAOI activity of the compound.

[0012] Summary

[0013] The invention recognizes that blocking the MAOI activity of phenelzine would allow the compound to be used as a scavenger of acrolein or other toxic aldehydes while also reducing orAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0014] PATENT APPLICATION

[0015] eliminating all of the side-effects of phenelzine associated with the MAOI portion of the molecule. Accordingly, the invention provides compounds in which the MAOI activity of phenelzine has been blocked, while maintaining the acrolein / toxic aldehyde scavenging ability of the compounds. Therefore, the compounds of the invention are able to effectively scavenge acrolein or other toxic aldehydes without the MAOI side-effects associated with phenelzine.

[0016] In certain aspects, the invention provides compositions that include a pharmaceutically acceptable carrier and a compound of formula I:

[0017]

[0018] R1

[0019] I

[0020] wherein:

[0021] R1is a straight or branched chain alkyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkenyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkyne optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a mono- or poly- cyclic ring structure optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3;

[0022] R1may also optionally be COR3, COOR3, or NR3;

[0023] R2is a straight or branched chain alkyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkenyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkyne optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a mono- or poly- cyclic ring structure optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3;

[0024] R2may also optionally be COR3, COOR3, or NR3and;Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0025] PATENT APPLICATION

[0026] R3is hydrogen; a straight or branched chain alkyl optionally substituted by one or more heteroatoms; a straight or branched chain alkenyl optionally substituted by one or more heteroatoms; a straight or branched chain alkyne optionally substituted by one or more heteroatoms; or a mono- or poly- cyclic ring structure optionally substituted by one or more heteroatoms.

[0027] In certain embodiments, R1is a straight or branched chain alkyl. In other embodiments, R1is a C1-C6alkyl. In other embodiments, R1is a C1-C3 branched chain alkyl. In other embodiments, R2is a C1-C6alkyl substituted by a monocyclic ring structure. In certain embodiments, the monocyclic ring structure is a phenyl group. In preferred embodiments, the compound of formula I is the compound of formula II:

[0028]

[0029] II.

[0030] In certain embodiments, the compositions of the invention may also include a pharmaceutically acceptable salt. An exemplary pharmaceutically acceptable salt is hydrochloride. In certain embodiments, the composition is formulated for oral administration. In certain embodiments, the composition is formulated as a single unit dosage.

[0031] The above compositions can be used for scavenging an alpha / beta unsaturated aldehyde compound (e.g., acrolein). Typically, the alpha / beta unsaturated aldehyde compound is associated with a disease, and by scavenging the alpha / beta unsaturated aldehyde compound the disease is treated, e.g., disease symptoms are reduced or eliminated. In certain embodiments, the disease is associated with degeneration of nervous system tissue. The compositions of the invention can also provide neuro-protection as well as have an analgesic effect.

[0032] Certain diseases, such as diseases associated with degeneration of nervous system tissue, as well as certain chemical substituents, are described for example in U. S. patent number 9,517,237 and U. S. patent number 8,946,220, the content of each of which is incorporated by reference herein in its entirety.Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0033] PATENT APPLICATION

[0034] Brief Description of the Drawings

[0035] FIGS. 1A-F show design of Phenelzine Analog (PhzA) and its mechanism of action. (A) The hypothetical binding mode of phenelzine in the active site of MAO-B. The crystal structure of human MAO-B was used as the enzyme 3-dimensional structure. The compound phenelzine was docked into the active site of MAO-B using GOLD 3.1 based on default parameters. A best binding mode was chosen from 10 docking poses according to the Gold-Score fitness function as the final binding conformation. Note Cl is pointing towards flavin. (B) Chemical structure of PhzA. (3 -methyl- l-phenylbutan-2-yl) hydrazine hydrochloride and proposed reaction between PhzA and acrolein. (C) Mass spectrum of crude acrolein - PhzA reaction products. A peak emerged at 217.51 (red) in the mass spectrum of reaction products, demonstrating the existence of the proposed products in the reaction product mixture, and indicating the occurrence of the proposed reaction between PhzA and acrolein. (D) MS-MS spectrum of compounds at peak 217. The compounds at peak 217.51 in MS spectrum were cleaved into smaller fragments via MS-MS for further structure identification. The characteristic peaks for the fragments were at 71.56, 91.44 and 105.30, respectively. The chemical structures of the fragments were shown on their peaks. Note that these fragments all stemmed from the proposed products. This spectrum verified the chemical identity of the proposed products. (E, F) PhzA loses the inhibitory effects to monoamine oxidases (MAO, subtype A and B). The dose-dependent curve indicated PhzA did not present any inhibitory effects to MAO-A and B. Selective MAO-A inhibitor clorgyline and MAO-B inhibitor selegiline were used as inhibitor controls (IC), respectively. All data are expressed as the mean ± SEM. Sample size = 3 for each concentration.

[0036] FIGS. 2A-B show acrolein elevation in the spinal cord and its dose-dependent reduction by Phenelzine Analog (PhzA) at 2 days post injury. (A) Representative western blot acrolein bands (50kDa) are shown above corresponding GAPDH bands (37kDa) for both Sham and SCI groups (top and bottom) and in SCI rats treated with PhzA at 5, 15, 30 mg / kg (Bottom). Note the elevation of acrolein in the SCI group 48 hours after injury and its reduction with PhzA treatment. All rats received either vehicle (Sham or SCI), or PhzA (SCI + PhzA) at different dosages daily until sacrifice at the 48-hour acute timepoint. (B) Quantitative analysis demonstrates that acrolein expression is significantly elevated after SCI. The application of Phenelzine analog, PhzA, resulted in a dosage-dependent acrolein reduction when compared to the SCI group. Note that the rats receiving 30 mg / kg PhzA had a similar level of acrolein to thatAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0037] PATENT APPLICATION

[0038] in the 15 mg / kg group. Therefore, 15 mg / kg was chosen to be used for the rest of the study to minimize the potential side-effects of the drug. Data is normalized to GAPDH for analysis. Data presented as the Mean ± SEM (n = 3-4). 2-tailed T-test was utilized with p-value <0.05 considered statistically significant (*). ** p < 0.01, *** p < 0.001.

[0039] FIGS. 3A-B show Phenelzine Analog (PhzA) mitigated acrolein elevation in the spinal cord at 28 days post injury. (A) Representative images of the transverse section of the spinal cord and (B) densitometry quantification of acrolein DAB staining showed a significant elevation of acrolein in the whole cord, white matter, and gray matter of the spinal cord after injury. PhzA treatment significantly reduced the acrolein accumulation compared to the injury-only (SCI) group in the whole spinal cord, both in the white and grey matter. Note that even with the administration of PhzA, the levels of acrolein in the whole cord as well as in the white matter of the injured spinal cord remained significantly elevated at 28 days after injury when compared to sham. Data presented as the Mean ± SEM (n = 6-7). 2-tailed T-test was utilized with p-value <0.05 considered statistically significant (*). * p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0040] FIGS. 4A-C show Phenelzine Analog (PhzA) alleviated astrocytes activation in the spinal cord at 28 days post injury. Immunocytochemistry was performed on the spinal cord sections immediately adjacent (caudal) to the injury epicenter. (A) Representative images demonstrated increased expression of reactive astrocytes with anti-GFAP (green) in the spinal cord after SCI and its mitigation by PhzA. Scale bar = 500 pm. (B) Higher magnification view in several regions (dorsal column, central canal, dorsal horn, ventral horn, and lateral column) revealed widespread glial scar formation in both gray and white matter. DAPI (blue) was used for nuclei counterstains. Scale bar = 500um. (C) Quantitative analysis of the mean fluorescent intensity of GFAP (% to sham baseline) indicated acrolein scavenging through PhzA treatment significantly reduced astrocytes activation and glial scar formation in various spinal cord regions at 28 days after injury. All data are presented as the mean ± SEM, n = 6-7. One-way ANOVA and Tukey’s multiple comparisons test were used to analyze differences among groups. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0041] FIGS. 5A-C show Phenelzine Analog (PhzA) reduced immune cell activation in the spinal cord at 28 days post injury. Immunocytochemistry was performed on the spinal cord sections immediately adjacent (caudal) to the injury epicenter. (A) Representative imagesAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0042] PATENT APPLICATION

[0043] demonstrated an increased expression of reactive microglia / macrophages with anti-Iba-1 (red) in the spinal cord after SCI and its mitigation by PhzA. Scale bar = 500 pm. (B) Higher magnification view in several regions (dorsal column, central canal, dorsal horn, ventral horn, and lateral column) revealed an increase in macrophage and microglia numbers, characterized by increased soma size and less ramified processes (second row, red arrows) at the several regions in both gray and white matter when compared to the resting state (first row, white arrows). DAPI (blue) was used for nuclei counterstains. (C) Quantitative analysis of the mean fluorescent intensity of Iba-1 (% to sham baseline) indicated acrolein scavenging through PhzA treatment significantly reduce microglial / macrophages activation in various spinal cord regions at 4 weeks after injury. All data are presented as the mean ± SEM, n = 6-7. One-way ANOVA and Tukey’s multiple comparisons test were used to analyze differences among groups. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0044] FIGS. 6A-B show Phenelzine Analog (PhzA) relieved locomotor and sensory impairments in rats through 28 days after SCI. Time course of SCI-induced movement and sensory dysfunctions. (A) Post-SCI lower limb locomotor function assessment by Basso, Beattie and Bresnahan (BBB) Score indicates significant motor functional deficits and its mitigation with the PhzA treatment within 4 weeks post injury. (B) Post-SCI hypersensitivity and its alleviation by PhzA. Pain threshold assessment by von Frey filament test (VF). Rats with SCI showed severe continuous mechanical nociception disturbance. PhzA treatment significantly mitigated such hyperalgesia in SCI rats from the 7 days after SCI. Error bars represent the mean ± SEM (n = 7-9). * represented the comparison between the injury and PhzA treatment groups.

[0045] 2 -tailed T-test was utilized with p-value <0.05 considered statistically significant (*). * p < 0.05, ** p < 0.01, **** p < 0.0001.

[0046] Detailed Description

[0047] Oxidative stress is widely recognized as a critical factor in the functional deficits after spinal cord injury (SCI). Oxidative stress and lipid peroxidation-derived aldehydes such as acrolein are known to play a key role in SCI pathology and have therefore

[0048] emerged as valuable therapeutic targets. The invention herein in certain aspectes introduces a novel phenelzine analogue (PhzA), designed to retain the acrolein scavenging capability ofAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0049] PATENT APPLICATION

[0050] phenelzine (Phz) while removing its undesirable monoamine oxidase (MAO) inhibition effects through structure-based modification. Using a rat model of contusion SCI, we showed that PhzA significantly reduced acrolein levels in both the acute and chronic stages of SCI with minimal MAO inhibition. In addition, PhzA reduced excessive microglial and astrocytic activation, dampening inflammation and gliosis. Furthermore, PhzA-treated rats exhibited significant improvements in motor function and reduction in mechanical hypersensitivity for up to 28 days post-injury compared to untreated rats. These findings further

[0051] underscore the crucial role of aldehydes in SCI pathology and strengthen the notion that acrolein could serve as an effective therapeutic target for mitigating post-SCI neurodegeneration. These results also indicate that the expansion of acrolein-scavenging drug discovery through structure-based modification of existing repurposed drugs, such as with Phz, is a viable strategy with the benefit of a likely accelerated path towards clinical application. This effort may also benefit a range of neuronal diseases and injuries beyond SCI where acrolein is implicated, advancing the health of millions of patients.

[0052] The invention generally relates to hydrazine based compounds and methods of use thereof for treatment of disease or neurological injury. In certain embodiments, the compounds scavenge alpha / beta unsaturated aldehyde compounds (such as acrolein). Generally, a scavenger is a chemical substance added to a mixture or sample or medium in order to remove or deactivate one or more molecules within the mixture or sample or medium. A scavenger can be administered to a patient to remove or de-activate one or more molecules circulating within the patient. In the context of the invention, the compositions include compounds that can remove acrolein or other toxic aldehyde from circulation within a body of a patient or de-activate acrolein or other toxic aldehyde within a body of a patient.

[0053] An alpha / beta unsaturated aldehyde is a functional group of a molecule, and may have the general formula of (O=CR)-C“=C|3-R. In this functional group, the carbonyl group is conjugated with an alkene. Unlike the case for simple carbonyls, a,β-unsaturated aldehyde functional groups are often attacked by nucleophiles at the 0 carbon. Exemplary compounds that include alpha / beta unsaturated aldehydes are acrolein, methylenedioxyamphetamine (MDA), or hydroxynonenal (HNE). Other exemplary alpha / beta unsaturated aldehydes are shown in Table 1 below.Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0054] PATENT APPLICATION

[0055] Table 1: Alpha / beta unsaturated aldehydes

[0056] Compound Name Compound Structure

[0057] Hex-2(trans)-enal

[0058] - ' ' D

[0059] N ona-2(trans), 6(ci s)-dienal

[0060] Oct-2-enal

[0061] 4,5-Epoxydec-2(trans)-enal

[0062] z / \ \

[0063] ^4 ' \

[0064] % — < J \

[0065] 2-Methylpent-2-enal

[0066] beta-Sinensal

[0067] 2-Methylprop-2-enal Q

[0068] or the precursor

[0069] 2-Methylallyl butyrate

[0070] Hexa-2(trans),4(trans)-dienal

[0071] --o

[0072] Deca-2(trans),4(trans)-dienal

[0073] 4-Methylpent-3 -en-2-one

[0074]

[0075] Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0076] PATENT APPLICATION

[0077] 7 -Methyl-3 -octenone-2

[0078] Pent- l-en-3 -one o

[0079] Oct-l-en-3-one

[0080] 2-Pentylbut- 1 -en-3 -one

[0081] A

[0082] -Methylhepta-3,5-dien-2-one 1

[0083] Pseudo-ionone

[0084] X-.x^ ^X.

[0085] /

[0086] p-Mentha-l,8-dien-7-al

[0087] 1

[0088] ,6,6-Trimethylcyclohexa-l,3- diene-1- carbaldehyde

[0089] o

[0090]

[0091] Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0092] PATENT APPLICATION

[0093] alpha-ionone G

[0094] AC

[0095] Allyl alpha-ionone Q

[0096] alpha-Damascone o

[0097] beta-Ionone epoxide

[0098] xx u

[0099] l(7),8-p-menthadien-2-one

[0100] or the precursor

[0101] xx, XX X8l(7),8-p-Menthadien-2-yl acetate

[0102] (mixture of (E) and (Z) isomers)

[0103] 3 -Methyl-2-cyclopenten- 1 -one

[0104] f / ?

[0105] 3,5, 5 -Trimethylcy clohex-2-en- 1 - ©

[0106] one

[0107] A

[0108]

[0109] Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0110] PATENT APPLICATION

[0111] 2,6-Dimethyl-9-(l- Jt

[0112] methylethylidene)- bicyclo[5.3.0]dec-2-en-4-one A YA /

[0113] or its precursor V. A- / \ J \ \~J Vetiverol \ beta-Ionone 0^

[0114] Maltol jj

[0115] beta-Damascone €

[0116] X. J

[0117] Nootkatone "'x

[0118] ,6,6-Trimethylcyclohex-2-en- 1, 4-dione

[0119] 1

[0120] Piperitenone oxide —

[0121] I.

[0122] 4-Phenylbut-3 -en-2-one G

[0123] pX..

[0124]

[0125] Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0126] PATENT APPLICATION

[0127] -(4-Methoxyphenyl)-4pent- 1 - Q

[0128] en-3-one X — i

[0129] - J / % - / X □ /

[0130] _ y

[0131] 2-Phenylcrotonaldehyde

[0132] X\ / /

[0133] 5-Ethyl-3-hydroxy-4- methylfuran-2(5H)-one

[0134] \ _ /

[0135] / 'OH

[0136] 3,4-Dimethyl-5-, O. pentylidenefuran-2(5H)-one NZ \ /

[0137] Furan-2(5H)-one,0.

[0138] z X o

[0139] \ /

[0140] -Hydroxy-2,5-dimethylfuran- 3(2H)-one

[0141] X.. X

[0142] TO / % SQ

[0143] 5-Methylfuran-3(2H)-one

[0144] x

[0145] ''b

[0146] 4-Acetyl-2,5-dimethylfuran- a,

[0147] 3(2H)-one "'<? — Y"

[0148] b

[0149] 3-(2-Furyl)acrylaldehydezo,

[0150] < V'x''ZzxG

[0151] X / /

[0152] V >‘1

[0153]

[0154] Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0155] PATENT APPLICATION

[0156] 4-(2-Furyl)but-3-en-2-one

[0157] 2-Acetyl-l,4,5,6- tetrahydropyridine

[0158] ... N,,1

[0159] 5-Methyl-2- O thiophenecarbaldehyde

[0160] / /

[0161] 7..........7

[0162] 3-Acetyl-2,5-dimethylthiophene

[0163] _

[0164]

[0165] With alpha / beta unsaturated aldehydes, the carbonyl group draws electrons away from the alkene, and the alkene group is, therefore, deactivated towards an electrophile, such as bromine or hydrochloric acid. As a general rule with asymmetric electrophiles, hydrogen attaches itself at the a-position in an electrophilic addition. On the other hand, these compounds are activated towards nucleophiles in nucleophilic conjugate addition. Since a, P-unsaturated compounds are electrophiles, many a, P-unsaturated carbonyl compounds are toxic, mutagenic and carcinogenic. DNA can attack the P carbon and thus be alkylated.

[0166] In certain embodiments, compounds that include alpha / beta unsaturated aldehydes may be generated in the body via metabolism of polyamine compounds. The term "polyamine" herein represents a straight-chain aliphatic hydrocarbon having two or more primary amino groups. Known biogenic polyamines may include, but are not limited to, putrescine, cadaverine, spermidine, spermine, 1,3-diaminopropane, caldine, homospermidine, 3 -aminopropylcadaverine, norspermine, thermospermine, caldopentamine, and so on. Meanwhile, preferred polyamines in the present invention may be putrescine, spermidine and spermine.Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0167] PATENT APPLICATION

[0168] The above polyamines may be metabolized by oxidation, acetylation, transamination and carbamoylation, and polyamine oxidase is the enzyme that involves in the oxidation of polyamine. The term "polyamine oxidase" herein represents an enzyme that oxidizes diamine or polyamine as a good substrate and generates hydrogen peroxide. Polyamine receives oxidative deamination by polyamine oxidase, thereby aldehyde compounds such as acrolein would be produced. The preferred aldehyde compound in the present invention may be acrolein, but is not so limited to it.

[0169] As used herein, alpha / beta unsaturated aldehyde compounds may also include adducts formed when an alpha / beta unsaturated aldehyde compound binds to another compound. For example, acrolein, an alpha / beta unsaturated aldehyde compound, is known to bind to proteins, such as alpha-synuclein. The complex formed when acrolein binds alpha-synuclein is considered an alpha / beta unsaturated aldehyde compound within the context of the invention because the alpha / beta unsaturated aldehyde function group of the acrolein molecule remains exposed for scavenging by compositions of the invention.

[0170] Diseases associated with abnormal acrolein or toxic aldehyde levels are described for example in U. S. patent number 9,517,237 and U. S. patent number 8,946,220, the content of each of which is incorporated by reference herein in its entirety. In certain embodiments, the diseases are diseases associated with degeneration of nervous system tissue, such as neuropathic pain related diseases, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, or multiple sclerosis. Other diseases include chemotherapy-induced peripheral neuropathic pain or hemorrhagic cystitis, COPD, stroke, alcohol induced liver disease, or kidney disease. Injuries that can be treated with the compositions of the invention include spinal cord injury or traumatic brain injury (TBI).

[0171] The invention provides compounds in which the MAOI activity of phenelzine has been blocked, while maintaining the ability of the compounds to scavenge alpha / beta unsaturated aldehyde compounds (e.g., acrolein). Therefore, the compounds of the invention are able to effectively scavenge acrolein or other toxic aldehydes (e.g., other alpha / beta unsaturated aldehyde compounds) without the MAOI side-effects associated with phenelzine.

[0172] In certain aspects, the invention provides compositions that include a pharmaceutically acceptable carrier and a compound of formula I:Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0173] PATENT APPLICATION

[0174]

[0175] I

[0176] wherein:

[0177] R1is a straight or branched chain alkyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkenyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkyne optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a mono- or poly- cyclic ring structure optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3;

[0178] R1may also optionally be COR3, COOR3, or NR3;

[0179] R2is a straight or branched chain alkyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkenyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkyne optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a mono- or poly- cyclic ring structure optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3;

[0180] R2may also optionally be COR3, COOR3, or NR3and;

[0181] R3is hydrogen; a straight or branched chain alkyl optionally substituted by one or more heteroatoms; a straight or branched chain alkenyl optionally substituted by one or more heteroatoms; a straight or branched chain alkyne optionally substituted by one or more heteroatoms; or a mono- or poly- cyclic ring structure optionally substituted by one or more heteroatoms.

[0182] In certain embodiments, R1is a straight or branched chain alkyl. In other embodiments, R1is a C1-C6alkyl. In other embodiments, R1is a C1-C3 branched chain alkyl. In other embodiments, R2is a C1-C6alkyl substituted by a monocyclic ring structure. In certainAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0183] PATENT APPLICATION

[0184] embodiments, the monocyclic ring structure is a phenyl group. In preferred embodiments, the compound of formula I is the compound of formula II:

[0185]

[0186] II.

[0187] In certain embodiments, the compositions of the invention may also include a pharmaceutically acceptable salt. An exemplary pharmaceutically acceptable salt is hydrochloride. In certain embodiments, the composition is formulated for oral administration. In certain embodiments, the composition is formulated as a single unit dosage.

[0188] Alkyl refers to an alkane missing one hydrogen. Exemplary alkyls include Methyl, Ethyl, Propyl, Butyl, Pentyl, Hexyl, Heptyl, Octyl, Nonyl, Decyl, Undecyl, or Dodecyl. As used herein, the alkyl may be a straight chain alkyl or a branched chain alkyl. As used herein, one or more of the carbon atoms may be substituted by one or more substituents.

[0189] Alkenyl refers to an alkyl group having one or more double bonds. Any of the above mentioned alkyl groups can also be transformed into alkenyl groups. An exemplary structure of

[0190]

[0191] an alkenyl group is. As used herein, the alkenyl may be a straight chain alkenyl or a branched chain alkenyl. As used herein, one or more carbon atoms of the alkenyl group may be substituted by one or more substituents.

[0192] Alkyne refers to an unsaturated hydrocarbon containing at least one carbon — carbon triple bond. The simplest acyclic alkynes with only one triple bond and no other functional groups form a homologous series with the general chemical formula CnH2n-2. As used herein, the alkenyl may be a straight chain alkenyl or a branched chain alkenyl. As used herein, one or more carbon atoms of the alkenyl group may be substituted by one or more substituents.

[0193] A heteroatom refers to any atom that is not carbon or hydrogen. Usually, the heteroatom indicates a non-carbon atom having replaced a carbon in the backbone of the molecular structure, e.g., of an alkyl, alkenyl, heteroalkyl, aryl, fused cycloalkyl, fused cycloalkenyl. Exemplary heteroatoms are nitrogen and sulfur.Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0194] PATENT APPLICATION

[0195] A mono- or poly- cyclic ring structure includes ring based moi eties, such as aryl, cycloalkyl, heterocycloalkyl, fused cycloalkyl, fused cycloalkenyl, fused heterocycloalkenyl, or heterocycloalkyl-aryl.

[0196] A cycloalkyl refers to a ring structure composed of single bonded carbon atoms. The ring structure can have anywhere from 3 to 20 atoms, such as 3, 4, 5, 6, 7, 8, 9, 10, etc. Exemplary cycloalkyl structures are

[0197]

[0198] As used herein, one or more of the carbon ring atoms may be substituted by one or more substituents.

[0199] A heterocycloalkyl refers a cycloalkyl in which one of the ring carbons is substituted by a

[0200]

[0201] N N

[0202] heteroatom. An exemplary heterocycloalkyl is

[0203]

[0204] . As used herein, one or more carbon ring atoms may be substituted by one or more substituents and / or the heteroatom may be substituted by one or more substituents.

[0205] Aryl (Ar) refers to any functional group or substituent derived from an aromatic ring, usually an aromatic hydrocarbon. Exemplary aryl groups are phenyl (Ph), naphthyl, thienyl, or

[0206] indolyl. An exemplary aryl ring is shown as

[0207]

[0208] Rv -. As used herein, one or more of the carbon ring atoms may be substituted by one or moJre substituents.

[0209] A fused cycloalkenyl refers to a fused ring structure having one or more double bonds. The fused structure can include two or more rings. Exemplary fused ring structures have between 6 and 20 carbon atoms, for example 8, 10, 12, 14, 16, or 18 carbon atoms. An

[0210] exemplary structure of a fused cycloakenyl is

[0211]

[0212] As used herein, one or more carbon ring atoms may be substituted by one or more substituents.Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0213] PATENT APPLICATION

[0214] A fused heterocycloalkenyl is a fused cycloalkenyl as described above in which one or more of the carbon ring atoms is substituted by a heteroatom. An exemplary structure of a fused

[0215] / ”1

[0216] h '

[0217]

[0218] ;H

[0219] heterocycloalkenyl is. As used herein, one or more carbon ring atoms may be substituted by one or more substituents and / or the heteroatom may be substituted by one or more substituents.

[0220] A fused heterocycloalkyl-aryl refers to a heterocycloalkyl group fused to an aryl group.

[0221] An exemplary fused heterocycloalkyl-aryls is CO. As used herein, one or more carbon ring atoms may be substituted by one or more substituents and / or the heteroatom may be substituted by one or more substituents.

[0222] The term "—COR3" refers to an aldehyde or ketone moiety where R3may be selected from the group consisting of hydrogen and C-Cs alkyl. When R3is hydrogen, the "—COR3" may be an aldehyde —COH. When R3is a C-Cs alkyl, the "—COR3" may be a ketone containing one to eight carbon atoms, where the alkyl chain may be a straight or branched alkyl chain, or saturated or unsaturated alkyl. Thus, the term "—COR3" includes —COH, — COCH3, — COCH2 CH3, -CO(CH2)2CH3, -CO(CH2)3CH3, -CO(CH2)4CH3, -CO(CH2)5CH3, -COCH(CH3)2, - COC(CH3)3, -COCH2 CH(CH3)2, -COCH2 C(CH3)3, -CO(CH2)2CH(CH3)2, -CO(CH2)2C(CH3)3, -COCH(CH3)(CH2)3 CH3, -COCH2CH(CH3)2CH2CH3, -CO(CH2)2CH(CH3)3 and the like, and includes their unsaturated counterparts.

[0223] The term “—COOR3” refers to a "carboxylate" or "carboxyl" or — COOH. The carboxyl group can form a carboxylic acid. A substituted carboxyl refers to -COOR3where R is alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group. For example, a substituted carboxyl group could be a carboxylic acid ester or a salt thereof (e.g., a carboxylate).

[0224] The term “-NR3” refers to an amine group where R3may be alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group.Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0225] PATENT APPLICATION

[0226] A pharmaceutical composition containing the active ingredient and a pharmaceutically acceptable carrier may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in U. S. Pat. 4,256,108, U. S. Pat. 4,166,452 and U. S. Pat. 4,265,874, to form osmotic therapeutic tablets for control release.

[0227] Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

[0228] Formulations may also include complexes of the parent (unionized) compounds with derivatives of P-cyclodextrin, especially hydroxypropyl-P-cyclodextrin.

[0229] An alternative oral formulation can be achieved using a controlled-release formulation, where the compound is encapsulated in an enteric coating.

[0230] Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as a naturally occurring phosphatide, for example lecithin, or condensation products of anAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0231] PATENT APPLICATION

[0232] alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxy cetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such a polyoxyethylene with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0233] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0234] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified, for example sweetening, flavoring and coloring agents, may also be present.

[0235] The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally occurring phosphatides, for example soya bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

[0236] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may beAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0237] PATENT APPLICATION

[0238] formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0239] Each active agent may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

[0240] For topical use, creams, ointments, jellies, solutions or suspensions are suitable. Topical application includes the use of mouth washes and gargles.

[0241] The compositions of the invention may be formulated in unit dosage form. The compositions of the invention include a therapeutically effective amount of one or more compounds described herein. In certain embodiments, the compositions of the invention include a therapeutically effective amount of one or more compounds described herein that is not therapeutically effective or clinically effective for treating depression or anxiety. In another embodiment, the compositions of the invention include a therapeutically effective amount of one or more compounds described herein that is at least about 2-fold, at least about 3-fold, at least about 4-fold, or at least about 5-fold lower than the therapeutically effective or clinically effective dose for treating depression or anxiety. In another embodiment, the compositions of the invention include a therapeutically effective amount of one or more compounds described herein that is at least about 10-fold, at least about 20-fold, at least about 30-fold, or at least about 50-fold lower than the therapeutically effective or clinically effective dose for treating depression or anxiety.

[0242] In another embodiment, the compositions of the invention include a therapeutically effective amount of one or more compounds described herein, such as the equivalent of aboutAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0243] PATENT APPLICATION

[0244] 0.01 mg / kg to about 2 mg / kg, about 0.01 mg / kg to about 1.5 mg / kg, about 0.01 mg / kg to about 1 mg / kg, or about 0.01 mg / kg to about 0.5 mg / kg, administered orally.

[0245] In another embodiment, the compositions of the invention include a therapeutically effective amount of one or more compounds described herein, such as the equivalent of about 0.05 mg / kg to about 2 mg / kg, about 0.05 mg / kg to about 1.5 mg / kg, about 0.05 mg / kg to about 1 mg / kg, or about 0.05 mg / kg to about 0.5 mg / kg, administered orally.

[0246] In another embodiment, the compositions of the invention include a therapeutically effective amount of one or more compounds described herein, such as the equivalent of about 0.1 mg / kg to about 2 mg / kg, about 0.1 mg / kg to about 1.5 mg / kg, about 0.1 about 0.1 mg / kg to about 1 mg / kg, or about 0.1 mg / kg to about 0.5 mg / kg, administered orally.

[0247] In another embodiment, the compositions of the invention include a therapeutically effective amount of one or more compounds described herein, such as the equivalent of about 0.5 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 3 mg / kg, about 0.5 mg / kg to about 2 mg / kg, about 0.5 mg / kg to about 1 mg / kg, administered orally.

[0248] In each of the foregoing embodiments, it is to be understood that the dose may be single or divided. In addition, it is to be understood that the therapeutically effective amount be administered following any of a wide variety of dosing schedules, including q.d., b.i.d., three times daily, four times daily, and the like.

[0249] Accordingly, an illustrative dosing schedule for an adult of average weight may be about 5 mg to 15 mg, p.o. twice, thrice, or four times daily, or about 5 mg to about 10 mg, p.o. twice, thrice, or four times daily.

[0250] In another embodiment, described herein are packages for daily administration of one or more compounds or compositions described herein according to the methods or uses described herein, including a unit dosage form as described above wherein the unit dosage form is a single or divided daily dose that sums to a daily amount of about 1 mg to about 50 mg of the compound, administered orally.

[0251] In another embodiment, described herein are packages for daily administration of one or more compounds or compositions described herein according to the methods or uses described herein, including a unit dosage form is a single or divided daily dose that sums to a daily amount of about 1 mg to about 40 mg of the compound, administered orally.Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0252] PATENT APPLICATION

[0253] In another embodiment, described herein are packages for daily administration of one or more compounds or compositions described herein according to the methods or uses described herein, including a unit dosage form is a single or divided daily dose that sums to a daily amount of about 1 mg to about 30 mg of the compound, administered orally.

[0254] In another embodiment, described herein are packages for daily administration of one or more compounds or compositions described herein according to the methods or uses described herein, including a unit dosage form is a single or divided daily dose that sums to a daily amount of about 1 mg to about 25 mg of the compound, administered orally.

[0255] In another embodiment, described herein are packages for daily administration of one or more compounds or compositions described herein according to the methods or uses described herein, including a unit dosage form is a single or divided daily dose that sums to a daily amount of about 1 mg to about 20 mg of the compound, administered orally.

[0256] In another embodiment, described herein are packages for daily administration of one or more compounds or compositions described herein according to the methods or uses described herein, including a unit dosage form is a single or divided daily dose that sums to a daily amount of about 1 mg to about 15 mg of the compound, administered orally.

[0257] In another embodiment, described herein are packages for daily administration of one or more compounds or compositions described herein according to the methods or uses described herein, including a unit dosage form is a single or divided daily dose that sums to a daily amount of about 1 mg to about 10 mg of the compound, administered orally.

[0258] It is to be understood that other routes of administration may be used, including buccal, sublingual, parenteral, and the like. It is appreciated herein that when other routes of administration that lead to higher bioavailability are used, the illustrative oral doses described herein will be reduced accordingly.

[0259] In addition to the foregoing illustrative dosages and dosing protocols, it is to be understood that an effective amount of any one or a mixture of the compounds described herein can be readily determined by the attending diagnostician or physician by the use of known techniques and / or by observing results obtained under analogous circumstances. In determining the effective amount or dose, a number of factors are considered by the attending diagnostician or physician, including, but not limited to the species of mammal, including human, its size, age, and general health, the specific disease or disorder involved, the degree of or involvement or theAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0260] PATENT APPLICATION

[0261] severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances.

[0262] In another embodiment, described herein are pharmaceutical compositions, unit doses, and unit dosage forms as described above further comprising one or more carriers, diluents, or excipients, or a combination thereof.

[0263] In making the pharmaceutical compositions of the compounds described herein, a therapeutically effective amount of one or more compounds in any of the various forms described herein may be mixed with one or more excipients, diluted by one or more excipients, or enclosed within such a carrier which can be in the form of a capsule, sachet, paper, or other container. Excipients may serve as a diluent, and can be solid, semi-solid, or liquid materials, which act as a vehicle, carrier or medium for the active ingredient. Thus, the formulation compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. The compositions may contain anywhere from about 0.1% to about 99.9% active ingredients, depending upon the selected dose and dosage form.

[0264] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxybenzoates; sweetening agents; and flavoring agents. The compositions can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art. It is appreciated that the carriers, diluents, and excipients used to prepare the compositions described herein are advantageously GRAS (generally regarded as safe) compounds.

[0265] In another embodiment, described herein is a method for treating a patient, the method comprising the step of administering to the patient a therapeutically effective amount of one orAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0266] PATENT APPLICATION

[0267] more compounds, as described herein, capable of scavenging acrolein or other alpha / beta unsaturated aldehyde compounds.

[0268] In each of the foregoing and following embodiments, it is to be understood that the formulae include and represent not only all pharmaceutically acceptable salts of the compounds, but also include any and all hydrates and / or solvates of the compound formulae. It is appreciated that certain functional groups form complexes and / or coordination compounds with water and / or various solvents, in the various physical forms of the compounds. Accordingly, the above formulae are to be understood to include and represent those various hydrates and / or solvates. In each of the foregoing and following embodiments, it is also to be understood that the formulae include and represent each possible isomer, such as stereoisomers and geometric isomers, both individually and in any and all possible mixtures. In each of the foregoing and following embodiments, it is also to be understood that the formulae include and represent any and all crystalline forms, partially crystalline forms, and non-crystalline and / or amorphous forms of the compounds.

[0269] Incorporation by Reference

[0270] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0271] Equivalents

[0272] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

[0273] EXAMPLES

[0274] Spinal cord injuries (SCI) result in profound motor, sensory, and autonomic functionalAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0275] PATENT APPLICATION

[0276] deficits, often rendering individuals incapable of performing routine daily activities. It is estimated that 27 million people are affected by SCI globally, contributing to an unemployment rate of approximately 60% among this population, which results in a profound social, economic, and emotional impact to SCI victims, care givers, and society at large. Despite intense social awareness and decades of research aiming to enhance post-injury functional recovery through proper management, effective therapies remain elusive, underscoring the urgent need for novel approaches through a deeper understanding of the injury mechanism.

[0277] It is known that the mechanical insult of SCI triggers a series of secondary biochemical reactions that intensify the initial trauma, and both mechanisms contribute to clinical outcomes. Among known post-SCI secondary injury mechanisms, oxidative stress plays a critical pathogenic role in functional loss. A substantial body of evidence indicates that acrolein, a toxic aldehyde that is markedly increased in SCI, plays a pivotal role in secondary injury 4-6. As both a product and catalyst of lipid peroxidation, acrolein damages neuronal tissues by degrading critical biomolecules, impairing mitochondrial function, compromising neuronal membrane integrity, and degrading myelin, thereby perpetuating oxidative stress and neuronal cellular destruction.

[0278] Given acrolein's critical role in post-SCI secondary injury, targeting this aldehyde with specific scavengers has emerged as a promising therapeutic strategy. We have initiated investigations to confirm the beneficial effects of anti-acrolein treatments in central nervous system (CNS) trauma and diseases using several repurposed acrolein scavengers, including hydralazine, phenelzine (Phz), and dimercaprol in SCI, multiple sclerosis (MS), and Parkinson’s disease (PD) models 16-28. These studies have demonstrated improved outcomes, underscoring the effectiveness of neuroprotection through acrolein scavenging.

[0279] However, while a proven, effective treatment for conditions where acrolein is implicated, the use of repurposed drugs is hindered by their intrinsic limitations and side effects unique to each drug, such as lowering blood pressure, inhibiting monoamine oxidase (MAO), and hyperalgesia. As such, strategies that can curtail unwanted side effects while preserving the acrolein-scavenging abilities of these drugs is warranted to facilitate their translation to human application.

[0280] Among these acrolein scavengers, Phz is a better candidate for optimization, offering multiple advantages. Phz is a proven, highly effective acrolein scavenger with a significantlyAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0281] PATENT APPLICATION

[0282] longer half-life (11.6 hrs) than hydralazine (~2 hrs) and dimercaprol (4 hrs) in vivo. However, Phz is also a MAO inhibitor commonly used as an antidepressant and anxiolytic. Such MAO inhibition in both the central and peripheral tissues can lead to significant side effects, including hypertensive crisis, serotonin syndrome, insomnia, and anxiety. Interestingly, the binding structure of a MAO-inhibitor complex is known by protein crystallization - a critical piece of information for structure-based modification. Furthermore, it is known that the functional group responsible for its acrolein-scavenging capability is the hydrazine group. Therefore, we hypothesized that it would be possible to eliminate Phz’s MAO-inhibitory properties without affecting its hydrazine group through chemical modification. As such, we proposed the addition of bulk groups to Phz to prevent its binding to MAO.

[0283] This led to the identification of a novel phenelzine analogue (PhzA), engineered to serve as an effective acrolein scavenger without MAO inhibition. Our studies show that the MAO-inhibition is largely nullified in PhzA, while the efficient acrolein neutralization function of the parent structure is preserved, with PhzA providing substantial neuroprotective benefits in rats with SCI. The current findings signify a new strategy in anti-acrolein drug discovery for significantly galvanizing the advancement of acrolein scavenging therapy.

[0284] Example 1: Structure-Based Modification

[0285] The crystal structure of human MAO-B was retrieved from the RCSB Protein Data Bank (PDB code: 2VRM). The crystallized ligand was removed, and the MAO-B structure was prepared by correcting corresponding atom types, removing crystallized water, and adding hydrogens in Sybyl (Certara USA, Inc., Princeton, NJ, USA). The compound phenelzine was docked into the active site of MAO-B using GOLD 3.1 (The Cambridge Crystallographic Data Centre, CCDC, NJ, USA) based on default parameters. The binding site was defined as all residues within 5 A of the N(5) atom of NAD. A total of 10 docking poses were returned, and the best docking solution according to the GoldScore fitness function was chosen as the binding conformation.

[0286] Example 2: Mass Spectrometry

[0287] The reaction between acrolein and phenelzine analog (PhzA) was confirmed by the 4000Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0288] PATENT APPLICATION QTRAP triple quadrupole / linear ion trap (LIT) hybrid Mass Spectrometer System (Sciex, Toronto, Canada). Briefly, PhzA and acrolein were incubated together for 2 hours in PBS in the dark before being collected for analysis. The solvent was evaporated and the products were reconstituted by isopropanol. The sample was diluted 100 folds by isopropanol and ionized through nanoESI to get a positive ion mode.

[0289] Example 3: Enzyme Activity Assay

[0290] Enzyme activity evaluations were used to test the MAO inhibitory function on both of two isoforms, MAO-A and MAO-B. Monoamine Oxidase A (MAO-A) and Monoamine Oxidase B (MAO-B) Inhibitor Screening Kits (Fluorometric) were obtained from BioVision, Inc.

[0291] (Milpitas, CA, USA). Briefly, enzymes and developers were reconstituted with the assay buffer. The MAO substrate was dissolved in ddH₂O. The MAO-A inhibitor Clorgyline and MAO-B inhibitor Selegiline were used as inhibitor controls. Inhibitor controls and the designed phenelzine analogue (PhzA) were diluted to 10X of the desired test concentration with ddH₂O before being transferred into assigned wells. The same volume of the assay buffer was also added into the assigned wells to serve as an enzyme control without inhibitors. Next, the enzyme solution was put into these wells and incubated for 10 min at 25°C. To check for the possible inhibitory effect of the test inhibitors on the developer, the enzyme solution was replaced with 10 mM H₂O₂. A mixture of the substrate, developer, probe and buffer was then added into each well. The fluorescence (Ex / Em = 535 / 587nm) was measured kinetically at 25°C for 10-30 min and fluorescence lines were generated. The slope of the lines was used to calculate relative inhibition and relative activity.

[0292] Example 4: Animal Experiment

[0293] Healthy adult male (weight 240 - 270g) Sprague-Dawley (SD) rats were purchased from Envigo (Indianapolis, USA). The animals were housed in a facility with a temperature and illumination control, with free access to food and water Ad libitum. Two animals were caged together. The animals were acclimated for at least 1 week before surgery. The rats were randomly divided into the experimental groups: Sham (T10 laminectomy only), SCI (T10 spinal cord injury) and PhzA treatment (SCI with PhzA treatment). All experimental proceduresAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0294] PATENT APPLICATION

[0295] mentioned herein were approved by the Animal Care and Use Committee at the Purdue University (PUCAC).

[0296] Example 5: Rat Spinal Cord Injury Model

[0297] All surgeries were conducted under standard aseptic conditions, with experimental procedures approved by the Purdue University Animal Care and Use Committee. Rats were anesthetized via intraperitoneal injection of a ketamine (80 mg / kg) and xylazine (10 mg / kg) cocktail. Anesthesia was confirmed by the absence of a withdrawal response to a rear foot pinch. After shaving and sanitizing the dorsal skin, a longitudinal incision was made to expose the vertebrae at the T10 level. A laminectomy was performed to access the spinal cord at this site. A moderate contusion injury was induced using the Infinite Horizon Impactor (IH impactor) with an impact force of 200 Kdyne. Following surgery, the animals were placed on a heating pad for recovery and administered NSAID Ketoprofen (10 mg / kg subcutaneously) once daily for three days. Bladders were manually expressed twice daily until reflexive control returned. For the sham group, only the laminectomy was performed at T10 without inducing a spinal cord contusion injury.

[0298] Example 6: Phenelzine Analog Treatment

[0299] Phenelzine analog [(3 -methyl- l-phenylbutan-2-yl) hydrazine hydrochloride] or PhzA (EN300-156135) was designed by our group and purchased from Enamine (NJ, USA). This is the first study on the efficacy of PhzA in SCI; therefore, we opted for a various therapeutic dosage experiment to select the ideal concentration before the long-term experiment. PhzA was dissolved in saline with the concentration of 3 mg / ml and administered through IP injection with corresponding dosages (5, 15, 30 mg / kg) once a day after the SCI. Rats in the Sham and SCI groups received a saline vehicle injection.

[0300] Example 7: Tissue Isolation

[0301] At Day 2 and 28 post-SCI, the rats were anesthetized and perfused intracardially with cold, oxygenated Kreb’s solution. The spinal cord was extracted and a half-inch segment centering the injury site was dissected. Samples for immunoblotting were directly stored in the -80°C and samples for immunohistochemistry were stored in 4% PFA for 24 hours, followed byAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0302] PATENT APPLICATION

[0303] 30% sucrose / PBS solution for 72 hours. The immunohistochemistry samples were frozen in OTC using a dry ice / isopentane slurry. Tissue samples were then cyro-sectioned (Thermo HM525NX) at 25 μm thickness and slices were stored in PBS containing 0.1% sodium azide in 4 °C before biochemical analysis.

[0304] Example 8: Western Blot

[0305] After perfusion, spinal cord tissue was harvested and stored at -80°C. The tissue was homogenized in IxRIPA buffer supplemented with a protease inhibitor cocktail, then centrifuged at 14,000 RPM for 30 minutes. Protein concentrations in the supernatant were determined using the PIERCE BCA Protein Assay Kit (Rockford, IL, USA) and an analytical plate reader (SPECTRAMAX; Molecular Devices, Sunnyvale, CA). Twenty micrograms of protein were mixed with 20% SDS, P-mercaptoethanol (BME), and Laemmli buffer, then loaded onto 15% Tris-HCL gels for electrophoresis. The separated proteins were transferred to a nitrocellulose membrane using the Power Blotter-Semi-dry Transfer System (Thermo Fisher Scientific Inc., MA, USA). The membrane was blocked with lx casein solution (Vector, SP-5020) at room temperature for 30 minutes, then incubated overnight at 4°C with primary antibodies against acrolein-lysine (StressMarq, SMC-504D) or GAPDH (ThermoFisher, PAI-987). The membranes were subsequently incubated with the appropriate secondary antibodies (Vector, BA-2000, #BA-1000) at room temperature for 45 minutes. After washing with 0.1% TBST solution, the membrane was treated with VECTASTAIN ABC-AmP reagent (Vector Laboratories, CA, USA) for 10 minutes to amplify the signal. DuoLuX substrate (Vector, SK-6605) was applied for chemiluminescent detection using the Azure C300 Western blot imaging system (Azure Biosystems, Dublin, CA). Band intensity was quantified using ImageJ software (NIH, USA) and normalized to GAPDH.

[0306] Example 9: DAB Immunohistochemistry

[0307] Spinal sections were washed in PBS solution then placed in 3% H2O2 / water to quench endogenous peroxidase activity. After washing three times in PBS, sections were transferred into the 10% normal goat serum for blocking for 1 hour at room temperature, and then sections were incubated overnight with an anti-acrolein-lysine antibody (StressMarq; SMC-504D) in 4 °C, followed by biotinylated secondary antibodies (Vector Laboratories), incubated in ABCAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0308] PATENT APPLICATION

[0309] avidin / biotin complex solution (Thermo ScientificTM; 32020) and developed using the DAB Peroxidase (HRP) Substrate Kit, 3,3 '-diaminobenzidine (PierceTM DAB Substrate Kit; PI34002) for 4 minutes. Sections were dried on a slide warmer and dehydrated in ascending series of ethanol / water solutions before being mounted with Paraclear (Polyscience; 22463) and toluene solution. Images were taken using a Nikon Eclipse Ti-2 microscope with a standard light camera (Digital Sight 10). Quantification was done using standard techniques for measuring DAB intensity on the ImageJ software.

[0310] Example 10: Immunofluorescence

[0311] Dissected spinal cord slices were washed in 1x PBS and permeabilized in a 1% PBS-Triton solution (Millipore-Sigma, USA) for 1 hour. Blocking was performed using a 0.1% PBS-Triton solution containing 10% normal donkey serum (Millipore-Sigma, USA). The slices were then incubated overnight at 4°C with primary antibodies against Iba-1 (Abeam, ab5076) and GFAP (Invitrogen, MA5-12023) diluted according to the manufacturers' recommendations. After incubation, tissue slices were stained with secondary antibodies (Anti-Mouse Alexa 488, Abeam, USA; Anti-Goat Alexa 594, Abeam, USA) and mounted on coverslips using antifade mounting medium with DAPI (Vector, H2000). Images were captured using a Nikon Eclipse Ti-2 AX confocal microscope. Imaging areas within the target regions were randomly selected, and signal intensity was calculated and averaged from at least three slices per animal. Fluorescence intensity quantification was performed using ImageJ software.

[0312] Example 11: Data Analysis and Statistics

[0313] Statistical analysis was performed using Prism 10. Multiple comparisons were carried out by ordinary One-Way ANOVA or Brown-Forsythe and Welch ANOVA tests. The Student's t-test was used when comparing only two groups, p < 0.05 was considered statistically significant, and the results were expressed as the mean ± SEM.

[0314] Example 12: Structure-Based Modification and Design Criteria

[0315] Based on the mechanism that phenelzine irreversibly inhibits MAO, one way to eliminate the MAO inhibitory activity is to decrease its binding affinity with the MAO active site. To investigate the binding mode of phenelzine in the active site of MAO, a molecular docking studyAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0316] PATENT APPLICATION

[0317] was carried out based on the crystal structure of MAO-B. The docking results showed that the terminal nitrogen of phenelzine’s hydrazine group was approaching the flavin on MAO-B and forming two hydrogen bonds with surrounding waters. The hydrogen in the central nitrogen of phenelzine’s hydrazine group pointed toward the N(5) of flavin, which is consistent with the proposed inactivation mechanism. The phenyl ring was situated in a small pocket surrounded by Glu206, Ilel98, Cysl72, Leul71, and Tyr326 (FIG. 1A).

[0318] Our strategy is to introduce a bulky group (i-propyl group) at the Cl position to form a compound named (3-methyl-l-phenylbutan-2-yl) hydrazine hydrochloride (FIG. IB). According to the docking results, Cl is pointing towards flavin. Incorporating a bulky group in this position will result in a seriously steric clash with flavin. This steric clash would prevent the hydrazine group from approaching flavin and therefore stop the initiation of enzyme inactivation.

[0319] Phenelzine Analog Mitigates Acrolein Surge and Eliminates MAO-Inhibitory Activity First, we used mass spectrometry (MS) to verify that PhzA maintains the ability to react with acrolein. The product mixture of acrolein and PhzA was collected after incubation. MS analysis showed that the ionized cation form of the proposed products had a molecular weight of 217.51 (FIG. 1C), demonstrating the existence of the proposed products in the reaction mixture and indicating the occurrence of the proposed reaction between PhzA and acrolein (FIG. IB). To further verify their chemical structures, the compounds at peak 217.51 were cleaved into small fragments via MS-MS (FIG. ID), revealing several characteristic peaks at 71.56, 91.44 (methylbenzene), and 105.30 (styrene), respectively. Notably, these fragments all originated from the proposed products, confirming the chemical identity of the peak observed at 217.51.

[0320] Next, we performed an enzymatic activity assay to test the inhibitory abilities of PhzA and Phz on both MAO-A and MAO-B. The selective MAO-A inhibitor clorgyline and MAO-B inhibitor selegiline were used as positive controls. The enzyme activities of MAO-A were 108.20 ± 0.96%, 111.97 ± 1.63%, 100.65 ± 3.41%, and 45.94 ± 1.40% with Phz concentrations of 1, 10, 100, and 1000 nM. Phenelzine did not show significant inhibitory effects until the concentration reached 1000 nM (1 pM). Meanwhile, the enzyme activities remained at 105.00 ± 2.31%, 107.44 ± 2.03%, 106.09 ± 1.28%, and 107.10 ± 1.17% under various concentrations of PhzA, indicating that PhzA did not inhibit MAO-A (FIG. IE). Similarly, Phz showed around 5% inhibition at a concentration of 1 pM, while PhzA did not show any inhibitory effect on MAO-B, with activities of 119.69 ± 7.40%, 109.99 ± 2.32%, 117.38 ± 9.00%, and 117.25 ± 6.69% at all concentrationsAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0321] PATENT APPLICATION

[0322] (FIG. IF). These findings suggest that PhzA retains its ability to clear acrolein while eliminating the MAO inhibitory mechanism that could cause adverse effects, making it an ideal experimental drug candidate.

[0323] Example 13: Dose-Dependent Acrolein Suppression by Phenelzine Analog after SCI at Acute Stage

[0324] Toxic reactive aldehydes, represented by acrolein, are generated by oxidative stress induced lipid peroxidation, which plays a particularly damaging role in secondary injury, causing cellular degeneration and functional loss in SCI39,40. Immunoblotting (FIG. 2A, top) and densitometry quantification (FIG. 2B) demonstrated a significant elevation (672%) of acroleinlysine adducts in the spinal cord compared with sham-operated rats (p < 0.001) at 48 hours after trauma. To evaluate the effectiveness of acrolein scavenging by PhzA in the rat SCI model, PhzA was administered at various concentrations (5, 15, and 30 mg / kg) once a day for 2 days post-SCI (n = 3-4 per group). Rats that received PhzA treatment showed a dose-dependent reduction in acrolein levels in the spinal cord (FIG. 2A, below). Notably, dosages of 15 mg / kg and 30 mg / kg significantly suppressed acrolein levels compared to the SCI-only group (48.3%, p < 0.01 and 41.8%, p < 0.05, respectively). These findings indicate that PhzA can efficiently reduce acrolein in rats within 48 hours after SCI. Moreover, we chose the dosage of 15 mg / kg for subsequent animal studies to minimize potential side effects from the drug administration, as the efficacy was similar between the 15 mg / kg and 30 mg / kg concentrations (p = 0.64).

[0325] Example 14: Phenelzine Analog Administration Lowers Acrolein Load in the Spinal Cord at Chronic Stage

[0326] Acrolein has been reported to persist at elevated levels for more than 2 weeks and continuously propagate degeneration in SCI. Therefore, after determining the ideal dosage of PhzA in the rat in-vivo model, we tested its anti-acrolein effect over a longer period using immunohistochemistry. PhzA was administered intraperitoneally once a day for the first 2 weeks after SCI. Acrolein levels, represented by the DAB signal intensity, were measured in the spinal cord at 28 days post-injury. Tissues in the SCI group displayed a prolonged acrolein load compared to the sham group (155.16 ± 7.51%, p < 0.0001) (FIG. 3B, left). The white matter in the spinal cord, which contains a high concentration of myelinated axons, showed a moreAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

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[0328] exaggerated acrolein buildup (181.95 ± 8.55%) (FIG. 3B, middle) compared to the gray matter (138.94 ± 5.88%) (FIG. 3B, right). However, treatment with PhzA significantly decreased acrolein levels in all parts of the spinal cord at 28 days post-SCI (p < 0.05, p < 0.01, and p < 0.01, respectively).

[0329] Example 15: Phenelzine Analog Protects Against Glial Scars Formation and Inflammation of SCI Rats

[0330] Overwhelming inflammatory and astrocytic responses in the CNS after injury lead to chronic inflammation and biochemical barriers, exacerbating the initial damage. We doublestained active microglia / macrophages with the Iba-1 marker and reactive astrocytes with the GFAP marker to assess their intensity in the spinal cord at 0.5 mm caudal to the epicenter. We also examined different regions of the neuroaxis, including the central canal, dorsal horn, ventral horn in the gray matter, and the ventral and lateral columns in the white matter.

[0331] Following the injury, we observed an increase in the number of reactive astrocytes, which developed thicker processes and exhibited a more ramified appearance morphologically in the spinal cord (FIGS. 4A-C) and all regions of interest (FIGS. 4B-C). This led to a significant increase in GFAP staining intensity across the entire spinal cord and all measured sub-areas (p < 0.01 to 0.0001). In comparison, PhzA treatment significantly reduced GFAP-positive gliosis at 28 days compared to the SCI group (FIGS. 4A-C).

[0332] Activated microglia / macrophages labeled with Iba-1 displayed a ramified morphology under normal conditions (FIG. 5B, first row, white arrows). In contrast, tissues from the SCI group contained widely spread amoeboid, immunologically activated microglia, characterized by increased soma size and less ramified processes (FIG. 5B, second row, red arrows).

[0333] Quantification of the Iba-1 signal intensity showed that microglia activation significantly increased in the spinal cord after 28 days (FIG. 5C, p < 0.0001). PhzA treatment significantly reduced the persistence of inflammation in the entire spinal cord, presenting more resting-state ramified microglia cells. When analyzed more regionally, most areas of the gray matter had recovered to baseline levels (sham). However, the signal in the lateral column (p < 0.001) and the dorsal column (p = 0.504) was reduced but remained elevated when compared to sham, indicating sustained neuroinflammation associated with oxidative stress in the lipid-rich white matter.Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0334] PATENT APPLICATION

[0335] Example 16: Phenelzine Analog relieved locomotor and sensory impairments after SCI The BBB locomotor rating scale was used to assess functional recovery and locomotor behavior in chronic SCI studies. Animals that suffered from SCI initially exhibited flaccid paralysis and scored 0-1 point immediately after the injury, with subsequent modest timedependent recovery over 28 days (FIG. 6A). Rats in the PhzA treatment group showed statistically significant improvement in lower-limb motor function at both 7 and 28 days compared to rats that did not receive any treatment (n = 7 per group, p < 0.05).

[0336] Neuropathic pain is a common symptom following SCI in both human patients and animal models. Mechanical allodynia was assessed using the von Frey filament test (VF) on the hind limbs before and from day 7 after SCI. Prior to sham and SCI surgery, the paw withdrawal thresholds of all animals reached the cut-off of 15 g. Rats that underwent spinal cord injury showed a significant drop in mechanical thresholds starting at day 7, indicating hypersensitivity to noxious stimuli. In contrast, animals receiving the PhzA treatment exhibited significant recovery from hyperreflexia, and this statistical significance was maintained throughout the study period (FIG. 6B). Therefore, PhzA appears to effectively alleviate both motor deficits and neuropathic pain following SCI.

[0337] Example 17: Ability of compounds to provide neuroprotection

[0338] We have shown that the phenelzine analog (3-methyl-l-phenylbutan-2-yl) (PhzA), which has limited MAO inhibition, could provide significant neuroprotection in a rat contusive SCI model. Similar to its parent compound, PhzA could significantly reduce acrolein by both 2 days (acute), and 28 days (chronic) after injury, suppressing both microglial activation and astrocyte proliferation, which are accompanied by significant improvements in motor and sensory function up to 28 days post-injury compared to the untreated injury group. To our knowledge, this is the first reported attempt to structurally optimize a known, repurposed acrolein scavenger to eliminate undesirable side effects while effectively retaining its acrolein scavenging capability. This effort has resulted in a novel effective scavenger, PhzA, which will likely achieve an accelerated path towards clinical application due to its close structural similarities with phenelzine (Phz), an FDAapproved medication. More importantly, such findings also underscoreAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

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[0340] the effectiveness of targeted, structure-based chemical modification to generate new acrolein scavengers, broadening the scope of drug discovery for novel anti-acrolein strategies.

[0341] While Phz has been demonstrated to be neuroprotective with effective acrolein scavenging and anti-inflammatory properties in various neurotrauma and neurological disease models, its original FDA-approved indication as a monoamine oxidase (MAO) inhibitor will likely present undesirable side effects for clinical anti-acrolein treatment. Specifically, Phz-mediated MAO inhibition can potentially lead to mania and other mood disorders, with additional side effects affecting the peripheral nervous system, complicating and potentially hindering the application of Phz as an acrolein scavenger33. This is of particular concern among CNS neurotrauma patients, in whom undesirable mental abnormalities and other unwanted side effects could be especially troublesome. Therefore, despite its excellent record of being an acrolein neutralizer, Phz in its original structure is unlikely to be translated to clinical application to safely and effectively combat acrolein toxicities. Motivated by the effective acrolein scavenging capability and this significant limitation of Phz, we have designed a strategy to introduce a bulk group (i-propyl group) at the side chain of the parent molecule of phenelzine (Cl position), aiming to present a steric clash with flavin and surrounding residues and therefore decrease its binding affinity with MAO. This hypothesis was tested with enzyme activity evaluations in the current study, confirming PhzA lacked MAO inhibitory function on both isoforms of this enzyme, MAO-A and MAO-B (FIGS. 1E-F). Importantly, with such a modification, the functional hydrazine group of Phz should be preserved to maintain its acrolein neutralizing effects. This theoretical reasoning was confirmed experimentally based on MS and MS-MS spectra in a cell-free, abiotic condition (FIGS. 1C-D). Taken together, the newly designed compound PhzA resulting from structure-based chemical modification is an effective acrolein scavenger that is free from MAO inhibition.

[0342] It is well established that after SCI, acrolein accumulates rapidly, peaking at 24 - 48 h post injury, and remains elevated for at least 14 days. In the current study, we have revealed that acrolein overload persists in the spinal cord, in both the white and gray matter, for up to 28 days, a significantly longer period than previously shown. This prolonged presence of acrolein further strengthens its critical, long-lasting, and detrimental role in post-SCI secondary injury.

[0343] Furthermore, this data also signifies a correspondent expanded therapeutic window forAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

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[0345] anti-acrolein intervention, spanning from the acute to chronic phase. As acrolein is known to harm neuronal cells and their processes, the persistent acrolein elevation in both white and gray matter could contribute to neuronal cell death as well as axonal and myelin damage, underlying the long-term, functional deficits observed after SCI. Clearly, PhzA, when applied immediately after injury and daily thereafter for 14 days, can significantly reduce acrolein accumulation in both the white and gray matter of the spinal cord, as assessed acutely (2 days post injury), and chronically (28 days post injury) (FIGS. 2A-B and 3A-B). This is consistent with the behavioral improvements resulting from PhzA application assessed during the same period (FIGS. 6A-B). As such, the ability of PhzA to suppress acrolein appears to be rapid and persistent, resulting in neuroprotective, motor, and sensory benefits at both the acute and chronic stages to effectively treat SCI.

[0346] Following SCI, it is known that increased ROS and elevated myeloperoxidase (MPO) activity contributes to the generation of acrolein through lipid peroxidation54,55. In particular, MPO, an enzyme found in immune cells like neutrophils, macrophages, and activated microglia cells, produces acrolein as a byproduct. SCI disrupts the blood-brain barrier, leading to significant macrophage infiltration and accumulation at the injury site, accompanied by activation and proliferation of resident microglia58. This results in localized, sustained inflammation, which is characterized by an increase in macrophage and microglia peaking around 7 and 60 days postinjury before gradually declining. Not only does this inflammation contribute to the generation of acrolein, but it is well documented that acrolein, a known pro-inflammatory aldehyde, could lead to the activation of macrophages / microglia cells and MPO. Therefore, the interplay of MPO, macrophages / microglia, and acrolein may contribute to a vicious cycle that underlines the prolonged activation of both inflammation and oxidative stress in SCI. The fact that PhzA can not only lower acrolein, but also dampen inflammation (FIGS. 5A-C), is consistent with the notion that acrolein is a critical factor in this vicious cycle, a causality of inflammation, and an effective therapeutic target to suppress post-SCI oxidative stress and inflammation.

[0347] In addition to immune cell involvement, astrocytes, the most abundant glial cells in the central nervous system, play a crucial pathological role in SCI through the formation of glial scars that act as both physical and chemical barriers to axonal regeneration, impeding functional recovery. In fact, several studies have indicated that both microglia and astrocytes could beAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

[0348] PATENT APPLICATION

[0349] activated in parallel and are correlated with the severity of SCI, suggesting a potential interaction between these two players of secondary injury mechanisms. Consistent with these observations, our data revealed an increasing expression / activation of both microglia (Iba1) and astrocytes (GFAP) at 28 days post-SCI, indicating sustained inflammation and astrocytic gliosis at the chronic stage (FIGS. 4A-C and 5A-C). Similarly, by targeting acrolein, PhzA treatment has significantly reduced glial scar formation in animals following SCI. This is expected, among other benefits, to promote axonal regrowth after SCI, further supporting the potential of acrolein scavenging as an effective therapeutic strategy.

[0350] Neuropathic pain resulting from spinal cord injury (SCI) has profound, long-term consequences, often exceeding the impact of other functional disabilities and severely diminishing patients’ quality of life. Chronic pain often emerges due to persistent inflammation, with more than half of SCI patients reporting persistent pain within a year of injury. Consistent with its anti-oxidative stress and anti-inflammatory effects, PhzA treatment significantly reduced pain at all time points (FIG. 6B), as assessed by Von Frey mechanical tests. It is known that acrolein can activate the pro-algesic transient receptor potential ankyrin 1 (TRPA1), while also upregulating the expression of TRPA1 in animal models of SCI and Parkinson’s disease.

[0351] Therefore, the PhzA-induced analgesic effect in SCI is likely through the decrease of the activation of TRPA1 by acrolein, as well as the lessening in TRPA1 expression from a reduction of acrolein. This data is comparable to other known acrolein scavengers that have been shown to alleviate hyperalgesia in the rodent SCI models with similar mechanisms.

[0352] It is worth noting that MAO inhibitors, such as moclobemide and selegiline, have been shown to reduce postoperative and sensory hypersensitivity in rodent models, independent of acrolein scavenging. Unlike its parent compound Phz, PhzA has minimal MAO inhibition yet still offers robust analgesic efficacy, suggesting that acrolein scavenging is likely the main reason for PhzA’s ability to mitigate post-SCI hypersensitivity. In fact, since PhzA and Phz demonstrated comparable pain alleviation effects upon a similar treatment regimen (15 mg / kg), 14 days immediately following injury21,2439,78 (FIG. 6B), it is reasonable to speculate that acrolein scavenging is also the main contributor for the analgesic effect of Phz. In further support of this anti-acrolein-mediated analgesic effect, hydralazine, which also possesses a hydrazine group for acrolein scavenging and has no MAO inhibition, could significantly reduce post-SCI neuropathic pain. Similar to sensory functional benefits, the assessment of locomotor functionAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768

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[0354] using the BBB score also revealed significant improvements in PhzA-treated rats at 7 and 28 days postinjury (FIG. 6A), which is consistent with the PhzA-mediated reduction of acrolein, microglia activation, and astrocytes gliosis (FIGS. 4A-C and 5A-C). These findings collectively support PhzA as a promising candidate for mitigating neurological deficits following SCI.

[0355] There are several additional investigations that need to be addressed if PhzA is to be developed for clinical applications. First, although PhzA has only a minor structural modification from Phz, which is clinically available, the safety and pharmacokinetic properties of PhzA, including half-life and blood-brain barrier permeability, need to be confirmed. Second, this study was conducted exclusively on male rats, owing to the higher incidence of SCI in males (male-to-female ratio of - 2:1). However, for greater clinical relevance, future studies should include female subjects. Third, the current study mainly focused on two time points (2 days and 28 days post-injury). Future investigations should conduct assessments at the intermediate (e.g., 7 days) and extended chronic time points (e.g., up to 6 months) to evaluate long-term neuroprotective effects on biochemical, morphological, and behavioral recovery. This would be beneficial due to evidence indicating the persistence of deficits at an extended timepoint, such as microglial accumulation found at 60 days. Lastly, future research is needed to further explore the molecular mechanisms underlying the neuroprotective effects of acrolein scavenging, such as its impact on oxidative stress signaling pathways and the polarization of microglia, macrophages, and astrocytes.

[0356] In summary, the novel compound PhzA has shown promising capabilities in scavenging acrolein, promoting motor functional recovery, and attenuating sensory disorders following spinal cord injury, all while avoiding the side effects typically associated with MAO inhibition. These findings not only highlight the potential of PhzA to serve as a novel therapeutic agent for SCI, but also underscores the broader relevance of targeting acrolein to treat various neurological conditions where acrolein-mediated toxicity is implicated. Furthermore, this study suggests that structure-based modification is a feasible and effective method for the development of novel acrolein scavengers.

Claims

Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768PATENT APPLICATIONWhat is claimed is:

1. A composition comprising a pharmaceutically acceptable carrier and a compound of formula I:Iwherein:R1is a straight or branched chain alkyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkenyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkyne optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a mono- or poly- cyclic ring structure optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3;R1may also optionally be COR3, COOR3, or NR3;R2is a straight or branched chain alkyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkenyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkyne optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a mono- or poly- cyclic ring structure optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3;R2may also optionally be COR3, COOR3, or NR3and;R3is hydrogen; a straight or branched chain alkyl optionally substituted by one or more heteroatoms; a straight or branched chain alkenyl optionally substituted by one or more heteroatoms; a straight or branched chain alkyne optionally substituted by one or moreAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768PATENT APPLICATIONheteroatoms; or a mono- or poly- cyclic ring structure optionally substituted by one or more heteroatoms.

2. The composition according to claim 1, wherein R1is a straight or branched chain alkyl.

3. The composition according to claim 2, wherein R1is a C1-C6alkyl.

4. The composition according to claim 3, wherein R1is a C1-C3 branched chain alkyl.

5. The composition according to claim 4, wherein R2is a C1-C6alkyl substituted by a monocyclic ring structure.

6. The composition according to claim 5, wherein the monocyclic ring structure is a phenyl group.

7. The composition according to claim 1, wherein the compound of formula I is the compound of formula II:II8. The composition according to claim 1, further comprising a pharmaceutically acceptable salt.

9. The composition according to claim 8, wherein the pharmaceutically acceptable salt is hydrochloride.

10. The composition according to claim 1, wherein the composition is formulated for oral administration.Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768PATENT APPLICATION11. The composition according to claim 1, wherein the composition is formulated as a single unit dosage.

12. A method for scavenging an alpha / beta unsaturated aldehyde compound from a patient, the method comprising:providing to a patient a compound of formula I:wherein:R1is a straight or branched chain alkyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkenyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkyne optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a mono- or poly- cyclic ring structure optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3;R1may also optionally be COR3, COOR3, or NR3;R2is a straight or branched chain alkyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkenyl optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a straight or branched chain alkyne optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3; a mono- or poly- cyclic ring structure optionally substituted by one or more heteroatoms, wherein one or more of the atoms may be optionally substituted by R3;R2may also optionally be COR3, COOR3, or NR3and;R3is hydrogen; a straight or branched chain alkyl optionally substituted by one or more heteroatoms; a straight or branched chain alkenyl optionally substituted by one or moreAttorney Docket No.: PURD- 166 / 01 WO 28593 / 768PATENT APPLICATIONheteroatoms; a straight or branched chain alkyne optionally substituted by one or more heteroatoms; or a mono- or poly- cyclic ring structure optionally substituted by one or more heteroatoms.

13. The method according to claim 12, wherein R1is a straight or branched chain alkyl.

14. The method according to claim 13, wherein R1is a C1-C6alkyl.

15. The method according to claim 14, wherein R1is a C1-C3 branched chain alkyl.

16. The method according to claim 15, wherein R2is a C1-C6alkyl substituted by a monocyclic ring structure.

17. The method according to claim 16, wherein the monocyclic ring structure is a phenyl group.

18. The method according to claim 12, wherein the compound of formula I is the compound of formula II:II19. The method according to claim 12, wherein the compound of formula I is formulated with a pharmaceutically acceptable salt.

20. The method according to claim 19, wherein the pharmaceutically acceptable salt is hydrochloride.

21. The method according to claim 12, wherein the acrolein is associated with a disease.Attorney Docket No.: PURD- 166 / 01 WO 28593 / 768PATENT APPLICATION22. The method according to claim 21, wherein the disease is associated with degeneration of nervous system tissue.

23. The method according to claim 12, wherein the alpha / beta unsaturated aldehyde compound is acrolein.

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